- Email: [email protected]
Mechanisms of macular edema: Beyond the surface
Accepted Manuscript Mechanisms of macular edema: Beyond the surface Alejandra Daruich, Alexandre Matet, Alexandre Moulin, Laura Kowalczuk, Michaël Nicolas, Alexandre Sellam, Pierre-Raphaël Rothschild, Samy Omri, Emmanuelle Gélizé, Laurent Jonet, Kimberley Delaunay, Yvonne De Kozak, Marianne Berdugo, Min Zhao, Patricia Crisanti, Francine Behar-Cohen PII:
S1350-9462(17)30075-7
DOI:
10.1016/j.preteyeres.2017.10.006
Reference:
JPRR 694
To appear in:
Progress in Retinal and Eye Research
Received Date: 30 July 2017 Revised Date:
24 October 2017
Accepted Date: 31 October 2017
Please cite this article as: Daruich, A., Matet, A., Moulin, A., Kowalczuk, L., Nicolas, Michaë., Sellam, A., Rothschild, Pierre.-Raphaë., Omri, S., Gélizé, E., Jonet, L., Delaunay, K., De Kozak, Y., Berdugo, M., Zhao, M., Crisanti, P., Behar-Cohen, F., Mechanisms of macular edema: Beyond the surface, Progress in Retinal and Eye Research (2017), doi: 10.1016/j.preteyeres.2017.10.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Mechanisms of macular edema: beyond the surface Alejandra Daruich,1,2* Alexandre Matet,2* Alexandre Moulin,2 Laura Kowalczuk,2 Michaël Nicolas,2 Alexandre Sellam,1 Pierre-Raphaël Rothschild,1 Samy Omri,1 Emmanuelle Gélizé,1 Laurent Jonet,1 Kimberley Delaunay,1 Yvonne De Kozak, 1 Marianne Berdugo,1 Min Zhao,1 Patricia Crisanti,1 Francine Behar-Cohen1,3,4
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* Both authors contributed equally to this work. 1
INSERM, UMRS1138, Team 17, From Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, 15 rue de l’Ecole de Médecine, 75006 Paris, France
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Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Avenue de France 15, 1004 Lausanne, Switzerland 3
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Ophtalmopole, Cochin Hospital, AP-HP, Assistance Publique Hôpitaux de Paris, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France
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Faculty of Biology and Medicine, University of Lausanne, Rue du Bugnon 21, 1011 Lausanne, Switzerland
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Corresponding author: Francine Behar-Cohen Inserm U1138, Team 17, Centre de Recherche des Cordeliers 15 rue de l’Ecole de Médecine 75006 Paris, France Tel : +33-144278169 E-mail: [email protected]
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Funding sources: This work was supported by the Agence Nationale de la Recherche, France (ANR-15-CE180032 “ROCK SUR MER”), by the Swiss National Science Foundation (#320030_156401), by the Faculty of Biology and Medicine Research Commission Fund, University of Lausanne, Switzerland, by the Abraham J. & Phyllis Katz Foundation, Atlanta, GA, and by the Lowy Medical Research Institute, San Diego, CA.
ACCEPTED MANUSCRIPT Mechanisms of macular edema: beyond the surface
ABSTRACT Macular edema consists of intra- or subretinal fluid accumulation in the macular region. It
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occurs during the course of numerous retinal disorders and can cause severe impairment of central vision. Major causes of macular edema include diabetes, branch and central retinal vein occlusion, choroidal neovascularization, posterior uveitis, postoperative inflammation and central serous chorioretinopathy. The healthy retina is maintained in a relatively
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dehydrated, transparent state compatible with optimal light transmission by multiple active and passive systems. Fluid accumulation results from an imbalance between processes
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governing fluid entry and exit, and is driven by Starling equation when inner or outer bloodretinal barriers are disrupted. The multiple and intricate mechanisms involved in retinal hydro-ionic homeostasis, their molecular and cellular basis, and how their deregulation lead to retinal edema, are addressed in this review. Analyzing the distribution of junction proteins
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and water channels in the human macula, several hypotheses are raised to explain why edema forms specifically in the macular region. “Pure” clinical phenotypes of macular edema, that result presumably from a single causative mechanism, are detailed. Finally, diabetic
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macular edema is investigated, as a complex multifactorial pathogenic example. This comprehensive review on the current understanding of macular edema and its mechanisms
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opens perspectives to identify new preventive and therapeutic strategies for this sightthreatening condition.
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Macula, edema, mechanisms, diabetes, retina, cysts
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ACCEPTED MANUSCRIPT ARTICLE HIGHLIGHTS
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Cells forming inner and outer blood-retinal barriers maintain retinal homeostasis
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Macular edema results from an imbalance between fluid entry and drainage
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mechanisms
Intraretinal accumulation of macromolecules osmotically attracts water and solutes
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The structural organization of the retina explains why edema develops in the macula
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A glymphatic system may be formed by AQP4 expression along macular Müller cells
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ACCEPTED MANUSCRIPT TABLE OF CONTENTS
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Introduction .............................................................................................................. 8 1.1 Histology of the retina and macula: brief overview........................................................ 8 1.2 General introduction to macular edema...................................................................... 10 1.2.1 Limitations to study macular edema ............................................................................... 11 1.2.2 Macular edema: an underestimated public health issue ................................................ 11
What controls the retinal hydro-ionic and osmotic homeostasis?............................. 12
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2.1 Regulation of fluid and molecule entry: retinal barriers ............................................... 13 2.1.1 Inner blood-retinal barrier............................................................................................... 13
2.1.2
Endothelial cells and their intercellular junctions ............................................................... 13 Transendothelial transport .................................................................................................. 15 Pericytes .............................................................................................................................. 16 Glial cells .............................................................................................................................. 17
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2.1.1.1 2.1.1.2 2.1.1.3 2.1.1.4
Outer blood-retinal barrier .............................................................................................. 18
2.1.2.1 2.1.2.2
Junctional complex of the retinal pigment epithelium ........................................................ 18 Outer limiting membrane .................................................................................................... 19
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2.2 Regulation of fluid exit: drainage mechanisms ............................................................ 20 2.2.1 Retinal glial Müller cells ................................................................................................... 22 2.2.2 Retinal pigment epithelium ............................................................................................. 24
Why does retinal edema form?................................................................................ 25 3.1 Increased retinal fluid entry ........................................................................................ 25 3.1.1 Increased fluid entry through the inner blood-retinal barrier ........................................ 25
Increased fluid entry through outer retinal barriers ....................................................... 33
3.1.2.1 3.1.2.2 3.1.2.3
Retinal pigment epithelium and choroidal vessel junction alteration ................................. 33 Retinal pigment epithelial cell death ................................................................................... 37 Outer limiting membrane disruption................................................................................... 37
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Junctional complex alteration.............................................................................................. 26 Enhanced transcellular permeability ................................................................................... 28 Loss of endothelial cells ....................................................................................................... 29 Loss of pericytes .................................................................................................................. 29 Role of leucocytes in inner blood-retinal barrier breakdown .............................................. 31 Neovascularization and vessel abnormalization.................................................................. 32
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3.1.1.1 3.1.1.2 3.1.1.3 3.1.1.4 3.1.1.5 3.1.1.6
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Why does edema specifically form in the macula? Several hypotheses..................... 43 4.1 4.2 4.3 4.4
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Alteration of junction proteins in the “Z-shaped” Müller cells zone.............................. 43 The “glymphatic” system hypothesis .......................................................................... 44 Increased interstitial pressure: vascular consequences ................................................ 46 The formation of “cysts” ............................................................................................. 46
Why does macular edema alter vision? .................................................................... 47 5.1 5.2 5.3
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3.2 Decreased drainage functions ..................................................................................... 38 3.2.1 Glial cells and neurosensory retina ................................................................................. 38 3.2.2 Retinal pigment epithelium ............................................................................................. 39 3.3 Protein leakage .......................................................................................................... 41
Evaluation of visual function in macular edema........................................................... 48 Acute effects of macular edema on visual function...................................................... 49 Long-term effects of macular edema on visual function ............................................... 50
What can we learn from pure phenotypes? ............................................................. 52 6.1 6.2
Irvine-Gass postoperative macular edema .................................................................. 52 Idiopathic macular telangiectasia type 1 ..................................................................... 53
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Idiopathic macular telangiectasia type 2 ..................................................................... 53 Paclitaxel-induced maculopathy ................................................................................. 54 MEK-inhibitor-associated maculopathy ....................................................................... 55 Hypoproteinemia ....................................................................................................... 56 Paracentral acute middle maculopathy ....................................................................... 56
Major pathways identified as causative of macular edema ...................................... 57
Inflammatory molecules .................................................................................................. 63
7.6.2.1 7.6.2.2 7.6.2.3 7.6.2.4 7.6.2.5 7.6.2.6 7.6.2.7
General mechanisms contributing to diabetic ME .................................................... 68
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The Polyol pathway .................................................................................................... 69 The AGE pathway ....................................................................................................... 69 Protein kinase C (PKC) activation ................................................................................ 70 Local renin-angiotensin system (RAS) .......................................................................... 70 Inflammation and oxidative stress .............................................................................. 71 Blood-retinal barrier dysfunction ................................................................................ 71 Drainage functions ..................................................................................................... 72 Kinetic of events in the diabetic GK rat model ............................................................. 72
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8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8
Concluding remarks and future directions................................................................ 75
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Complement components ................................................................................................... 63 Tumor Necrosis Factor-α (TNF-α) ........................................................................................ 64 Interleukin-1β (IL-1β)........................................................................................................... 66 Interleukin-6 (IL-6) ............................................................................................................... 67 Interleukin-8 (IL-8) ............................................................................................................... 67 Intercellular Adhesion Molecule 1 (ICAM-1) ....................................................................... 68 Transforming growth factor-ß (TGF-β) ................................................................................ 68
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Microglia/macrophages ....................................................................................................... 61 Retinal Müller glial cells ....................................................................................................... 62 Astrocytes ............................................................................................................................ 62 Retinal pigment epithelial cells ............................................................................................ 62
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7.6.1.1 7.6.1.2 7.6.1.3 7.6.1.4
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7.1 VEGF/PGF pathway .................................................................................................... 57 7.2 Kinin/kallikrein system ............................................................................................... 58 7.3 Angiopoietin-2 (Ang2)/Tie2/α /α3β /α β1 pathway ................................................................. 59 7.4 Renin-angiotensin system and angiotensin-converting enzyme ................................... 59 7.5 The mineralocorticoid pathway .................................................................................. 59 7.6 Inflammation and inflammatory mediators with pro-edematous effects...................... 60 7.6.1 Resident immune-competent cells.................................................................................. 61
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AGEs: Advanced glycation end-products AMD: age-related macular degeneration Ang2: Angiopoietin 2 AQP: aquaporin AT: angiotensin II receptor BDNF: Brain-derived neurotrophic factor BIGH3: Transforming growth factor-ß-induced protein Ig-h3 C: complement factor Cav-1: caveolin-1 CRALBP: cellular retinaldehyde-binding protein DAG: diacyglycerol ENAC: epithelial sodium channel ERK: extracellular signal-regulated kinase GFAP: glial fibrillary acid protein GS: glutamine synthetase GSK-3ß: Glycogen synthase kinase 3 ß HBMvEC: human brain microvascular endothelial cells hESC: human embryonic stem cell HIF: hypoxia inducible factor HUVEC: human umbilical vein endothelial cell line cell ICAM: Intercellular adhesion molecule IL: interleukin ILM: inner limiting membrane INL: inner nuclear layer iNOS: inducible nitric oxide synthase IPL: inner plexiform layer JAM: junctional adhesion molecule JNK: c-Jun N-terminal kinase Kir: K+-inward rectifying KKS: Kinin/kallikrein system LPS: lipopolysaccharide MAPK: mitogen-activated protein kinase MCP-1: Monocyte chemotactic protein 1 ME: macular edema MLC: myosin light-chain MMP: matrix metalloproteinase MR: mineralocorticoid receptor NF-kB: nuclear factor-kB NGF: nerve growth factor Nrf2: Nuclear factor erythroid 2-related factor 2 OCT: optical coherence tomography OLM: outer limiting membrane ONL: outer nuclear layer OPL: outer plexiform layer PAR2: protease activated receptor 2 PCR: polymerase chain reaction PDGF: platelet-derived growth factor PEDF: pigment epithelium-derived factor PGF: placental growth factor PI3K: phosphatidylinositol 3-kinase PIP3: phosphatidylinositol-3,4,5-trisphosphate PKC: protein kinase C RMG cells: retinal Müller glial cells
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ABBREVIATIONS
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ROCK-1: Rho-associated protein Kinase 1 RPE: retinal pigment epithelium SHP-1: Src homology-2 domain-containing phosphatase-1 STAT3: Signal transducer and activator of transcription 3 STZ: streptozotocin TGF: transforming growth factor Tie: tyrosine kinase with immunoglobulin and EGF homology domains TLR: Toll-like receptor TNF: tumor necrosis factor TNFR: TNF receptor TRAF: TNF receptor associated factors TRPV4: Transient receptor potential cation channel subfamily vanilloid member 4 VEGF-R: vascular endothelial growth factor receptor VEGF: vascular endothelial growth factor ZO-1: zonula occludens-1
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1 Introduction 1.1
Histology of the retina and macula: brief overview
The neural retina is located in the posterior segment of the eye, between the vitreous
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anteriorly and the sclera posteriorly, and lies on the retinal pigment epithelium (RPE) that is closely associated with the choroid (Figure 1). The neural retina is a multi-layer specialized tissue, composed from the innermost to the outermost layer by the inner limiting membrane (ILM) which is the basal membrane of the retinal Müller glial (RMG) cells, the nerve fiber
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layer, the ganglion cell layer, the inner plexiform layer (IPL) that contains synapses between
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ganglion cells and interneurons (bipolar, amacrine, and horizontal cells), the inner nuclear layer (INL), formed by nuclei of bipolar, amacrine, horizontal and RMG cells, the outer plexiform layer (OPL), composed by synaptic connections between visual neurons (or photoreceptors) and interneurons, the outer nuclear layer (ONL) formed by nuclei of cone and rod photoreceptors, and the inner and outer segments of the photoreceptors (Hogan et
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al., 1971). The outer limiting membrane (OLM) is formed by the cellular connections between RMG cells and photoreceptor inner segments. Photoreceptor outer segments are in close contact with RPE microvilli, but no molecular adhesion or junction system maintains the
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neural retina attached to the RPE. The RPE lies on the Bruch’s membrane, which outermost part forms the basal membrane of the choriocapillaris, the innermost vascular layer of the
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choroid.
The retina is vascularized by two independent vascular beds, the retinal and choroidal vasculatures. The larger retinal vessels, branches of the central retinal artery and vein, lay below the ILM, and are surrounded by astrocytes, pericytes and RMG cells. Between precapillary arterioles and post-capillary venules, the retinal capillary network is arranged in three layers. The superficial, intermediate and deep plexuses are located in the nerve fiber and ganglion cell layers, at the inner border of the IPL, and at the inner and outer border of the INL, respectively (Bonnin et al., 2015; Hwang et al., 2016; Park et al., 2016; Rizzolo et al., 2011) (Figure 2; see Supplementary Methods and Supplementary Table 1 for details of 8
ACCEPTED MANUSCRIPT immunostaining techniques employed for Figure 2 and all subsequent Figures). Around the optic nerve head, a radial capillary network lays within the nerve fiber layer, along the temporal superior and inferior retinal vessels (Henking and De Oliveira, 1967). Since the outer retinal layers are avascular, the choroidal vessels supply nutrients and oxygen to the
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high energy-demanding photoreceptors. Sympathetic regulation is present in the choroidal vasculature but not in retinal vessels. The major part of the choroidal space is occupied by vessels, organized in three vascular layers. The choriocapillaris, a thin interconnected
capillary network is the innermost layer, the medium- and small-sized vessels form the
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intermediate Sattler's layer, and large arteries derived from the short posterior ciliary arteries and large lumen veins form the outermost Haller's layer. The supra-choroid is the virtual
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space separating Haller's layer from the sclera. Numerous non-vascular cell types, such as melanocytes, macrophages/microglia and mast cells lay around choroidal vessels. Lymphatic-like vessels have been described in the human choroid, located in the innermost vascular layers external to the fenestrated vessels of the choriocapillaris. Yet, this important
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finding remains to be confirmed (Nickla and Wallman, 2010), since all classical lymphatic markers were not detected in the choroid (Schrödl et al., 2015). In the normal retina, no lymphatic vessel has been identified.
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Three types of glial cells are present in the neurosensory retina: astrocytes, located in the inner retina, at the vitreal interface and around vessels, microglial cells, which in physiologic
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conditions are ramified cells, located only in the inner retina and around retinal vessels, and RMG cells. RMG cells are the only cells spanning the entire thickness of the neurosensory retina, ensuring contact between all neuronal cell types, retinal vessels and the vitreous cavity (Figure 2). The macula is a highly specialized region of the retina responsible for fine visual acuity. It is located temporal to the optic nerve head and can be recognized on fundus visualization by its yellow color, due to the accumulation of the xanthophyll pigments lutein and zeaxanthin (Figure 3A and B). The average retinal thickness is 210 microns in adult eyes but the thickness varies in different regions. From 100 µm in the extreme periphery, retinal thickness increases to 450µm at the border of the macula and then decreases to 130µm at
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ACCEPTED MANUSCRIPT the center of the foveal depression, where inner retinal layers are displaced laterally. The center of the fovea is indeed exclusively composed of cone photoreceptors and specific foveal RMG cells. In the fovea, cones are densely packed, thinner, and have elongated outer segments, as compared to cones of the peripheral retina. The center of the macula is
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generally avascular, surrounded by circularly arranged capillaries delimitating the foveal avascular zone, a central 400-500-µm-diameter area. During development, the formation of
(Provis et al., 2013).
General introduction to macular edema
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the normal foveal morphology is closely related to the presence of a foveal avascular zone
Macular edema (ME) is defined as an abnormal increase of fluid volume in the macula. Extracellular fluid can infiltrate retinal layers, accumulate in cavities commonly referred to as “cysts” (without certitude that a “membrane” delimitates these cavities), or collect in the subretinal space, where it is referred to as subretinal fluid (Figure 3C and D). An increase of
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intracellular fluid volume (cell swelling) may also occur (Kohno et al., 1983; Yanoff et al., 1984), often associated with extracellular fluid. In physiologic conditions, fluid entry and exit are tightly regulated to maintain a balanced hydration state compatible with retinal
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homeostasis, necessary for tissue transparency and light transmission. ME results from an
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imbalance between fluid entry and exit, two multifactorial processes frequently deregulated in retinal diseases. Conditions in which one mechanism predominates are of particular interest when trying to discriminate the respective roles of fluid entry versus fluid exit in the formation of ME. Changes in retinal hydration state interfere with photon transmission and disturb vision. Long-standing ME may lead to permanent retinal structural damages. Visual consequences of macular edema mostly depend on structural alterations induced by intraretinal or subretinal fluid accumulation, with a worse prognosis linked to alterations of outer retinal structures, such as OLM or photoreceptor segments (Otani et al., 2010;
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ACCEPTED MANUSCRIPT Wakabayashi et al., 2009), and to the neural disorganization of inner retinal layers (Sun et al., 2015). Independently from its etiology, ME is per se a threat for central vision.
1.2.1
Limitations to study macular edema
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Retinal imaging methods developed in the last two decades, and particularly optical coherence tomography (OCT) (Puliafito et al., 1995), have brought major advances in the study and understanding of ME, allowing in vivo longitudinal, functional and microstructural analysis of the human macula (Figure 3B). Indeed, no laboratory animal model can fully
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recapitulate the macular edema phenotype since rodents, lagomorphs and other species commonly used for pathophysiology studies do not have a macula. Non-human primates
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have a macula (Figure 3C) but can rarely be employed for research purposes, given the increasing ethical, regulatory and financial constraints. In this context, human ocular specimens are particularly valuable, but the availability of post-enucleation or post-mortem eyes is extremely limited, and fixation/dehydration procedures necessary for tissue
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preparation may interfere with ME pathology. To circumvent these limitations, numerous groups have studied the biological composition of aqueous, and less frequently vitreous humor, to identify molecular pathways associated with ME (Hillier et al., 2017; Jung et al.,
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2014; McAuley et al., 2014; Owen and Hartnett, 2013; Wen et al., 2015; Zhang et al., 2014). This strategy proved effective in the past, being the basis of anti-vascular endothelial growth
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factor (VEGF) development for retinal diseases (Aiello et al., 1994; Malecaze et al., 1994). However, since the advent of these revolutionary agents more than a decade ago, no novel optimal therapeutic target has emerged from this type of analysis.
1.2.2
Macular edema: an underestimated public health issue
ME is a major cause of visual impairment in the course of metabolic, vascular and inflammatory retinal diseases. It affects worldwide around 7 million subjects due to diabetes (Figure 4) (Yau et al., 2012) and 3 million subjects due to vein occlusions (Figure 5) (Rogers et al., 2010). It accounts for 40% of visual impairment in patients with uveitis (Figure 6E and 11
ACCEPTED MANUSCRIPT F) (Rothova et al., 1996). In industrialized countries, 5% of individuals older than 60 years of age have ME due to neovascular age-related macular degeneration (AMD) (Figure 6A and B) (Pennington and DeAngelis, 2016). Besides being a major cause of visual impairment of retinal origin, ME is also among the most accessible to treatment. The currently approved
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intraocular anti-angiogenic and glucocorticoid agents indicated for several common retinal conditions (diabetic ME, central and branch retinal vein occlusion, and neovascular AMD), have been approved for their effect on ME reduction and subsequent visual acuity gain.
Taking into account the major impediment of ME on quality of life and working ability, these
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treatments are cost-effective, but still represent an important burden on health care systems (Hodgson et al., 2016; Ross et al., 2016). A better understanding of ME, an extremely
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frequent and vision-threatening manifestation of many retinal disorders, is thus crucial to elaborate new concepts and subsequent innovative therapeutic strategies. Indeed, beyond a common single phenotype, macular edema results from multiple, complex and not fully understood mechanisms.
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The aim of this review is to look at ME ‘beyond the surface’, recapitulating in depth the current knowledge on the intricate molecular and cellular mechanisms involved in retinal hydro-ionic homeostasis, and how their deregulation leads to ME. This review includes a
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specific emphasis on diabetic ME, and finally opens future research directions.
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2 What controls the retinal hydro-ionic and osmotic homeostasis?
In physiologic conditions, different mechanisms maintain the retina in a transparent and relatively dehydrated state. Barriers limit fluid entry and active drainage systems allow fluid exit while creating extracellular molecular gradients important for hydration homeostasis. The main glial drainage mechanisms controlling retinal hydro-ionic homeostasis are summarized in the Video 1.
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2.1
Regulation of fluid and molecule entry: retinal barriers
Fluid and molecules of variable size can differentially enter the neurosensory retina from the vitreous, from retinal vessels, or from the choroid through the RPE, via the subretinal space.
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This process is tightly controlled by structures forming the inner and the outer blood-retinal barriers and by the osmotic gradients across these barriers (Bradbury and Lightman, 1990;
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Törnquist et al., 1990).
Inner blood-retinal barrier
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The inner blood-retinal barrier plays a major role in controlling fluid entry into the retina. It is ensured by the tight-junctions between endothelial cells of retinal vessels, dynamically regulated by a neuro-glio-vascular cross-talk involving astrocytes and RMG cells (Fruttiger, 2007; Klaassen et al., 2013; Sorrentino et al., 2016), and interactions with pericytes and smooth muscle cells (Figure 2).
Endothelial cells and their intercellular junctions
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2.1.1.1
Endothelial cells of retinal capillaries are connected by molecular complexes, consisting of tight junctions (zonula occludens), adherens junctions (zonula adherens) and gap junctions
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(Figure 2 B and C). Besides their role in cell-to-cell adhesion, these complexes regulate the contact inhibition for endothelial cell division, cell survival, polarity and paracellular
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permeability (Klaassen et al., 2013). Endothelial cells of the retina and brain have the highest number of tight-junction strands, a lack of fenestrations, low pinocytic activity and the presence of highly complex continuous tight junctions, which establishes a physical barrier to water and hydrosoluble molecules. The exact transcellular resistance of the retinal vascular endothelium is not known but is thought to be similar to the one measured in the brain vascular endothelium, reaching 1000-1500 Ohm.cm2, much higher than the outer retinal barrier resistance (Crone and Olesen, 1982). The transcellular resistance reflects the flux of small ions but does not measure the paracellular size selectivity or the vesicular transcytosis
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ACCEPTED MANUSCRIPT of larger molecules. The smallest intercellular gaps form a selective barrier to the paracellular passage of molecules with radius higher than 3 nm, with differential size selectivity between different molecular components of the tight junctions. For more details, readers should refer to a recent extensive review on the inner blood-retinal barrier (Klaassen et al., 2013). We will
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here summarize the major molecular players involved, focusing on the newest findings.
Endothelial tight junctions are complex structures formed by multiple transmembrane,
scaffolding and signaling proteins. The most important transmembrane proteins are occludin,
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claudin-1, -2 and -5 (Russ et al., 1998; Tian et al., 2014) and junction adhesion molecules (JAMs) A and C (Economopoulou et al., 2009; Orlova et al., 2006; Saker et al., 2014) (Figure
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2 B). The cytoplasmic tails of occludin and claudins are linked to the actin cytoskeleton by scaffolding proteins. For instance, zona occludens-1 (ZO-1) that belongs to the PDZ domain proteins (Campbell and Humphries, 2012; Hosoya and Tachikawa, 2012), is responsible for anchoring transmembrane and receptor proteins to cytoskeletal components. Other molecular components of endothelial tight junctions may also be present in retinal capillaries,
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such as vascular endothelial cell-specific adhesion molecule (VCAM), poliovirus receptorrelated-1 (Nectin) and tight junction protein-2 (zona occludens-2, ZO-2) (Klaassen et al., 2013). Recent observations have highlighted that polarity proteins are present in endothelial
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cells, as in epithelia, and that they play major roles in vessel formation and maintenance. These proteins include pals1/PATJ and PAR3, PAR6, and crumbs-3 (CRB3) interacting with
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VE-cadherin (Brinkmann et al., 2016). Atypical protein kinase C (PKC) and particularly PKCζ, that phosphorylates junction proteins and is part of the PAR3 complex, is crucial for the formation and maintenance of the blood-brain barrier (Sewduth et al., 2017). In the human retina, this complex has been recognized as a regulator of junction proteins in endothelial cells (Song et al., 2014) and as a major actor in VEGF-receptor-mediated endocytosis, determining the active growth and maturation of vessels submitted to VEGF (Nakayama et al., 2013). In developing retinal vessels, the formation of tight junctions in between endothelial cells depends on their cellular interactions with pericytes and astrocytes (Kim et
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The adherens junctions are constituted by VE-Cadherin (Navaratna et al., 2007), ß-
Catenin (Russ et al., 1998) and N-Cadherin/Angiomotin-like protein 1 (AmotL1t) (Figure 2B). The gap junctions consist of a hemi-channel (or connexon) formed by six connexins
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(cx). Gap junctions mediate electrical and chemical communication between cells and allow the free passage of small molecules (<1 kDa). Amongst the various connexins, cx7, cx40
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and cx43 are widely expressed in the retina, and particularly in retinal endothelial cells (Danesh-Meyer et al., 2016). Notably, cx30.2 is involved in the modulation of vascular
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permeability (Manasson et al., 2013).
The different types of endothelial junctions are functionally and structurally linked. For example, the presence of adherens junctions stimulates the formation of tight junctions, as exemplified by the observation that VE-cadherin at adherens junctions induces claudin-5
2.1.1.2
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expression (Taddei et al., 2008).
Transendothelial transport
Transport across the endothelium is highly regulated by active membrane transporters and
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by vesicular transport. Caveolae-mediated transcytosis is defined by the migration of plasma membrane vesicles from one side of the cell to the other, and/or the formation of a pore
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resulting from vesicular fusion. Caveolin-1 (Cav-1), the major protein component of caveolae is expressed in the developing and mature retinal vessels and in the choroidal vasculature (Gu et al., 2014a). Transcytosis allows plasma macromolecules, such as albumin, transferrin, insulin, lipoproteins, and possibly immunoglobulins, to penetrate from the circulation into tissues. It is reduced in the retina, as compared to other organs (Anderson, 2008; Predescu et al., 2004; Red-Horse and Ferrara, 2007). In contrast, this mechanism seems important in driving macromolecules out of the retina into the circulation. Indeed, vesicular transport of albumin from the retina towards the circulation is higher than the reverse, as reflected by the higher vesicle density at the abluminal side of retinal vessels (Hofman et al., 2000, 2001). 15
ACCEPTED MANUSCRIPT The directional regulation of albumin transport remains incompletely understood. Yet, it should be of utmost importance in the maintenance of protein gradients in the retina, and subsequent fluid movements. Interestingly, Cav-1 knock-out mice show changes in the expression of mural cell markers (down-regulation of NG2 and up-regulation of αSMA), of the
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junction protein claudin-5, and reduced nitric oxide (NO)-dependent vasodilation, demonstrating that caveola-mediated transport may have broader regulating effects (Gu et al., 2014a). In the developing retina, the inner blood-retinal barrier properties are
progressively acquired concomitantly to the reduction of endothelial transcytosis (Chow and
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Gu, 2017).
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2.1.1.3 Pericytes
Pericytes are specialized mural cells located at the abluminal surface of retinal veins and capillaries. They share their basement membrane with endothelial cells, and are covered by an external basal membrane (Figure 2 B and C). Pericytes contribute to the regulation of the inner blood-retinal barrier, the microvascular blood flow through their contractile properties,
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and to angiogenesis. There is recent evidence that brain, retinal and choroidal pericytes derive, at least in part, from neural crests (Trost et al., 2016). Myeloid-derived cells could also contribute to pericyte coverage, particularly during retinal injury and repair. In the retina,
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pericyte density with respect to endothelial cells reaches a 1:1 ratio and pericyte coverage of human retinal capillaries is as high as 94%, as compared to 11% in the choriocapillaris
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(Chan-Ling et al., 2011). In the rat retina, the ratio of pericyte plasma membrane length in contact with the vascular circumference, to the outer circumference of the endothelial cell tube is around 40%, as compared to 20-30% in various brain regions (Frank et al., 1987). Adherens junctions connect the cytoskeleton of pericytes to endothelial cells, partly through cadherins, allowing molecular mechanical signaling between pericytes and endothelial cells. Angiomotin like-1 (AmotL1), scaffold protein integrating polarity, junctional, and cytoskeletal cues that modulate cellular shape, was recently identified as an essential effector of the Ncadherin mediated endothelial/pericyte junctional complex (Zheng et al., 2016). The pericyte-
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ACCEPTED MANUSCRIPT derived lipidic mediator, sphingosine-1-phosphate, up-regulates N-cadherin and VE-cadherin expression and down-regulates angiopoietin-2 (Ang-2), modulating the inner blood-retinal barrier in physiologic and pathologic conditions (McGuire et al., 2011). Gap junctions, particularly cx43 also contributes to the molecular communication between pericytes and
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endothelial cells (Danesh-Meyer et al., 2016; Li et al., 2003). The crucial role of pericytes in blood-brain barrier formation and maintenance has been demonstrated using mouse
mutants. While increased expression of ZO-1 correlated with enhanced pericyte coverage (Kim et al., 2009), impaired platelet-derived growth factor (PDGF)-B/PDGF-Receptor-b
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signaling, reduced pericyte coverage, together with endothelial hyperplasia, vascular
abnormalization and microaneurysm formation (Lindahl et al., 1997). VEGF over-expression
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by the hyperplasic endothelium was associated with enhanced trans-endothelial permeability (Hellström et al., 2001). Increased vascular permeability in pericyte-depleted vessels results from indirect over-expression of vasoactive molecules intervening in both tight junctions and vesicle trafficking (Daneman et al., 2010). Glial cells
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2.1.1.4
The glial cells of the retina include macroglia, composed of RMG cells and astrocytes, and microglia (Figure 2A, C and D). Their processes wrap around retinal capillaries, forming a
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glia limitans. Retinal arterioles, venules, and capillaries are closely ensheathed by macroglia. The superficial retinal vasculature is ensheathed by both astrocytes and RMG cells (Figure
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2B and C), whilst the deep vascular plexus is ensheathed solely by RMG cells (Figure 2 A and E) (Schnitzer, 1988). This differential distribution of glial cells around the superficial and deep plexuses, may have important functional consequences for the pathophysiology of ME. Macroglial cells are key players in the dynamic neuro-glio-vascular unit and regulate a wide range of endothelial cell functions. Cultured retinal glial cells stimulate pericyte growth in vitro (Ikuno et al., 2002). The cell-cell contact of astrocytes and endothelial cells promotes the differentiation of endothelial cells, allowing the maturation of the inner blood-retinal barrier (Jiang et al., 1995; Yao et al., 2014). Astrocytes and RMG cells both have the ability to
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ACCEPTED MANUSCRIPT induce the formation of competent barriers by vascular endothelial cells (Tout et al., 1993; Gardner et al., 1997). They also stabilize the tight junctions between endothelial vascular cells, and play fundamental roles in local immune responses and immune-surveillance (Dong and Benveniste, 2001). Microglia also contributes to the formation and maintenance of the
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inner blood-retinal barrier mostly through production of soluble factors, but also through vesicular communication (Checchin et al., 2006; Vecino et al., 2016) and cx43 gap-junctions (Danesh-Meyer et al., 2016). Outer blood-retinal barrier
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2.1.2
The outer blood-retinal barrier is classically defined as the intercellular junction complex of
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the RPE, separating the neurosensory retina from the choroidal circulation. The OLM also contributes to the outer blood-retinal barrier function (Omri et al., 2010), and to its disruption in pathological conditions.
2.1.2.1
Junctional complex of the retinal pigment epithelium
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The junctional complex of the RPE is formed, as in other highly polarized epithelia, by tight, adherens and gap junctions (Figure 7). The transepithelial resistance is much lower than the resistance of the inter-endothelial junctions at the inner retinal barrier (Rizzolo, 1997, 2007;
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Rizzolo et al., 2011; S. Peng et al., 2011). However, the estimated transepithelial resistance of human adult RPE (around 80 Ohm.cm2) efficiently prevents water and protein entry from
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the choroid to the subretinal space, and allows water exit towards the choroid, following an osmotic gradient (Radius and Anderson, 1980; Stern et al., 1980; Kirchhof and Ryan, 1993). The RPE junction complex also participates to the regulation of RPE cell shape, polarity and proliferation control through the interaction with specific cytoskeletal, adapter and effector proteins. This complex has been detailed in a comprehensive review (Rizzolo et al., 2011). The tight junctions are mostly formed by occludins (Phillips et al., 2008), claudins (S. Peng et al., 2011), principally claudin-19 and 3 (Peng et al., 2016; Rizzolo et al., 2011), ZO-1 (Konari et al., 1995) and the JAM proteins JAM-A (Mandell et al., 2007) and JAM-C. JAM-C regulates
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ACCEPTED MANUSCRIPT the recruitment of N-cadherin and ZO-1, and is involved in the polarization of RPE cells (Daniele et al., 2007; Economopoulou et al., 2009). Recently, crumbs-2 (CRB2) was shown to participate to tight junctions at the apico-lateral RPE cell membrane (Paniagua et al., 2015). The adherens junctions contain mostly E- and N-cadherins (Burke et al., 1999; Burke,
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2008; McKay et al., 1997). In chicken RPE, the adherens junctions can form a complex with α-actin and vinculin (Sandig and Kalnins, 1988). Regarding gap junctions, the presence of connexin-43 has been reported on rat RPE (Tibber et al., 2007).
Junction proteins and actin cytoskeleton distribution and functions are regulated by kinases
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of Rho/Rac and atypical PKC families. While diurnal outer segment phagocytosis requires Rac1 activation (Mao and Finnemann, 2012), pathological remodeling of RPE involves
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mostly ROCK-1 activation, leading to subsequent junction destabilization (Lee et al., 2008). The non-classical, calcium-independent, atypical protein kinase PKCζ, associated with the PAR3/PAR6 complex within occludin loops, phosphorylates occludin, thereby promoting the assembly of tight junctions (Figure 7 A and B) (Omri et al., 2013). The polarized organization
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of RPE cells creates differential compartments. RPE apical microvilli are in contact with the immune privileged subretinal milieu, while RPE basal and lateral cell membranes are in contact with a protein-enriched fluid emanating from the choroid. This choroid-derived media
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provides critical compounds for retinal metabolism, including the retinol-binding protein
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needed for retinol uptake and essential to the visual pigment cycle.
2.1.2.2
Outer limiting membrane
The OLM is formed by heterotypic tight-like and adherens junctions, located at the interface between RMG cells and photoreceptor inner segments (Figure 7A and C-E). The tight-like junctions are mostly composed of occludin, JAM-B, JAM-C, and ZO-1 (Daniele et al., 2007; Omri et al., 2010), and notably lack claudins, an essential tight junction protein (Alves et al., 2014). Adherens junctions of the OLM are composed of cadherin/catenin, interacting with adaptor proteins such as Crumbs homologue 1 (CRB1) (Gosens et al., 2008) and ZO-1
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ACCEPTED MANUSCRIPT (Pearson et al., 2010). The exact role of the OLM in the regulation of fluid movements across the macula is not fully understood. Earlier studies have demonstrated that the OLM is a barrier to protein diffusion. Biotinylated protein probes of known Stokes radius were used to determine the pore size of the OLM on ex vivo rabbit retina (Bunt-Milam et al., 1985). The
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Stokes radius is the effective theoretical radius of a given molecule in a hydrated state that influences its movement in solution. This experiment estimated the pore radius of the OLM junction between 30 and 36 Å, much smaller than albumin and globulins. The OLM thus serves as an important barrier to free protein diffusion across the retina from the inner retinal
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layers to the subretinal space, and vice-versa. This observation should be considered when
2.2
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therapeutic proteins are injected into the vitreous to target sub-RPE pathological processes.
Regulation of fluid exit: drainage mechanisms
Active and passive mechanisms continuously drain water, ions and other molecules from the retina to the vitreous, retinal vessels, and the choroid through the subretinal space. Indeed,
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the tight regulation of pH, and concentration gradients of ions and proteins across retinal and choroidal layers, and the clearance of metabolic products are essential for retinal homeostasis and function. Different cell types are thus equipped with numerous transporters
critical.
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(which have only been partly identified), which appropriate expression and distribution is
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Before analyzing the cellular functions involved in transports, the tissues resistance to fluid and protein diffusion should be recalled. The neurosensory retina opposes a weak resistance to the passage of water from the subretinal space to the vitreous and vice versa (Marmor, 1985; Marmor et al., 1985; Orr et al., 1986), but is not permeable to compounds of high molecular weight. Part of the aqueous humor secreted by the ciliary body hydrates the vitreous cavity, and is pumped by the RPE through the retina, to be drained into the choroid vessels. There are several obstacles to the diffusion of molecules in the retina. High molecular weight molecules (>150kD) do not diffuse though the ILM, and the plexiform layers
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ACCEPTED MANUSCRIPT are the layers of lowest hydric conductivity, constituting barriers to the diffusion of macromolecules. But the main resistance to their passage through the retina is due to the OLM barrier (see 2.1.2.2). Indeed, after subretinal injection, FITC-dextran 10S (Stokes radius: 23 Å) diffuses rapidly into
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the vitreous, but 70S- and 150S- dextrans (Stokes radius: 60 and 85 Å, respectively) mostly remain in the subretinal space due to the OLM barrier (Marmor et al., 1985). The kinetics of water and macromolecules injected under the macula has never been studied and could show significant differences with kinetics observed in animal models lacking a macula
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(Marmor et al., 1985), related to the specific cellular architecture and physiology of the
macula. Movement of molecules between photoreceptors might be influenced, depending on
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their physico-chemical properties, by interactions with glycosaminoglycans and glycoproteins in the interphotoreceptor matrix. The Bruch’s membrane does not oppose a high resistance to the passage of macromolecules between the choriocapillaris and the RPE. The greatest resistance to fluid movement across Bruch’s membrane occurs at its inner collagenous layer,
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and could increase with ageing (Starita et al., 1997).
The protein concentration is higher in the choroid, compared to the retina and vitreous, and the retina is less permeable to water than the RPE (Kirchhof and Ryan, 1993). Therefore, the
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osmotic movement of water from the vitreous to the choroid, partly depending on the maintenance of cellular barriers, contributes to maintain the neurosensory retina permanently
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attached to the RPE and “dehydrated”. The exact mechanisms regulating the protein gradient from the retina to the choroid is not fully understood. Recent work has demonstrated that the majority of albumin is not transported across choriocapillaris endothelial cell fenestrations but through transcytosis via caveolae (Nakanishi et al., 2016). In aged mice, reduced caveolae transport and leaky tight-junctions between endothelial cells may alter the retina/choroid protein gradients (Nakanishi et al., 2016).
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ACCEPTED MANUSCRIPT 2.2.1
Retinal glial Müller cells
As described in 1.1 and extensively reviewed (Bringmann et al., 2006; Reichenbach and Bringmann, 2013), RMG cells are specialized macroglial cells unique to the retina that span its entire thickness from the inner limiting membrane (ILM) to the photoreceptor inner
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segments (Figure 1 and 2). Nuclei of RMG cells are located at the level of the inner nuclear layer. RMG extend their endfeet frontally on the ILM which form their basal membrane, and send perivascular extension, where their basal membrane is fused with the basal membrane of the capillary endothelium (Hogan et al., 1971). RMG cells are the sole mediator of the
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macroglia-vascular interaction at the deep capillary plexus, where astrocytes are absent (Schnitzer, 1988) (Figure 2). RMG cells are responsible for a range of critical functions. They
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are structural backbones for the retinal architecture, act as optic fibers for photon guidance toward photoreceptor outer segments (Franze et al., 2007), regulate the vascular, neuronal and extracellular milieu homeostasis (ions, water, neurotransmitter molecules, and pH), and contribute to metabolic and immune regulation (Bringmann et al., 2006). Numerous ion and
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aqueous channels are expressed in RMG cells, varying among species (Pannicke et al., 2016). The membrane conductance of RMG cells is mostly regulated by inward and outward K+ currents. Different species-specific inward-rectifier potassium ion (Kir) channels have
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specific spatial distribution within RMG cells, allowing the buffering of extracellular K+ released by the photoreceptor and neuronal activity. In the rodent retina, Kir2.1 is
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predominantly located in the membrane domains of RMG cells in contact with retinal neurons, while Kir4.1protein is located at their endfeet and surrounds retinal blood vessels (Kofuji et al., 2002) (Video 1). Aquaporins (AQP) are a major class of small membrane proteins that facilitate water transport across plasma membranes in response to osmotic gradients, contributing to intra- and extracellular hydro-ionic regulation. The human retina expresses all AQPs transcripts (AQP0 to AQP12) but AQP2, AQP8 and AQP11 could not be identified at the protein level (Schey et al., 2014; Verkman et al., 2008). Perivascular AQP4 and Kir4.1 have been shown to act as a macromolecular complex with α-syntrophin and the short dystrophin isoform Dp71 (Claudepierre et al., 2000; Sene et al., 2009; Verkman et al.,
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ACCEPTED MANUSCRIPT 2008). But whether a functional interaction exists between AQP4 and Kir4.1, that would account for hydro-ionic clearance of neuronal activity metabolic waste remains controversial. Recently, transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4), a non-selective calcium cationic channel activated by osmotic, mechanical and chemical
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stimuli has been identified as another important actor in the coupling with AQP4. Indeed, in hypo-osmotic conditions, water influx through AQP4 would drive a calcium influx via TRPV4 in the glial endfoot, regulating the expression of AQP4 and Kir4.1 genes, and subsequently controlling the cell volume increase (Jo et al., 2015). RMG swelling that participates to
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retinal edema and degeneration is inhibited by endothelins (Vogler et al., 2013), brain-
derived neurotrophic factor (BDNF) (Berk et al., 2015), nerve growth factor (NGF) (Garcia et
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al., 2014), osteopontin (Wahl et al., 2013), VEGF and glutamate (Brückner et al., 2012). Many other ion channels (K+, Na+, Ca2+) are expressed along the RMG cell membrane in a polarized and regional arrangement, that varies amongst species (Pannicke et al., 2016), limiting the clinical application of certain animal model observations.
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Whether RMG cells can play a role in the transport of proteins across the normal retina has not been specifically investigated. Several studies have analyzed the fate of intravitreally injected molecules. Interestingly, bevacizumab is a 150-kD protein with a hydrodynamic
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diameter of approximately 15 Å, according to the Stokes-Einstein equation, and therefore is not expected to cross the OLM (see 2.1.2.2). But following intravitreal administration it was
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detected in the subretinal space (Dib et al., 2008), and was identified within RMG cells using immunohistochemistry (Shahar et al., 2006). Protein transport through RMG cells can thus not be excluded since these glial cells do have phagocytic activities (Stolzenburg et al., 1992) and the capacity to transport peptidic particles from the vitreous to the subretinal space (Normand et al., 2005). Moreover, RMG cells also express caveolin-1, involved in the vesicular transport of proteins (Gu et al., 2014b). In the case of blood retinal barrier disruption, RMG cells in situ can phagocytize blood-derived proteins like IgG (Iandiev et al., 2008).
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ACCEPTED MANUSCRIPT 2.2.2
Retinal pigment epithelium
The structure and function of the RPE, like other epithelia, depend on its apico-basal polarization, which occurs through linking asymmetrically intercellular junctions to the cytoskeleton of cells (Gibson and Perrimon, 2003). Differential polarized insertion of
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channels, transporters, and related proteins into apical and baso-lateral membranes is essential for the regulation of RPE cell volume, pH and trans-epithelial transports. Alteration of the RPE cell junctional complex results in the dysfunction of polarized ion and water
channels, favoring the accumulation of fluid in the subretinal space. Reciprocally, inhibition of
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the apical Na+/K+-ATPase alters the tight junction structure and increases the permeability in cultured human RPE cells (Rajasekaran et al., 2003). The outward molecular movement
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across the RPE is largely dependent on active ionic transport (~70%), while a smaller fraction (~30%) may be the result of the higher oncotic pressure in the choroid than in the retina (Negi and Marmor, 1986a, 1986b), a mechanism discussed in 4.2.1. The RPE actively removes water from the subretinal space at a rate ranging from 1.4 to 11
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µl/cm2/h (Tsuboi and Pederson, 1988), that is driven by an active trans-epithelial Cl- gradient. The apical Na+/K+-ATPase actively enhances intracellular K+, recycled by Kir7.1, inducing a Na+ gradient and an increased basolateral chlore conductance. The increase of intracellular
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Cl- concentration is associated with an acidification of the cells, which is regulated by different Cl- channels located at the basolateral membrane (e.g. bestrophin, which is volume-
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and Ca2+-dependent). These Cl- channels facilitate the efflux of Cl- ions from the cells, osmotically driving also water through aquaporin channels. Thus, there is an active water flux from the subretinal space towards the choroid through the RPE (Reichhart and Strauss, 2014). The involvement of the HCO3-/Cl- exchanger at the basolateral membrane links the water transport to the pH regulation and to the transport of lactate, resulting from the intense metabolic activity of photoreceptors. Trans-epithelial water efflux is thus linked to chloride conductance and lactate transport. These mechanisms of ions transport in RPE cells have been recently reviewed, and will not be further detailed here (Reichhart and Strauss, 2014). Acidification of the apical RPE membrane by anhydrase carbonic inhibition (e.g. using
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ACCEPTED MANUSCRIPT acetazolamide) increases water transport through RPE cells (Marmor and Maack, 1982; Yamamoto and Steinberg, 1992). The presence of functional AQP1 in the RPE has been subject to controversies (Verkman et al., 2008). In rabbit eyes with experimental subretinal blebs created in vivo, cGMP enhanced
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the water transport towards the RPE (Marmor and Negi, 1986), an effect consistent with AQP1-mediated net fluid flux increase, that was confirmed in human RPE cultures (Baetz et al., 2012). Functional AQP1 and AQP11 were recently described in human pluripotent stemcell-derived retinal pigmented epithelial cells (Juuti-Uusitalo et al., 2013). In addition to AQP1
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there are other subtypes of AQP in the RPE, as detailed in 3.2.2.
The RPE plays a major role in the fluid homeostasis of the outer retina and the choroid. As a
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highly polarized epithelium, any cytoskeletal stress could not only alter the junctions but also the proper distribution of membrane transporters and result in sub-retinal fluid accumulation.
3 Why does retinal edema form?
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Retinal edema is a consequence of an imbalance between fluid entry, fluid exit and retinal hydraulic conductivity. In most retinal diseases, macular edema is multifactorial and results from multiple, intricate mechanisms, but in certain specific conditions, one single of these
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mechanisms predominates, allowing a better analysis of each component. Figure 8
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summarizes these different intricate mechanisms.
3.1
3.1.1
Increased retinal fluid entry Increased fluid entry through the inner blood-retinal barrier
Main mechanisms leading to an increase of the inner blood-retinal barrier permeability are: •
Alteration of the intercellular junction proteins through down-regulation, change in phosphorylation state, or loss of membrane anchoring components (Antonetti et al., 1999; Klaassen et al., 2013).
•
Increased trans-endothelial transport.
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ACCEPTED MANUSCRIPT •
Loss of cells constituting the barrier (endothelial cells, pericytes, macroglial cells).
Vascular barrier properties can be altered within mature retinal vessels, but also due to the proliferation of immature neovessels.
Junctional complex alteration
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3.1.1.1
Loss of the functional and/or structural integrity of tight-junctions between endothelial cells increases vascular permeability to water, solutes and proteins (Radius and Anderson, 1980). Various experimental conditions (in cell cultures or experimental animals) mimicking
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pathological environments favoring ME, such as diabetes or inflammation, can alter junction
•
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proteins.
High glucose concentrations induce the down-regulation of claudin-5, occludin, JAM-
A, and ZO-1 in human retinal endothelial cells (Saker et al., 2014; Stewart et al., 2016; Tien et al., 2013) and reduces VE-cadherin phosphorylation via an increase in Ang-2 (Rangasamy et al., 2011). A proteolytic degradation of VE-cadherin is also observed in the retina of STZ-
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induced diabetic rats (Navaratna et al., 2007). Endoplasmic reticulum (ER) stress also causes down-regulation of claudin-5 (Adachi et al., 2011). •
Cytokines acts on tight-junctions through direct and indirect regulations. Tumor
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necrosis factor α (TNF-α) decreases the expression of ZO-1 and claudin-5, and alters their
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subcellular distribution in bovine retinal endothelial cells through PKCζ-mediated NF-κB activation (Aveleira et al., 2010). Transforming growth factor-ß (TGF-ß), secreted by glial cells, stimulates the production of matrix metalloproteinase (MMP)-9 (Behzadian et al., 2001). In turn, MMP-9 degrades occludin, as shown in the retinas of diabetic animals, in which both MMP-2 and MMP-9 expression is induced (Giebel et al., 2005). In patients with uveitis-related ME, angiography with fluorescein-conjugated dextrans showed that 4-kD, 20kD but not 150-kD molecules can cross the ruptured inner blood retinal barrier at the macula, whereas no passage was observed in healthy controls (Atkinson et al., 1991). This demonstrates that in inflammatory conditions the inner retinal vessel permeability is
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ACCEPTED MANUSCRIPT selectively increased. •
Activation of the VEGF/placental growth factor (PGF) pathways, secondary to hypoxia
and hyperglycemia, induce retinal vascular permeability by direct effects on endothelial tight and adherens junctions. PGF favors the degradation of ZO-1 and VE-cadherin (Huang et al.,
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2015). VEGF-induced permeability results from multiple convergent effects: phosphorylation, ubiquitination and internalization of occludin by activation of PKC-β (Antonetti et al., 1999; Harhaj et al., 2006; X. Liu et al., 2016; Murakami et al., 2012) and Src-family kinases (Scheppke et al., 2008) and down-regulation of occludin through ß-catenin pathway
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regulation (Behzadian et al., 2003). VEGF induces the ß-arrestin-2-dependent endocytosis of VE-cadherin and its removal from adherens junctions (Gavard and Gutkind, 2006) and
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increases transglutaminase-2 activity, which produces stress fiber formation through Rhokinase activation and VE-cadherin disruption (Lee et al., 2016). •
The VEGF/pigment epithelium-derived factor (PEDF) balance plays also a major role
in retinal vascular permeability. PEDF produced by RMG cells (Hauck et al., 2014) inhibits
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VEGF-induced permeability through several mechanisms, including: γ-secretase activity (Ablonczy et al., 2009); regulation of VEGF receptors with VE-cadherin and β-catenin, and their subsequent phosphorylation (Cai et al., 2011); blocking of the VEGF-induced ERK/P38
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and GSK3-ß transduction pathways without direct effect on VEGF receptor-2 (VEGFR-2) phosphorylation, inhibiting the activation of β-catenin (Yang et al., 2010). Moreover, PEDF
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inhibits the permeability induced by advanced glycation end-products (AGEs) via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the up-regulation of ZO-1 (Sheikpranbabu et al., 2010). •
Endothelial cell permeability is also influenced by glial cells. The barrier function of
endothelial cells co-cultured with RMG cells is enhanced in normoxic conditions, but is impaired under hypoxia (Tretiach et al., 2005). In the streptozotocin (STZ)-diabetic rat model, glial fibrillary acidic protein (GFAP) expression is altered in retinal glial cells, together with a reduction and redistribution of occludin in endothelial cells (Barber et al., 2000). The role of glial cells in the maintenance of vascular barrier integrity was further demonstrated by 27
ACCEPTED MANUSCRIPT selective glial cell depletion models. Intravitreal injection of a glial toxin, DL- α-aminoadipic acid (DL-α-AAA), led to increased expression of VEGF and a reduced expression of claudin5 (Shen et al., 2010). The specific ablation of RMG cells using a Müller-specific variant of AAV, that delivered a photo-inducible toxic protein, induced retinal remodeling, vascular
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abnormalization and leakage (Byrne et al., 2013). Microglial activation contributes to the breakdown of inner blood retinal barrier through pro-inflammatory cytokines and VEGF
production. For example, IL-6 produced by microglia under high glucose induces the down-
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regulation of occludin and ZO-1 and the production of VEGF through signal transducer and activator of transcription 3 (STAT3) activation (Yun et al., 2016b). Minocycline, known to
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deactivate microglia, prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury (Abcouwer et al., 2013). 3.1.1.2
Enhanced transcellular permeability
Increased inner blood-retinal barrier permeability can result from transcytosis mechanisms. In
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vivo studies in non-human primates showed that VEGF-induced permeability results from increased transendothelial protein transport, through caveolae involving endothelial nitric oxide regulation, rather than from tight-junctions opening or increased fenestrations (Feng et al., 1999; Hofman et al., 2000). The presence of plasmalemma vesicle-associated protein
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(PLVAP) in retinal capillaries has been associated with VEGF-induced blood retinal barrier
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permeability in diabetic macular edema (Wisniewska-Kruk et al., 2016). The observation that the genetic ablation of Cav-1 induces breakdown of the inner bloodretinal barrier without alterations of junction protein expression (Gu et al., 2014a) suggest that alteration of macromolecules vesicular transport in the retina could enhance the barrier permeability. This mechanism has been recently considered in the central nervous system as an important contributor to blood-brain barrier disruption without alteration of junctional structures (De Bock et al., 2016).
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ACCEPTED MANUSCRIPT 3.1.1.3
Loss of endothelial cells
Endothelial cell death has been described in models of oxygen-induced retinopathy (Beauchamp et al., 2001; Gu et al., 2003; Yamada et al., 1999). It was also reported in a model of branch retinal vein occlusion, where early endothelial cell death in capillaries
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upstream the occluded vessels, was associated with vasogenic edema and hemorrhages, followed by a proliferation of endothelial cells (Dominguez et al., 2015). In STZ-induced
diabetic rats, leukocyte-mediated Fas-FasL-dependent endothelial cell apoptosis induced a breakdown of the inner blood-retinal barrier (Joussen et al., 2002). Several factors can cause
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endothelial cell death. For instance, TNF-α can contribute through apoptosis (Behl et al.,
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2008), necrosis (Claudio et al., 1994), decreased cx43-mediated glio-vascular communication (Muto et al., 2014), and photoreceptor-released pro-apoptotic factors (Tonade et al., 2016). In diabetes, endothelial activation and subsequent leucocyte- and monocyte-induced cell death results from either pro-inflammatory bone marrow circulating cells (Li et al., 2012), or increased stiffness of vessel basal membrane secondary to
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oxidative-stress-induced collagen cross-linking (Yang et al., 2016). More chronic endothelial dysfunction and metabolic stress could induce cytoskeletal remodeling and non-apoptotic cell death (van Gorp et al., 1999). It could also cause non-apoptotic cell blebbing, as observed in
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endothelial cells from Goto-Kakizaki (GK) type 2 diabetic rats (Rothschild et al., 2017). These cellular mechanisms explain the co-occurrence of capillary occlusion, increased permeability
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and hemorrhages.
3.1.1.4 Loss of pericytes Pericyte loss, considered as a mechanistic hallmark of early diabetic retinopathy, results from reduced adhesion of pericytes, which might be more sensitive than endothelial cells to microenvironment alterations (Li et al., 1996, p. 39). Yet, the exact mechanisms responsible for pericyte loss are not completely understood. Several factors contribute to pericyte death in diabetic conditions such accumulation of AGEs, hypoxia, increased levels of reactive
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ACCEPTED MANUSCRIPT oxygen species contributing to oxidative stress, rapid glycemia variations (Shojaee et al., 1999), macrophages/microglia activation and action of matrix metalloproteinases. AGEs induce the decrease of the Bcl-2/Bax ratio and the activation of caspase-3, via oxidative stress (Chen et al., 2006). The Ang-2/Tie-2, VEGF/Flt-1 (VEGF receptor-1, VEGFR-1) and
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the PDGF-B/PDGF-ß pathways have been incriminated in pericyte cell death (Ejaz et al., 2008). The regulation of Ang1/Tie2 pathway is important in vascular development and
particularly in maturation and stabilization of vessels (Felcht et al., 2012; Park et al., 2003). But, in hyperglycemic conditions, the effect of Ang-2 produced by endothelial cells on
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pericyte apoptosis could be mediated via the integrin-α3 and -β1 (Park et al., 2014).
Circulating VEGF and PGF, secreted by tumors or delivered by adenoviral vectors, induce
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the ablation of pericytes from the mature retinal vasculature through the Flt-1 (VEGFR-1)mediated signaling pathway, leading to increased vascular leakage (Cao et al., 2010). Interestingly, only VEGFR-1 and not VEGFR-2 is up-regulated by ischemia through activation of the hypoxia-inducible factor-1α (HIF-1α) promoter (Gerber et al., 1997). In
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addition, alteration of the PDGF signaling pathway, for instance in hyperglycemic conditions, contributes not only to decreased pericyte coverage but also to pericyte death. Hyperglycemia-induced dephosphorylation of PDGF-receptor-ß results in pericyte apoptosis,
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via the activation of PKC-∂and p38α mitogen-activated protein kinase (MAPK), increasing the expression of the Src homology-2 domain-containing phosphatase-1 (SHP-1) (Geraldes
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et al., 2009).
In the diabetic retina, Ca2+/calmodulin-dependent protein kinase II has been also implicated in inducible nitric oxide synthase (iNOS)-related pericyte loss (Kim et al., 2011). More recently, it is has been suggested that during diabetic retinopathy, human retinal pericyte apoptosis could be mediated by macrophages, TGF-β and the pro-apoptotic BIGH3 protein (transforming growth factor-ß-induced protein ig-h3) (Betts-Obregon et al., 2016). Increased expression of MMP-2 by pericytes in elevated glucose conditions, and in the diabetic retina,
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ACCEPTED MANUSCRIPT induces retinal pericyte apoptosis/anoikis by loss of contact with an appropriate extracellular matrix (Yang et al., 2007). In the rat model of branch retinal vein occlusion mentioned above, pericyte death was also observed in capillaries upstream the occluded vessel, and occurred in a delayed manner
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through a non-caspase-dependent apoptotic mechanism (Dominguez et al., 2015).
3.1.1.5 Role of leucocytes in inner blood-retinal barrier breakdown
High glucose, nitrosative/oxidative stress, and elevated VEGF levels (but not PGF) induce
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the attachment of monocytes and leukocytes to the vascular wall (leukostasis), by the upregulation of intercellular adhesion molecule 1 (ICAM-1) (Huang et al., 2015; Joussen et al.,
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2002; Leal et al., 2007). Leukostasis is accompanied by blood-retinal barrier dysfunction (Edens and Parkos, 2000; Moore et al., 2003). Leukocyte diapedesis also induces a transient loss in the tight junction proteins occludin-1 and claudin-3 (Xu et al., 2005). Neutralization of ICAM-1 and CD18 prevents leukocyte adhesion and retinal endothelial cell death (Joussen et
dysfunction.
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al., 2001) suggesting that leukostasis directly induces endothelial cell death and barrier
The role of macrophages in microangiopathy has not been demonstrated but in diabetic rat
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retinas, levels of chemokine ligand 2 (CCL2) are increased, inducing monocyte macrophage infiltration in retinal tissues (Rangasamy et al., 2014). It was shown recently that cytokine-
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activated endothelial cells recruit specific types of human monocyte subsets, which in turn can activate endothelial cells and induce neutrophil recruitment (Chimen et al., 2017). Leukostasis was enhanced by TNF-α, but reduced by IL-6. Thus, activation of tissue monocytes is involved in neutrophil recruitment through endothelial cell activation, via a cytokine-dependent regulatory pathway. This is in line with previous observations in rodent models of ischemic retinopathy demonstrating that TNF-α mediates leukostasis induced by VEGF, IL-1ß, and platelet-activating factor. But while TNF-α ablation suppressed vascular permeability induced by platelet-activating factor, it did not affect VEGF- or IL1β-induced
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ACCEPTED MANUSCRIPT permeability, showing that leukostasis and permeability may not be directly linked (Vinores et al., 2007). Multiple mechanisms involving circulating cells, endothelial cells, pericytes, and glial cells can lead to increased barrier permeability without structural vascular alterations. Yet, these
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mechanisms may also lead to structural damages to the retinal microvasculature, subsequently to occlusion, hemorrhages, leakage and vascular remodeling. As opposed to experimental conditions in which the role of single factors can be assessed, multiple intricate pathways and cell functions are involved in the complex pathogenesis of ME. In addition,
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potential compensatory mechanisms may be activated, which further complicates their study.
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3.1.1.6 Neovascularization and vessel abnormalization
Most active neovessels, either originating from the choroidal or retinal vasculature, manifest with leakage or diffusion on fluorescein angiography. This leakage results from the weakness of their barrier properties. VEGF, a major inducer of retinal neovascularization, also induces
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phosphorylation of occludin, which is required for VEGF-induced endothelial permeability. Expression of occludin mutated at the phosphorylation site inhibits angiogenesis in cell culture models and in vivo (X. Liu et al., 2016). Cell division can thus not be dissociated from
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cell junction opening. Pericyte coverage defect and non-mature neurovascular glial coupling can favor leakage of neovessels. After anti-VEGF treatment, choroidal neovessels in age-
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related macular degeneration show reduced leakage on fluorescein angiography (Avery et al., 2006). Retinal vessel abnormalization, as occurring in micro- or macro-aneurysms can be associated with focal leakage, resulting at least partly from VEGF/PGF-induced permeability (Nguyen et al., 2016; Witmer et al., 2003), cx43 and caveolin-1 decrease (Gu et al., 2014a; Shoja et al., 2007), and PDGF-B-mediated pericyte alterations (Lindahl et al., 1997). Choroidal neovascularization, when developing anterior to the RPE, creates a physical RPE barrier rupture, whilst it rather exerts multifactorial stress on the RPE when developing under the RPE (e.g. inflammatory, mechanical, proteolytic stresses), that also contribute to RPE barrier dysfunction (D’Amore, 1994; Gao et al., 2016; Zhang and Ma, 2007). Subretinal and 32
ACCEPTED MANUSCRIPT intraretinal fluid accumulation in neovascular macular disorders result not only from the abnormal neovessel itself, but also from the associated outer blood-retinal barrier rupture.
3.1.2
Increased fluid entry through outer retinal barriers
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Because barrier integrity and functions of polarized ion/water channels are closely linked, it is difficult to determine in which proportion subretinal fluid results from increased entry from the choroid, and from insufficient removal by the RPE.
Retinal pigment epithelium and choroidal vessel junction alteration
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3.1.2.1
A focal disruption of RPE intercellular junctions may by itself be the site of fluid entry from the
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choroidal vessels, as shown in central serous chorioretinopathy (Figure 6G and H) with focal or multifocal fluid leakage sites visible on fluorescein angiography (Daruich et al., 2015b; Pryds et al., 2010). Indeed, contrarily to retinal vessels, choroidal capillaries are fenestrated, allowing fluid passage, that in physiologic conditions is stopped by the tight junctions between RPE cells. In case of enhanced permeability of choroidal vessels, as observed in a
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range of pathologic conditions such as CSCR, disruption of RPE intercellular junctions favors fluid to enter into the sub-retinal space. Moreover, underlying choroidal vascular abnormalities may also alter Bruch’s membrane integrity. Subretinal fluid accumulation is
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possibly further exacerbated by mechanical stress induced by choroidal pressure on the RPE, that may alter RPE polarity, and subsequently its fluid drainage capacity.
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Chronic oxidative and/or metabolic stress can also result in subtle and diffuse RPE barrier dysfunction, as observed during diabetes (Y.-H. Chen et al., 2012; Omri et al., 2013; Xu et al., 2011). Serous detachment of the macula, suggestive of RPE barrier breakdown, is observed in approximately half of cystoid ME cases secondary to uveitis (Grajewski et al., 2016) or branch retinal vein occlusion (Celık et al., 2016), and a third of diabetic ME cases (Shereef et al., 2014; Vujosevic et al., 2017). This suggests that inner and outer blood-retinal barrier breakdown may occur simultaneously, and that among mechanisms leading to ME, RPE dysfunction is probably underestimated. Yet, subretinal detachments in ME may not be
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ACCEPTED MANUSCRIPT caused by RPE disruption, since RPE junction barrier seems to be highly resistant to acute hypoxia/ischemia in experimental conditions (Kaur et al., 2008). In addition, it is not clear to which extent the RPE, depending on choroidal blood supply, is affected by retinal nonperfusion. The processes involved in RPE barrier alteration in vivo are not fully understood
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and have drawn less attention than those involved in inner barrier breakdown. The following mechanisms are involved in RPE barrier dysfunction, and will be detailed below: release of pro-inflammatory mediators, VEGF-mediated enhanced permeability, MMP proteolytic activities, cytoskeleton regulatory proteins, and mast cell degranulation.
Several pro-inflammatory mediators, mostly cytokines, were shown to exert direct
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•
effects on the expression and/or subcellular localization of junction proteins in vitro. For
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example, TNF-α directly influences ZO-1 distribution in polarized RPE cells through the p38 MAPK (Shirasawa et al., 2013) and IL-1β pathways, increasing intercellular permeability through occludin down-regulation and claudin-1 up-regulation (Abe et al., 2003). In cultured RPE cells from retinas of rats from the “PVG” strain, susceptible to development of
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experimental uveitis, lipopolysaccharide associated with IFN-γ and TNF-α induce alterations of ZO-1 and actin cytoskeleton, while NO was found to maintain the barrier integrity (Zech et al., 1998). Oxidative stress also disrupts RPE barrier integrity through NF-κB activation
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(Inumaru et al., 2009). These pro-inflammatory mediators are involved in uveitis-related ME, but also in ME of other causes, as illustrated by the elevated levels of several cytokines
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detected in ocular media from patients with ME (Tables 1-3). Yet, RPE cells also produce in vivo molecules that potentially prevent TNF-α- or IL-1β-induced barrier breakdown, such as IL1-receptor agonist (IL1-RA) and soluble TNF receptors. •
Permeability of the RPE may be increased by angiogenic molecules of the VEGF
family. VEGF-A, secreted at the baso-lateral side of RPE cells in physiologic conditions, can favor the alteration of RPE barrier function in pathologic conditions. In rodents exposed to acute light damage, photoreceptor cell death can be prevented by inhibition of VEGFinduced RPE barrier breakdown. This observation suggests that VEGF produced in response
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ACCEPTED MANUSCRIPT to photic stress contributes to photoreceptor cell death secondary to RPE barrier rupture (Cachafeiro et al., 2013). This mechanism may explain the rupture of the RPE barrier occurring in other causes of retinal injury, and leading to ME. PGF, up-regulated under hypoxic conditions or insulin exposure, modifies the subcellular distribution of occludin
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through MEK signaling pathway in vitro, and in vivo in a rat model overexpressing PGF (Kowalczuk et al., 2011; Miyamoto et al., 2007). Whether the effect of VEGF on RPE
permeability involves VEGF receptor 1 or 2 is controversial (Ablonczy et al., 2009; Miyamoto et al., 2007). Both receptors were found in RPE from human eye specimens (Chen et al.,
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1997). In transgenic mice overexpressing VEGF in photoreceptors, a focal increase of RPE permeability was observed, and manifested by RPE tight junction alterations and
•
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degenerative changes of RPE cells (Vinores et al., 1999).
Activation of matrix metalloproteinases, particularly MMP-9, can also contribute to
RPE junction protein alterations. MMP-9 is activated by oxidative and nitrosative stress in the diabetic retina (Giebel et al., 2005; Kowluru et al., 2012), and by amyloid-β, leading to the
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proteolytic degradation of occludin and overall disruption of the tight junction complex (Cao et al., 2013; Giebel et al., 2005; Navaratna et al., 2007). In addition, MMP-1 increased activity is involved in the action of TNF-α on RPE barrier integrity (Li et al., 2010). The polarity/adhesion/barrier functions of highly polarized epithelia such as the RPE
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•
are closely regulated by small kinases and GTPases, particularly calcium-independent
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atypical PKCs (Citi et al., 2014; Etienne-Manneville and Hall, 2003), as mentioned in 2.1.2.1. Recently, the concept of "zonular signalosome" has been proposed, highlighting the close interaction between junction proteins (zonulae occludens and adherens) and the cytoskeletal organization, through Rho GTPases and their effectors (Citi et al., 2014). •
Mast cells are another important actor of outer barrier alteration. Mast cells, abundant
along choroidal vessels (Figure 9A), particularly arterioles, play a major role in ocular inflammation (Godfrey, 1987), being the first activated cells in experimental autoimmune uveitis (de Kozak et al., 1981). Mast cells produce numerous factors such as biogenic amines (e.g. histamine), proteases (e.g. chymase, tryptase), angiogenin, proteoglycans, 35
ACCEPTED MANUSCRIPT cytokines (e.g. TNF-α, IL-4, IL-6, IL-33, IL-15), chemokines (e.g. CCL5, IL-8, Monocyte chemotactic protein 1 (MCP-1), eotaxin), growth factors (e.g. NGF), peptides, prostaglandins, leukotrienes, and complement factors. Histamine is not only a potent vasodilator but also a major player in microvascular disease.
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In primary endothelial cells in vitro, ~53% of all VEGF-regulated transcripts were also regulated by histamine, including genes involved in tight junction formation (i.e. claudin 5) and expression of pro-angiogenic transcription factors affecting endothelial cells survival, migration and tube formation (Laakkonen et al., 2017).
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Substances released experimentally after substance-P-induced mast cell degranulation
include complement C3a and C5a fragments, and endothelin 1, and might differ from those
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released after anti-IgE-induced degranulation (Gaudenzio et al., 2016). In patients with branch retinal vein occlusion, circulating endothelin-1 has been detected, and anti-VEGF treatment has been associated with reduced circulating endothelin-1 levels (Kida et al., 2016) suggesting a potential indirect role on mast cell degranulation. Hypoxia induces the release
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of IL-8 and TNF-α by mast cells via HIF-1α, and catalyzes the formation of histamine (Nizet and Johnson, 2009). Oxidative stress also induces mast cell degranulation (Chelombitko et al., 2016). Tryptase released upon mast cells degranulation cleaves the protease activated
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receptor 2 (PAR2) present at microglia surface, resulting in microglia activation (Park et al., 2010). We have shown recently that the local degranulation of mast cells using sub-
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conjunctival injection of compound 48/80, a synthetic polymer and known inducer of mast cell degranulation (de Kozak et al., 1983), resulted in serous retinal detachments and pigment epithelial detachments in rodents (Figure 9 B and C), and that its inhibition by cromoglycate could alleviate the retinal consequences of mast cell degranulation (Bousquet et al., 2015; Steptoe et al., 1994). Histamine overproduction per se does not seem to cause any retinal abnormalities in transgenic mice (Greferath et al., 2014). Yet, the complex cocktail of active agents released by mast cells degranulation massively disrupted the outer retinal barrier. Taking into account the large number of mast cells present in the choroid, their proximity with vessels and 36
ACCEPTED MANUSCRIPT microglia (Forrester et al., 2010), and the presence of known inducers of mast cells degranulation in the choroid (particularly C5a), mast cells is likely an underestimated contributor to outer retinal permeability in several retinal diseases. 3.1.2.2
Retinal pigment epithelial cell death
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Whether RPE cell death per se contributes to subretinal fluid accumulation remains to be demonstrated. Indeed, large surgical resection of RPE and choroid in eyes with neovascular AMD that underwent free autologous RPE-choroid graft, before the anti-VEGF therapy era, did not induce retinal or subretinal fluid accumulation (van Zeeburg et al., 2012). In
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geographic atrophy, characterized by RPE cell death (Hanus et al., 2015), macular edema does not occur unless neovascularization is associated (Dunaief et al., 2002; Kaneko et al.,
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2011; Kim et al., 2002). In both examples, note that the underneath choroidal vascularization is either absent or reduced. On the other hand, subretinal fluid is commonly associated with areas of RPE atrophy as observed in chronic central serous chorioretinopathy (Daruich et al., 2015b), often adjacent to dilated large choroidal vessels. In this disorder, choroidal vascular
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hyperpermeability, visible on mid-phase indocyanine green angiography, may cause choroidal edema, manifesting for instance as “loculation” of fluid in the posterior choroid (Spaide and Ryan, 2015). Choroidal edema may presumably alter the retina-to-choroid
3.1.2.3
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osmotic gradient, providing an explanation for the accumulation of fluid under the retina. Outer limiting membrane disruption
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OLM barrier alteration favors large Stoke radius proteins movement towards the subretinal space. As the OLM is also critical for the polarization and structural maintenance of RMG cells and photoreceptors, its destabilization favors photoreceptor misalignment and degeneration. In chronic retinal diseases, such as diabetic retinopathy, disruption of junction protein complexes occurs in the retinal vascular endothelium, the RPE and at the OLM, with differential severity and kinetics. From a clinical point of view, OLM disruption on OCT has been correlated with poorer visual acuity in diabetic ME and retinal vein occlusion (Ito et al., 2013; Murakami et al., 2016). It had also been linked to poor responses to anti-VEGF
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ACCEPTED MANUSCRIPT therapy in wet AMD (Zandi et al., 2017), vein occlusion (Chatziralli et al., 2017) and diabetic ME (Ashraf et al., 2016; Muftuoglu et al., 2017).
3.2
Decreased drainage functions
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As detailed in 2.2, RPE and RMG cells actively drain water and osmolytes from the retina to the systemic circulation, or the vitreous. Hydro-ionic transport across cells is ensured by ion and water channels distributed in a synchronized manner with cell polarization, depending on intercellular signaling, and by various transporter molecules. The hydro-ionic transports can
3.2.1
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dysfunction, or in response to extracellular signals.
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be altered due to RPE and/or RMG cell death, cell metabolic suffering, cytoskeleton
Glial cells and neurosensory retina
RMG cells rapidly respond to any change in their microenvironment and develop strong resistances to injury because of their glycogen reserve, as well as their anti-oxidant and
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detoxification capacities, for instance via glutamate uptake. In response to injury RMG cells rarely undergo apoptosis and rather dedifferentiate into pluripotent retinal progenitors with the potential of regenerating retinal neurons, as evidenced for instance in zebrafish studies
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(Goldman, 2014). However this process is limited and poorly effective in mammals (Hamon et al., 2016). Any retinal stimulus induces a reactive state of RMG cells, identified
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experimentally by the increased expression of intermediate filaments contributing to the cytoskeleton, such as GFAP (Lewis and Fisher, 2003). Uncontrolled healing processes can turn into gliosis (Bringmann et al., 2006; Bringmann and Wiedemann, 2012), which may have deleterious structural and functional consequences. Such dedifferentiation is associated with a reduction of potassium conductance due to decreased expression and/or mislocation of Kir4.1 channels (Reichenbach et al., 2007). Ion and water channel expression in astrocytes, involved in the osmoregulation of the inner retina is also altered in pathologic conditions (Iandiev et al., 2006). Since it is admitted that water transport is tightly coupled to spatial K+-buffering currents, any 38
ACCEPTED MANUSCRIPT inhibition of either water or potassium transport mechanisms leads to consequences on the other. Reduction of potassium conductance has been observed rapidly in models of diabetes (Fletcher et al., 2007; Pannicke et al., 2006), retinal vein occlusion (Rehak et al., 2009), various forms of retinal degeneration (Dinet et al., 2012; Lassiale et al., 2016), and uveitis
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(Eberhardt et al., 2011; Feng et al., 2013; Liu et al., 2007; Zhao et al., 2011). The disturbance of potassium homeostasis contributes to neuronal cell death and ischemia.
Indeed, the perivascular release of potassium plays an important role in regulating regional blood flow, in response to changes in neuronal activity (Paulson and Newman, 1987). Table
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4 summarizes the changes in K+ and water channels observed in different rodent models of retinal diseases.
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In response to ischemic stress, the aquaglyceroporin AQP9, belonging to a subgroup of aquaporins transporting water and other osmoregulators such as glycerol and urea (HaraChikuma and Verkman, 2006), is increased in the neurosensory retina (Hollborn et al., 2012). In the STZ-induced diabetes rat model, AQP1 is increased while AQP6 and AQP11 are
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decreased (Hollborn et al., 2011). In the Torii type 2-diabetes rat model, AQP0 is increased at the level of the nerve fiber layer (Fukuda et al., 2011). In addition, the transport of osmoregulator amino acids such as GABA and taurine is also involved in the formation of
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al., 2004).
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retinal edema (Pasantes-Morales et al., 1999), in both RMG and RPE cells (El-Sherbeny et
3.2.2
Retinal pigment epithelium
The proper polarization of the RPE and subsequent adequate localization of ion and water channels requires a synchronized cross-talk between extracellular signals and cell cytoskeleton. This cross-talk requires the activation of different kinases involved in signal transduction, such as those belonging to the MEK/ERK and RhoA/ROCK pathways (Caplan, 1997; Citi et al., 2014; Schevzov et al., 2015). In several clinical situations, RPE transport dysfunction is associated with or favors fluid accumulation in the subretinal space. Multiple, rapid-onset transient serous retinal 39
ACCEPTED MANUSCRIPT detachments have been described after systemic administration of MEK inhibitors for metastatic cancer, without observation of leakage on fluorescein angiography (Urner-Bloch et al., 2014). This clinical observation suggests a cellular RPE dysfunction, rather than a focal junction breakdown, as detailed in 6.4. In vitro, over-activation of Rho kinase stimulates
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actin cytoskeleton contraction in RPE cells, and subsequent cell mobility (Zheng et al., 2004). In an in vivo rat diabetic mode, ROCK inhibition reduces RPE leakage (Rothschild et al., 2017).
In addition, aquaporins are also critical for the drainage functions of RPE cells. AQP1
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contributes to the transepithelial water transport (Stamer et al., 2003), that is enhanced by the natriuretic peptide (Baetz et al., 2012). AQP1 is overexpressed to compensate fluid
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transport reduction above drusen in eye specimens with early or advanced AMD (Tran et al., 2016), and in the diabetic retina (Hollborn et al., 2011). It is down-regulated in response to UV-induced oxidative stress (Jiang et al., 2009). AQP9 is also overexpressed in vitro by RPE cells under chemical hypoxia, oxidative stress, VEGF, and high glucose (Hollborn et al.,
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2012).
In STZ-induced type 1 diabetes, AQP5, AQP9, AQP11, and AQP12 were over-expressed in the RPE, whilst AQP0 was reduced (Hollborn et al., 2011). The alternative complement
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pathway contributes to physiologic transport of ions and macromolecules through pore formation such as C5b-8. This mechanism may be underestimated in the context of AMD
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(Farkas et al., 2002).
In summary, any retinal stress can induce the activation of glial cells and modify the expression and cellular distribution of water channels in RMG and RPE cells. Subsequently, the distribution of ions and other osmolytes across the retina is also altered, resulting in ME. In this context, ageing, accompanied by cumulative oxidative stress, is associated per se to a reduction of potassium conductance in RMG cells, explaining that ME is favored by older age (Bringmann et al., 2003). To date, the full spectrum of molecular signals that control hydroionic retinal homeostasis is incompletely understood, and other crucial pathways have also
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ACCEPTED MANUSCRIPT been identified. For instance, the gluco- and mineralocorticoid receptors stimulated by endogenous or therapeutic steroids have been shown to control the expression and distribution of AQP4 and Kir4.1, in both healthy and inflammatory rat retina (Zhao et al.,
3.3
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2011).
Protein leakage
In addition to inner and outer blood-retinal barrier breakdown, allowing fluid entry, permanent
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fluid accumulation in the retina depends on several factors that may differ according to
clinical etiologies of ME. When barrier functions are no longer acting, the Starling equation
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governs fluid movements between different compartments.
The Starling equation allows to calculate the movement of fluid across capillary membranes, integrating the effects of hydrostatic and oncotic forces (referred as “Starling forces”). The net fluid movement across capillaries results from diffusion, filtration and pinocytosis, but the Starling equation only refers to filtration parameters. The direction and extent of passive
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water exchanges between the retinal and choroidal extravascular compartments, and the intravascular compartments (formed by the retinal capillaries, the choriocapillaris and large choroidal vessels), is mostly determined by their relative hydrostatic and oncotic pressures.
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The intra-capillary hydrostatic force is a positive pressure generated by cardiac contractions, while the interstitial hydrostatic pressure may reach negative values in certain tissues, as a
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result of lymphatic drainage and muscle contraction (Aukland and Reed, 1993). In the retina, absence of lymphatic vessels could be at least in part compensated by active transepithelial fluid transport across the RPE toward the choriocapillaris (Marmor et al., 1985; Nickla and Wallman, 2010). The exact values of hydrostatic pressure in the different layers of the neurosensory retina are not known, but are expected to be also negative in physiological state. The Starling equation reads as follows: Jv = Kf × ([Pc - Pi] - σ × [πc - πi])
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ACCEPTED MANUSCRIPT Where: • Jv is the net fluid movement between compartments, • Kf is a proportionality constant called the filtration coefficient,
• Pc is the capillary hydrostatic pressure • Pi is the interstitial hydrostatic pressure • πc is the capillary oncotic pressure
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• πi is the interstitial oncotic pressure
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• ([Pc - Pi] - σ × [πc - πi]) is the net driving force,
• σ is the reflection coefficient
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Due to the low protein concentration of the vitreous, the presence of retinal barriers, and the structure and function of the choroidal vasculature, a protein gradient is established from the vitreous to the choroid, allowing a permanent outflow of water towards the choroid. As mentioned in 1.1. Any increase in retinal protein concentration within interstitial retinal tissue
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will cause an accumulation of water driven by osmotic forces. A moderate increase in vascular leakage with increased pinocytic transport, but no rupture of the endothelial tight junctions (and consequently no major protein leakage within the neurosensory retina), may not lead to the formation of retinal edema. On the other hand, intense protein and lipid
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extravasation is associated with focal edema around the so-called “exudates”.
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The OLM constitutes a filter to macromolecular diffusion from the subretinal space to the outer nuclear layer (Marmor et al., 1985; Marmor, 1990). If the OLM is preserved, proteins may accumulate in the retina, but remain inner to the OLM. Accumulation of proteins within the photoreceptor layer may cause neuronal toxicity (Cachafeiro et al., 2013). Rupture of the OLM allows proteins to diffuse into the subretinal space, causing subsequent RPE hyperosmotic stress and potential additional damages (Willermain et al., 2014). OLM disruption on OCT is associated with worse visual prognosis in conditions manifesting with ME, as detailed in 3.1.2.3. Depending on the microenvironment, proteins, lipids and glycoproteins may form low soluble aggregates. These exudates may persist or require
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ACCEPTED MANUSCRIPT months to regress, as observed clinically.
4 Why does edema specifically form in the macula? Several
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hypotheses
We provide below a list of several mechanisms involved in ME, some of which are
4.1
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on current biological and translational evidence.
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consensually admitted, and some others being advanced as hypothetic mechanisms, based
Alteration of junction proteins in the “Z-shaped” Müller cells zone
The functional specialization of the fovea, ensuring precision vision, results from several
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anatomical specificities. They include high cone density, reduced vascularity with absence of retinal vessels in the visual axis, and centrifugal displacement of cone axons and specialized cone RMG cells. RMG cell density is about 5 times higher in the fovea as compared to the periphery (Distler and Dreher, 1996). The cone-projected axons are intertwined with RMG
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cells and follow a centrifugal course towards the inner layers at the edges of the fovea. In this
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area, RMG cells are highly elongated as compared to peripheral RMG cells, reaching several hundred micrometers (Figure 10A and B). As their processes draw from ILM to the OLM, the foveal RMG cells follow a “Z-shape” trajectory (Bringmann et al., 2004; Matet et al., 2015). They run vertically from their endfeet at the ILM to the OPL, turn inwards towards the foveal center across the Henle fiber layer where they reach almost horizontally the foveal pit, before adopting again a vertical diving direction towards the OLM (Figure 10A). Along this Z-shaped trajectory, RMG prolongations are closely bound to photoreceptor axons by junction proteins such as ZO-1 (Matet et al., 2015) (Figure 10B), preventing high Stoke radius molecules, notably proteins, to accumulate in this specific region.
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ACCEPTED MANUSCRIPT In pathologic conditions altering the organization and stability of junction proteins (e.g. ZO-1), this “molecular filter” may become leaky, allowing proteins to accumulate between axons and Z-shaped RMG cells. This alteration would attract oncotically-driven fluid, subsequently leading to the formation of “cysts”, as observed in cystoid ME. Interestingly, the radial
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organization of cysts around the fovea (Figure 3E) corresponds to the area where RMG cells and ZO-1 co-localize (Figure 10B), reinforcing this hypothesis. In addition, in the human foveola, we have identified GFAP-positive cells, highly expressing AQP4, but poorly expressing glutamine synthetase and CRALBP, forming a “glial roof” and projecting
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extensions toward the Henle fiber layer (Figure 10F inset, yellow arrows). The exact nature of these cells and their role in water drainage from the fovea and in central cysts formation
4.2
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remains to be determined.
The “glymphatic” system hypothesis
In the retina, like in the brain, no lymphatic system has been identified to clear waste
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products, such as proteins, water and other metabolic byproducts. Water and hydosoluble compounds do not diffuse freely across vessels (Cunha-Vaz, 2017) since in addition to tightjunctions in endothelial cells, the water channel AQP-1 is not present (Motulsky et al., 2010).
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In the brain, AQP4 localized in perivascular end-feet of astrocytes allows the formation of low-resistance pathways for fluid movement between paravascular spaces and interstitium.
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Indeed, although AQP4 is located in astrocytes around vessels in the brain, it does not allow water to enter the brain vessels, creating a paravascular flow, recognized as the “glymphatic pathway” since like the lymphatic system it constitutes a waste clearance pathway for interstitial solutes and proteins (Iliff et al., 2012; Nakada et al., 2017). In the rodent retina, AQP4 is highly expressed in RMG cells, specifically located at the RMG endfeet, at the ILM and around vessels, and at the optic nerve head (Amann et al., 2016). We have analyzed the distribution of AQP4 and RMG in healthy post-mortem human retinas including the maculae (Figure 11) on sections and on flat-mounted specimen. As expected,
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ACCEPTED MANUSCRIPT AQP4 is located around all retinal vessels in all plexuses (Figure 11A), and in astrocytes and RMG endfeet in the superficial inner retina. However, AQP4 co-localizes with glutamine synthetase only at the level of the deep capillary plexus, where no astrocytes but only RMG cells are present (Figure 2 A and E, and Figure 11F).
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At the fovea, where no vessels are present, AQP4 is highly expressed along central RMG cells (Figure 11 A, B-D) that poorly express GS (Figure 11 C). In the Henle fiber layer, Zshaped RMG cells highly express AQP4 (Figure 11A, dotted contour, and 11D inset). In flatmounted macula from non-human primate and from human, RMG cells (labeled with
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glutamine synthetase and with CRALBP respectively) follow the axon organization towards the optic disc (Figure 12A and Supplementary Figure 1). AQP4 is concentrated in the fovea
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(Figure 12A) and form patches along RMG cells towards the optic nerve head (Figure 12C and D). Higher magnification allows the identification of “channel-like” structures surrounded by AQP4 (Figure 12E and inset).
We raise the hypothesis that like in the brain, AQP4 could create a permanent solute flow
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between RMG cells and a hydraulic path, around the vessels towards the optic nerve head (Figure 13). A glymphatic pathway could thus exist also in the retina and particularly the macula for the clearance of waste solutes and proteins. At the optic nerve head, previous
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studies had identified a defect in blood-retinal barrier integrity, allowing proteins to be drained from the retina (Flage, 1977). More recently, a glymphatic pathway, driven by AQ4 has been
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described at the optic nerve connected with the cerebrospinal fluid, allowing transport of protein below 70KD (Mathieu et al., 2017). In case of excess of protein leakage and overload of the glymphatic drainage of proteins along the Z shaped Müller cells, proteins accumulation will retain water and cause CME. In retinal diseases such as diabetic retinopathy, AQP4 expression and/or localization is modified (Table 4), which could disrupt this potential “glymphatic pathway” and promote protein accumulation within the neurosensory retina, favoring fluid accumulation according to Starling equation. In a human diabetic retina, AQP4 expression is reduced in the fovea (Figure 14 A-C) and increased outside the macula (Figure 14 D-F). Further studies should
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ACCEPTED MANUSCRIPT explore this hypothesis that still remains speculative.
4.3
Increased interstitial pressure: vascular consequences
Increase in protein concentration inside the neuroretina subsequently raises the interstitial
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pressure, reducing capillary blood flow. In particular, the downstream and terminal deep retinal capillary plexus may be more susceptible to flow disruption, since its intravascular pressure is lower than in the superficial plexus. Because Kir4.1 channels of RMG cells are
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mislocalized in several animal models of retinal vein occlusion and diabetic retinopathy
(Table 4), and because regulation of the potassium microenvironment at the deep capillary
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plexus only relies on RMG cells, and not astrocytes (as discussed in 2.1.1.4 and Figures 2 and 11), the local decrease in perivascular potassium concentration is expected to reduce the vascular flow (Longden et al., 2016) (Figure 15). Subsequently, drainage by the putative glymphatic system (see 4.2 and Figure 13) along non-perfused vessels may decrease, creating an amplifying mechanism. The recent clinical observation that cystoid ME of
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vascular origin correlates with areas of reduced flow in the deep capillary plexus (Mané et al., 2016; Spaide, 2016) is a possible manifestation of these mechanisms.
The formation of “cysts”
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4.4
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Very few anatomical eyes specimens presenting ME have been studied, and most of them were analyzed with conventional staining of macular sections, without immunohistochemistry. The description of cystoid ME reveals that the empty cavities begin to form in the Henle fiber layer and can extend over time to other layers. In some cases, the central RMG cell cone seemed absent (Wolter, 1981). Three eyes with cystoid macular edema diagnosed on fluorescein angiography were examined using photonic and electron microscopy, showing widespread swelling and necrosis of RMG cells without enlargement of intercellular spaces, suggesting that cysts are caused by degeneration of RMG cells (Fine and Brucker, 1981; Yanoff et al., 1984). Whether retinal cysts result from degenerating RMG
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ACCEPTED MANUSCRIPT cells remains a subject of debate, but the role of RMG cells in the formation of cystoid macular edema is certain. If a “glymphatic” system exists in the retina, the loss of central RMG cells would prevent the central drainage system to function and permanent recurrence
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of edema.
Depending on the primary mechanism and etiology of ME, different fluid topography and associated signs may be observed on OCT, which may differentiate the causative diagnoses (Munk et al., 2014). For example, in purely retinal vascular disorders such as aneurysmal
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telangiectasia (macular telangiectasia type 1), focal ME tends to localize in the vicinity of vascular lesions (Figure 16). In diabetic ME, leakage from the deep capillary plexus induces
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fluid migration in the outer plexiform layer whereas more superficial plexus leakage induces fluid to accumulate in the inner nuclear layer (Byeon et al., 2012). Interestingly, subretinal fluid accumulation is frequently associated with ME due to diabetes or branch retinal vein occlusion (Celık et al., 2016; Noma et al., 2011b; Ota et al., 2013; Shereef et al., 2014), as
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discussed in 3.1.2.1. This observation suggests that early alteration of outer retinal barriers occurs, as observed in animal models of diabetic retinopathy (Desjardins et al., 2016; Omri et al., 2013; Xu et al., 2011).
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Intracellular edema cannot be detected clinically but has been observed on pathological analyses of eyes with ME (Kohno et al., 1983; Yanoff et al., 1984). It may localize within RPE
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cells, RMG cells, and in neurons, as a consequence of metabolic disturbances. How does intracellular edema contribute to the clinically significant ME remains unclear, but it makes no doubt that intracellular edema contributes per se to neuronal toxicity and to extracellular fluid volume increase.
5 Why does macular edema alter vision? ME is a severe complication of numerous retinal disorders because it affects the central vision. This visual impairment can be variable, and results from multiple mechanisms.
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ACCEPTED MANUSCRIPT Moreover, vision loss may be reversed by ocular therapy or control of favoring systemic factors in recent ME of short-duration, but may become irreversible in long-standing cases.
5.1
Evaluation of visual function in macular edema
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Additionally to central vision loss, usually experienced as a relative central scotoma affecting far- and near-vision, patients with ME often also complain of metamorphopsia (Achiron et al., 2015), reading difficulties (Munk et al., 2013), impaired stereopsis, or disturbed color vision. Visual acuity testing, a subjective method based on letter recognition assessing the
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resolution ability of the fovea, is the most widely employed test, both in the clinical setting
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and as validated clinical research endpoint. However, visual acuity does not reflect all alterations affecting central vision, such as perifoveal scotoma or reading speed reduction. Microperimetry is a subjective test that estimates the contrast sensitivity of the macular area, and can detect perceived or non-perceived micro-scotoma. The contrast sensitivity is altered in the presence of ME, correlates with retinal thickness and visual acuity (Okada et al., 2006;
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Vingolo et al., 2016), and shows a spatial correlation with macular lesions such as outer or inner retinal cysts, serous retinal detachments and hard exudates (Deák et al., 2010). Microperimetry testing also assesses retinal fixation, impaired in diabetic ME (Carpineto et
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al., 2007), and aggravated when hard exudates are present (Vujosevic et al., 2008). Reading speed, another subjective test evaluating near vision, correlates with central macular
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thickness (Kiss et al., 2006; Suñer et al., 2013), and central microperimetry (Edington et al., 2017).
The influence of structural changes induced by ME on visual function has been comprehensively reviewed (Tomkins-Netzer et al., 2015). Retinal contrast sensitivity on microperimetry, as well as reading speed testing, are useful adjuncts to visual acuity testing to evaluate visual impairment caused by diabetic, vascular or uveitic ME. The function of the macula can also be assessed objectively by multifocal electroretinography, recording the retinal electric activity upon focal stimulation at multiple macular locations. Multifocal electroretinography studies showed that this activity is altered in diabetic ME, and that the 48
ACCEPTED MANUSCRIPT degree of signal alteration correlates with retinal thickness and visual acuity (Tehrani et al., 2015; Yamamoto et al., 2001). Interestingly, electroretinography also showed that alterations of neuronal activity affecting all retinal layers preceded retinal thickening (Nagesh et al., 2016), in line with the notion that ME
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results from severe physiological changes disturbing the normal retinal metabolism, before the development of clinically-patent ME. Noteworthy, certain presentations of subretinal or intraretinal fluid may not - or very mildly - alter vision, such as early diabetic ME or acute
subretinal fluid is not the sole cause of visual loss in ME.
Acute effects of macular edema on visual function
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5.2
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central serous chorioretinopathy. This supports the concept that the presence of cysts or
Several mechanisms at the subcellular level, and other related to the architecture and vascular supply of the retina, explain why fluid accumulation disturbs visual function. Several acute effects of ME are reversible, when fluid resorbs spontaneously during disease course
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or as response to therapy, as evidenced by the more favorable functional outcome after earlier therapeutic intervention on ME of various causes in clinical practice. ME may disturb the normal path of light from the inner retinal surface to the photoreceptor outer segments,
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where the light signal is captured, and triggers the phototransduction cascade. First, retinal thickening induces a loss in normal retinal transparency. Second, a change in the refractive
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index of retinal layers, as well as an increased light diffraction may occur secondary to hydroionic deregulation, and protein accumulation in the retinal interstitium. Finally, disruption of the normal RMG cell architecture in the central macula, due to intracellular swelling or extracellular fluid excess, may alter light transmission. The role of RMG cells in light guidance having been established for peripheral cells but not for the foveal glial cells, whether this mechanism play a major role in vision alteration remains questionable (Agte et al., 2011; Reichenbach and Bringmann, 2013). These physical effects of ME are probably reversed after ME resolution and explain the good visual recovery in the absence of major alterations of retinal structure. 49
ACCEPTED MANUSCRIPT ME is also accompanied by vascular changes, either causative or secondary, that have also been linked to the degree of visual impairment. The recent advent of OCT angiography allows to image non-invasively details of the perifoveal vasculature. Foveal avascular zone enlargement, frequently observed in diabetic ME, has been identified as an independent
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factor of visual acuity loss (Balaratnasingam et al., 2016). Capillary density of the superficial and deep capillary plexuses is correlated to visual acuity levels in many retinal vascular
disorders manifesting with ME, such as diabetic retinopathy (Samara et al., 2017), central and branch retinal vein occlusion (Samara et al., 2016), or type 1 idiopathic macular
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telangiectasia (Matet et al., 2016). In particular, there is increasing evidence from clinical imaging studies that alterations of the deep retinal capillary plexus are more pronounced
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than those affecting the superficial plexus, as mentioned in 4.2.3. In addition, areas of deep capillary plexus dropout co-localize with cystoid cavities in diabetic ME (Mané et al., 2016) and vascular ME from other causes such as vein occlusion, macular irradiation, and macular telangiectasia type 1 (Spaide, 2016). Moreover, there is no reconstitution of the disrupted
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deep capillary plexus when ME resolves, and in case of recurrence edema tends to recur in the same locations (Spaide, 2016). The retinal vasculature, and particularly the deep plexus, is essential for the metabolic support of the high energy-demanding inner retinal neural
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network. Therefore, disruption of this vasculature provides a plausible explanation for the impaired retinal neural activity present in ME, as evidenced by multifocal electroretinography.
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Acute visual decrease may also occur indirectly when ME is localized nasal to the fovea and disrupts the normal neural signal transmission in the papillomacular nerve fiber bundle (Konieczka et al., 2016).
5.3
Long-term effects of macular edema on visual function
Long standing ME induces irreversible neural retinal damage and glial reactions, as detailed in 3.2.1, which persist even after ME resolution. Definitive photoreceptor alterations manifest clinically with outer nuclear layer thinning, outer segment atrophy and disruption of the ellipsoid zone signal on OCT. Alterations of this hyperreflective band have been correlated 50
ACCEPTED MANUSCRIPT with the degree of visual dysfunction in ME of various causes, such as diabetes (Maheshwary et al., 2010; Otani et al., 2010), branch (Akagi-Kurashige et al., 2014; Ota et al., 2007, 2008b) or central retinal vein occlusion (Ota et al., 2008a), posterior uveitis (Tortorella et al., 2015), Irvine-Gass syndrome (Hunter et al., 2014), epiretinal membranes
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(Fang et al., 2016) and retinitis pigmentosa (Oishi et al., 2009). Alterations of cone arrangement regularity, potentially linked to irreversible photoreceptor damage, have been observed in diabetic ME using adaptive optics imaging (Lammer et al., 2016; Nesper et al., 2017). Intraretinal ME is variably associated with subretinal detachment, depending on its
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etiology and on the time course of ME. Subretinal detachment may occur early, for instance in retinal vein occlusions, or later, for instance in diabetic ME, as intraretinal fluid is
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progressively drained by the RPE or as RPE barrier dysfunction occurs (Omri et al., 2011, 2013; Xia and Rizzolo, 2017). While short-duration detachment of the neurosensory retina from the RPE may not have functional consequences after resolution, long-standing detachments may lead to photoreceptor damage, as exemplified by the more severe visual
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sequelae in chronic versus acute central serous chorioretinopathy (Daruich et al., 2015b). Hard exudate deposition within the neuroretina have long been recognized as a marker of worse visual prognosis (Coscas and Gaudric, 1984), and studies combining OCT and
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microperimetry have confirmed that they co-localize with areas of decreased retinal sensitivity (Deák et al., 2010).
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In diabetic ME, long-standing enlargement of the foveal avascular zone can lead to irreversible foveal ischemia, clinically described as “ischemic maculopathy” on fluorescein, and recently OCT angiography. This complication of advanced ME leads to irreversible foveal thinning affecting all layers on OCT. In particular, thinning of photoreceptor outer segment, ellipsoid zone (Lee et al., 2013) and ganglion cell layer (Byeon et al., 2009) has been identified following ischemic diabetic ME. Finally, chronic ME can also induce permanent visual loss, due to structural disorganization of retinal inner layers, or “DRIL”, persisting after ME resolution. DRIL is defined as the absence of detectable boundaries on OCT between the ganglion cell-inner plexiform layer
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ACCEPTED MANUSCRIPT complex, the inner nuclear layer, and the outer nuclear layer. DRIL reflects the degree of cellular damage accompanying ME, and its morphological extension has been correlated to the degree of visual loss (Radwan et al., 2015; Sun et al., 2014).
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6 What can we learn from pure phenotypes? Besides the most frequent causes of ME extensively investigated in the literature, such as diabetes, retinal vein occlusions or uveitis, there are specific conditions manifesting with
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intraretinal fluid accumulation, in which one single cellular mechanism predominates. From a translational perspective, these presentations contribute to our understanding of the
6.1
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underlying mechanisms and their clinical expression.
Irvine-Gass postoperative macular edema
Pseudophakic macular edema, also referred to as Irvine-Gass syndrome, is a complication of
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cataract removal or other intraocular procedures (Figure 6 C-D). It is characterized by the release of pro-inflammatory mediators triggered by the surgical “trauma”, that induces bloodocular barrier breakdown (Grzybowski et al., 2016). Although its exact pathogenesis is not
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fully elucidated, it clearly occurs after barrier alteration, and can be therefore considered a pure phenotype. Amongst associated factors suspected to predispose for postoperative
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macular edema, including vitreous traction and inflammation, authors have suggested a role of choroidal blood flow changes and instability (Nicholls, 1954; Welch and Cooper, 1958). Reduction in choroidal blood flow velocity and volume was indeed shown to occur in pseudophakic macular edema (Miyake et al., 2007). Interestingly Irvine-Gass macular edema frequently presents with subclinical optic disc swelling, manifesting on fluorescein angiography as late disc hyperfluorescence (Figure 6C). Whether impaired glymphatic pathway drainage, discussed in 4.2, could be involved in the pathogenesis of pseudophakic macular edema should therefore be evaluated.
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ACCEPTED MANUSCRIPT 6.2
Idiopathic macular telangiectasia type 1
Macular telangiectasia type 1, also termed “aneurysmal macular telangiectasia”, is a retinal vascular disorder of congenital or developmental origin that affects mostly men. It manifests
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during early adulthood or later with multiple exudative dilations of perifoveal capillaries, termed telangiectasia, that cause macular edema. It may be associated with areas of
perifoveal non-perfusion, and in some cases with localized peripheral non-perfusion (Gass
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and Blodi, 1993; Yannuzzi et al., 2006). Since it is unilateral in most cases, and may extend beyond the macula, it has been linked to the spectrum of Coats disease (Cahill et al., 2001).
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Macular telangiectasia type 1 is thought to originate from a vascular abnormality affecting retinal and macular capillaries, without other structural alterations than the presence of cystoid cavities. In particular, there is no metabolic or inflammatory precipitating factors, no RPE or OLM alteration, and good visual function is maintained, except at sites of photoreceptor damage associated with long-standing ME (Takayama et al., 2012). MacTel 1
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can therefore be considered a “pure” phenotype. The molecular basis of this disorder has not been elucidated, but we recently proposed that vessel abnormalities might be a consequence of non-perfusion foci (Matet et al., 2016), and that this effect may be mediated
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by the PGF/VEGFR-1 pathway (Kowalczuk et al., 2016). Leakage from perifoveal telangiectasia is the main feature visible on fluorescein angiography, where it appears more
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intense than in idiopathic macular telangiectasia type 2 (MacTel 2) (Figure 16), and usually correlates with areas of retinal thickening and intraretinal cysts. This leakage is responsible for chronic intraretinal exudation of fluid and proteins. We hypothesize that this protein accumulation over time is responsible for ME, due to focal alteration of the normal protein gradient in the retina.
6.3
Idiopathic macular telangiectasia type 2
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ACCEPTED MANUSCRIPT MacTel 2 is a rare macular disease associating subtle leakage from perifoveal retinal telangiectasia, macular pigment loss, intraretinal cavitations, crystalline deposits, focal disruption of the retinal layers, and photoreceptor loss (Charbel Issa et al., 2013). Intraretinal cavitations have a particular appearance on OCT. They harbor square edges, a thin remnant
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of inner limiting membrane when affecting the inner retina, and are not associated with retinal thickening. This latter feature suggests that they result from loss of tissue replaced by fluid, rather than from true exudation (Oh et al., 2014) (Figure 16). The disease manifests during the fourth-to-sixth decades, and affected subjects often present diabetes mellitus or
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hypertension (Clemons et al., 2013).
The molecular origin of the disorder is not known, and some familial cases have been
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identified, suggesting possible genetic factors (Gillies et al., 2009). Regarding the involved cell type, a defect in RMG function or loss of RMG cells is thought to be the primary pathogenic factor leading to the MacTel 2 phenotype. RMG cell loss has been observed in MacTel 2 eyes (Powner et al., 2010, 2013), and in two proposed rodent models
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recapitulating part of MacTel type 2 phenotype (Shen et al., 2012; Zhao et al., 2015). Recent data obtained with OCT angiography showed advanced deep capillary plexus abnormalities in Mactel 2, with outer retinal invasion and possible capillary proliferation from the deep
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capillary plexus to the outer retina (Gaudric et al., 2015; Spaide et al., 2015, 2016). These manifestations might result from a deregulation of endothelial growth exerted by RMG cells.
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As discussed above (see 2.2.1), only RMG cells interact and control the deep capillary plexus, explaining why loss of normal RMG cell function would primarily affect this plexus. Finally, the absence of intraretinal cystoid cavities and retinal thickening, in the absence of choroidal neovascularization that may complicate the course of Mactel 2, may be explained by the low leakage intensity from altered endothelial junctions in the deep plexus, that would not allow the massive passage of large proteins.
6.4
Paclitaxel-induced maculopathy
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ACCEPTED MANUSCRIPT Patients receiving paclitaxel, a chemotherapy drug of the taxane family, preventing microtubule assembly, may develop bilateral cystoid ME (Ehlers et al., 2013). This adverse event presents with intraretinal cystoid cavities on OCT, and as silent ME on fluorescein angiography, where no leakage is observed, either from the retinal vessels or the RPE
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(Georgakopoulos et al., 2012; Joshi and Garretson, 2007). Although its exact mechanism is not understood, RMG cell dysfunction has been hypothesized, by analogy with MacTel 2, and based on electrophysiological observations (Nakao et al., 2016). Therefore, paclitaxel maculopathy belongs to the “pure” phenotypes listed in this chapter. We suggest that
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inhibition of microtubule polymerization and intracellular RMG transport could cause fluid to accumulate in RMG cells. If confirmed, this unique clinical presentation would demonstrate
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that intracellular fluid accumulation in the retina can cause cystoid ME. Consistently, antiVEGF agents were not efficient in treatment of paclitaxel-induced ME, and the only therapeutic option to date is discontinuation of paclitaxel therapy.
MEK-inhibitor-associated maculopathy
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6.5
Serous macular detachments and cystoid macular edema have been reported during treatment by mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors (e.g.
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Binimetinib, Trametinib) for cutaneous melanoma (Duncan et al., 2015; McCannel et al., 2014; Niro et al., 2015; van Dijk et al., 2015) (Figure 16, E-F). This effect is reversible and
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may originate from either acute RPE toxicity by inhibition of the MAPK pathway that lies downstream of the fibroblast growth factor receptor, a key actor for the maintenance of RPE integrity (van der Noll et al., 2013). But the exact mechanisms might be more complex since ERK activation takes place in the mature neurosensory retina (van Dijk et al., 2016). The upregulation of AQP1 in RPE induced by MEK inhibitor suggest a compensatory mechanism to hyperosmotic pressure in the subretinal space (Stamer et al., 2003).
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ACCEPTED MANUSCRIPT 6.6
Hypoproteinemia
ME can also develop in patients with pronounced hypoproteinemia, due to severe renal failure (Williams et al., 2006), deficient protein intake or other causes. This mechanism may also contribute to manifestations of advanced diabetic ME, such as hard exudate deposits,
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that may improve after initiation of hemodialysis (Matsuo, 2006). Several observations of serous macular detachment due to hypoproteinemia, for instance during the course of
nephrotic syndrome of various origins, have been reported (Bilge et al., 2016; Izzedine et al.,
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2014) (Figure 16, I-J). We suggest that disruption of the normal protein gradients between the vascular and the retinal compartments induced by low proteinemia, leads to an
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impairment of water exit mechanisms, and to the development of ME or subretinal fluid accumulation. Furthermore, this “pure” mechanism is a supportive example for the putative critical role of proteins in retinal hydro-ionic homeostasis.
Paracentral acute middle maculopathy
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6.7
Paracentral acute middle maculopathy is a recently described entity consisting in focal hyperreflective intraretinal lesions at the level of the inner nuclear layer, visible on OCT
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(Figure 16, K-L). These lesions are usually associated to a relative thickening of the inner nuclear layer, and originate from focal retinal ischemia (Sarraf et al., 2013). They develop in
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the context of retinal vascular occlusions, either arterial (Bonini Filho et al., 2015; Christenbury et al., 2015) or venous (Ghasemi Falavarjani et al., 2017; Sarraf et al., 2013). OCT angiography demonstrated that focal decrease in perfusion of the deep capillary network, located at the outer border of the inner nuclear layer, is the probable cause of this phenomenon (Nemiroff et al., 2016). This focal deregulation of fluid homeostasis in the retina results purely from cellular hypoxia in an ischemic microenvironment secondary to vascular occlusive disease and therefore represents a purely ischemic mechanism of ME.
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7 Major pathways identified as causative of macular edema 7.1
VEGF/PGF pathway
The VEGF family includes VEGF-A, -B, -C, -D, -E and PGF. Among all factors, the
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overexpression of PGF and VEGF-A causes increased vascular permeability (Witmer et al., 2003). VEGF-A binds to two high-affinity tyrosine kinase receptors, VEGFR-1 (also known as flt-1) and VEGFR-2 (also known as kinase insert domain-containing receptor or KDR), while
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PGF binds with high affinity only to VEGFR-1. Whether VEGF-A influences vascular permeability via VEGFR-1 or 2 remains controversial. It has been shown in human
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endothelial cells that VEGF-A mutants binding selectively to VEGFR-2 are able to induce vascular permeability in vivo, whereas VEGF-A mutants selectively binding to VEGFR-1 do not exert these effects (Gille et al., 2001; Keyt et al., 1996). However, activation of the VEGFR-1 pathway by PGF/VEGF-A signaling may be involved in other retinal vascular cell types such as pericytes and vascular smooth muscle cells (Luttun et al., 2002). VEGF-A
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mediates vascular hyper-permeability by increasing both paracellular and transcellular permeability. VEGF-A regulates endothelial junction proteins through VEGFR-2 activation (Kim et al., 2009). For instance, in endothelial vascular cells, VEGF-A induces occludin
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phosphorylation via activation of PKCβ (Harhaj et al., 2006; Murakami et al., 2012) and
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occludin down-regulation via an increase of free cytosolic ß-catenin and up-regulation of uPAR (Behzadian et al., 2003). Loss of VE-cadherin by endocytosis has been also implicated in paracellular hyperpermeability via VEGFR-2 (Gavard and Gutkind, 2006). The VEGF/VEGFR-2 pathway also increases transcellular permeability in retinal endothelial cell by transcytosis involving caveolae and a NOS-dependent mechanism (Feng et al., 1999; Hofman et al., 2000). Clinically, aqueous humor and vitreous levels of VEGF-A, PGF, and VEGFR-1 and -2 are elevated in eyes with ME due to diabetes (Table 1) and retinal vein occlusion (Tables 2 and 3) (Funatsu et al., 2003; Noma et al., 2015).
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7.2
Kinin/kallikrein system
Kinins, including bradykinin and kallidin are polypeptides with natural vasodilator and endothelial protector properties. They are produced by the action of kallikreins on kininogens,
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produced by the liver and released into the plasma. There are two types of kallikrein, the tissue and plasma kallikreins. The angiotensin converting enzyme (ACE) belongs to the kinin inactivating enzymes that regulate kinin activity, linking the renin-angiotensin system (RAS) to the kinin/kallikrein system (KKS) (Regoli and Gobeil, 2017; Wilkinson-Berka and Fletcher,
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2004). Effects of kinins are mediated by two related G-protein-coupled receptors, the
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bradykinin receptors, B1R and B2R. Both receptors are expressed in the normal retina and choroid (Ma et al., 1996), but vasodilation may be mediated by B2R that is predominantly expressed in the vascular endothelium, GCL, INL and ONL (Yasuyoshi et al., 2000). However, B1R is over-expressed in response to oxidative stress in the central nervous system, in a type-2-diabetes rat model (Dias et al., 2015), and in post-mortem eyes of
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patients with type 1 or 2 diabetes (Bhat et al., 2014). Cumulative evidence has emerged that the KKS system is activated and exerts pro-edematous and pro-inflammatory effects in diabetic retinopathy. Retinal vascular permeability was increased in animal models using
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intravitreal injection of plasma kallikrein or bradykinin and was reduced after KKS inhibition using either transgenic animals or antagonists, as exhaustively reviewed (Bhat et al., 2014).
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Specific B1R antagonists have been developed and were found efficient in reducing retinal vascular permeability and thickening (Lawson et al., 2005). However, differential and paradoxical effects of bradykinins might be observed, mediated by either B1R or B2R, depending on the pathologic conditions (Sang et al., 2016; Shi et al., 2016). At the cellular level, bradykinin induces vasodilation through the activation of calcium-dependent eNOS and prostacycline via B2R, and induces vasodilation and permeability through the up-regulation of iNOS via B1R. B2R activation can also induce permeability through Src activation pathway (Liu and Feener, 2013). Ultimately, hyper-permeability is, at least in part, mediated by the down-regulation of claudin-5 through a calcium-induced calcium-release mechanism (Zhou 58
ACCEPTED MANUSCRIPT et al., 2014) and by the phosphorylation of cadherin (Liu and Feener, 2013). Finally, the KKS interacts with other pathways involved in the pathogenesis of ME, such as the complement factor system, as illustrated by the occurrence of angioedema due to over-
7.3
Angiopoietin-2 (Ang2)/Tie2/α3β1 pathway
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bradykinin activity and mutations in C1-inhibitor coding genes (Reshef et al., 2016).
The Ang2/Tie-2 pathway is mostly involved in vessel stabilization and proliferation. But it also intervenes in retinal vascular permeability through the neuro-glio-vascular cross-talk. As
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detailed above (3.1.1.3), Ang2 induces pericyte apoptosis through Tie2 and α3β1 binding.
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Ang2 also induces apoptosis of retinal astrocytes through avβ5 binding (Yun et al., 2016a). Novel therapeutic strategies are developed to regulate the angiopioetin-2/Tie2 pathway (Saharinen et al., 2017).
7.4
Renin-angiotensin system and angiotensin-converting enzyme
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The renin-angiotensin system (RAS), particularly via the angiotensin-converting enzyme (ACE), is involved in the progression of diabetic retinopathy (Fletcher et al., 2010). In a recent meta-analysis collecting 21 randomized clinical trials with 13,823 participants, RAS
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inhibition was associated to a reduced risk of progression and an increased regression rate of diabetic retinopathy. Moreover, a polymorphism in the gene encoding ACE is associated
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with the risk of developing diabetic retinopathy, particularly in the type 2 diabetic Asian population (Fletcher et al., 2010). Angiotensin-2 exerts its action via the angiotensin-2 receptors type 1 (AT1) and 2 (AT2) that are differentially expressed in endothelial cells or glial cells. Angiotensin exerts vasoconstriction and pro-angiogenic effects but no direct effect is known on vascular permeability (Fletcher et al., 2010).
7.5
The mineralocorticoid pathway
The mineralocorticoid receptor (MR) is expressed in the retina and particularly in RMG cells
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ACCEPTED MANUSCRIPT and in endothelial cells of the retinal and choroidal vasculature. Aldosterone, the specific MR ligand, regulates the expression and distribution of several ion and water channels in RMG cells (such as AQP4, Kir4.1, αENac) and induces retinal thickening through alteration of glial drainage mechanisms (Zhao et al., 2010). On the other hand, MR activation in the choroidal
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vasculature induces vasodilation and permeability through up-regulation of the endothelial vasodilatory K+ channel KCa2.3, expressed in choroidal but not in retinal vessels (Zhao et al., 2012). In vascular endothelial cells, activation of the MR pathway induced an increased paracellular permeability through activation of the Rho kinase and subsequent actin cytoskeleton
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stress (Kirsch et al., 2013). MR over-activation is the putative pathogenic mechanism accounting for the efficacy of oral MR antagonist therapy in patients with central serous
7.6
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chorioretinopathy (Daruich et al., 2015b).
Inflammation and inflammatory mediators with pro-edematous effects
Inflammation is a protective response of the innate immune system programmed to neutralize harmful stimuli, and initiate a healing process restoring tissue homeostasis. The
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retina is an immune-privileged organ due to the blood-retinal barriers, and the absence of lymphatic vessels or dendritic cells. Yet, local activation of immune competent resident cells such as microglia/macrophages, choroidal mast cells but also astrocytes, RMG and RPE
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cells occurs in response to any injury. When immune resident cells are activated locally without barrier rupture, no sign of inflammation is clinically detected, a silent process termed
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“para-inflammation”. Histamine, VEGF and permeating cytokines, produced by resident immune cells, together with reduced protective mechanisms, contribute to blood-retinal barriers breakdown. ME is one of the cardinal signs of retinal inflammation and should be considered as an “inflammatory sign”. Numerous cytokines, chemokines and permeating factors have been repeatedly found elevated in ocular fluids of eyes with ME, some studies identifying correlations with retinal thickness. Tables 1-3 recapitulate the factors measured in diabetic ME, central and branch vein occlusions, respectively. However, the increased level of a given cytokine is not
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ACCEPTED MANUSCRIPT sufficient to establish a causative pathogenic link. Indeed, many endogenous neutralizing mechanisms are also involved, for instance via soluble receptors which have not been systematically measured in these studies. Moreover, secretion of these inflammatory mediators could also result from ME. Whether inflammation causes ME in a priori non-
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inflammatory retinal diseases remains a subject of debate, but inflammatory pathways are clearly involved, as evidenced by the clinical efficacy of intravitreal corticosteroid agents in ME secondary to diabetes (Boyer et al., 2014; Daruich et al., 2015a), or vein occlusions
(Haller et al., 2011). On the other hand, subclinical retinal chronic inflammation can certainly
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create environmental conditions for ME to develop. Submitted to chronic oxidative,
metabolic, degenerative stress, retinal cells activate pathways that can each favor the
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occurrence of blood barrier function alterations and/or alter physiologic drainage mechanisms. In all these processes, ageing acts as a favoring factor. It precipitates homeostasis disruption, since ocular tissue are modified with advancing age, as illustrated by the increasing deposits in Bruch’s membrane, the basal membrane of the RPE, and the
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decreased density of capillary endothelial cell and pericytes. A list of growth factors and inflammatory mediators potentially involved in retinal or choroidal vascular permeabilization
7.6.1
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is provided in Table 5.
Resident immune-competent cells
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7.6.1.1 Microglia/macrophages Microglia recruitment and activation occurs in response to any retinal stress or insult. Microglia migrate from their physiologic perivascular location to the outer retina causing alteration of RPE functions, release of several pro-permeabilizing factors (particularly TNF, IL-1, NO and VEGF), and reducing the production of anti-inflammatory mediators at the proximity of the retinal vasculature (Grigsby et al., 2014; Vecino et al., 2016; Wang and Wong, 2014). The chemokine, CCL2 (also referred to as MCP-1), is elevated in the ocular
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ACCEPTED MANUSCRIPT media of patients with ME. CCL2 is produced by inflammatory cells, resident microglia, but also by RMG and RPE cells under IL1-β produced by microglia (Natoli et al., 2017).
7.6.1.2 Retinal Müller glial cells
include, among others:
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RMG cells, in response to metabolic or ischemic stress show multiple responses. These
up-regulation of the intermediate filament protein, GFAP,
•
reduced potassium conductance (reduced drainage mechanisms and capillary
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•
dilation)
modified location and expression of AQPs, including AQP4 which reduces drainage
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•
mechanisms (Nicchia et al., 2016) •
production of pro-permeabilizing cytokines such as VEGF, IL-6 and MCP-1 (Eastlake et al., 2016; Liu et al., 2015) and other chemokines (Rutar et al., 2015) activation of the renin-angiotensin pathway (Wilkinson-Berka et al., 2010)
•
activation of the small GTPase Rho (Tura et al., 2009)
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•
In addition, RMG swelling has been reported in response to metabolic stress (Bringmann et
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al., 2004, 2006; Bringmann and Wiedemann, 2012).
7.6.1.3 Astrocytes
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High glucose conditions lead to the production of oxidative stress and induced the production of TNF-α and IL1-β and iNOS through activation of NF-κB. Enhanced Nuclear factor erythroid 2-related factor 2 (Nrf2) translocation and the production of anti-oxidant enzymes was associated with protection against oxidative-stress induced apoptosis (Shin et al., 2014).
7.6.1.4 Retinal pigment epithelial cells RPE cells are posterior uveal cells that express Toll-like receptors (TLR), components of the innate immunity (Kumar et al., 2004) and, P2X7 receptor (Guha et al., 2013), activated by
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ACCEPTED MANUSCRIPT mast cells degranulation and inflammation. RPE can produce a large number of cytokines and chemokines and closely interact with choroidal macrophages/microglia and mast cells. Activation of P2X7 receptor and TLR receptors expressed in RPE cells induces differential responses. Photo-oxidative stress, aging, oxidization of lipids, impairment of autophagy,
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accumulation of β-amyloid, iron loading or Alu RNA, activation of inflammasome through P2X7 receptor (Guha et al., 2013), lead to several metabolic changes contributing to AMD pathogenesis. These changes include the release of pro-inflammatory cytokines, with polarized apical secretion of IL-1β and IL-18, and the basal down-regulation of VEGF
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(Brandstetter et al., 2015; Gelfand et al., 2015; Gnanaguru et al., 2016; J. Liu et al., 2016;
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Mohr et al., 2015; Tarallo et al., 2012). Via TLR-4 activation, LPS induces NF-κB activation, subsequent inflammatory cascade (Wang et al., 2017) and disruption of the epithelial barrier (Zech et al., 1998). The expression of TLR-4 in RPE cells is enhanced by C5a, which induces the production of IL-6 and IL-8 (Zhu et al., 2015). The sub-retinal microglial accumulation contributes to the pro-inflammatory mediator production by RPE cells. We have
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shown that transcytosis of activated microglia/ macrophages through RPE cells contributes to cell trafficking without alteration RPE tight junction (Omri et al., 2011) (Video 3). Other mechanisms of macrophage/microglia elimination by apoptosis regulated by interaction of
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CFH with CD47 have been demonstrated (Calippe et al., 2017). Their impairment could play important role of pathogenic sub-retinal inflammation that takes place in numerous “a priori”
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non-inflammatory retinal diseases.
7.6.2
Inflammatory molecules
7.6.2.1 Complement components The complement system plays important roles in macular edema through mechanisms related to its state of activation. In example, C1 inhibitor is an important regulator of the kinininduced permeability. The anaphylatoxins C3a and C5a induce hyper-permeability through degranulation of mast cells, present in the choroid. In addition, C5a but not C3a induces
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ACCEPTED MANUSCRIPT direct endothelial cell retraction and increases paracellular permeability on human endothelial cells in vitro through PI3K and Src kinase pathway (Schraufstatter et al., 2002). Preclinical experimental studies suggest that mostly TNF and IL-1ß may have direct effect on
7.6.2.2 Tumor Necrosis Factor-α (TNF-α)
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retinal barrier integrity.
TNF-α is one of earliest cytokines with permeating properties being released at the site of inflammation as it is the only cytokine stored in mast cells granules that can be immediately
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released by mast cells degranulation (Gu et al., 2015). The role of TNF-α on vascular
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permeability has been demonstrated by in vitro studies on various endothelial cell types, including brain and retinal microvascular cells, as well as ex vivo and in vivo. Depending on the micro-environment and on its concentration, TNF-α can induce vascular permeability but also endothelial cell death as detailed in 3.1.1.3. Herein, the main mechanisms of TNF-αinduced permeability will be summarized, with an emphasis on its effects on brain and retinal
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endothelial cells.
On cultured human umbilical vein endothelial cell line cells (HUVECs), TNF-α induced permeability through activation of RhoA/ROCK pathway, contraction of actin cytoskeleton
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and reduced VE-Cadherin (J. Peng et al., 2011; Wei et al., 2017). Depending on the applied concentration, TNF-α induced either junction disruption at low dose (identified by claudin-5
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delocalization), or apoptosis at higher concentrations (Miyazaki et al., 2017). On mouse brain endothelial cells, TNF-α repressed claudin-5 expression through NF-kB signaling (Aslam et al., 2012). Using human brain microvascular endothelial cells (HBMvECs), both TNF-α and IL-6 induced permeability through decreased expression, phosphorylation and cytoplasmic translocation of ZO-1 (Rochfort and Cummins, 2015). In bovine retinal endothelial cells, the down-regulation and translocation of ZO-1 and claudin-5 was preceded by PKCζ phosphorylation (Aveleira et al., 2010). On tridimensional chips modelling the multicellular blood-brain barrier component, TNF-α induced the production of G-CSF in presence of
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ACCEPTED MANUSCRIPT endothelial cells and pericytes, and the increase of IL-6 and IL8 in the presence of astrocytes or pericytes. However, this was not observed in endothelial cells cultured alone, suggesting that TNF-α initiates the production of other cytokines at the glio-vascular interface. These results are consistent with observations that TNF-α does not directly disrupt brain endothelial
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cell junctions when co-cultured with astrocytes, but that junctions were disrupted when cocultured astrocytes were pre-exposed to TNF-α-conditioned media (Chaitanya et al., 2011). In addition, TNF-α leads to endothelial barrier disruption by exacerbating several other mechanisms. For instance, polymorphonuclear endothelial transcytosis, increasing
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intercellular permeability, occurs through ICAM-1 and is enhanced by TNF-α (Wong et al., 2007). TNF-α contributes to ischemic VEGF-induced leukostasis but does not seem to
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intervene directly in VEGF-induced retinal permeability, suggesting that inhibition of both pathways could be synergistic (Vinores et al., 2007). Different observations were made in lipopolysaccharide (LPS)-induced uveitis, where anti-TNF-α suppressed leukostasis, retinal vascular leakage, and apoptosis (Koizumi et al., 2003). In addition, TNF-α produced by LPS-
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stimulated microglia was also able to disrupt brain microvascular cells barrier, strengthening the notion that the glio-vascular cross-talk regulates TNF-α-induced permeability (Nishioku et al., 2010).
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TNF-α not only disrupts the inner blood-retinal barrier but also increases RPE permeability. In rodent RPE cells, TNF-α directly influences ZO-1 distribution (Shirasawa et al., 2013; Zech
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et al., 1998), while in human embryonic stem cell (hESC)-derived RPE cells, TNF-α-induced permeability is associated with increased expression of MMP1, MMP2 (Juuti-Uusitalo et al., 2015) and MMP9 (Wang et al., 2012). In contact with RPE, activated macrophages and microglia produce TNF-α in models of AMD, such as the Cxcr5-knock-out mouse (Huang et al., 2017, p. 5; Müller et al., 2003). TNF-α is involved in the close interaction between RPE and macrophages. Indeed, co-cultures of macrophages and RPE produce more MCP-1, IL-6, and VEGF, as compared to separate cultures. This effect is mediated by the production of TNF-α by macrophages, negatively regulated by contact with RPE cells (Yamawaki et al.,
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ACCEPTED MANUSCRIPT 2016). On the other hand, TNF-α induces ICAM-1 expression and promotes monocyte adhesion through TNF receptor-1 (TNFR1) activation and the PKCδ/JNK1/2/c-Jun pathway (Lee et al., 2015). Finally, the interaction of TNF-α and VEGF is complex and may depend on the inflammatory
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status of the RPE. TNF-α decreases VEGF secretion in highly polarized RPE cells but increases it in non-polarized RPE cells, related to the cross-talk between the c-Jun N-
terminal kinase (JNK) and NF-κB pathways (Terasaki et al., 2013). TNF-α also enhances VEGF expression in a model of induced choroidal neovascularization (Wang et al., 2016).
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To summarize, the role of TNF-α in RPE permeability is complex. It implies direct effects through MMP activation, and indirect effects through a controlled cross-talk with
7.6.2.3 Interleukin-1β (IL-1β)
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macrophage/microglia, suggesting differential effects in the normal and pathologic retina.
The intravitreal administration of IL-1β induces a reversible retinal inflammatory response. It
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is accompanied by inner blood-retinal barrier breakdown and leukocyte recruitment, through retinal endothelial transcytosis. This effect is probably mediated by histamine in a biphasic time-dependent pattern, with barrier opening observed 4-8 hours and 24-48 hours post-
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injection (Bamforth et al., 1997). In the rabbit retina, intravitreal injection of IL-1β disrupted endothelial cell junctions (Claudio et al., 1994; Rosenbaum et al., 1987). VEGF can
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contribute to the effect of IL-1β on vessels, as shown in transgenic mice overexpressing IL1β in the lens, in which an early peak of retinal VEGF expression was concomitant to bloodretinal barrier breakdown and inflammatory cell infiltration (Vinores et al., 2003). Similarly, in human brain astrocytes, IL-1β induces the expression of HIF-1α and VEGF-A (Argaw et al., 2006). However, several in vitro experiments have demonstrated direct effects of IL-1β on endothelial cells. After endothelial cells stimulation by IL-1β, over 2500 genes were differentially expressed, including genes associated with apoptosis, cell cycle, NF-kB cascade, chemotaxis, and immune response. Interestingly, claudin-1 was up-regulated, but
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ACCEPTED MANUSCRIPT claudin-5 and occludin were down-regulated (Williams et al., 2008). In brain microvascular endothelial cells, IL-1β down-regulated the expression occludin, which increased permeability, when submitted to hypoxic, but not normoxic conditions (Yamagata et al., 2004). This suggests that the mechanisms of IL-1β on permeability may differ according to
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the pathologic conditions. IL-1β-induced permeability could be driven by: •
PKC-θ-dependent phosphorylation of ZO-1 (Rigor et al., 2012)
•
alterations in the f-actin cytoskeleton due to the phosphorylation of MLCs by ROCK
•
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activation, counter-regulated by neuregulin-1β (Wu et al., 2016)
decreased VE-cadherin expression, counter-regulated by BDNF (Matsuda et al.,
•
melatonin (Yuan et al., 2011).
7.6.2.4 Interleukin-6 (IL-6)
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2015)
Aqueous humor and vitreous levels of IL-6 are elevated in eyes with ME associated to retinal vein occlusion or diabetes (Koss et al., 2012; Noma et al., 2009, 2015; Owen and Hartnett,
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2013). Recently, it has been found that IL-6 produced by microglia induces STAT3 activation in retinal endothelial cells under high glucose conditions, down-regulating occludin and ZO-1, and producing VEGF (Yun et al., 2016b). In human brain microvascular endothelial cells, IL-6
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decreases the expression of VE-cadherin, occludin and claudin-5 (Rochfort et al., 2014).
7.6.2.5 Interleukin-8 (IL-8) Aqueous humor and vitreous levels of IL-8 are also elevated in patients with ME associated to retinal vein occlusion or diabetes (Fonollosa et al., 2010; Noma et al., 2015; Owen and Hartnett, 2013; Sonoda et al., 2014). Human endothelial vascular cells treated with increased concentration of IL-8 showed a down-regulation of tight junction proteins, including occludin, claudin-5 and ZO-1 (Yu et al., 2013). In human microvascular endothelial cells, permeability induced by IL-8 required the activation of VEGFR-2 (Petreaca et al., 2007). IL-1β potently
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ACCEPTED MANUSCRIPT stimulates IL-8 expression in human RMG cells, mainly through the p38 MAPK and ERK1/2 pathways (Liu et al., 2015).
7.6.2.6 Intercellular Adhesion Molecule 1 (ICAM-1)
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Vitreous level of ICAM-1 are elevated in patients with ME associated to retinal vein occlusion (Noma et al., 2011a, 2013, 2014) or diabetes (Funatsu et al., 2005, 2009). Aqueous humor level of ICAM-1 have been correlated with the height of subretinal fluid in DME (Zhu et al., 2014). In a rat model, increased leukostasis, vascular permeability and capillary non-
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expression in the retina (Miyamoto et al., 2000).
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perfusion after intravitreal VEGF injections was associated with up-regulation of ICAM-1
7.6.2.7 Transforming growth factor-ß (TGF-β)
TGF-ß secreted by glial cells stimulates the production of MMP-9, which in turn degrades
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occludin, contributing to inner blood-retinal barrier disruption, as detailed in 3.1.1.1.
In summary, any chronic retinal stress induces multiple subclinical molecular and cellular inflammatory responses, creating a favorable environment for ME to develop. In this context,
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ME appears clinically upon an additive acute event (such as hypertension), or by reaching the threshold of compensatory mechanisms. When ME occurs, breakdown of blood retinal
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barrier leads to the infiltration of circulating cells, that together with activated resident cells amplify these inflammatory reactions.
8 General mechanisms contributing to diabetic ME Chronic hyperglycemia activates cardinal pathways, recognized as main contributors to diabetes-induced damages at cell, tissue and organ levels (Figure 17). In hyperglycemic conditions, retinal cells are subjected to nutrient excess, causing mitochondrial and endoplasmic reticulum stress (Wellen and Thompson, 2010). When early compensatory
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ACCEPTED MANUSCRIPT mechanisms are overwhelmed cellular damage may occur. We will briefly review the mechanisms involved, most of which were previously mentioned in specific paragraphs. Increased fluid entry and impaired fluid drainage are both involved in diabetic ME (Figure 17). The main cellular mechanisms dysregulated in diabetic conditions and leading to ME are
8.1
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summarized in the Video 2.
The Polyol pathway
Upon long-lasting exposure to glucose excess, the hexokinase enzyme becomes saturated
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and glucose is derived toward an alternative glycolysis pathway through sorbitol and fructose
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production. The level of cellular NADPH is lowered, leading to oxidative stress. Sorbitol does not enter the cell membrane creating creating osmotic stress, and it induces nitrogen glycation of proteins, contributing to the accumulation of AGEs.
8.2
The AGE pathway
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Non-enzymatic glucose-protein reactions integrate a heterogeneous group of irreversible molecular adducts called AGEs, whose normal accumulation with ageing is exacerbated by diabetes. AGEs increase fibrosis, and activates inflammation through the formation of
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collagen crosslinks, and by interaction with the AGE-receptor (RAGE), which impairs calcium handling. AGEs have been associated with diabetic microangiopathy (Madonna et al., 2017).
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In experimental diabetic conditions in vitro and in vivo, AGEs were shown to induce retinal pericytes apoptosis (Denis et al., 2002; Yamagishi et al., 1995) and accumulate in RMG cells. These processes cause hydro-ionic channels dysfunction, oxidative stress and GFAP activation (Curtis et al., 2011; Lecleire-Collet et al., 2005), as well as RMG cell inflammatory responses through RAGE activation (Zong et al., 2010). Exposure of RPE cells to AGEs also activated inflammatory response via the NF-kB and JAK-STAT pathways (Lin et al., 2013).
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ACCEPTED MANUSCRIPT 8.3
Protein kinase C (PKC) activation
There are multiple isoforms of PKC. The conventional PKCs (PKC-α, -β1, -β2, and -γ) are activated by phosphatidylserine, calcium, and diacyglycerol (DAG). Novel PKCs (PKC-δ, -ε, θ, and -η) are activated by phosphatidylserine and DAG but not by calcium, and atypical
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PKCs (such as PKC-ζ) are not activated by calcium or diacylglycerol (DAG) but by phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3), which is mainly produced by the
phosphoinositide 3-kinase (PI-3 kinase) (Idris et al., 2001). As a consequence of impaired glycolysis and glucose overload, DAG synthesis increases in cells and activates conventional
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and novel PKCs, particularly isoforms β δ, ε, and γ. Activation of the DAG/PKC pathway has
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been associated with endothelial permeability and dysfunction, vasoconstriction, angiogenesis, leukostasis and inflammation (Aiello, 2002; Das Evcimen and King, 2007; Geraldes and King, 2010). Under hyperglycemic conditions, the activation of PKC-δ induces retinal vascular cell death (Geraldes et al., 2009). Atypical PKCζ, activated in response to TNF-α in endothelial and RPE cells in diabetic conditions, contributes to tight-junction
al., 2017).
Local renin-angiotensin system (RAS)
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8.4
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destabilization and cell polarity alteration (Aveleira et al., 2010; Omri et al., 2013; Sewduth et
In addition to the systemic RAS regulating vascular and extravascular concentrations of Na+
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and K+, and maintaining blood volume (Patel et al., 2017), local RAS pathways participate in the homeostasis of vascularized organs such as kidney and retina, and are activated in diabetic conditions (Giese and Speth, 2014). For instance, in the diabetic retina, activation of the local RAS and increased retinal levels of angiotensin-2 can contribute to ME via its receptor AT1, and the downstream pathological chain of events, as detailed in 7.4. This pathway has also indirect effects on other mechanisms involved in diabetic ME such as oxidative stress, production of AGEs, polyol pathway, and PKCs (Steckelings et al., 2009).
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Inflammation and oxidative stress
Recent investigations have shown that chronic hyperglycemia induces oxidative stress and inflammation in the retina, which constitute early processes in the development of diabetic ME (Arroba and Valverde, 2017; Roy et al., 2017; Yu et al., 2015). As detailed in 7.6, local
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inflammation is a major and permanent pathogenic mechanism contributing to diabetic ME. Involved pathways are summarized in Figure 18. Inflammation is not only a consequence of barrier dysfunction, but also an early local mechanism contributing to barrier alteration and leukostasis. As a consequence of the activation of pathways hereby listed from paragraphs
8.6
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8.1 to 8.5, all retinal functions preventing the formation of edema in the retina are altered.
Blood-retinal barrier dysfunction
Hyperglycemia induces a degradation of junctional complex partners, including occludin, claudin, JAM-A, ZO-1 (Saker et al., 2014; Stewart et al., 2016; Tien et al., 2013; Wang et al., 2015) and VE-cadherin (Navaratna et al., 2007; Rangasamy et al., 2011). VEGF contributes
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to diabetes-induced vascular permeability by the rapid phosphorylation of occludin and ZO-1, and by reducing occludin expression in endothelial cells (Antonetti et al., 1998, 1999). VEGF could also stimulate stress fiber formation and VE-cadherin disruption in the diabetic retina
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(Lee et al., 2016). The endothelial barrier is further disrupted by pro-inflammatory cytokines, such as TGF-ß released by glial cells (Behzadian et al., 2001), and interleukin-6 by activated
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microglia (Yun et al., 2016b). Leukostasis induced by hyperglycemia, nitrosative/oxidative stress, VEGF and TNF-α, favors endothelial permeability (Huang et al., 2015; Joussen et al., 2002; Leal et al., 2007; Vinores et al., 2007). It also potentially leads to retinal capillary endothelial cell apoptosis (Joussen et al., 2001). Among hallmarks of diabetic ME, the inner blood-retinal barrier rupture is characterized by important pericyte loss. As mentioned in section 3.1.1.3, several pathways contributing to pericyte loss have been identified. These pathways include Ang-2 via integrin-α3 and -β1 (Cai et al., 2008; Pfister et al., 2008), PEDF signaling (Geraldes et al., 2009),
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ACCEPTED MANUSCRIPT Ca2+/calmodulin-dependent protein kinase II via inducible nitric oxide synthase (iNOS) (Kim et al., 2011), TGF-β and the pro-apoptotic BIGH3 protein (Betts-Obregon et al., 2016). Junctional complexes at the level of the outer blood-retinal barrier, are also altered by hyperglycemia, especially via the activation of metalloproteinases such as MMP-9 by
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oxidative and nitrosative stress (Cao et al., 2013; Giebel et al., 2005, 2005; Kowluru et al., 2012; Navaratna et al., 2007). The OLM, that contributes to the outer blood/retinal barrier, is also damaged in diabetic ME, because of tight junction disruption due to the activation of PKC-ζ by hyperglycemia (Omri et al., 2013).
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More advanced events in the pathophysiology of diabetic ME, such as microaneurysms or neovascularization, contribute to its persistence and are controlled by angiogenic factors,
8.7
Drainage functions
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such as VEGF (Witmer et al., 2003) and PGF (Kowalczuk et al., 2011).
In RMG cells, diabetes alters the cellular distribution and expression of aquaporins (Fukuda
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et al., 2010), and reduces potassium conductance (Pannicke et al., 2006). Alterations in the expression pattern of aquaporins has also been described in the RPE of diabetic animal
8.8
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models (Hollborn et al., 2011).
Kinetic of events in the diabetic GK rat model
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The Goto Kakizaki (GK) rat is a spontaneous non-obese type 2 diabetic rat model, recognized as one of the most appropriate to study complication of type 2 diabetes mellitus (Akash et al., 2013). GK rats spontaneously develop hyperglycemia at about 3 months after birth, that persists for lifetime. Retinopathy progressively develops in the GK rat (Gong et al., 2016) and clear signs of microangiopathy can be detected after one year (Figure 19E). Vascular permeability, the hallmark of blood-retinal barrier breakdown was correlated to iNOS expression and activity in the GK rat retina (Carmo et al., 2000) and associated with decreased pericytes coverage, beginning at 8 months (Agardh et al., 1997). Accordingly,
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ACCEPTED MANUSCRIPT VEGF levels increased in the retina of GK rats at 7 months (Sone et al., 1997). We have previously characterized the early retinal changes occurring in GK rats from the onset of the hyperglycemia to the development of microangiopathy (Omri et al., 2013). Note that the GK rat is a spontaneously mutant albino Wistar rat. Unfortunately, fluorescein angiography
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cannot be performed very well in the GK rats. Thus, vascular abnormalities were evaluated by vessel labeling on flat-mounted retina after animal sacrifice and could not be followed longitudinally in vivo. The first clinical sign detectable on OCT and on histologically observed on retinal sections is an increase in choroidal thickness due to choroidal vessels dilation,
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including capillaries, observed as early as 4 months of age (Figure 19 A-D). In the
neurosensory retina, at the onset of hyperglycemia (at 3 months), a significant increase in
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the expression of Kir4.1 and AQP9 was detected. In the Wistar normoglycemic rat, Kir4.1 and AQP4 expression increase between 3 and 6 months and then remain stable. But in the GK diabetic rats, following increase in AQP4 expression at 3 months, the levels of expression of Kir4.1 and AQP4 remain significantly lower than in the Wistar control rat
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(Figure 20 B and D). Immunolocalization confirmed the decreased expression of Kir4.1 and AQP4 in the diabetic GK rat with a more pronounced reduced distribution in the INL and around the vessels (Figure 20 A and C). The aquaglyceroporin AQP9 remains highly up-
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regulated at all time points (Figure 20 E). The retinal thickness increases particularly in the outer retinal layers as early as 8 months and with progresses with longer diabetes duration
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(18 months), demonstrating that retinal edema occurs in GK rats after long-term hyperglycemia.
Alterations of the outer blood-retinal barrier, both at the RPE and OLM, occur early at the onset of hyperglycemia and progress between 6 and 12 months. The earlier changes in the RPE is the membrane translocation and activation of ROCK-1, which phosphorylates components of the actin cytoskeleton, inducing apical constrictions and stress fibers, and that can eventually lead to later non-apoptotic membrane cell blebbing in RPE cells (Figure 21 E, G and H). Polydispersity of RPE cell size and multinucleated syncytium are observed (Figure 21 F, H and I). The atypical PKCζ, which is associated to junction proteins at the cell
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ACCEPTED MANUSCRIPT membrane, translocates in the cytoplasma together with occludin (Figure 22), due to the down-regulation of the regulating N-terminal part of the kinase. This process induces its overactivation and the subsequent deregulation of junction stability (Omri et al., 2013). With prolonged hyperglycemia, PKCζ activity and junction proteins expression decrease. In
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addition, there is a reduction in the transcytosis mechanism through RPE cells. This reduces the elimination of activated microglia from the subretinal space towards the choroid, and subsequent production of pro-inflammatory mediators (Figure 23 and Video 3) (Omri et al., 2011). At 12 months, focal leakage from the choroid to the subretinal space demonstrates
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the functional alteration of the RPE barrier (Rothschild et al., 2017). At the OLM, similar changes are observed leading to alteration of the tight-like junction barrier between
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photoreceptor segments and RMG cell apices. Occludin and ZO-1 are removed from the OLM junction plaques and are internalized into RMG cells (Figure 24), suggesting that OLM alterations may occur earlier at the molecular level, than detected clinically by the loss of the OLM hyperreflective band on OCT (X. Chen et al., 2012). At 18-24 months of age, retinal
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vascular occlusions and enhanced permeability are associated with vascular endothelial cell blebbing (Rothschild PR et al Sci Report 2017). In summary, although no rodent animal model can recapitulate all the features of a human
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retinal disease, the GK diabetic rat develops many features of human diabetic retinopathy. The kinetics of events in this model show that alterations in the choroid may precede
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clinically detectable retinopathy, as in humans (Gupta et al., 2017; Tavares Ferreira et al., 2017). Microangiopathy can be detected in the GK rat but appears as a late phenomenon after alterations occur in retinal neurons, RPE and RMG cells. Importantly, early and continuous modifications of the RPE polarity and cytoskeleton, can have important consequences on transport and on the sub-retinal micro-environment (Philp et al., 2003; Shirasawa et al., 2013). In addition, microglial activation and sub-retinal accumulation of microglia contribute to photoreceptor inflammatory injury (Grigsby et al., 2014; Omri et al., 2011). These important non-clinically detectable retinal alterations, present long before
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ACCEPTED MANUSCRIPT macular edema is clinically detected, may explain the inconstant visual recovery after resolution of ME in diabetic patients.
9 Concluding remarks
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Homeostasis of a tissue is secured by the proper regulation of its energetic metabolism, its hydro-ionic balance, and intercellular communications. As a consequence of acute or chronic stress, edema may occur and spontaneously resolve through lymphatic drainage as tissue
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homeostasis is restored. In the retina, like in brain parenchyma, there are no lymphatic
vessels, and limited protein and fluid drainage takes place in normal conditions because
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barriers actively maintain the local micro-environment from any uncontrolled entry. The retina and particularly the macula are functionally extremely sensitive to any change in their hydration state to allow the proper transmission of light to photoreceptors. In addition, any disturbance of the local microenvironment subsequently threatens the structure and neural function of the macular region. Thus, regulatory mechanisms specific to the macula should
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be present. We have hypothesized that a “glymphatic system”, closely similar to mechanisms observed in the brain, is an important and unappreciated mechanism of fluid and protein drainage from the macula. While extensive research has been conducted to decipher the
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mechanisms of barrier disruption and to identify molecules inducing or preventing the formation of edema, little attention has been paid to the exact molecular and cellular
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specificity of the macula and the fovea. Yet, no animal or cellular model can equal the knowledge gained by analysing the structure of the human macula. With the development of novel imaging technologies, major improvements of the in vivo structural analysis of the macula should be expected. In parallel, molecular and metabolic analysis of cells composing the macula should be explored in normal and pathologic conditions using more sophisticated omics technologies. The return to basic knowledge revisited by multidisciplinary technological tools will most probably expand our vision on macular edema and help us dive below its surface. With this new knowledge, innovative therapies will emerge.
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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS This work was supported by the Agence Nationale de la Recherche, France (ANR-15-CE180032 “ROCK SUR MER”), by the Swiss National Science Foundation (#320030_156401), by the Faculty of Biology and Medicine Research Commission Fund, University of Lausanne,
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Switzerland, by the Abraham J. & Phyllis Katz Foundation, Atlanta, GA and by the Lowy Medical Research Institute, San Diego, CA. The authors do not have any conflicting interest related to the content of this work.
The authors wish to acknowledge Michèle Savoldelli for electronic microscopy image, and
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Gunilla Norrgren (Allergan, Dublin, Ireland) for technical support in video production.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Schematic representation of retinal layers and main retinal cell types. Histology section of a human retina at the mid-periphery, stained with hematoxylin-eosin. Color drawings of the different neuronal cell types and the retinal glial Müller cells (RMG) are
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superimposed on the histologic picture.
ILM= Inner Limiting Membrane; GCL= Ganglion Cell Layer; NFL= nerve fiber layer; IPL= Inner Plexiform Layer; INL= Inner Nuclear Layer; OPL= Outer Plexiform Layer; ONL= Outer
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Nuclear Layer; OLM= outer limiting membrane; IS= Inner Segment; OS= Outer Segment;
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RPE= Retinal Pigment Epithelium.
Figure 2. Cellular and molecular components of the inner blood-retinal barrier. A. Human retinal section labeled for AQP4 that surrounds the vessels green), glutamine synthetase (GS, retinal Müller glial cells, red), and Dapi (nuclei, blue), showing that retinal glial Müller cells expressing AQP4, ensheath retinal capillaries at the level of the superficial
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and deep capillary plexuses (superior and inferior insets, respectively). In the deep capillary plexus (low inset) AQP4 and GS co-stain the vessel wall. B. Junctional complex between retinal vascular endothelial cells, forming the inner retinal
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barrier. Tight junctions are formed by occludin, claudins 1,2 and 5, JAM-A and C, and are linked to the actin cytoskeleton by ZO-1. Adherens junctions are formed by VE-cadherin, ß-
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catenin and N-cadherin. Gap junctions are formed by hemi-channels of 6 connexins. The pericyte-endothelial cell junction is composed mainly by N-cadherin and connexin-43. C. Schematic representation of the neuro-glio-vascular unit forming the inner blood-retinal barrier, composed by vascular endothelial cells, pericytes (p), retinal glial Müller (RMG) cells, astrocytes (a), microglia (mc). Retinal glial Müller cell projections are present at the level of all retinal vascular plexuses (superficial, SCP; intermediate, ICP and deep, DCP), while astrocytes are only present at the level of the superficial plexus.
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ACCEPTED MANUSCRIPT D. Astrocyte labelling by GFAP surrounding retinal vessels on a human retinal flat-mount shows that in the superficial vascular layers, astrocytes are the major glial component of the gliovascular unit. E. Human retinal section labelled by GFAP (astrocytes, and endfeet of RMG cells, red) and
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collagen IV (vessels, green) showing astrocyte labelling at the level of the superficial vascular plexus (left inset) and absence of astrocytes labeling at the level of the deep
vascular plexus (right inset). In the deep capillary plexus, the main glial component of the neuro-gliovascular unit is the RMG cell.
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GCL=ganglion cell layer; INL= inner nuclear layer; HFL= Henle fiber layer; ONL= outer
nuclear layer; OLM= outer limiting membrane; RMG= retinal Müller glial cell; a= astrocyte,
intermediate capillary plexus
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mc= microglia; SCP= superficial capillary plexus; DCP= deep capillary plexus; ICP=
Figure 3. Morphology of the macula in physiological conditions and with edema.
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A. Histological retino-choroidal section of a normal human macula. B. Macular optical coherence tomography section of a normal human retina and choroid (subfoveal choroidal thickness, arrow)
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C. Histological retino-choroidal section of the macula from a non-human primate (Macaca fascicularis) showing intraretinal and subretinal fluid.
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D. Montage from the same image, with superimposed representation of the intraretinal course of cone photoreceptors (pink), and elongated retinal Müller glial cells (green) and Muller cone cells (light green) E. Optical coherence tomography of a human macula presenting intraretinal and subretinal fluid. Note the smaller, round cystoid cavities at the level of the inner nuclear layer, and the larger, elongated cavities at the level of the outer plexiform/Henle fiber layer. F. En face optical coherence tomography of the same eye showing the radial distribution of the two types of cystoid hyporeflective cavities. Images E-F: courtesy Bruno Lumbroso.
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Figure 4. Multimodal clinical imaging of diabetic macular edema A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in a 70-year old woman with type 1 diabetes and severe nonproliferative diabetic retinopathy. On
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OCT, multiple intraretinal hyperreflective dots, extended cystoid edema, hard exudates and focal attenuation of the ellipsoid zone suggesting photoreceptor suffering are visible.
C-D: OCT and midphase FA in a 65-year old man with type 2 diabetes and moderate
nonproliferative diabetic retinopathy. OCT shows cystoid cavities and hyperreflective dots.
leakage from exudative microanevrysms.
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FA suggests a possible diffuse leakage from the outer blood retinal barrier, in addition to
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E-F: OCT and midphase FA in a 56-year old woman with type 2 diabetes exhibiting minimal nonproliferative diabetic retinopathy. OCT shows intraretinal cystoid cavities and subretinal detachment.
G-I: OCT and midphase FA in a 42-year old woman with type 1 diabetes and untreated
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hypertension. FA shows moderate proliferative diabetic retinopathy with a neovascular lesion on the superior temporal arcade, a crescent-shape inferior retro-hyaloidal hemorrhage, and panretinal peripheral photocoagulation scars (G). OCT showed macular cystoid edema (H).
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Due to the preserved visual acuity and the untreated hypertension, the initial management consisted in blood pressure control, which resulted in the regression of macular edema 3
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months later (I).
Figure 5. Multimodal clinical imaging of macular edema secondary to retinal vein occlusions
A-B: OCT and midphase FA in a 49-year old woman with inferior-temporal branch retinal vein occlusion. FA shows increased venous tortuosity and dilation with flame-shaped hemorrhages in the affected sector, extending to the capillary network drained by the occluded branch. OCT shows macular cystoid edema with discrete subfoveal detachment.
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ACCEPTED MANUSCRIPT C-D: OCT and midphase FA in a 53-year old woman with superior-temporal branch retinal vein occlusion. Wide-field FA shows increased venous tortuosity and dilation in the corresponding drainage territory, and extended peripheral capillary nonperfusion. Cystoid edema affecting the temporal aspect of the macula is visible on OCT with accumulation of
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subretinal fluid at the fovea. E-F: OCT and midphase FA in a 74-year-old woman with inferior hemi-retinal vein occlusion. FA shows a tortuous, dilated and hyperpermeable inferior hemi-retinal venous network. OCT demonstrates intraretinal cystoid cavities and extended inner retinal hyperreflectivity,
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corresponding to paracentral acute middle maculopathy reflecting the decreased perfusion of the deep retinal plexus.
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G-H: OCT and midphase FA in a 56-year-old man with central retinal vein occlusion. FA shows diffuse dot-shaped hemorrhages and moderate venous tortuosity, and cystoid macular edema is visible on OCT.
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(Images A-D courtesy Aude Ambresin)
Figure 6. Multimodal clinical imaging of macular edema secondary to neovascularization, inflammation or other causes.
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A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in an 83-year-old woman with age-related macular degeneration and type 3 choroidal
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neovascularization, leading to focal retinal thickening and intraretinal edema. C-D: OCT and midphase FA in a 76-year-old woman with chronic postoperative macular edema (Irvine-Gass syndrome) following posterior segment surgery for dislocated intraocular lens. FA shows diffuse outer blood-retinal barrier rupture manifesting as multiple pinpoints, and fluorescein filling of the cystoid cavities. OCT shows intraretinal cystoid edema and subretinal fluid. E-F: OCT and midphase FA in a 29-year-old man with macular edema secondary to posterior uveitis in the context of Behcet disease. FA shows diffuse outer blood-retinal barrier rupture while cystoid macular edema with subfoveal detachment is visible on OCT.
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ACCEPTED MANUSCRIPT G-H: OCT and midphase FA in a 54-year-old man with chronic central serous chorioretinopathy. FA shows an area of retinal pigment epithelium atrophy manifesting as a window defect, and fluorescein filling of a large pigment epithelial detachment. This epithelial lesion is visible on OCT, together with intraretinal cystoid cavities, a classical finding in
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chronic forms of the disease. I-J: OCT and midphase FA in a 40-year-old man with radiation maculopathy following ocular irradiation in childhood for retinoblastoma. FA shows the presence of multiple exudative
telangiectasia and progressive filling of cystoid cavities, clearly visualized on OCT, together
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with an irregular ellipsoid zone at the fovea indicating photoreceptor alteration.
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(Images I-J: courtesy Francis Munier)
Figure 7. Cellular and molecular components of the outer blood-retinal barrier. A. Junctional complex of the retinal pigment epithelium (RPE) formed by tight and adherens junctions. Junctional complex of the outer limiting membrane (OLM) formed by tight and
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adherens junctions between retinal Müller glial cells and photoreceptor inner segments B. Confocal microscopy of non-human primate retinal pigment epithelium showing labeling of occludin (green) and PKC-ζ (red), within occludin loops at the tight junction.
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C. Rat retinal section at the level of the outer limiting membrane showing the coimmunolabeling for zonula occludens-1 (ZO-1, red) and glutamine synthetase (GS, retinal
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Müller glial cells, green), particularly around the cones (nuclei labelled by Dapi, blue). D. Primate retinal section at the level of the outer limiting membrane showing the coimmunolabeling for zonula occludens-1 (ZO-1, red) and peanut agglutinin (PNA, cone photoreceptors, green), showing the expression of ZO-1 in the cones. Nuclei are labelled by dapi (blue). E. Rat retina section microscopy of the outer limiting membrane showing the coimmunolabeling for occludin (green) and glutamine synthetase (GS, retinal Müller glial cells, green). Nuclei are labelled by dapi (blue). A and C-E: adapted with permission from (Omri et al., 2010) 128
ACCEPTED MANUSCRIPT GS= glutamine synthetase; PKC= protein kinase C; ZO-1= zonula occludens-1; PNA= peanut agglutinin
Figure 8. Pathophysiological mechanisms leading to macular edema
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Macular edema results from an imbalance between fluid entry and fluid exit, leading to intraretinal or subretinal fluid accumulation, driven by Starling equation.
Figure 9. Role of choroidal mast cells in outer blood-retinal barrier alteration.
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A. Nomarsky phase-contrast microscopy of rat flat-mounted choroid (left) showing resident
cells (right) with hexagonal shape.
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mast cells (white arrows) along choroidal vessels, and binucleated retinal pigment epithelial
B. Optical coherence tomography showing in vivo serous retinal detachment in a rat eye, 6 hours after subconjunctival administration of compound 48/80 resulting in the degranulation of mast cells.
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C. Histo-resin section showing ex vivo serous retinal detachment in a rodent eye, 6 hours after subconjunctival administration of compound 48/80 resulting in the degranulation of mast cells. Vasodilation of choroidal vessels (red star) and RPE swelling and detachment are
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associated with sub-retinal fluid. Inset: Detached RPE cell. D. Flat-mounted retinal pigment epithelium immunostaining by occludin (green) and Dapi
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(blue) showing enlarged retinal pigment epithelial cells with irregular occludin labeling, cytoplasmic translocation (white stars) and junction disruption (white arrow) as a consequence of mast cell degranulation. Images A, B and D: courtesy Yvonne de Kozak. Image B: adapted with permission from (Bousquet et al., 2015).
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ACCEPTED MANUSCRIPT Figure 10. Macular immunohistochemistry and schematic representation A. Schematic representation of macular Müller glial cells at the macula (blue lines) showing their lateral displacement and characteristic Z shape, from the outer limiting membrane (red) to the inner limiting membrane (green)
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B. Immunohistochemistry at the level of the Henle layer, showing the co-labeling of RMG cells with glutamine synthetase (GS, red) and with zonula occludens-1 (ZO-1, red).
C. Flat-mounted macula with GS (red) and ZO-1 immunostaining (red) showing the radial organization of retinal Müller glial cells around the fovea in the Henle layer.
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D. Schematic representation of the nerve fibers from the macula to the optic nerve head (red).
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E. Glutamine synthetase (GS) labeling (red) of retinal Müller glial cells around the fovea on flat-mounted retina. Around the fovea, retinal Müller glial cells are radially oriented. F. Section of human macula stained with GFAP (red). Astrocytes are distributed at the innermost retinal layers around vessels and in the superficial inner nuclear layer.
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At the fovea, GFAP-positive cells form a roof and send extensions toward the Henle layer (inset and yellow arrows). GS labelling shows an intense labelling at the Henle fiber layer where retinal Müller glial cells are densified and have a Z-shaped orientation and then
(Inset).
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vertically oriented all over the retina. Note that at the fovea, the glial cells poorly express GS
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D: adapted with permission from (Matet et al., 2015) RMG= retinal Müller glial cells; GS= glutamine synthetase; GFAP= glial fibrillary acidic protein; ZO-1= zonula occludens-1; ILM= Inner Limiting Membrane; NFL= nerve fiber layer; GCL= Ganglion Cell layer; INL= Inner Nuclear Layer; OPL= Outer Plexiform Layer; ONL= Outer Nuclear Layer; OLM= outer limiting membrane
Figure 11. AQP4 immunostaining in the normal macula A. Transversal section of a normal human macula immuno-stained with AQP4 (green), glutamine synthetase (GS, red) and Dapi (blue).
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ACCEPTED MANUSCRIPT AQP4 is located is astrocytes and retinal Müller glial cells, surrounding vessels. At the fovea, where no vessels are present (central continuous-contour inset and B-D), AQP4 is highly expressed along the central foveal Müller cone cells (B) that poorly express GS (C). At the Henle layer, retinal Müller glial cells highly express AQP4 along the in the Z-shaped retinal
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Müller glial cells (D and magnified inset). Outside the avascular zone (A, dotted-contour inset and E-G); AQP4 is located in astrocytes and endfeet of retinal Müller glial cells, and along the vertical course of retinal Müller glial cells. AQP4 is highly expressed around the vessels in all capillary plexuses. In the superficial plexus, AQP4 co-localizes partly with GS, but in the
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deep plexus, AQP4 completely co-localizes with GS (F, upper and lower insets, respectively).
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GS= glutamine synthetase
Figure 12. AQP4 immunostaining in retinal Müller glial cells on flat-mounted human macula and the glymphatic hypothesis
A. Flat-mounted human macula with glutamine synthetase (GS, blue) and AQP4 (green) co-
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staining. At low magnification, AQP4 is concentrated in the macula and along RMG cells that follow the interpapillo-macular fibers.
B. Higher magnification (inset in A) showing the concentration of AQP4 around the fovea.
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C-D. Higher magnification (lower inset in B) shows that AQP4 distribution follows a structured pattern around a network composed of small channels running in parallel (C), and
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corresponding to the orientation of glial cells stained by GS (D). E. Identification of a similar channel pattern in the upper inset of B. F. Schematic hypothesis of AQP4-retinal Müller glial cell glymphatic drainage (green dotted line) from the macula towards the optic nerve head (yellow arrows). GS= glutamine synthetase; AQP= aquaporin
Figure 13. Schematic representation of the hypothetical glymphatic system. The distribution of aquaporin channels at retinal glial Müller cell membrane, allows water drainage along vessels, or “paravascular” drainage (A) and results in a radial network of
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ACCEPTED MANUSCRIPT draining channels between retinal glial Müller cells (B), orientated towards the optic nerve head.
Figure 14. Confocal microscopy of a human diabetic retina labelled with AQP4
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A-C: Staining for aquaporin 4 (AQP4, green), glutamine synthetase (GS, red) and Dapi (blue) showing a reduced expression of AQP4 in the perifoveal region together with reduced GS expression of the Z-shaped RMG cells. AQP4 is rather concentrated only around vessels. Outside the macula (Figure A, small inset and D, E), AQP4 is highly co-expressed with GS
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AQP= aquaporin; GS= glutamine synthetase
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along RMG cells, and is particularly concentrated around vessels (F, arrow).
Figure 15. Schematic representation of gliovascular changes in diabetic macular edema and correlation with OCT-A changes
A. In the normal retina, Kir4.1 (orange channels) is located around vessels expressed by
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astrocytes and retinal Müller glial cells. Microglia surrounds vessels. B. In the diabetic retina, Kir4.1 distribution is altered in retinal Müller glial cells, leading to Kir4.1 channel loss around vessels in the deep capillary plexus, since there are no astrocytes
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in the deep capillary plexus. In the more superficial vessels Kir 4.1 is still located around vessels, expressed by astrocytes. Since potassium intervenes in the regulation of blood flow,
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flow decreases in the deep capillary plexus. Retinal Müller glial cells are hypertrophic, and Kir 4.1 is mislocalized at their apices. Activated microglia migrate in the sub-retinal space. C: Optical coherence tomography angiography of a normal retina. D: coherence tomography angiography of diabetic macular edema showing focal defects in the deep capillary network adjacent to hyporeflective cystoid edema spaces. RMG= retinal Müller glial cell; a= astrocyte, mc= microglia; OLM= outer limiting membrane; SCP= superficial capillary plexus; DCP= deep capillary plexus; ICP= intermediate capillary plexus”
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ACCEPTED MANUSCRIPT Figure 16. Multimodal clinical imaging of “pure” macular edema phenotypes. A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in a 45-year-old man with idiopathic macular telangiectasia type 1 showing intraretinal cystoid edema and intraretinal exudates of pure vascular origin, related to an intense leakage from
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perifoveal telangiectasia and hyperpermeable capillaries. C-D: OCT and midphase FA in a 72-year-old man with idiopathic macular telangiectasia type 2 showing intraretinal cavitations without retinal thickening, and focal loss of the normal
retinal layer structure, resulting from a cellular neuro-vasculo-glial degenerative process of
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unknown origin. There is a mild fluorescein leakage from fine perifoveal telangiectasia, but intraretinal cavities result from tissue loss and not from exudation.
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E-F: OCT and midphase FA in a 48-year-old man treated by Trametinib, a mitogen-activated protein kinase kinase (MEK) inhibitor, for metastatic melanoma, showing marked cystoid macular edema and retinal thickening, but absence of vascular fluorescein leakage. A moderate filling of intraretinal cavities is visible on FA. Adapted with permission (Duncan et
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al., 2015).
G-H: OCT and midphase FA in a 59-year-old woman with receiving Paclitaxel (formulated as albumin-bound nanoparticles) for metastatic breast cancer, showing cystoid macular edema
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but absence of fluorescein leakage. Adapted with permission (Ehlers et al., 2013). I-J: OCT and midphase FA in a 69-year-old man with hypoproteinemia due to nephrotic
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syndrome, showing subretinal fluid accumulation and absence of fluorescein leakage. K-L: OCT in a 44-year-old woman with inferior hemi-central retinal vein occlusion complicated by paracentral middle acute middle maculopathy, showing a focal hyperreflectivity on OCT localized across several relatively thickened layers, from the inner nuclear to the Henle fiber layers, with a focal hyporeflectivity on infrared reflectance. These changes indicate intraretinal fluid accumulation related to focal cellular ischemia due to a focal perfusion decrease in the deep capillary plexus. E-F: adapted with permission from (Duncan et al., 2015). G-H: adapted with permission from (Ehlers et al., 2013)
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Figure 17. Mechanisms induced by hyperglycemia in the retina leading to diabetic macular edema.
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Figure 18. Implication of inflammation in diabetic macular edema. A. Schematic view of inflammation, aging and oxidative stress as contributors to diabetic macular edema.
B. Pathways activated in hyperglycemic conditions triggering inflammation
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AGE= advanced glycation endproducts; RAGE= receptor for advanced glycation
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endproducts; PKC= protein kinase C; ROS= reactive oxygen species
Figure 19. Kinetics of retinopathy in the Goto-Kakizaki rat model A-D Histologic paraffin sections of rat retina stained with hemalun-eosin A: Retina from a one-year-old Wistar rat, with normoglycemic control
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B-D: Histology of Goto-Kakizaki rat retinas at 4, 8 and 18 months showing progressive retinal thickening with fluid accumulation in the photoreceptor outer segment layer (white arrows) and outer nuclear layer (yellow arrows). Progressive dilation of choroidal vessels is also
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visible (black arrows and blue stars).
E: Lectin-labelled flat-mounted retina of a Goto-Kakizaki rat at 18 months showing clear
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signs of retinal microangiopathy
WS= Wistar rat; GK= Goto Kakizaki rat; INL= inner nuclear layer; ONL= outer nuclear layer
Figure 20. Kir4.1 and aquaporin expression kinetics in the Goto-Kakizaki diabetic rat model A and C: immunostaining on retinal cryosections from Wistar control rats and Goto-Kakizaki diabetic rats with Kir4.1 (A) or AQP4 (C) labeling, showing decreased Kir4.1 and AQP4 fluorescence in the outer retina and in the retinal glial Müller cell processes surrounding the retinal vessels.
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ACCEPTED MANUSCRIPT B, D and E: time-dependent evolution of Kir4.1 (B), AQP4 (D) and aquaglyceroporin AQP9 (E) expression in the retina of Goto-Kakizaki diabetic rats, compared to age-matched control Wistar rats, using quantitative PCR (HPRT1 and 18S serving as internal controls). Data were expressed as mean ± standard error of the mean (n=6).
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*, P<0.05; **, P<0.01, ***, P>0.001. WS= Wistar rat; GK= Goto Kakizaki rat; GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; AQP= aquaporin.
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Figure 21. Retinal pigment epithelium changes in the Goto-Kakizaki diabetic rat model In the Wistar non-diabetic rat, phalloidin stains the actin cytoskeleton that borders the inner
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region of tight junctions (B) and shows a regular mosaic pattern of the retinal pigment epithelium. ROCK-1 is localized in the cytoplasma (C). On electron microscopy, RPE cells have a regular morphology.
In the Goto-Kakizaki rat, at 6 months, apical constriction of the cytoskeleton (E, as compared
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to A; and F, dotted circle) is associated with membrane translocation of ROCK-1 (G) and formation of membrane blebs (G inset and H, yellow dotted line). At one year, large syncytium can be observed (H, yellow arrows) together with areas of disrupted intercellular
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junctions in the diabetic Goto-Kakizaki model (J, arrow) as compared to Wistar rat (I). WS= Wistar rat; GK= Goto Kakizaki rat; POS= photoreceptor outer segments; RPE= retinal
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pigment epithelium
Figure 22. Schematic representation of hyperglycemia-induced changes in the PAR3/PAR6/PKCζ complex in retinal pigment epithelial cells of Goto-Kakizaki diabetic rats The PAR-6/PKCζ/PAR-3 scaffold complex is involved in tight junction (TJ) formation and maintenance. Alteration of any of those junctional partners leads to mislocalization of the others and to tight junction disruption. Hyperglycemia induces a biphasic activation of PKCζ. Higher (GK 6 months) and lower panels (GK 12 months): the PKCζ-PT410/ PKCζ ratio, in
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ACCEPTED MANUSCRIPT western blotting, shows that PKCζ activity increases significantly at 6 months (~ +40%) and then decreases at 12 months (~ -60%) after diabetes onset, compared to control Wistar rats. Upper panels: In 6-month-old controls, PKCζ staining (red) appeared as focal spots within loops formed by occludin protrusions (green). During hyperglycemia, 6 months after diabetes
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onset, phosphorylation of PKCζ (PKCζ-P) by PDK1 (Pyruvate Dehydrogenase Kinase 1) increases, inducing ROCK-1 phosphorylation (ROCK1-P). ROCK1 activation induces PAR3 phosphorylation, disrupting the PAR-3 / PKCζ interaction and thereby suppressing the activity of the PAR-6/PKCζ/PAR-3 complex.
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Lower panel: twelve months after diabetes onset, the PKCζ staining associated to tight
junctions was either barely or not detectable. The decrease of PKCζ activity leads to tight
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junction opening and occludin internalization from the cell membrane to the cytoplasm, further contributing to tight junction disruption and outer retinal barrier disruption. Administration of a PKCζ inhibitor induces similar changes.
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GK= Goto-Kakizaki rat; PKC= protein kinase C
Figure 23. Transcytosis of microglia/macrophages through retinal pigment epithelial cells
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A. Semi-thin section of rat retinal pigment epithelium showing the formation of a “pore” resulting from the invagination of the apical towards the basal cell membrane (arrow).
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B and C: Electron microscopy imaging of a retinal pigment epithelial cells showing the formation of a “pore” (B), inducing the lateral displacement of a cell nucleus (C, arrow). D: tridimensional representation of a microglial cell transcytosis (arrow) from the subretinal space through a retinal pigment epithelial cell. A and C: Adapted with permission from (Omri et al., 2011)
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ACCEPTED MANUSCRIPT Figure 24. Changes in the outer limiting membrane in the Goto-Kakizaki diabetic rat model Flat-mounted retinas imaged by confocal microscopy at the level of the outer limiting membrane (A-D) and retinal sections magnified around the outer limiting membrane (E-F).
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A-B: Labeling for glutamine synthetase (GS, red), a marker of retinal Müller glial cells, shows that the outer limiting membrane network formed by retinal Müller glial cells is disrupted in the diabetic model.
C-D: Additional labelling for occludin, that is expressed between retinal Müller glial cells, and
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between these cells and photoreceptors, shows a focal disruption of occludin staining in the diabetic model (C, arrow).
limiting membrane (arrows).
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E-F: Staining for PKCζ on retinal sections shows delocalization of PKCζ from the outer
Adapted with permission from (Omri et al., 2010) and (Omri et al., 2013) WS= Wistar rat; GK= Goto Kakizaki rat; CTL= control (WS rat); DIA= diabetic GK rat; PKC=
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protein kinase C; GS= glutamine synthetase
Supplementary Figure 1. Radial distribution of retinal glial Müller cells in a non-human
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primate retina.
A. Flat-mounted retina from a Macaca fascicularis, labelled with glutamine synthetase,
head.
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showing the radial orientation of retinal glial Müller cells from the fovea to the optic nerve
B. Flat-mounted retina from a normal post mortem human donor eye, labelled with CRALBP, showing the radial orientation of retinal glial Müller cells around to fovea and towards the optic nerve head. GS= glutamine synthetase; F= fovea, ONH= optic nerve head
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ACCEPTED MANUSCRIPT VIDEO LEGENDS
Video 1. Mechanisms of hydro-ionic homeostasis in the retina.
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Video 2. Mechanisms of diabetic macular edema
Video 3. Transcytosis across retinal pigment epithelial cells allowing microglia
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migration from the retina to the choroid
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TABLE 1. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with diabetic macular edema and reported in the literature. Patients, No. (Controls, No.)
Correlation with central macular thickness (+/-) _ _
Reference
23 (22) 13 (14)
Fold increase Patients vs Controls 3.6 × NS*
Interleukin 3 (IL-3)
Aqueous Vitreous
Multiplex Multiplex
Interleukin 6 (IL-6)
Aqueous
Multiplex
23 (22)
10.4 ×
_
(Jonas et al., 2012)
Vitreous
ELISA
26 (12)
19.8 ×
_
(Funatsu et al., 2003)
Vitreous Vitreous
ELISA Multiplex
53 (15) 92 (83)
10.2 × 14.4 ×
Yes (+) _
(Funatsu et al., 2009) (Yoshimura et al., 2009)
Aqueous
Multiplex Multiplex
18 (16) 13 (14)
2.3 × NS*
No _
(Lee et al., 2012) (Ghodasra et al., 2016)
Interleukin 8 (IL-8)
Aqueous Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex Multiplex
23 (22) 92 (83) 18 (16) 13 (14)
10.3 × 8.9 × 2.3 × NS*
_ _ Yes (+) _
(Jonas et al., 2012) (Yoshimura et al., 2009) (Lee et al., 2012) (Ghodasra et al., 2016)
Interleukin 13 (IL-13)
Aqueous
Multiplex
18 (16)
No
(Lee et al., 2012)
Epidermal Growth Factor (EGF)
Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex
0.9 × NS*
_ _ _
(Ghodasra et al., 2016) (Jonas et al., 2012) (Yoshimura et al., 2009)
Aqueous Vitreous Aqueous Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex ELISA Multiplex ELISA
18 (16) 13 (14) 23 (22) 20 (8) 23 (22) 20 (8)
NS NS* 1.9 × 3.6 × 2.4 × 0.025 ×
No _ _ _ _ _
(Lee et al., 2012) (Ghodasra et al., 2016) (Jonas et al., 2012) (Patel et al., 2006) (Jonas et al., 2012) (Patel et al., 2006)
Aqueous
ELISA
42 (23)
1.8 ×
_
(Kocabora et al., 2016)
Transforming Growth Factor ß (TGF-ß)
Tumor Necrosis Factor alpha (TNF-α α)
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Hepatocyte Growth Factor (HGF)
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Technique
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Ocular media
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Biological factor (acronym)
1.7 × NS
(Jonas et al., 2012) (Ghodasra et al., 2016)
1
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Platelet Derived Growth Factor (PDGFAB) Platelet Derived Growth Factor (PDGFBB) InterCellular Adhesion Molecule 1 (ICAM-1, CD54)
Vascular cell adhesion molecule 1 (VCAM) C-X-C motif chemokine 10 (CXCL-10) (=Interferon gamma-induced protein 10, IP10) Chemokine ligand 2 (CCL2) (=monocyte chimoattractant protein 1, MCP-1)
(Jonas et al., 2012) (Jonas et al., 2012) (Funatsu et al., 2003) (Funatsu et al., 2005) (Funatsu et al., 2005) (Patel et al., 2006) (Funatsu et al., 2009)
Multiplex ELISA Multiplex ELISA ELISA ELISA ELISA Multiplex Multiplex ELISA
92 (83) 15 (15) 18 (16) 36 (6) 20 (8) 53 (15) 15 (15) 18 (16) 13 (14) 15 (15)
2.1 ×
_ _ No Yes (-) _ Yes (-) _ No _ _
(Yoshimura et al., 2009) (Praidou et al., 2011) (Lee et al., 2012) (Funatsu et al., 2005) (Patel et al., 2006) (Funatsu et al., 2009) (Praidou et al., 2011) (Lee et al., 2012) (Ghodasra et al., 2016) (Praidou et al., 2011)
Vitreous
ELISA
15 (15)
0.56 ×
_
(Praidou et al., 2011)
Aqueous Vitreous
Multiplex ELISA
23 (22) 33 (13)
3.2 × 2.5 ×
_ Yes (+)
(Jonas et al., 2012) (Funatsu et al., 2005)
Vitreous
ELISA
53 (15)
2.2 ×
Yes (+)
(Funatsu et al., 2009)
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_ _ _ Yes (+) Yes (+) _ Yes (+)
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Vitreous Vitreous Aqueous Vitreous Vitreous Vitreous Vitreous Aqueous Vitreous Vitreous
7.9 × 2× 46 × 60.5 × 58 × 4.5 × 30.5 × NS 7.6 × 2.8 × 0.15 × NS
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23 (22) 23 (22) 26 (12) 36 (8) 33 (13) 20 (8) 53 (15)
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Platelet Derived Growth Factor (PDGFAA)
Multiplex Multiplex ELISA ELISA ELISA ELISA ELISA
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Pigment Epithelium Derived Factor (PEDF)
Aqueous Aqueous Vitreous Vitreous Vitreous Vitreous Vitreous
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Placental Growth Factor (PGF) Vascular Endothelial Growth Factor (VEGF)
0.13 × 2.4 × 1.5 × NS*
Aqueous
Multiplex
23 (22)
2.7 ×
_
(Jonas et al., 2012)
Aqueous Vitreous
Multiplex Multiplex
23 (22) 13 (14)
1.4 × NS*
_ _
(Jonas et al., 2012) (Ghodasra et al., 2016)
Aqueous Vitreous Vitreous
Multiplex ELISA Multiplex
23 (22) 53 (15) 92 (83)
2.1 × 2.6 × 15.8 ×
_ Yes (+) _
(Jonas et al., 2012) (Funatsu et al., 2009) (Yoshimura et al., 2009)
2
Multiplex Multiplex
18 (16) 23 (22)
2.5 × 1.6 ×
No _
(Lee et al., 2012) (Jonas et al., 2012)
Aqueous Vitreous Aqueous
Multiplex ELISA Multiplex
23 (22) 20 (8) 23 (22)
6.1 × NS
_ _ _
(Jonas et al., 2012) (Patel et al., 2006) (Jonas et al., 2012)
Vitreous
ELISA
20 (8)
0.67 ×
_
(Patel et al., 2006)
Vitreous
Multiplex
92 (83)
NS
_
(Yoshimura et al., 2009)
4.1 ×
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Plasminogen activator inhibitor-1 (PAI1) Soluble fms-like tyrosine kinase-1 (sFlt1) (=soluble vascular endothelial growth factor 1, sVEGFR-1) Tumor Necrosis Factorα α (TNF-α α)
Aqueous Aqueous
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C-X-C motif chemokine ligand 9 (CXCL-9) or Monokine induced by γinterferon (MIG) Matrix metallopeptidase 9 (MMP-9)
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* Multivariate analysis with adjustment on all biological factors assessed NS= non-significant; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
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TABLE 2. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with macular edema due to central retinal vein occlusion and reported in the literature. Technique
Patients, No. (Controls, No.)
Interleukin-1ß (IL-1ß)
Aqueous
Multiplex
Interleukin 6 (IL-6)
Vitreous
No
(Feng et al., 2013)
Multiplex
13 (83)
81.4 ×
_
(Yoshimura et al., 2009)
Vitreous
ELISA
27 (16)
47.9 ×
Yes (+)
(Noma et al., 2009)
Aqueous
Multiplex
5 (13)
39.6 ×
_
(Funk et al., 2009)
Vitreous
CBA
35 (14)
10.4 ×
_
(Koss et al., 2012)
Aqueous
Multiplex
18 (10)
2.3 ×
No
(Feng et al., 2013)
Vitreous
Multiplex
13 (83)
35.3 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
5 (13)
63.8 ×
_
(Funk et al., 2009)
Aqueous
Multiplex
18 (10)
1.3 ×
No
(Feng et al., 2013)
Aqueous
Multiplex
18 (10)
2.1 ×
No
(Feng et al., 2013)
Vitreous
Multiplex
13 (83)
14.7 ×
_
(Yoshimura et al., 2009)
Vitreous
ELISA
27 (16)
7×
Yes (+)
(Noma et al., 2009)
Aqueous
Multiplex
5 (13)
23.7 ×
_
(Funk et al., 2009)
CBA
35 (14)
39.8 ×
_
(Koss et al., 2012)
Multiplex
18 (10)
1.6 ×
No
(Feng et al., 2013)
Multiplex
18 (10)
1.9 ×
No
(Feng et al., 2013)
Multiplex
5 (13)
4.4 ×
_
(Funk et al., 2009)
Vitreous Aqueous Basic fibroblast growth factor (bFGF) C-X-C motif chemokine 10 (CXCL-10) or Interferon gamma-
Aqueous Aqueous
M AN U
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Transforming Growth Factor ß (TGF-ß) Vascular Endothelial Growth Factor (VEGF)
Reference
AC C
Interleukin 8 (IL-8)
Correlation with CMT (+/-)
8 (10)
Fold increase Patients vs Controls 1.4 ×
RI PT
Ocular media
SC
Biological factor (acronym)
ACCEPTED MANUSCRIPT
induced protein 10 (IP10) 13 (83)
28.8 ×
Aqueous
Multiplex
5 (13)
5.6 ×
Vitreous
CBA
35 (14)
4×
Aqueous
Multiplex
5 (13)
2.2 ×
Aqueous
Multiplex
18 (10)
2.1 ×
_
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Multiplex
SC
Platelet Derived Growth Factor (PDGFAA) Serum Amyloid A (SAA)
Vitreous
M AN U
Chemokine ligand 2 (CCL2) or monocyte chimoattractant protein 1 (MCP-1)
(Yoshimura et al., 2009)
_
(Funk et al., 2009)
_
(Koss et al., 2012)
_
(Funk et al., 2009)
(Feng et al., 2013)
AC C
EP
TE D
CBA= cytometric bead array; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
ACCEPTED MANUSCRIPT
Technique
Patients, No. (Controls, No.)
Correlation with CMT (+/-)
Reference
8 (10)
Fold increase Patients vs Controls 2.1 ×
IL-1 beta (Interleukin 1 beta) IL-6 (Interleukin 6) Master inflammatory cytokine
Aqueous
Multiplex
No
(Feng et al., 2013)
Vitreous
Multiplex
30 (83)
5.4 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
38 (18)
1.8 ×
Yes (+)
(Kaneda et al., 2011)
Aqueous
Multiplex
12 (16)
NS
No
(Lee et al., 2012)
Vitreous
CBA
43 (14)
NS
_
(Koss et al., 2012)
Vitreous
CBA
43 (28)
NS
No
(Pfister et al., 2013)
Aqueous
Multiplex
8 (10)
2.3 ×
No
(Feng et al., 2013)
Vitreous
ELISA
28 (17)
9.6 ×
Yes (+)
(Noma et al., 2014)
Vitreous
Multiplex
30 (83)
16.4 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
38 (18)
7.1 ×
Yes (+)
(Kaneda et al., 2011)
Aqueous
Multiplex
12 (16)
2.1 ×
Yes (+)
(Lee et al., 2012)
Aqueous
Multiplex
8 (10)
1.3 ×
No
(Feng et al., 2013)
IL-12 (Interleukin 12)
Aqueous
Multiplex
38 (18)
2.2 ×
No
(Kaneda et al., 2011)
IL-15 (Interleukin 15)
Aqueous
Multiplex
38 (18)
1.6 ×
No
(Kaneda et al., 2011)
IL-17 (Interleukin 17)
Aqueous
Multiplex
38 (18)
1.8 ×
No
(Kaneda et al., 2011)
IL-23 (Interleukin 23) TGF beta (Transforming Growth Factor Beta) VEGF
Aqueous
Multiplex
38 (18)
2.6 ×
No
(Kaneda et al., 2011)
Multiplex
8 (10)
2.1 ×
No
(Feng et al., 2013)
Multiplex
30 (83)
NS
_
(Yoshimura et al., 2009)
Aqueous
Vitreous
TE D
EP
AC C
IL-8 (Interleukin 8)
SC
Ocular media
M AN U
Biological factor (acronym)
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TABLE 3. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with macular edema due to branch retinal vein occlusion and reported in the literature.
2.4 ×
No
(Lee et al., 2012)
Aqueous
Multiplex
38 (18)
NS
No
(Kaneda et al., 2011)
Vitreous
Multiplex
43 (14)
23.1 ×
_
(Koss et al., 2012)
Vitreous
Multiplex
43 (28)
23.1 ×
Aqueous
Multiplex
8 (10)
1.6 ×
Vitreous
Multiplex
28 (17)
48.3 ×
Aqueous
Multiplex
8 (10)
1.7 ×
Vitreous
ELISA
28 (17)
2.4 ×
Vitreous
Multiplex
30 (83)
Aqueous
Multiplex
38 (18)
Aqueous
Multiplex
12 (16)
Vitreous
CBA
43 (14)
Vitreous
CBA
Vitreous
ELISA
Vitreous
ELISA
Aqueous
Multiplex
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12 (16)
(Pfister et al., 2013)
No
(Feng et al., 2013)
Yes (+)
(Noma et al., 2014)
No
(Feng et al., 2013)
Yes (+)
(Noma et al., 2014)
4.4 ×
_
(Yoshimura et al., 2009)
NS
No
(Kaneda et al., 2011)
NS
No
(Lee et al., 2012)
2.4 ×
_
(Koss et al., 2012)
43 (28)
2.4 ×
No
(Pfister et al., 2013)
28 (17)
3×
Yes (+)
(Noma et al., 2014)
28 (17)
1.9 ×
Yes (+)
(Noma et al., 2014)
2.2 ×
No
(Feng et al., 2013)
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No
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sVEGFR2 (soluble Vascular Endothelial Growth Factor Receptor 2) SAA (Serum Amyloid A)
Multiplex
EP
bFGF (Basic fibroblast growth factor) sICAM-1 (soluble InterCellular Adhesion Molecule 1) or CD54 CCL2 (Chemokine ligand 2) or MCP-1 (Monocyte chimoattractant protein 1)
Aqueous
AC C
(Vascular Endothelial Growth Factor)
SC
ACCEPTED MANUSCRIPT
8 (10)
CBA= cytometric bead array; NS= non-significant; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
ACCEPTED MANUSCRIPT
Identified mechanism(s)
Vascular endothelial growth factor-A (VEGF-A) / VEGF-R2
Junction integrity alteration
Occludin phosphorylation via PKCβ activation
SC
Biological factor / receptor
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TABLE 5. Major growth factors and inflammatory mediators potentially involved in retinal and choroidal vascular permeabilization
Occludin downregulation by increasing free cytosolic ß-catenin, and upregulating u-PAR
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VE-cadherin endocytosis via Rac activation/ß-arrestin-2
VE-cadherin disruption via increased transglutaminase-2 activity by raising intracellular Ca and ROS concentrations Up-regulation of ICAM-1 expression: leukostasis
(Miyamoto et al., 2000)
Occludin internalization and ubiquitinization via Src-family kinases
(Scheppke et al., 2008)
Transcellular permeability
Transcytosis in caveolae via an eNOS-dependent mechanism
(Feng et al., 1999)0) (Hofman et al., 2000) (Wisniewska-Kruk et al., 2016)
Junction integrity alteration
Changes in occludin distribution in RPE cells MEK signaling pathway
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Degradation of ZO-1 and VE-cadherin
Bradykinin (BK) / B2R and B1R
Tie-2 receptor α3β1-Integrin
EP
Loss of pericytes
(Lee et al., 2016)
(Miyamoto et al., 2007) (Otrock et al., 2007) (Kowalczuk et al., 2011) (Huang et al., 2015) (Hammes et al., 2004) (Cai et al., 2008) (Pfister et al., 2008) (Park et al., 2014)
Loss of astrocytes
avβ5 binding
Junction integrity alteration Vasodilatation and hyperpermeability Junction integrity alteration
Reduces VE-cadherin by phosphorylation
(Rangasamy et al., 2011)
eNOS-mediated B1R stimulation iNOS-mediated B2R stimulation Src kinase-mediated VE-cadherin phosphorylation
(Kita et al., 2015)
Claudin-5 down-regulation
(Zhou et al., 2014)
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Angiopoietin-2 (Ang-2)
(Antonetti et al., 1999) (Harhaj et al., 2006) (Murakami et al., 2012) (Liu et al., 2016) (Behzadian et al., 2003) (Gavard and Gutkind, 2006)
2+
Induces plasmalemma vesicle-associated protein formation Placental growth factor (PGF) / VEGFR-1
Reference
(Yun et al., 2016a)
(Liu and Feener, 2013)
Angiotensin II
Pericyte migration
-
(Wilkinson-Berka, 2006)
Stimulates VEGF
(Gilbert et al., 2000)
Aldosterone / MR
Junction integrity alteration Choroidal permeability
Up-regulation of the endothelial vasodilatory K channel KCa2.3 in choroidal endothelial cells and
(Zhao et al., 2012)
ACCEPTED MANUSCRIPT
adjacent smooth muscle cell relaxation (Zhao et al., 2010) (Gaudenzio et al., 2016)
Decreased ZO-1 and claudin-5 expression and alteration of their sub-cellaular distribution in retinal endothelial cells Via PKC-mediated NF-kB activation ZO-1 disruption in RPE cells Via p38 MAPK Increased VEGF and ICAM
(Aveleira et al., 2010)
Apoptosis
(Behl et al., 2008)
Retraction of endothelial cells Junction integrity alteration
Endothelial cell death
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Regulates the expression and distribution of ion and water channels in RMG cells Histamine-mediated vasodilation Release of biogenic amines (e.g. histamine), proteases, angiogenin, cytokines (e.g. TNF-α,) chemokines (e.g. CCL5, IL-8, MCP-1, eotaxin) Activation of PI3K and Src kinase pathways (C5a)
SC
Tumor necrosis factor-α (TNF-α) / TNF-Rs
Decreased drainage Mast cell degranulation
Necrosis
Interleukin-1ß (IL1-ß)
Junction integrity alteration
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Complement components (C3a and C5a)
(Schraufstatter et al., 2002)
(Shirasawa et al., 2013) (Penfold et al., 2002)
(Claudio et al., 1994)
Decreased cx43-mediated glio-vascular communication
(Muto et al., 2014)
Recruitment of leukocytes Histamine production VEGF-A production
(Bamforth et al., 1997)
Occludin down-regulation under hypoxic conditions
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PKC-θ-dependent phosphorylation of ZO-1
(Vinores et al., 2003) (Yamagata et al., 2004) (Rigor et al., 2012)
Alterations in the f-actin cytoskeleton due to the phosphorylation of MLCs by ROCK activation
(Wu et al., 2016)
Decreased VE-cadherin expression
(Matsuda et al., 2015)
Junction integrity alteration Pericyte loss
Occludin degradation via MMP-9 production
(Behzadian et al., 2001)
-
(Betts-Obregon et al., 2016)
Interleukin-6 (IL-6)
Junction integrity alteration
Down-regulation of occludin and ZO-1 VEGF production by STAT3 activation
(Yun et al., 2016b)
Decreases the expression of VE-cadherin, occludin and claudin-5
(Rochfort et al., 2014)
Junction integrity alteration
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IL-8
EP
Transforming growth factor-ß (TGF-ß)
Down-regulation of tight junction proteins, including occludin, claudin-5 and ZO-1
(Yu et al., 2013)
VEGF= vascular endothelial growth factor; VEGFR-2= kinase insert domain-containing receptor or KDR; PGF= placental growth factor; VEGFR-1 (Flt-1)= VEGF receptor-1 (fms-like tyrosine kinase-1); MAPK= mitogen-activated protein kinase; PACAP= pituitary adenylate cyclase-activating polypeptide; VIP= vasoactive intestinal peptide; B1R= bradykinin B1 receptor; B2R= bradykinin B2 receptor; eNOS= endothelial nitric oxide synthase; iNOS= inducible nitric oxide synthase; pKal= plasma kallikrein; PCK= protein kinase C; ROCK= Rho-associated protein kinase; NO= nitric oxide; MMP= matrix metalloproteinase; IL=interleukin; uPAR= urokinase plasminogen activator receptor; MR= mineralocorticoid receptor; ROS= reactive oxygen species; TNF= tumor necrosis factor; TNF-Rs= TNF receptors; cx43= connexin-43; MLC= myosin light chain
ACCEPTED MANUSCRIPT
Kir4.1 expression Immunohistochemistry
Ischemiareperfusion
RT-PCR
Immunohistochemistry
Decreased
Slight reduction in IPL (D1-7)
Decreased
Lewis Rat
Decreased around vessels and at RMG cell endfeet
-
Horse
Absent
Decreased
Wistar rats
Globally decreased. Absent at OLM and perivascular membranes (6 months)
-
Wistar rats
-
Long Evans rats
Decreased. Absent at ILM and around vessels. Mislocalized in IPL
Absent around vessels and at RMG cell endfeet
Reference
Westernblot Decreased
RT-PCR Unchanged
(Liu et al., 2007)
-
Unchanged
(Zhao et al., 2011)
-
Absent at RMG cells in inner layers. Mislocalized at ONL and OLM
-
-
(Eberhardt et al., 2011)
-
-
-
-
(Pannicke et al., 2006)
Absent at superficial capillary plexus. Present in NFL and GCL. Not altered at deep capillary plexus (6 months) Slightly increased at IPL
-
-
(Iandiev et al., 2007)
Slightly decreased (D7)
Unaltered -
(Hollborn et al., 2011) (Pannicke et al., 2004)
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Decreased (D1) Disappeared (D3-7)
-
-
Decreased (D7)
-
EP
Equine recurrent autoimmune uveitis Streptozotocyninduced diabetes
Westernblot Decreased
Wistar rats
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Endotoxininduced uveitis
AQP4 expression
SC
Animal
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Disease model
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TABLE 4. Changes in the membrane channels Kir4.1 and Aquaporin-4 expression and localization in animal models of retinal disorders relevant to study cellular mechanisms of macular edema
Long Evans rats
Absent at superficial vascular plexus. Normal at deep vascular plexus (D7)
-
-
-
-
-
(Iandiev et al., 2006a)
Long Evans rats C57BL/6 mice
-
-
-
Decreased (D7)
-
-
Slightly increased at IPL
Decreased D7 -
(Iandiev et al., 2006b)
Decreased around vessels
Decreased (D7) -
(Hirrlinger et al., 2010)
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Wistar rats
-
Decreased (D7-14)
Decreased (D7-14)
-
Decreased (D7-14)
Decreased (D7-14)
(Dibas et al., 2010)
Branch Retinal Vein Occlusion
Long Evans rats
Decreased
-
Decreased (D1-3)
Not altered (except in cysts)
-
Decreased (D1-3)
(Rehak et al., 2009)
Light induce damage
Pigmented C57BL/6SV129 mice
Increased in outer retina
-
-
Increased in outer retina
-
-
(Iandiev et al., 2008)
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Optic nerve crush
AC C
EP
TE D
M AN U
SC
AQP4= Aquaporin-4; RT-PCR= real-time polymerase chain reaction; D= day; RMG cells= retinal Müller glial cells; ILM= inner limiting membrane; NFL= nerve fiber layer; GCL= ganglion cell layer; IPL= inner plexiform layer; ONL= outer nuclear layer; OLM= outer limiting membrane Time of observations after model induction is indicated between parentheses
ED
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M AN US C
R
PT ED
M AN US
AC C
EP TE D
M AN US C
RI P
CC
EP TE D
M AN US C
RI P
AC C
EP
TE D M AN US C
RI P
EP TE D
M AN US C
ED
M AN
EP TE D
M AN US C
R
CE PT ED
M AN US C
RI
PT ED
M AN US
PT ED
M AN US
D
M AN
PT ED
M AN US C
EP TE D
M AN US C
AC C
EP
TE D M AN US C
RI P
PT ED
M AN US
CC
EP TE D
M AN US C
RI
EP TE D
M AN US C
R
EP TE D
M AN US C
R
EP TE D
M AN US C
EP TE D
M AN US C
PT ED
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ED
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ACCEPTED MANUSCRIPT ARTICLE HIGHLIGHTS •
Cells forming inner and outer blood-retinal barriers maintain retinal homeostasis
•
Macular edema results from an imbalance between fluid entry and drainage mechanisms Intraretinal accumulation of macromolecules osmotically attracts water and solutes
•
The structural organization of the retina explains why edema develops in the macula
•
A glymphatic system may be formed by AQP4 expression along macular Müller cells
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SC
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•
S1350-9462(17)30075-7
DOI:
10.1016/j.preteyeres.2017.10.006
Reference:
JPRR 694
To appear in:
Progress in Retinal and Eye Research
Received Date: 30 July 2017 Revised Date:
24 October 2017
Accepted Date: 31 October 2017
Please cite this article as: Daruich, A., Matet, A., Moulin, A., Kowalczuk, L., Nicolas, Michaë., Sellam, A., Rothschild, Pierre.-Raphaë., Omri, S., Gélizé, E., Jonet, L., Delaunay, K., De Kozak, Y., Berdugo, M., Zhao, M., Crisanti, P., Behar-Cohen, F., Mechanisms of macular edema: Beyond the surface, Progress in Retinal and Eye Research (2017), doi: 10.1016/j.preteyeres.2017.10.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Mechanisms of macular edema: beyond the surface Alejandra Daruich,1,2* Alexandre Matet,2* Alexandre Moulin,2 Laura Kowalczuk,2 Michaël Nicolas,2 Alexandre Sellam,1 Pierre-Raphaël Rothschild,1 Samy Omri,1 Emmanuelle Gélizé,1 Laurent Jonet,1 Kimberley Delaunay,1 Yvonne De Kozak, 1 Marianne Berdugo,1 Min Zhao,1 Patricia Crisanti,1 Francine Behar-Cohen1,3,4
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* Both authors contributed equally to this work. 1
INSERM, UMRS1138, Team 17, From Physiopathology of Ocular Diseases to Clinical Development, Université Paris Descartes Sorbonne Paris Cité, Centre de Recherche des Cordeliers, 15 rue de l’Ecole de Médecine, 75006 Paris, France
2
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Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Avenue de France 15, 1004 Lausanne, Switzerland 3
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Ophtalmopole, Cochin Hospital, AP-HP, Assistance Publique Hôpitaux de Paris, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France
4
Faculty of Biology and Medicine, University of Lausanne, Rue du Bugnon 21, 1011 Lausanne, Switzerland
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Corresponding author: Francine Behar-Cohen Inserm U1138, Team 17, Centre de Recherche des Cordeliers 15 rue de l’Ecole de Médecine 75006 Paris, France Tel : +33-144278169 E-mail: [email protected]
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Funding sources: This work was supported by the Agence Nationale de la Recherche, France (ANR-15-CE180032 “ROCK SUR MER”), by the Swiss National Science Foundation (#320030_156401), by the Faculty of Biology and Medicine Research Commission Fund, University of Lausanne, Switzerland, by the Abraham J. & Phyllis Katz Foundation, Atlanta, GA, and by the Lowy Medical Research Institute, San Diego, CA.
ACCEPTED MANUSCRIPT Mechanisms of macular edema: beyond the surface
ABSTRACT Macular edema consists of intra- or subretinal fluid accumulation in the macular region. It
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occurs during the course of numerous retinal disorders and can cause severe impairment of central vision. Major causes of macular edema include diabetes, branch and central retinal vein occlusion, choroidal neovascularization, posterior uveitis, postoperative inflammation and central serous chorioretinopathy. The healthy retina is maintained in a relatively
SC
dehydrated, transparent state compatible with optimal light transmission by multiple active and passive systems. Fluid accumulation results from an imbalance between processes
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governing fluid entry and exit, and is driven by Starling equation when inner or outer bloodretinal barriers are disrupted. The multiple and intricate mechanisms involved in retinal hydro-ionic homeostasis, their molecular and cellular basis, and how their deregulation lead to retinal edema, are addressed in this review. Analyzing the distribution of junction proteins
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and water channels in the human macula, several hypotheses are raised to explain why edema forms specifically in the macular region. “Pure” clinical phenotypes of macular edema, that result presumably from a single causative mechanism, are detailed. Finally, diabetic
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macular edema is investigated, as a complex multifactorial pathogenic example. This comprehensive review on the current understanding of macular edema and its mechanisms
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opens perspectives to identify new preventive and therapeutic strategies for this sightthreatening condition.
1
ACCEPTED MANUSCRIPT KEYWORDS:
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Macula, edema, mechanisms, diabetes, retina, cysts
2
ACCEPTED MANUSCRIPT ARTICLE HIGHLIGHTS
•
Cells forming inner and outer blood-retinal barriers maintain retinal homeostasis
•
Macular edema results from an imbalance between fluid entry and drainage
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mechanisms
Intraretinal accumulation of macromolecules osmotically attracts water and solutes
•
The structural organization of the retina explains why edema develops in the macula
•
A glymphatic system may be formed by AQP4 expression along macular Müller cells
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EP
TE D
M AN U
SC
•
3
ACCEPTED MANUSCRIPT TABLE OF CONTENTS
1
Introduction .............................................................................................................. 8 1.1 Histology of the retina and macula: brief overview........................................................ 8 1.2 General introduction to macular edema...................................................................... 10 1.2.1 Limitations to study macular edema ............................................................................... 11 1.2.2 Macular edema: an underestimated public health issue ................................................ 11
What controls the retinal hydro-ionic and osmotic homeostasis?............................. 12
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2
2.1 Regulation of fluid and molecule entry: retinal barriers ............................................... 13 2.1.1 Inner blood-retinal barrier............................................................................................... 13
2.1.2
Endothelial cells and their intercellular junctions ............................................................... 13 Transendothelial transport .................................................................................................. 15 Pericytes .............................................................................................................................. 16 Glial cells .............................................................................................................................. 17
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2.1.1.1 2.1.1.2 2.1.1.3 2.1.1.4
Outer blood-retinal barrier .............................................................................................. 18
2.1.2.1 2.1.2.2
Junctional complex of the retinal pigment epithelium ........................................................ 18 Outer limiting membrane .................................................................................................... 19
3
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2.2 Regulation of fluid exit: drainage mechanisms ............................................................ 20 2.2.1 Retinal glial Müller cells ................................................................................................... 22 2.2.2 Retinal pigment epithelium ............................................................................................. 24
Why does retinal edema form?................................................................................ 25 3.1 Increased retinal fluid entry ........................................................................................ 25 3.1.1 Increased fluid entry through the inner blood-retinal barrier ........................................ 25
Increased fluid entry through outer retinal barriers ....................................................... 33
3.1.2.1 3.1.2.2 3.1.2.3
Retinal pigment epithelium and choroidal vessel junction alteration ................................. 33 Retinal pigment epithelial cell death ................................................................................... 37 Outer limiting membrane disruption................................................................................... 37
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3.1.2
Junctional complex alteration.............................................................................................. 26 Enhanced transcellular permeability ................................................................................... 28 Loss of endothelial cells ....................................................................................................... 29 Loss of pericytes .................................................................................................................. 29 Role of leucocytes in inner blood-retinal barrier breakdown .............................................. 31 Neovascularization and vessel abnormalization.................................................................. 32
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3.1.1.1 3.1.1.2 3.1.1.3 3.1.1.4 3.1.1.5 3.1.1.6
4
Why does edema specifically form in the macula? Several hypotheses..................... 43 4.1 4.2 4.3 4.4
5
Alteration of junction proteins in the “Z-shaped” Müller cells zone.............................. 43 The “glymphatic” system hypothesis .......................................................................... 44 Increased interstitial pressure: vascular consequences ................................................ 46 The formation of “cysts” ............................................................................................. 46
Why does macular edema alter vision? .................................................................... 47 5.1 5.2 5.3
6
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3.2 Decreased drainage functions ..................................................................................... 38 3.2.1 Glial cells and neurosensory retina ................................................................................. 38 3.2.2 Retinal pigment epithelium ............................................................................................. 39 3.3 Protein leakage .......................................................................................................... 41
Evaluation of visual function in macular edema........................................................... 48 Acute effects of macular edema on visual function...................................................... 49 Long-term effects of macular edema on visual function ............................................... 50
What can we learn from pure phenotypes? ............................................................. 52 6.1 6.2
Irvine-Gass postoperative macular edema .................................................................. 52 Idiopathic macular telangiectasia type 1 ..................................................................... 53
4
ACCEPTED MANUSCRIPT 6.3 6.4 6.5 6.6 6.7
7
Idiopathic macular telangiectasia type 2 ..................................................................... 53 Paclitaxel-induced maculopathy ................................................................................. 54 MEK-inhibitor-associated maculopathy ....................................................................... 55 Hypoproteinemia ....................................................................................................... 56 Paracentral acute middle maculopathy ....................................................................... 56
Major pathways identified as causative of macular edema ...................................... 57
Inflammatory molecules .................................................................................................. 63
7.6.2.1 7.6.2.2 7.6.2.3 7.6.2.4 7.6.2.5 7.6.2.6 7.6.2.7
General mechanisms contributing to diabetic ME .................................................... 68
9
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The Polyol pathway .................................................................................................... 69 The AGE pathway ....................................................................................................... 69 Protein kinase C (PKC) activation ................................................................................ 70 Local renin-angiotensin system (RAS) .......................................................................... 70 Inflammation and oxidative stress .............................................................................. 71 Blood-retinal barrier dysfunction ................................................................................ 71 Drainage functions ..................................................................................................... 72 Kinetic of events in the diabetic GK rat model ............................................................. 72
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8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8
Concluding remarks and future directions................................................................ 75
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8
Complement components ................................................................................................... 63 Tumor Necrosis Factor-α (TNF-α) ........................................................................................ 64 Interleukin-1β (IL-1β)........................................................................................................... 66 Interleukin-6 (IL-6) ............................................................................................................... 67 Interleukin-8 (IL-8) ............................................................................................................... 67 Intercellular Adhesion Molecule 1 (ICAM-1) ....................................................................... 68 Transforming growth factor-ß (TGF-β) ................................................................................ 68
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7.6.2
Microglia/macrophages ....................................................................................................... 61 Retinal Müller glial cells ....................................................................................................... 62 Astrocytes ............................................................................................................................ 62 Retinal pigment epithelial cells ............................................................................................ 62
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7.6.1.1 7.6.1.2 7.6.1.3 7.6.1.4
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7.1 VEGF/PGF pathway .................................................................................................... 57 7.2 Kinin/kallikrein system ............................................................................................... 58 7.3 Angiopoietin-2 (Ang2)/Tie2/α /α3β /α β1 pathway ................................................................. 59 7.4 Renin-angiotensin system and angiotensin-converting enzyme ................................... 59 7.5 The mineralocorticoid pathway .................................................................................. 59 7.6 Inflammation and inflammatory mediators with pro-edematous effects...................... 60 7.6.1 Resident immune-competent cells.................................................................................. 61
5
ACCEPTED MANUSCRIPT
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AGEs: Advanced glycation end-products AMD: age-related macular degeneration Ang2: Angiopoietin 2 AQP: aquaporin AT: angiotensin II receptor BDNF: Brain-derived neurotrophic factor BIGH3: Transforming growth factor-ß-induced protein Ig-h3 C: complement factor Cav-1: caveolin-1 CRALBP: cellular retinaldehyde-binding protein DAG: diacyglycerol ENAC: epithelial sodium channel ERK: extracellular signal-regulated kinase GFAP: glial fibrillary acid protein GS: glutamine synthetase GSK-3ß: Glycogen synthase kinase 3 ß HBMvEC: human brain microvascular endothelial cells hESC: human embryonic stem cell HIF: hypoxia inducible factor HUVEC: human umbilical vein endothelial cell line cell ICAM: Intercellular adhesion molecule IL: interleukin ILM: inner limiting membrane INL: inner nuclear layer iNOS: inducible nitric oxide synthase IPL: inner plexiform layer JAM: junctional adhesion molecule JNK: c-Jun N-terminal kinase Kir: K+-inward rectifying KKS: Kinin/kallikrein system LPS: lipopolysaccharide MAPK: mitogen-activated protein kinase MCP-1: Monocyte chemotactic protein 1 ME: macular edema MLC: myosin light-chain MMP: matrix metalloproteinase MR: mineralocorticoid receptor NF-kB: nuclear factor-kB NGF: nerve growth factor Nrf2: Nuclear factor erythroid 2-related factor 2 OCT: optical coherence tomography OLM: outer limiting membrane ONL: outer nuclear layer OPL: outer plexiform layer PAR2: protease activated receptor 2 PCR: polymerase chain reaction PDGF: platelet-derived growth factor PEDF: pigment epithelium-derived factor PGF: placental growth factor PI3K: phosphatidylinositol 3-kinase PIP3: phosphatidylinositol-3,4,5-trisphosphate PKC: protein kinase C RMG cells: retinal Müller glial cells
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ABBREVIATIONS
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ROCK-1: Rho-associated protein Kinase 1 RPE: retinal pigment epithelium SHP-1: Src homology-2 domain-containing phosphatase-1 STAT3: Signal transducer and activator of transcription 3 STZ: streptozotocin TGF: transforming growth factor Tie: tyrosine kinase with immunoglobulin and EGF homology domains TLR: Toll-like receptor TNF: tumor necrosis factor TNFR: TNF receptor TRAF: TNF receptor associated factors TRPV4: Transient receptor potential cation channel subfamily vanilloid member 4 VEGF-R: vascular endothelial growth factor receptor VEGF: vascular endothelial growth factor ZO-1: zonula occludens-1
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1 Introduction 1.1
Histology of the retina and macula: brief overview
The neural retina is located in the posterior segment of the eye, between the vitreous
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anteriorly and the sclera posteriorly, and lies on the retinal pigment epithelium (RPE) that is closely associated with the choroid (Figure 1). The neural retina is a multi-layer specialized tissue, composed from the innermost to the outermost layer by the inner limiting membrane (ILM) which is the basal membrane of the retinal Müller glial (RMG) cells, the nerve fiber
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layer, the ganglion cell layer, the inner plexiform layer (IPL) that contains synapses between
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ganglion cells and interneurons (bipolar, amacrine, and horizontal cells), the inner nuclear layer (INL), formed by nuclei of bipolar, amacrine, horizontal and RMG cells, the outer plexiform layer (OPL), composed by synaptic connections between visual neurons (or photoreceptors) and interneurons, the outer nuclear layer (ONL) formed by nuclei of cone and rod photoreceptors, and the inner and outer segments of the photoreceptors (Hogan et
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al., 1971). The outer limiting membrane (OLM) is formed by the cellular connections between RMG cells and photoreceptor inner segments. Photoreceptor outer segments are in close contact with RPE microvilli, but no molecular adhesion or junction system maintains the
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neural retina attached to the RPE. The RPE lies on the Bruch’s membrane, which outermost part forms the basal membrane of the choriocapillaris, the innermost vascular layer of the
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choroid.
The retina is vascularized by two independent vascular beds, the retinal and choroidal vasculatures. The larger retinal vessels, branches of the central retinal artery and vein, lay below the ILM, and are surrounded by astrocytes, pericytes and RMG cells. Between precapillary arterioles and post-capillary venules, the retinal capillary network is arranged in three layers. The superficial, intermediate and deep plexuses are located in the nerve fiber and ganglion cell layers, at the inner border of the IPL, and at the inner and outer border of the INL, respectively (Bonnin et al., 2015; Hwang et al., 2016; Park et al., 2016; Rizzolo et al., 2011) (Figure 2; see Supplementary Methods and Supplementary Table 1 for details of 8
ACCEPTED MANUSCRIPT immunostaining techniques employed for Figure 2 and all subsequent Figures). Around the optic nerve head, a radial capillary network lays within the nerve fiber layer, along the temporal superior and inferior retinal vessels (Henking and De Oliveira, 1967). Since the outer retinal layers are avascular, the choroidal vessels supply nutrients and oxygen to the
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high energy-demanding photoreceptors. Sympathetic regulation is present in the choroidal vasculature but not in retinal vessels. The major part of the choroidal space is occupied by vessels, organized in three vascular layers. The choriocapillaris, a thin interconnected
capillary network is the innermost layer, the medium- and small-sized vessels form the
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intermediate Sattler's layer, and large arteries derived from the short posterior ciliary arteries and large lumen veins form the outermost Haller's layer. The supra-choroid is the virtual
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space separating Haller's layer from the sclera. Numerous non-vascular cell types, such as melanocytes, macrophages/microglia and mast cells lay around choroidal vessels. Lymphatic-like vessels have been described in the human choroid, located in the innermost vascular layers external to the fenestrated vessels of the choriocapillaris. Yet, this important
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finding remains to be confirmed (Nickla and Wallman, 2010), since all classical lymphatic markers were not detected in the choroid (Schrödl et al., 2015). In the normal retina, no lymphatic vessel has been identified.
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Three types of glial cells are present in the neurosensory retina: astrocytes, located in the inner retina, at the vitreal interface and around vessels, microglial cells, which in physiologic
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conditions are ramified cells, located only in the inner retina and around retinal vessels, and RMG cells. RMG cells are the only cells spanning the entire thickness of the neurosensory retina, ensuring contact between all neuronal cell types, retinal vessels and the vitreous cavity (Figure 2). The macula is a highly specialized region of the retina responsible for fine visual acuity. It is located temporal to the optic nerve head and can be recognized on fundus visualization by its yellow color, due to the accumulation of the xanthophyll pigments lutein and zeaxanthin (Figure 3A and B). The average retinal thickness is 210 microns in adult eyes but the thickness varies in different regions. From 100 µm in the extreme periphery, retinal thickness increases to 450µm at the border of the macula and then decreases to 130µm at
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ACCEPTED MANUSCRIPT the center of the foveal depression, where inner retinal layers are displaced laterally. The center of the fovea is indeed exclusively composed of cone photoreceptors and specific foveal RMG cells. In the fovea, cones are densely packed, thinner, and have elongated outer segments, as compared to cones of the peripheral retina. The center of the macula is
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generally avascular, surrounded by circularly arranged capillaries delimitating the foveal avascular zone, a central 400-500-µm-diameter area. During development, the formation of
(Provis et al., 2013).
General introduction to macular edema
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the normal foveal morphology is closely related to the presence of a foveal avascular zone
Macular edema (ME) is defined as an abnormal increase of fluid volume in the macula. Extracellular fluid can infiltrate retinal layers, accumulate in cavities commonly referred to as “cysts” (without certitude that a “membrane” delimitates these cavities), or collect in the subretinal space, where it is referred to as subretinal fluid (Figure 3C and D). An increase of
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intracellular fluid volume (cell swelling) may also occur (Kohno et al., 1983; Yanoff et al., 1984), often associated with extracellular fluid. In physiologic conditions, fluid entry and exit are tightly regulated to maintain a balanced hydration state compatible with retinal
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homeostasis, necessary for tissue transparency and light transmission. ME results from an
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imbalance between fluid entry and exit, two multifactorial processes frequently deregulated in retinal diseases. Conditions in which one mechanism predominates are of particular interest when trying to discriminate the respective roles of fluid entry versus fluid exit in the formation of ME. Changes in retinal hydration state interfere with photon transmission and disturb vision. Long-standing ME may lead to permanent retinal structural damages. Visual consequences of macular edema mostly depend on structural alterations induced by intraretinal or subretinal fluid accumulation, with a worse prognosis linked to alterations of outer retinal structures, such as OLM or photoreceptor segments (Otani et al., 2010;
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1.2.1
Limitations to study macular edema
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Retinal imaging methods developed in the last two decades, and particularly optical coherence tomography (OCT) (Puliafito et al., 1995), have brought major advances in the study and understanding of ME, allowing in vivo longitudinal, functional and microstructural analysis of the human macula (Figure 3B). Indeed, no laboratory animal model can fully
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recapitulate the macular edema phenotype since rodents, lagomorphs and other species commonly used for pathophysiology studies do not have a macula. Non-human primates
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have a macula (Figure 3C) but can rarely be employed for research purposes, given the increasing ethical, regulatory and financial constraints. In this context, human ocular specimens are particularly valuable, but the availability of post-enucleation or post-mortem eyes is extremely limited, and fixation/dehydration procedures necessary for tissue
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preparation may interfere with ME pathology. To circumvent these limitations, numerous groups have studied the biological composition of aqueous, and less frequently vitreous humor, to identify molecular pathways associated with ME (Hillier et al., 2017; Jung et al.,
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2014; McAuley et al., 2014; Owen and Hartnett, 2013; Wen et al., 2015; Zhang et al., 2014). This strategy proved effective in the past, being the basis of anti-vascular endothelial growth
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factor (VEGF) development for retinal diseases (Aiello et al., 1994; Malecaze et al., 1994). However, since the advent of these revolutionary agents more than a decade ago, no novel optimal therapeutic target has emerged from this type of analysis.
1.2.2
Macular edema: an underestimated public health issue
ME is a major cause of visual impairment in the course of metabolic, vascular and inflammatory retinal diseases. It affects worldwide around 7 million subjects due to diabetes (Figure 4) (Yau et al., 2012) and 3 million subjects due to vein occlusions (Figure 5) (Rogers et al., 2010). It accounts for 40% of visual impairment in patients with uveitis (Figure 6E and 11
ACCEPTED MANUSCRIPT F) (Rothova et al., 1996). In industrialized countries, 5% of individuals older than 60 years of age have ME due to neovascular age-related macular degeneration (AMD) (Figure 6A and B) (Pennington and DeAngelis, 2016). Besides being a major cause of visual impairment of retinal origin, ME is also among the most accessible to treatment. The currently approved
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intraocular anti-angiogenic and glucocorticoid agents indicated for several common retinal conditions (diabetic ME, central and branch retinal vein occlusion, and neovascular AMD), have been approved for their effect on ME reduction and subsequent visual acuity gain.
Taking into account the major impediment of ME on quality of life and working ability, these
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treatments are cost-effective, but still represent an important burden on health care systems (Hodgson et al., 2016; Ross et al., 2016). A better understanding of ME, an extremely
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frequent and vision-threatening manifestation of many retinal disorders, is thus crucial to elaborate new concepts and subsequent innovative therapeutic strategies. Indeed, beyond a common single phenotype, macular edema results from multiple, complex and not fully understood mechanisms.
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The aim of this review is to look at ME ‘beyond the surface’, recapitulating in depth the current knowledge on the intricate molecular and cellular mechanisms involved in retinal hydro-ionic homeostasis, and how their deregulation leads to ME. This review includes a
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specific emphasis on diabetic ME, and finally opens future research directions.
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2 What controls the retinal hydro-ionic and osmotic homeostasis?
In physiologic conditions, different mechanisms maintain the retina in a transparent and relatively dehydrated state. Barriers limit fluid entry and active drainage systems allow fluid exit while creating extracellular molecular gradients important for hydration homeostasis. The main glial drainage mechanisms controlling retinal hydro-ionic homeostasis are summarized in the Video 1.
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2.1
Regulation of fluid and molecule entry: retinal barriers
Fluid and molecules of variable size can differentially enter the neurosensory retina from the vitreous, from retinal vessels, or from the choroid through the RPE, via the subretinal space.
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This process is tightly controlled by structures forming the inner and the outer blood-retinal barriers and by the osmotic gradients across these barriers (Bradbury and Lightman, 1990;
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Törnquist et al., 1990).
Inner blood-retinal barrier
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The inner blood-retinal barrier plays a major role in controlling fluid entry into the retina. It is ensured by the tight-junctions between endothelial cells of retinal vessels, dynamically regulated by a neuro-glio-vascular cross-talk involving astrocytes and RMG cells (Fruttiger, 2007; Klaassen et al., 2013; Sorrentino et al., 2016), and interactions with pericytes and smooth muscle cells (Figure 2).
Endothelial cells and their intercellular junctions
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2.1.1.1
Endothelial cells of retinal capillaries are connected by molecular complexes, consisting of tight junctions (zonula occludens), adherens junctions (zonula adherens) and gap junctions
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(Figure 2 B and C). Besides their role in cell-to-cell adhesion, these complexes regulate the contact inhibition for endothelial cell division, cell survival, polarity and paracellular
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permeability (Klaassen et al., 2013). Endothelial cells of the retina and brain have the highest number of tight-junction strands, a lack of fenestrations, low pinocytic activity and the presence of highly complex continuous tight junctions, which establishes a physical barrier to water and hydrosoluble molecules. The exact transcellular resistance of the retinal vascular endothelium is not known but is thought to be similar to the one measured in the brain vascular endothelium, reaching 1000-1500 Ohm.cm2, much higher than the outer retinal barrier resistance (Crone and Olesen, 1982). The transcellular resistance reflects the flux of small ions but does not measure the paracellular size selectivity or the vesicular transcytosis
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ACCEPTED MANUSCRIPT of larger molecules. The smallest intercellular gaps form a selective barrier to the paracellular passage of molecules with radius higher than 3 nm, with differential size selectivity between different molecular components of the tight junctions. For more details, readers should refer to a recent extensive review on the inner blood-retinal barrier (Klaassen et al., 2013). We will
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here summarize the major molecular players involved, focusing on the newest findings.
Endothelial tight junctions are complex structures formed by multiple transmembrane,
scaffolding and signaling proteins. The most important transmembrane proteins are occludin,
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claudin-1, -2 and -5 (Russ et al., 1998; Tian et al., 2014) and junction adhesion molecules (JAMs) A and C (Economopoulou et al., 2009; Orlova et al., 2006; Saker et al., 2014) (Figure
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2 B). The cytoplasmic tails of occludin and claudins are linked to the actin cytoskeleton by scaffolding proteins. For instance, zona occludens-1 (ZO-1) that belongs to the PDZ domain proteins (Campbell and Humphries, 2012; Hosoya and Tachikawa, 2012), is responsible for anchoring transmembrane and receptor proteins to cytoskeletal components. Other molecular components of endothelial tight junctions may also be present in retinal capillaries,
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such as vascular endothelial cell-specific adhesion molecule (VCAM), poliovirus receptorrelated-1 (Nectin) and tight junction protein-2 (zona occludens-2, ZO-2) (Klaassen et al., 2013). Recent observations have highlighted that polarity proteins are present in endothelial
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cells, as in epithelia, and that they play major roles in vessel formation and maintenance. These proteins include pals1/PATJ and PAR3, PAR6, and crumbs-3 (CRB3) interacting with
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VE-cadherin (Brinkmann et al., 2016). Atypical protein kinase C (PKC) and particularly PKCζ, that phosphorylates junction proteins and is part of the PAR3 complex, is crucial for the formation and maintenance of the blood-brain barrier (Sewduth et al., 2017). In the human retina, this complex has been recognized as a regulator of junction proteins in endothelial cells (Song et al., 2014) and as a major actor in VEGF-receptor-mediated endocytosis, determining the active growth and maturation of vessels submitted to VEGF (Nakayama et al., 2013). In developing retinal vessels, the formation of tight junctions in between endothelial cells depends on their cellular interactions with pericytes and astrocytes (Kim et
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The adherens junctions are constituted by VE-Cadherin (Navaratna et al., 2007), ß-
Catenin (Russ et al., 1998) and N-Cadherin/Angiomotin-like protein 1 (AmotL1t) (Figure 2B). The gap junctions consist of a hemi-channel (or connexon) formed by six connexins
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(cx). Gap junctions mediate electrical and chemical communication between cells and allow the free passage of small molecules (<1 kDa). Amongst the various connexins, cx7, cx40
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and cx43 are widely expressed in the retina, and particularly in retinal endothelial cells (Danesh-Meyer et al., 2016). Notably, cx30.2 is involved in the modulation of vascular
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permeability (Manasson et al., 2013).
The different types of endothelial junctions are functionally and structurally linked. For example, the presence of adherens junctions stimulates the formation of tight junctions, as exemplified by the observation that VE-cadherin at adherens junctions induces claudin-5
2.1.1.2
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expression (Taddei et al., 2008).
Transendothelial transport
Transport across the endothelium is highly regulated by active membrane transporters and
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by vesicular transport. Caveolae-mediated transcytosis is defined by the migration of plasma membrane vesicles from one side of the cell to the other, and/or the formation of a pore
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resulting from vesicular fusion. Caveolin-1 (Cav-1), the major protein component of caveolae is expressed in the developing and mature retinal vessels and in the choroidal vasculature (Gu et al., 2014a). Transcytosis allows plasma macromolecules, such as albumin, transferrin, insulin, lipoproteins, and possibly immunoglobulins, to penetrate from the circulation into tissues. It is reduced in the retina, as compared to other organs (Anderson, 2008; Predescu et al., 2004; Red-Horse and Ferrara, 2007). In contrast, this mechanism seems important in driving macromolecules out of the retina into the circulation. Indeed, vesicular transport of albumin from the retina towards the circulation is higher than the reverse, as reflected by the higher vesicle density at the abluminal side of retinal vessels (Hofman et al., 2000, 2001). 15
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junction protein claudin-5, and reduced nitric oxide (NO)-dependent vasodilation, demonstrating that caveola-mediated transport may have broader regulating effects (Gu et al., 2014a). In the developing retina, the inner blood-retinal barrier properties are
progressively acquired concomitantly to the reduction of endothelial transcytosis (Chow and
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Gu, 2017).
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2.1.1.3 Pericytes
Pericytes are specialized mural cells located at the abluminal surface of retinal veins and capillaries. They share their basement membrane with endothelial cells, and are covered by an external basal membrane (Figure 2 B and C). Pericytes contribute to the regulation of the inner blood-retinal barrier, the microvascular blood flow through their contractile properties,
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and to angiogenesis. There is recent evidence that brain, retinal and choroidal pericytes derive, at least in part, from neural crests (Trost et al., 2016). Myeloid-derived cells could also contribute to pericyte coverage, particularly during retinal injury and repair. In the retina,
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pericyte density with respect to endothelial cells reaches a 1:1 ratio and pericyte coverage of human retinal capillaries is as high as 94%, as compared to 11% in the choriocapillaris
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(Chan-Ling et al., 2011). In the rat retina, the ratio of pericyte plasma membrane length in contact with the vascular circumference, to the outer circumference of the endothelial cell tube is around 40%, as compared to 20-30% in various brain regions (Frank et al., 1987). Adherens junctions connect the cytoskeleton of pericytes to endothelial cells, partly through cadherins, allowing molecular mechanical signaling between pericytes and endothelial cells. Angiomotin like-1 (AmotL1), scaffold protein integrating polarity, junctional, and cytoskeletal cues that modulate cellular shape, was recently identified as an essential effector of the Ncadherin mediated endothelial/pericyte junctional complex (Zheng et al., 2016). The pericyte-
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ACCEPTED MANUSCRIPT derived lipidic mediator, sphingosine-1-phosphate, up-regulates N-cadherin and VE-cadherin expression and down-regulates angiopoietin-2 (Ang-2), modulating the inner blood-retinal barrier in physiologic and pathologic conditions (McGuire et al., 2011). Gap junctions, particularly cx43 also contributes to the molecular communication between pericytes and
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endothelial cells (Danesh-Meyer et al., 2016; Li et al., 2003). The crucial role of pericytes in blood-brain barrier formation and maintenance has been demonstrated using mouse
mutants. While increased expression of ZO-1 correlated with enhanced pericyte coverage (Kim et al., 2009), impaired platelet-derived growth factor (PDGF)-B/PDGF-Receptor-b
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signaling, reduced pericyte coverage, together with endothelial hyperplasia, vascular
abnormalization and microaneurysm formation (Lindahl et al., 1997). VEGF over-expression
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by the hyperplasic endothelium was associated with enhanced trans-endothelial permeability (Hellström et al., 2001). Increased vascular permeability in pericyte-depleted vessels results from indirect over-expression of vasoactive molecules intervening in both tight junctions and vesicle trafficking (Daneman et al., 2010). Glial cells
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2.1.1.4
The glial cells of the retina include macroglia, composed of RMG cells and astrocytes, and microglia (Figure 2A, C and D). Their processes wrap around retinal capillaries, forming a
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glia limitans. Retinal arterioles, venules, and capillaries are closely ensheathed by macroglia. The superficial retinal vasculature is ensheathed by both astrocytes and RMG cells (Figure
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2B and C), whilst the deep vascular plexus is ensheathed solely by RMG cells (Figure 2 A and E) (Schnitzer, 1988). This differential distribution of glial cells around the superficial and deep plexuses, may have important functional consequences for the pathophysiology of ME. Macroglial cells are key players in the dynamic neuro-glio-vascular unit and regulate a wide range of endothelial cell functions. Cultured retinal glial cells stimulate pericyte growth in vitro (Ikuno et al., 2002). The cell-cell contact of astrocytes and endothelial cells promotes the differentiation of endothelial cells, allowing the maturation of the inner blood-retinal barrier (Jiang et al., 1995; Yao et al., 2014). Astrocytes and RMG cells both have the ability to
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inner blood-retinal barrier mostly through production of soluble factors, but also through vesicular communication (Checchin et al., 2006; Vecino et al., 2016) and cx43 gap-junctions (Danesh-Meyer et al., 2016). Outer blood-retinal barrier
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2.1.2
The outer blood-retinal barrier is classically defined as the intercellular junction complex of
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the RPE, separating the neurosensory retina from the choroidal circulation. The OLM also contributes to the outer blood-retinal barrier function (Omri et al., 2010), and to its disruption in pathological conditions.
2.1.2.1
Junctional complex of the retinal pigment epithelium
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The junctional complex of the RPE is formed, as in other highly polarized epithelia, by tight, adherens and gap junctions (Figure 7). The transepithelial resistance is much lower than the resistance of the inter-endothelial junctions at the inner retinal barrier (Rizzolo, 1997, 2007;
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Rizzolo et al., 2011; S. Peng et al., 2011). However, the estimated transepithelial resistance of human adult RPE (around 80 Ohm.cm2) efficiently prevents water and protein entry from
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the choroid to the subretinal space, and allows water exit towards the choroid, following an osmotic gradient (Radius and Anderson, 1980; Stern et al., 1980; Kirchhof and Ryan, 1993). The RPE junction complex also participates to the regulation of RPE cell shape, polarity and proliferation control through the interaction with specific cytoskeletal, adapter and effector proteins. This complex has been detailed in a comprehensive review (Rizzolo et al., 2011). The tight junctions are mostly formed by occludins (Phillips et al., 2008), claudins (S. Peng et al., 2011), principally claudin-19 and 3 (Peng et al., 2016; Rizzolo et al., 2011), ZO-1 (Konari et al., 1995) and the JAM proteins JAM-A (Mandell et al., 2007) and JAM-C. JAM-C regulates
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2008; McKay et al., 1997). In chicken RPE, the adherens junctions can form a complex with α-actin and vinculin (Sandig and Kalnins, 1988). Regarding gap junctions, the presence of connexin-43 has been reported on rat RPE (Tibber et al., 2007).
Junction proteins and actin cytoskeleton distribution and functions are regulated by kinases
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of Rho/Rac and atypical PKC families. While diurnal outer segment phagocytosis requires Rac1 activation (Mao and Finnemann, 2012), pathological remodeling of RPE involves
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mostly ROCK-1 activation, leading to subsequent junction destabilization (Lee et al., 2008). The non-classical, calcium-independent, atypical protein kinase PKCζ, associated with the PAR3/PAR6 complex within occludin loops, phosphorylates occludin, thereby promoting the assembly of tight junctions (Figure 7 A and B) (Omri et al., 2013). The polarized organization
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of RPE cells creates differential compartments. RPE apical microvilli are in contact with the immune privileged subretinal milieu, while RPE basal and lateral cell membranes are in contact with a protein-enriched fluid emanating from the choroid. This choroid-derived media
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provides critical compounds for retinal metabolism, including the retinol-binding protein
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needed for retinol uptake and essential to the visual pigment cycle.
2.1.2.2
Outer limiting membrane
The OLM is formed by heterotypic tight-like and adherens junctions, located at the interface between RMG cells and photoreceptor inner segments (Figure 7A and C-E). The tight-like junctions are mostly composed of occludin, JAM-B, JAM-C, and ZO-1 (Daniele et al., 2007; Omri et al., 2010), and notably lack claudins, an essential tight junction protein (Alves et al., 2014). Adherens junctions of the OLM are composed of cadherin/catenin, interacting with adaptor proteins such as Crumbs homologue 1 (CRB1) (Gosens et al., 2008) and ZO-1
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ACCEPTED MANUSCRIPT (Pearson et al., 2010). The exact role of the OLM in the regulation of fluid movements across the macula is not fully understood. Earlier studies have demonstrated that the OLM is a barrier to protein diffusion. Biotinylated protein probes of known Stokes radius were used to determine the pore size of the OLM on ex vivo rabbit retina (Bunt-Milam et al., 1985). The
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Stokes radius is the effective theoretical radius of a given molecule in a hydrated state that influences its movement in solution. This experiment estimated the pore radius of the OLM junction between 30 and 36 Å, much smaller than albumin and globulins. The OLM thus serves as an important barrier to free protein diffusion across the retina from the inner retinal
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layers to the subretinal space, and vice-versa. This observation should be considered when
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therapeutic proteins are injected into the vitreous to target sub-RPE pathological processes.
Regulation of fluid exit: drainage mechanisms
Active and passive mechanisms continuously drain water, ions and other molecules from the retina to the vitreous, retinal vessels, and the choroid through the subretinal space. Indeed,
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the tight regulation of pH, and concentration gradients of ions and proteins across retinal and choroidal layers, and the clearance of metabolic products are essential for retinal homeostasis and function. Different cell types are thus equipped with numerous transporters
critical.
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(which have only been partly identified), which appropriate expression and distribution is
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Before analyzing the cellular functions involved in transports, the tissues resistance to fluid and protein diffusion should be recalled. The neurosensory retina opposes a weak resistance to the passage of water from the subretinal space to the vitreous and vice versa (Marmor, 1985; Marmor et al., 1985; Orr et al., 1986), but is not permeable to compounds of high molecular weight. Part of the aqueous humor secreted by the ciliary body hydrates the vitreous cavity, and is pumped by the RPE through the retina, to be drained into the choroid vessels. There are several obstacles to the diffusion of molecules in the retina. High molecular weight molecules (>150kD) do not diffuse though the ILM, and the plexiform layers
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ACCEPTED MANUSCRIPT are the layers of lowest hydric conductivity, constituting barriers to the diffusion of macromolecules. But the main resistance to their passage through the retina is due to the OLM barrier (see 2.1.2.2). Indeed, after subretinal injection, FITC-dextran 10S (Stokes radius: 23 Å) diffuses rapidly into
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the vitreous, but 70S- and 150S- dextrans (Stokes radius: 60 and 85 Å, respectively) mostly remain in the subretinal space due to the OLM barrier (Marmor et al., 1985). The kinetics of water and macromolecules injected under the macula has never been studied and could show significant differences with kinetics observed in animal models lacking a macula
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(Marmor et al., 1985), related to the specific cellular architecture and physiology of the
macula. Movement of molecules between photoreceptors might be influenced, depending on
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their physico-chemical properties, by interactions with glycosaminoglycans and glycoproteins in the interphotoreceptor matrix. The Bruch’s membrane does not oppose a high resistance to the passage of macromolecules between the choriocapillaris and the RPE. The greatest resistance to fluid movement across Bruch’s membrane occurs at its inner collagenous layer,
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and could increase with ageing (Starita et al., 1997).
The protein concentration is higher in the choroid, compared to the retina and vitreous, and the retina is less permeable to water than the RPE (Kirchhof and Ryan, 1993). Therefore, the
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osmotic movement of water from the vitreous to the choroid, partly depending on the maintenance of cellular barriers, contributes to maintain the neurosensory retina permanently
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attached to the RPE and “dehydrated”. The exact mechanisms regulating the protein gradient from the retina to the choroid is not fully understood. Recent work has demonstrated that the majority of albumin is not transported across choriocapillaris endothelial cell fenestrations but through transcytosis via caveolae (Nakanishi et al., 2016). In aged mice, reduced caveolae transport and leaky tight-junctions between endothelial cells may alter the retina/choroid protein gradients (Nakanishi et al., 2016).
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Retinal glial Müller cells
As described in 1.1 and extensively reviewed (Bringmann et al., 2006; Reichenbach and Bringmann, 2013), RMG cells are specialized macroglial cells unique to the retina that span its entire thickness from the inner limiting membrane (ILM) to the photoreceptor inner
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segments (Figure 1 and 2). Nuclei of RMG cells are located at the level of the inner nuclear layer. RMG extend their endfeet frontally on the ILM which form their basal membrane, and send perivascular extension, where their basal membrane is fused with the basal membrane of the capillary endothelium (Hogan et al., 1971). RMG cells are the sole mediator of the
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macroglia-vascular interaction at the deep capillary plexus, where astrocytes are absent (Schnitzer, 1988) (Figure 2). RMG cells are responsible for a range of critical functions. They
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are structural backbones for the retinal architecture, act as optic fibers for photon guidance toward photoreceptor outer segments (Franze et al., 2007), regulate the vascular, neuronal and extracellular milieu homeostasis (ions, water, neurotransmitter molecules, and pH), and contribute to metabolic and immune regulation (Bringmann et al., 2006). Numerous ion and
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aqueous channels are expressed in RMG cells, varying among species (Pannicke et al., 2016). The membrane conductance of RMG cells is mostly regulated by inward and outward K+ currents. Different species-specific inward-rectifier potassium ion (Kir) channels have
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specific spatial distribution within RMG cells, allowing the buffering of extracellular K+ released by the photoreceptor and neuronal activity. In the rodent retina, Kir2.1 is
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predominantly located in the membrane domains of RMG cells in contact with retinal neurons, while Kir4.1protein is located at their endfeet and surrounds retinal blood vessels (Kofuji et al., 2002) (Video 1). Aquaporins (AQP) are a major class of small membrane proteins that facilitate water transport across plasma membranes in response to osmotic gradients, contributing to intra- and extracellular hydro-ionic regulation. The human retina expresses all AQPs transcripts (AQP0 to AQP12) but AQP2, AQP8 and AQP11 could not be identified at the protein level (Schey et al., 2014; Verkman et al., 2008). Perivascular AQP4 and Kir4.1 have been shown to act as a macromolecular complex with α-syntrophin and the short dystrophin isoform Dp71 (Claudepierre et al., 2000; Sene et al., 2009; Verkman et al.,
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ACCEPTED MANUSCRIPT 2008). But whether a functional interaction exists between AQP4 and Kir4.1, that would account for hydro-ionic clearance of neuronal activity metabolic waste remains controversial. Recently, transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4), a non-selective calcium cationic channel activated by osmotic, mechanical and chemical
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stimuli has been identified as another important actor in the coupling with AQP4. Indeed, in hypo-osmotic conditions, water influx through AQP4 would drive a calcium influx via TRPV4 in the glial endfoot, regulating the expression of AQP4 and Kir4.1 genes, and subsequently controlling the cell volume increase (Jo et al., 2015). RMG swelling that participates to
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retinal edema and degeneration is inhibited by endothelins (Vogler et al., 2013), brain-
derived neurotrophic factor (BDNF) (Berk et al., 2015), nerve growth factor (NGF) (Garcia et
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al., 2014), osteopontin (Wahl et al., 2013), VEGF and glutamate (Brückner et al., 2012). Many other ion channels (K+, Na+, Ca2+) are expressed along the RMG cell membrane in a polarized and regional arrangement, that varies amongst species (Pannicke et al., 2016), limiting the clinical application of certain animal model observations.
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Whether RMG cells can play a role in the transport of proteins across the normal retina has not been specifically investigated. Several studies have analyzed the fate of intravitreally injected molecules. Interestingly, bevacizumab is a 150-kD protein with a hydrodynamic
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diameter of approximately 15 Å, according to the Stokes-Einstein equation, and therefore is not expected to cross the OLM (see 2.1.2.2). But following intravitreal administration it was
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detected in the subretinal space (Dib et al., 2008), and was identified within RMG cells using immunohistochemistry (Shahar et al., 2006). Protein transport through RMG cells can thus not be excluded since these glial cells do have phagocytic activities (Stolzenburg et al., 1992) and the capacity to transport peptidic particles from the vitreous to the subretinal space (Normand et al., 2005). Moreover, RMG cells also express caveolin-1, involved in the vesicular transport of proteins (Gu et al., 2014b). In the case of blood retinal barrier disruption, RMG cells in situ can phagocytize blood-derived proteins like IgG (Iandiev et al., 2008).
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ACCEPTED MANUSCRIPT 2.2.2
Retinal pigment epithelium
The structure and function of the RPE, like other epithelia, depend on its apico-basal polarization, which occurs through linking asymmetrically intercellular junctions to the cytoskeleton of cells (Gibson and Perrimon, 2003). Differential polarized insertion of
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channels, transporters, and related proteins into apical and baso-lateral membranes is essential for the regulation of RPE cell volume, pH and trans-epithelial transports. Alteration of the RPE cell junctional complex results in the dysfunction of polarized ion and water
channels, favoring the accumulation of fluid in the subretinal space. Reciprocally, inhibition of
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the apical Na+/K+-ATPase alters the tight junction structure and increases the permeability in cultured human RPE cells (Rajasekaran et al., 2003). The outward molecular movement
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across the RPE is largely dependent on active ionic transport (~70%), while a smaller fraction (~30%) may be the result of the higher oncotic pressure in the choroid than in the retina (Negi and Marmor, 1986a, 1986b), a mechanism discussed in 4.2.1. The RPE actively removes water from the subretinal space at a rate ranging from 1.4 to 11
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µl/cm2/h (Tsuboi and Pederson, 1988), that is driven by an active trans-epithelial Cl- gradient. The apical Na+/K+-ATPase actively enhances intracellular K+, recycled by Kir7.1, inducing a Na+ gradient and an increased basolateral chlore conductance. The increase of intracellular
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Cl- concentration is associated with an acidification of the cells, which is regulated by different Cl- channels located at the basolateral membrane (e.g. bestrophin, which is volume-
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and Ca2+-dependent). These Cl- channels facilitate the efflux of Cl- ions from the cells, osmotically driving also water through aquaporin channels. Thus, there is an active water flux from the subretinal space towards the choroid through the RPE (Reichhart and Strauss, 2014). The involvement of the HCO3-/Cl- exchanger at the basolateral membrane links the water transport to the pH regulation and to the transport of lactate, resulting from the intense metabolic activity of photoreceptors. Trans-epithelial water efflux is thus linked to chloride conductance and lactate transport. These mechanisms of ions transport in RPE cells have been recently reviewed, and will not be further detailed here (Reichhart and Strauss, 2014). Acidification of the apical RPE membrane by anhydrase carbonic inhibition (e.g. using
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ACCEPTED MANUSCRIPT acetazolamide) increases water transport through RPE cells (Marmor and Maack, 1982; Yamamoto and Steinberg, 1992). The presence of functional AQP1 in the RPE has been subject to controversies (Verkman et al., 2008). In rabbit eyes with experimental subretinal blebs created in vivo, cGMP enhanced
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the water transport towards the RPE (Marmor and Negi, 1986), an effect consistent with AQP1-mediated net fluid flux increase, that was confirmed in human RPE cultures (Baetz et al., 2012). Functional AQP1 and AQP11 were recently described in human pluripotent stemcell-derived retinal pigmented epithelial cells (Juuti-Uusitalo et al., 2013). In addition to AQP1
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there are other subtypes of AQP in the RPE, as detailed in 3.2.2.
The RPE plays a major role in the fluid homeostasis of the outer retina and the choroid. As a
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highly polarized epithelium, any cytoskeletal stress could not only alter the junctions but also the proper distribution of membrane transporters and result in sub-retinal fluid accumulation.
3 Why does retinal edema form?
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Retinal edema is a consequence of an imbalance between fluid entry, fluid exit and retinal hydraulic conductivity. In most retinal diseases, macular edema is multifactorial and results from multiple, intricate mechanisms, but in certain specific conditions, one single of these
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mechanisms predominates, allowing a better analysis of each component. Figure 8
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summarizes these different intricate mechanisms.
3.1
3.1.1
Increased retinal fluid entry Increased fluid entry through the inner blood-retinal barrier
Main mechanisms leading to an increase of the inner blood-retinal barrier permeability are: •
Alteration of the intercellular junction proteins through down-regulation, change in phosphorylation state, or loss of membrane anchoring components (Antonetti et al., 1999; Klaassen et al., 2013).
•
Increased trans-endothelial transport.
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ACCEPTED MANUSCRIPT •
Loss of cells constituting the barrier (endothelial cells, pericytes, macroglial cells).
Vascular barrier properties can be altered within mature retinal vessels, but also due to the proliferation of immature neovessels.
Junctional complex alteration
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3.1.1.1
Loss of the functional and/or structural integrity of tight-junctions between endothelial cells increases vascular permeability to water, solutes and proteins (Radius and Anderson, 1980). Various experimental conditions (in cell cultures or experimental animals) mimicking
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pathological environments favoring ME, such as diabetes or inflammation, can alter junction
•
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proteins.
High glucose concentrations induce the down-regulation of claudin-5, occludin, JAM-
A, and ZO-1 in human retinal endothelial cells (Saker et al., 2014; Stewart et al., 2016; Tien et al., 2013) and reduces VE-cadherin phosphorylation via an increase in Ang-2 (Rangasamy et al., 2011). A proteolytic degradation of VE-cadherin is also observed in the retina of STZ-
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induced diabetic rats (Navaratna et al., 2007). Endoplasmic reticulum (ER) stress also causes down-regulation of claudin-5 (Adachi et al., 2011). •
Cytokines acts on tight-junctions through direct and indirect regulations. Tumor
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necrosis factor α (TNF-α) decreases the expression of ZO-1 and claudin-5, and alters their
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subcellular distribution in bovine retinal endothelial cells through PKCζ-mediated NF-κB activation (Aveleira et al., 2010). Transforming growth factor-ß (TGF-ß), secreted by glial cells, stimulates the production of matrix metalloproteinase (MMP)-9 (Behzadian et al., 2001). In turn, MMP-9 degrades occludin, as shown in the retinas of diabetic animals, in which both MMP-2 and MMP-9 expression is induced (Giebel et al., 2005). In patients with uveitis-related ME, angiography with fluorescein-conjugated dextrans showed that 4-kD, 20kD but not 150-kD molecules can cross the ruptured inner blood retinal barrier at the macula, whereas no passage was observed in healthy controls (Atkinson et al., 1991). This demonstrates that in inflammatory conditions the inner retinal vessel permeability is
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ACCEPTED MANUSCRIPT selectively increased. •
Activation of the VEGF/placental growth factor (PGF) pathways, secondary to hypoxia
and hyperglycemia, induce retinal vascular permeability by direct effects on endothelial tight and adherens junctions. PGF favors the degradation of ZO-1 and VE-cadherin (Huang et al.,
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2015). VEGF-induced permeability results from multiple convergent effects: phosphorylation, ubiquitination and internalization of occludin by activation of PKC-β (Antonetti et al., 1999; Harhaj et al., 2006; X. Liu et al., 2016; Murakami et al., 2012) and Src-family kinases (Scheppke et al., 2008) and down-regulation of occludin through ß-catenin pathway
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regulation (Behzadian et al., 2003). VEGF induces the ß-arrestin-2-dependent endocytosis of VE-cadherin and its removal from adherens junctions (Gavard and Gutkind, 2006) and
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increases transglutaminase-2 activity, which produces stress fiber formation through Rhokinase activation and VE-cadherin disruption (Lee et al., 2016). •
The VEGF/pigment epithelium-derived factor (PEDF) balance plays also a major role
in retinal vascular permeability. PEDF produced by RMG cells (Hauck et al., 2014) inhibits
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VEGF-induced permeability through several mechanisms, including: γ-secretase activity (Ablonczy et al., 2009); regulation of VEGF receptors with VE-cadherin and β-catenin, and their subsequent phosphorylation (Cai et al., 2011); blocking of the VEGF-induced ERK/P38
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and GSK3-ß transduction pathways without direct effect on VEGF receptor-2 (VEGFR-2) phosphorylation, inhibiting the activation of β-catenin (Yang et al., 2010). Moreover, PEDF
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inhibits the permeability induced by advanced glycation end-products (AGEs) via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the up-regulation of ZO-1 (Sheikpranbabu et al., 2010). •
Endothelial cell permeability is also influenced by glial cells. The barrier function of
endothelial cells co-cultured with RMG cells is enhanced in normoxic conditions, but is impaired under hypoxia (Tretiach et al., 2005). In the streptozotocin (STZ)-diabetic rat model, glial fibrillary acidic protein (GFAP) expression is altered in retinal glial cells, together with a reduction and redistribution of occludin in endothelial cells (Barber et al., 2000). The role of glial cells in the maintenance of vascular barrier integrity was further demonstrated by 27
ACCEPTED MANUSCRIPT selective glial cell depletion models. Intravitreal injection of a glial toxin, DL- α-aminoadipic acid (DL-α-AAA), led to increased expression of VEGF and a reduced expression of claudin5 (Shen et al., 2010). The specific ablation of RMG cells using a Müller-specific variant of AAV, that delivered a photo-inducible toxic protein, induced retinal remodeling, vascular
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abnormalization and leakage (Byrne et al., 2013). Microglial activation contributes to the breakdown of inner blood retinal barrier through pro-inflammatory cytokines and VEGF
production. For example, IL-6 produced by microglia under high glucose induces the down-
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regulation of occludin and ZO-1 and the production of VEGF through signal transducer and activator of transcription 3 (STAT3) activation (Yun et al., 2016b). Minocycline, known to
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deactivate microglia, prevents retinal inflammation and vascular permeability following ischemia-reperfusion injury (Abcouwer et al., 2013). 3.1.1.2
Enhanced transcellular permeability
Increased inner blood-retinal barrier permeability can result from transcytosis mechanisms. In
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vivo studies in non-human primates showed that VEGF-induced permeability results from increased transendothelial protein transport, through caveolae involving endothelial nitric oxide regulation, rather than from tight-junctions opening or increased fenestrations (Feng et al., 1999; Hofman et al., 2000). The presence of plasmalemma vesicle-associated protein
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(PLVAP) in retinal capillaries has been associated with VEGF-induced blood retinal barrier
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permeability in diabetic macular edema (Wisniewska-Kruk et al., 2016). The observation that the genetic ablation of Cav-1 induces breakdown of the inner bloodretinal barrier without alterations of junction protein expression (Gu et al., 2014a) suggest that alteration of macromolecules vesicular transport in the retina could enhance the barrier permeability. This mechanism has been recently considered in the central nervous system as an important contributor to blood-brain barrier disruption without alteration of junctional structures (De Bock et al., 2016).
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ACCEPTED MANUSCRIPT 3.1.1.3
Loss of endothelial cells
Endothelial cell death has been described in models of oxygen-induced retinopathy (Beauchamp et al., 2001; Gu et al., 2003; Yamada et al., 1999). It was also reported in a model of branch retinal vein occlusion, where early endothelial cell death in capillaries
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upstream the occluded vessels, was associated with vasogenic edema and hemorrhages, followed by a proliferation of endothelial cells (Dominguez et al., 2015). In STZ-induced
diabetic rats, leukocyte-mediated Fas-FasL-dependent endothelial cell apoptosis induced a breakdown of the inner blood-retinal barrier (Joussen et al., 2002). Several factors can cause
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endothelial cell death. For instance, TNF-α can contribute through apoptosis (Behl et al.,
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2008), necrosis (Claudio et al., 1994), decreased cx43-mediated glio-vascular communication (Muto et al., 2014), and photoreceptor-released pro-apoptotic factors (Tonade et al., 2016). In diabetes, endothelial activation and subsequent leucocyte- and monocyte-induced cell death results from either pro-inflammatory bone marrow circulating cells (Li et al., 2012), or increased stiffness of vessel basal membrane secondary to
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oxidative-stress-induced collagen cross-linking (Yang et al., 2016). More chronic endothelial dysfunction and metabolic stress could induce cytoskeletal remodeling and non-apoptotic cell death (van Gorp et al., 1999). It could also cause non-apoptotic cell blebbing, as observed in
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endothelial cells from Goto-Kakizaki (GK) type 2 diabetic rats (Rothschild et al., 2017). These cellular mechanisms explain the co-occurrence of capillary occlusion, increased permeability
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and hemorrhages.
3.1.1.4 Loss of pericytes Pericyte loss, considered as a mechanistic hallmark of early diabetic retinopathy, results from reduced adhesion of pericytes, which might be more sensitive than endothelial cells to microenvironment alterations (Li et al., 1996, p. 39). Yet, the exact mechanisms responsible for pericyte loss are not completely understood. Several factors contribute to pericyte death in diabetic conditions such accumulation of AGEs, hypoxia, increased levels of reactive
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ACCEPTED MANUSCRIPT oxygen species contributing to oxidative stress, rapid glycemia variations (Shojaee et al., 1999), macrophages/microglia activation and action of matrix metalloproteinases. AGEs induce the decrease of the Bcl-2/Bax ratio and the activation of caspase-3, via oxidative stress (Chen et al., 2006). The Ang-2/Tie-2, VEGF/Flt-1 (VEGF receptor-1, VEGFR-1) and
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the PDGF-B/PDGF-ß pathways have been incriminated in pericyte cell death (Ejaz et al., 2008). The regulation of Ang1/Tie2 pathway is important in vascular development and
particularly in maturation and stabilization of vessels (Felcht et al., 2012; Park et al., 2003). But, in hyperglycemic conditions, the effect of Ang-2 produced by endothelial cells on
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pericyte apoptosis could be mediated via the integrin-α3 and -β1 (Park et al., 2014).
Circulating VEGF and PGF, secreted by tumors or delivered by adenoviral vectors, induce
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the ablation of pericytes from the mature retinal vasculature through the Flt-1 (VEGFR-1)mediated signaling pathway, leading to increased vascular leakage (Cao et al., 2010). Interestingly, only VEGFR-1 and not VEGFR-2 is up-regulated by ischemia through activation of the hypoxia-inducible factor-1α (HIF-1α) promoter (Gerber et al., 1997). In
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addition, alteration of the PDGF signaling pathway, for instance in hyperglycemic conditions, contributes not only to decreased pericyte coverage but also to pericyte death. Hyperglycemia-induced dephosphorylation of PDGF-receptor-ß results in pericyte apoptosis,
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via the activation of PKC-∂and p38α mitogen-activated protein kinase (MAPK), increasing the expression of the Src homology-2 domain-containing phosphatase-1 (SHP-1) (Geraldes
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et al., 2009).
In the diabetic retina, Ca2+/calmodulin-dependent protein kinase II has been also implicated in inducible nitric oxide synthase (iNOS)-related pericyte loss (Kim et al., 2011). More recently, it is has been suggested that during diabetic retinopathy, human retinal pericyte apoptosis could be mediated by macrophages, TGF-β and the pro-apoptotic BIGH3 protein (transforming growth factor-ß-induced protein ig-h3) (Betts-Obregon et al., 2016). Increased expression of MMP-2 by pericytes in elevated glucose conditions, and in the diabetic retina,
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ACCEPTED MANUSCRIPT induces retinal pericyte apoptosis/anoikis by loss of contact with an appropriate extracellular matrix (Yang et al., 2007). In the rat model of branch retinal vein occlusion mentioned above, pericyte death was also observed in capillaries upstream the occluded vessel, and occurred in a delayed manner
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through a non-caspase-dependent apoptotic mechanism (Dominguez et al., 2015).
3.1.1.5 Role of leucocytes in inner blood-retinal barrier breakdown
High glucose, nitrosative/oxidative stress, and elevated VEGF levels (but not PGF) induce
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the attachment of monocytes and leukocytes to the vascular wall (leukostasis), by the upregulation of intercellular adhesion molecule 1 (ICAM-1) (Huang et al., 2015; Joussen et al.,
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2002; Leal et al., 2007). Leukostasis is accompanied by blood-retinal barrier dysfunction (Edens and Parkos, 2000; Moore et al., 2003). Leukocyte diapedesis also induces a transient loss in the tight junction proteins occludin-1 and claudin-3 (Xu et al., 2005). Neutralization of ICAM-1 and CD18 prevents leukocyte adhesion and retinal endothelial cell death (Joussen et
dysfunction.
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al., 2001) suggesting that leukostasis directly induces endothelial cell death and barrier
The role of macrophages in microangiopathy has not been demonstrated but in diabetic rat
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retinas, levels of chemokine ligand 2 (CCL2) are increased, inducing monocyte macrophage infiltration in retinal tissues (Rangasamy et al., 2014). It was shown recently that cytokine-
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activated endothelial cells recruit specific types of human monocyte subsets, which in turn can activate endothelial cells and induce neutrophil recruitment (Chimen et al., 2017). Leukostasis was enhanced by TNF-α, but reduced by IL-6. Thus, activation of tissue monocytes is involved in neutrophil recruitment through endothelial cell activation, via a cytokine-dependent regulatory pathway. This is in line with previous observations in rodent models of ischemic retinopathy demonstrating that TNF-α mediates leukostasis induced by VEGF, IL-1ß, and platelet-activating factor. But while TNF-α ablation suppressed vascular permeability induced by platelet-activating factor, it did not affect VEGF- or IL1β-induced
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ACCEPTED MANUSCRIPT permeability, showing that leukostasis and permeability may not be directly linked (Vinores et al., 2007). Multiple mechanisms involving circulating cells, endothelial cells, pericytes, and glial cells can lead to increased barrier permeability without structural vascular alterations. Yet, these
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mechanisms may also lead to structural damages to the retinal microvasculature, subsequently to occlusion, hemorrhages, leakage and vascular remodeling. As opposed to experimental conditions in which the role of single factors can be assessed, multiple intricate pathways and cell functions are involved in the complex pathogenesis of ME. In addition,
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potential compensatory mechanisms may be activated, which further complicates their study.
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3.1.1.6 Neovascularization and vessel abnormalization
Most active neovessels, either originating from the choroidal or retinal vasculature, manifest with leakage or diffusion on fluorescein angiography. This leakage results from the weakness of their barrier properties. VEGF, a major inducer of retinal neovascularization, also induces
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phosphorylation of occludin, which is required for VEGF-induced endothelial permeability. Expression of occludin mutated at the phosphorylation site inhibits angiogenesis in cell culture models and in vivo (X. Liu et al., 2016). Cell division can thus not be dissociated from
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cell junction opening. Pericyte coverage defect and non-mature neurovascular glial coupling can favor leakage of neovessels. After anti-VEGF treatment, choroidal neovessels in age-
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related macular degeneration show reduced leakage on fluorescein angiography (Avery et al., 2006). Retinal vessel abnormalization, as occurring in micro- or macro-aneurysms can be associated with focal leakage, resulting at least partly from VEGF/PGF-induced permeability (Nguyen et al., 2016; Witmer et al., 2003), cx43 and caveolin-1 decrease (Gu et al., 2014a; Shoja et al., 2007), and PDGF-B-mediated pericyte alterations (Lindahl et al., 1997). Choroidal neovascularization, when developing anterior to the RPE, creates a physical RPE barrier rupture, whilst it rather exerts multifactorial stress on the RPE when developing under the RPE (e.g. inflammatory, mechanical, proteolytic stresses), that also contribute to RPE barrier dysfunction (D’Amore, 1994; Gao et al., 2016; Zhang and Ma, 2007). Subretinal and 32
ACCEPTED MANUSCRIPT intraretinal fluid accumulation in neovascular macular disorders result not only from the abnormal neovessel itself, but also from the associated outer blood-retinal barrier rupture.
3.1.2
Increased fluid entry through outer retinal barriers
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Because barrier integrity and functions of polarized ion/water channels are closely linked, it is difficult to determine in which proportion subretinal fluid results from increased entry from the choroid, and from insufficient removal by the RPE.
Retinal pigment epithelium and choroidal vessel junction alteration
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3.1.2.1
A focal disruption of RPE intercellular junctions may by itself be the site of fluid entry from the
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choroidal vessels, as shown in central serous chorioretinopathy (Figure 6G and H) with focal or multifocal fluid leakage sites visible on fluorescein angiography (Daruich et al., 2015b; Pryds et al., 2010). Indeed, contrarily to retinal vessels, choroidal capillaries are fenestrated, allowing fluid passage, that in physiologic conditions is stopped by the tight junctions between RPE cells. In case of enhanced permeability of choroidal vessels, as observed in a
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range of pathologic conditions such as CSCR, disruption of RPE intercellular junctions favors fluid to enter into the sub-retinal space. Moreover, underlying choroidal vascular abnormalities may also alter Bruch’s membrane integrity. Subretinal fluid accumulation is
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possibly further exacerbated by mechanical stress induced by choroidal pressure on the RPE, that may alter RPE polarity, and subsequently its fluid drainage capacity.
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Chronic oxidative and/or metabolic stress can also result in subtle and diffuse RPE barrier dysfunction, as observed during diabetes (Y.-H. Chen et al., 2012; Omri et al., 2013; Xu et al., 2011). Serous detachment of the macula, suggestive of RPE barrier breakdown, is observed in approximately half of cystoid ME cases secondary to uveitis (Grajewski et al., 2016) or branch retinal vein occlusion (Celık et al., 2016), and a third of diabetic ME cases (Shereef et al., 2014; Vujosevic et al., 2017). This suggests that inner and outer blood-retinal barrier breakdown may occur simultaneously, and that among mechanisms leading to ME, RPE dysfunction is probably underestimated. Yet, subretinal detachments in ME may not be
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ACCEPTED MANUSCRIPT caused by RPE disruption, since RPE junction barrier seems to be highly resistant to acute hypoxia/ischemia in experimental conditions (Kaur et al., 2008). In addition, it is not clear to which extent the RPE, depending on choroidal blood supply, is affected by retinal nonperfusion. The processes involved in RPE barrier alteration in vivo are not fully understood
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and have drawn less attention than those involved in inner barrier breakdown. The following mechanisms are involved in RPE barrier dysfunction, and will be detailed below: release of pro-inflammatory mediators, VEGF-mediated enhanced permeability, MMP proteolytic activities, cytoskeleton regulatory proteins, and mast cell degranulation.
Several pro-inflammatory mediators, mostly cytokines, were shown to exert direct
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•
effects on the expression and/or subcellular localization of junction proteins in vitro. For
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example, TNF-α directly influences ZO-1 distribution in polarized RPE cells through the p38 MAPK (Shirasawa et al., 2013) and IL-1β pathways, increasing intercellular permeability through occludin down-regulation and claudin-1 up-regulation (Abe et al., 2003). In cultured RPE cells from retinas of rats from the “PVG” strain, susceptible to development of
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experimental uveitis, lipopolysaccharide associated with IFN-γ and TNF-α induce alterations of ZO-1 and actin cytoskeleton, while NO was found to maintain the barrier integrity (Zech et al., 1998). Oxidative stress also disrupts RPE barrier integrity through NF-κB activation
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(Inumaru et al., 2009). These pro-inflammatory mediators are involved in uveitis-related ME, but also in ME of other causes, as illustrated by the elevated levels of several cytokines
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detected in ocular media from patients with ME (Tables 1-3). Yet, RPE cells also produce in vivo molecules that potentially prevent TNF-α- or IL-1β-induced barrier breakdown, such as IL1-receptor agonist (IL1-RA) and soluble TNF receptors. •
Permeability of the RPE may be increased by angiogenic molecules of the VEGF
family. VEGF-A, secreted at the baso-lateral side of RPE cells in physiologic conditions, can favor the alteration of RPE barrier function in pathologic conditions. In rodents exposed to acute light damage, photoreceptor cell death can be prevented by inhibition of VEGFinduced RPE barrier breakdown. This observation suggests that VEGF produced in response
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ACCEPTED MANUSCRIPT to photic stress contributes to photoreceptor cell death secondary to RPE barrier rupture (Cachafeiro et al., 2013). This mechanism may explain the rupture of the RPE barrier occurring in other causes of retinal injury, and leading to ME. PGF, up-regulated under hypoxic conditions or insulin exposure, modifies the subcellular distribution of occludin
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through MEK signaling pathway in vitro, and in vivo in a rat model overexpressing PGF (Kowalczuk et al., 2011; Miyamoto et al., 2007). Whether the effect of VEGF on RPE
permeability involves VEGF receptor 1 or 2 is controversial (Ablonczy et al., 2009; Miyamoto et al., 2007). Both receptors were found in RPE from human eye specimens (Chen et al.,
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1997). In transgenic mice overexpressing VEGF in photoreceptors, a focal increase of RPE permeability was observed, and manifested by RPE tight junction alterations and
•
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degenerative changes of RPE cells (Vinores et al., 1999).
Activation of matrix metalloproteinases, particularly MMP-9, can also contribute to
RPE junction protein alterations. MMP-9 is activated by oxidative and nitrosative stress in the diabetic retina (Giebel et al., 2005; Kowluru et al., 2012), and by amyloid-β, leading to the
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proteolytic degradation of occludin and overall disruption of the tight junction complex (Cao et al., 2013; Giebel et al., 2005; Navaratna et al., 2007). In addition, MMP-1 increased activity is involved in the action of TNF-α on RPE barrier integrity (Li et al., 2010). The polarity/adhesion/barrier functions of highly polarized epithelia such as the RPE
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•
are closely regulated by small kinases and GTPases, particularly calcium-independent
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atypical PKCs (Citi et al., 2014; Etienne-Manneville and Hall, 2003), as mentioned in 2.1.2.1. Recently, the concept of "zonular signalosome" has been proposed, highlighting the close interaction between junction proteins (zonulae occludens and adherens) and the cytoskeletal organization, through Rho GTPases and their effectors (Citi et al., 2014). •
Mast cells are another important actor of outer barrier alteration. Mast cells, abundant
along choroidal vessels (Figure 9A), particularly arterioles, play a major role in ocular inflammation (Godfrey, 1987), being the first activated cells in experimental autoimmune uveitis (de Kozak et al., 1981). Mast cells produce numerous factors such as biogenic amines (e.g. histamine), proteases (e.g. chymase, tryptase), angiogenin, proteoglycans, 35
ACCEPTED MANUSCRIPT cytokines (e.g. TNF-α, IL-4, IL-6, IL-33, IL-15), chemokines (e.g. CCL5, IL-8, Monocyte chemotactic protein 1 (MCP-1), eotaxin), growth factors (e.g. NGF), peptides, prostaglandins, leukotrienes, and complement factors. Histamine is not only a potent vasodilator but also a major player in microvascular disease.
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In primary endothelial cells in vitro, ~53% of all VEGF-regulated transcripts were also regulated by histamine, including genes involved in tight junction formation (i.e. claudin 5) and expression of pro-angiogenic transcription factors affecting endothelial cells survival, migration and tube formation (Laakkonen et al., 2017).
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Substances released experimentally after substance-P-induced mast cell degranulation
include complement C3a and C5a fragments, and endothelin 1, and might differ from those
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released after anti-IgE-induced degranulation (Gaudenzio et al., 2016). In patients with branch retinal vein occlusion, circulating endothelin-1 has been detected, and anti-VEGF treatment has been associated with reduced circulating endothelin-1 levels (Kida et al., 2016) suggesting a potential indirect role on mast cell degranulation. Hypoxia induces the release
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of IL-8 and TNF-α by mast cells via HIF-1α, and catalyzes the formation of histamine (Nizet and Johnson, 2009). Oxidative stress also induces mast cell degranulation (Chelombitko et al., 2016). Tryptase released upon mast cells degranulation cleaves the protease activated
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receptor 2 (PAR2) present at microglia surface, resulting in microglia activation (Park et al., 2010). We have shown recently that the local degranulation of mast cells using sub-
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conjunctival injection of compound 48/80, a synthetic polymer and known inducer of mast cell degranulation (de Kozak et al., 1983), resulted in serous retinal detachments and pigment epithelial detachments in rodents (Figure 9 B and C), and that its inhibition by cromoglycate could alleviate the retinal consequences of mast cell degranulation (Bousquet et al., 2015; Steptoe et al., 1994). Histamine overproduction per se does not seem to cause any retinal abnormalities in transgenic mice (Greferath et al., 2014). Yet, the complex cocktail of active agents released by mast cells degranulation massively disrupted the outer retinal barrier. Taking into account the large number of mast cells present in the choroid, their proximity with vessels and 36
ACCEPTED MANUSCRIPT microglia (Forrester et al., 2010), and the presence of known inducers of mast cells degranulation in the choroid (particularly C5a), mast cells is likely an underestimated contributor to outer retinal permeability in several retinal diseases. 3.1.2.2
Retinal pigment epithelial cell death
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Whether RPE cell death per se contributes to subretinal fluid accumulation remains to be demonstrated. Indeed, large surgical resection of RPE and choroid in eyes with neovascular AMD that underwent free autologous RPE-choroid graft, before the anti-VEGF therapy era, did not induce retinal or subretinal fluid accumulation (van Zeeburg et al., 2012). In
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geographic atrophy, characterized by RPE cell death (Hanus et al., 2015), macular edema does not occur unless neovascularization is associated (Dunaief et al., 2002; Kaneko et al.,
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2011; Kim et al., 2002). In both examples, note that the underneath choroidal vascularization is either absent or reduced. On the other hand, subretinal fluid is commonly associated with areas of RPE atrophy as observed in chronic central serous chorioretinopathy (Daruich et al., 2015b), often adjacent to dilated large choroidal vessels. In this disorder, choroidal vascular
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hyperpermeability, visible on mid-phase indocyanine green angiography, may cause choroidal edema, manifesting for instance as “loculation” of fluid in the posterior choroid (Spaide and Ryan, 2015). Choroidal edema may presumably alter the retina-to-choroid
3.1.2.3
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osmotic gradient, providing an explanation for the accumulation of fluid under the retina. Outer limiting membrane disruption
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OLM barrier alteration favors large Stoke radius proteins movement towards the subretinal space. As the OLM is also critical for the polarization and structural maintenance of RMG cells and photoreceptors, its destabilization favors photoreceptor misalignment and degeneration. In chronic retinal diseases, such as diabetic retinopathy, disruption of junction protein complexes occurs in the retinal vascular endothelium, the RPE and at the OLM, with differential severity and kinetics. From a clinical point of view, OLM disruption on OCT has been correlated with poorer visual acuity in diabetic ME and retinal vein occlusion (Ito et al., 2013; Murakami et al., 2016). It had also been linked to poor responses to anti-VEGF
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ACCEPTED MANUSCRIPT therapy in wet AMD (Zandi et al., 2017), vein occlusion (Chatziralli et al., 2017) and diabetic ME (Ashraf et al., 2016; Muftuoglu et al., 2017).
3.2
Decreased drainage functions
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As detailed in 2.2, RPE and RMG cells actively drain water and osmolytes from the retina to the systemic circulation, or the vitreous. Hydro-ionic transport across cells is ensured by ion and water channels distributed in a synchronized manner with cell polarization, depending on intercellular signaling, and by various transporter molecules. The hydro-ionic transports can
3.2.1
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dysfunction, or in response to extracellular signals.
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be altered due to RPE and/or RMG cell death, cell metabolic suffering, cytoskeleton
Glial cells and neurosensory retina
RMG cells rapidly respond to any change in their microenvironment and develop strong resistances to injury because of their glycogen reserve, as well as their anti-oxidant and
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detoxification capacities, for instance via glutamate uptake. In response to injury RMG cells rarely undergo apoptosis and rather dedifferentiate into pluripotent retinal progenitors with the potential of regenerating retinal neurons, as evidenced for instance in zebrafish studies
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(Goldman, 2014). However this process is limited and poorly effective in mammals (Hamon et al., 2016). Any retinal stimulus induces a reactive state of RMG cells, identified
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experimentally by the increased expression of intermediate filaments contributing to the cytoskeleton, such as GFAP (Lewis and Fisher, 2003). Uncontrolled healing processes can turn into gliosis (Bringmann et al., 2006; Bringmann and Wiedemann, 2012), which may have deleterious structural and functional consequences. Such dedifferentiation is associated with a reduction of potassium conductance due to decreased expression and/or mislocation of Kir4.1 channels (Reichenbach et al., 2007). Ion and water channel expression in astrocytes, involved in the osmoregulation of the inner retina is also altered in pathologic conditions (Iandiev et al., 2006). Since it is admitted that water transport is tightly coupled to spatial K+-buffering currents, any 38
ACCEPTED MANUSCRIPT inhibition of either water or potassium transport mechanisms leads to consequences on the other. Reduction of potassium conductance has been observed rapidly in models of diabetes (Fletcher et al., 2007; Pannicke et al., 2006), retinal vein occlusion (Rehak et al., 2009), various forms of retinal degeneration (Dinet et al., 2012; Lassiale et al., 2016), and uveitis
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(Eberhardt et al., 2011; Feng et al., 2013; Liu et al., 2007; Zhao et al., 2011). The disturbance of potassium homeostasis contributes to neuronal cell death and ischemia.
Indeed, the perivascular release of potassium plays an important role in regulating regional blood flow, in response to changes in neuronal activity (Paulson and Newman, 1987). Table
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4 summarizes the changes in K+ and water channels observed in different rodent models of retinal diseases.
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In response to ischemic stress, the aquaglyceroporin AQP9, belonging to a subgroup of aquaporins transporting water and other osmoregulators such as glycerol and urea (HaraChikuma and Verkman, 2006), is increased in the neurosensory retina (Hollborn et al., 2012). In the STZ-induced diabetes rat model, AQP1 is increased while AQP6 and AQP11 are
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decreased (Hollborn et al., 2011). In the Torii type 2-diabetes rat model, AQP0 is increased at the level of the nerve fiber layer (Fukuda et al., 2011). In addition, the transport of osmoregulator amino acids such as GABA and taurine is also involved in the formation of
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al., 2004).
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retinal edema (Pasantes-Morales et al., 1999), in both RMG and RPE cells (El-Sherbeny et
3.2.2
Retinal pigment epithelium
The proper polarization of the RPE and subsequent adequate localization of ion and water channels requires a synchronized cross-talk between extracellular signals and cell cytoskeleton. This cross-talk requires the activation of different kinases involved in signal transduction, such as those belonging to the MEK/ERK and RhoA/ROCK pathways (Caplan, 1997; Citi et al., 2014; Schevzov et al., 2015). In several clinical situations, RPE transport dysfunction is associated with or favors fluid accumulation in the subretinal space. Multiple, rapid-onset transient serous retinal 39
ACCEPTED MANUSCRIPT detachments have been described after systemic administration of MEK inhibitors for metastatic cancer, without observation of leakage on fluorescein angiography (Urner-Bloch et al., 2014). This clinical observation suggests a cellular RPE dysfunction, rather than a focal junction breakdown, as detailed in 6.4. In vitro, over-activation of Rho kinase stimulates
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actin cytoskeleton contraction in RPE cells, and subsequent cell mobility (Zheng et al., 2004). In an in vivo rat diabetic mode, ROCK inhibition reduces RPE leakage (Rothschild et al., 2017).
In addition, aquaporins are also critical for the drainage functions of RPE cells. AQP1
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contributes to the transepithelial water transport (Stamer et al., 2003), that is enhanced by the natriuretic peptide (Baetz et al., 2012). AQP1 is overexpressed to compensate fluid
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transport reduction above drusen in eye specimens with early or advanced AMD (Tran et al., 2016), and in the diabetic retina (Hollborn et al., 2011). It is down-regulated in response to UV-induced oxidative stress (Jiang et al., 2009). AQP9 is also overexpressed in vitro by RPE cells under chemical hypoxia, oxidative stress, VEGF, and high glucose (Hollborn et al.,
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2012).
In STZ-induced type 1 diabetes, AQP5, AQP9, AQP11, and AQP12 were over-expressed in the RPE, whilst AQP0 was reduced (Hollborn et al., 2011). The alternative complement
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pathway contributes to physiologic transport of ions and macromolecules through pore formation such as C5b-8. This mechanism may be underestimated in the context of AMD
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(Farkas et al., 2002).
In summary, any retinal stress can induce the activation of glial cells and modify the expression and cellular distribution of water channels in RMG and RPE cells. Subsequently, the distribution of ions and other osmolytes across the retina is also altered, resulting in ME. In this context, ageing, accompanied by cumulative oxidative stress, is associated per se to a reduction of potassium conductance in RMG cells, explaining that ME is favored by older age (Bringmann et al., 2003). To date, the full spectrum of molecular signals that control hydroionic retinal homeostasis is incompletely understood, and other crucial pathways have also
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ACCEPTED MANUSCRIPT been identified. For instance, the gluco- and mineralocorticoid receptors stimulated by endogenous or therapeutic steroids have been shown to control the expression and distribution of AQP4 and Kir4.1, in both healthy and inflammatory rat retina (Zhao et al.,
3.3
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2011).
Protein leakage
In addition to inner and outer blood-retinal barrier breakdown, allowing fluid entry, permanent
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fluid accumulation in the retina depends on several factors that may differ according to
clinical etiologies of ME. When barrier functions are no longer acting, the Starling equation
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governs fluid movements between different compartments.
The Starling equation allows to calculate the movement of fluid across capillary membranes, integrating the effects of hydrostatic and oncotic forces (referred as “Starling forces”). The net fluid movement across capillaries results from diffusion, filtration and pinocytosis, but the Starling equation only refers to filtration parameters. The direction and extent of passive
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water exchanges between the retinal and choroidal extravascular compartments, and the intravascular compartments (formed by the retinal capillaries, the choriocapillaris and large choroidal vessels), is mostly determined by their relative hydrostatic and oncotic pressures.
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The intra-capillary hydrostatic force is a positive pressure generated by cardiac contractions, while the interstitial hydrostatic pressure may reach negative values in certain tissues, as a
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result of lymphatic drainage and muscle contraction (Aukland and Reed, 1993). In the retina, absence of lymphatic vessels could be at least in part compensated by active transepithelial fluid transport across the RPE toward the choriocapillaris (Marmor et al., 1985; Nickla and Wallman, 2010). The exact values of hydrostatic pressure in the different layers of the neurosensory retina are not known, but are expected to be also negative in physiological state. The Starling equation reads as follows: Jv = Kf × ([Pc - Pi] - σ × [πc - πi])
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ACCEPTED MANUSCRIPT Where: • Jv is the net fluid movement between compartments, • Kf is a proportionality constant called the filtration coefficient,
• Pc is the capillary hydrostatic pressure • Pi is the interstitial hydrostatic pressure • πc is the capillary oncotic pressure
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• πi is the interstitial oncotic pressure
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• ([Pc - Pi] - σ × [πc - πi]) is the net driving force,
• σ is the reflection coefficient
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Due to the low protein concentration of the vitreous, the presence of retinal barriers, and the structure and function of the choroidal vasculature, a protein gradient is established from the vitreous to the choroid, allowing a permanent outflow of water towards the choroid. As mentioned in 1.1. Any increase in retinal protein concentration within interstitial retinal tissue
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will cause an accumulation of water driven by osmotic forces. A moderate increase in vascular leakage with increased pinocytic transport, but no rupture of the endothelial tight junctions (and consequently no major protein leakage within the neurosensory retina), may not lead to the formation of retinal edema. On the other hand, intense protein and lipid
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extravasation is associated with focal edema around the so-called “exudates”.
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The OLM constitutes a filter to macromolecular diffusion from the subretinal space to the outer nuclear layer (Marmor et al., 1985; Marmor, 1990). If the OLM is preserved, proteins may accumulate in the retina, but remain inner to the OLM. Accumulation of proteins within the photoreceptor layer may cause neuronal toxicity (Cachafeiro et al., 2013). Rupture of the OLM allows proteins to diffuse into the subretinal space, causing subsequent RPE hyperosmotic stress and potential additional damages (Willermain et al., 2014). OLM disruption on OCT is associated with worse visual prognosis in conditions manifesting with ME, as detailed in 3.1.2.3. Depending on the microenvironment, proteins, lipids and glycoproteins may form low soluble aggregates. These exudates may persist or require
42
ACCEPTED MANUSCRIPT months to regress, as observed clinically.
4 Why does edema specifically form in the macula? Several
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hypotheses
We provide below a list of several mechanisms involved in ME, some of which are
4.1
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on current biological and translational evidence.
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consensually admitted, and some others being advanced as hypothetic mechanisms, based
Alteration of junction proteins in the “Z-shaped” Müller cells zone
The functional specialization of the fovea, ensuring precision vision, results from several
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anatomical specificities. They include high cone density, reduced vascularity with absence of retinal vessels in the visual axis, and centrifugal displacement of cone axons and specialized cone RMG cells. RMG cell density is about 5 times higher in the fovea as compared to the periphery (Distler and Dreher, 1996). The cone-projected axons are intertwined with RMG
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cells and follow a centrifugal course towards the inner layers at the edges of the fovea. In this
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area, RMG cells are highly elongated as compared to peripheral RMG cells, reaching several hundred micrometers (Figure 10A and B). As their processes draw from ILM to the OLM, the foveal RMG cells follow a “Z-shape” trajectory (Bringmann et al., 2004; Matet et al., 2015). They run vertically from their endfeet at the ILM to the OPL, turn inwards towards the foveal center across the Henle fiber layer where they reach almost horizontally the foveal pit, before adopting again a vertical diving direction towards the OLM (Figure 10A). Along this Z-shaped trajectory, RMG prolongations are closely bound to photoreceptor axons by junction proteins such as ZO-1 (Matet et al., 2015) (Figure 10B), preventing high Stoke radius molecules, notably proteins, to accumulate in this specific region.
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ACCEPTED MANUSCRIPT In pathologic conditions altering the organization and stability of junction proteins (e.g. ZO-1), this “molecular filter” may become leaky, allowing proteins to accumulate between axons and Z-shaped RMG cells. This alteration would attract oncotically-driven fluid, subsequently leading to the formation of “cysts”, as observed in cystoid ME. Interestingly, the radial
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organization of cysts around the fovea (Figure 3E) corresponds to the area where RMG cells and ZO-1 co-localize (Figure 10B), reinforcing this hypothesis. In addition, in the human foveola, we have identified GFAP-positive cells, highly expressing AQP4, but poorly expressing glutamine synthetase and CRALBP, forming a “glial roof” and projecting
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extensions toward the Henle fiber layer (Figure 10F inset, yellow arrows). The exact nature of these cells and their role in water drainage from the fovea and in central cysts formation
4.2
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remains to be determined.
The “glymphatic” system hypothesis
In the retina, like in the brain, no lymphatic system has been identified to clear waste
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products, such as proteins, water and other metabolic byproducts. Water and hydosoluble compounds do not diffuse freely across vessels (Cunha-Vaz, 2017) since in addition to tightjunctions in endothelial cells, the water channel AQP-1 is not present (Motulsky et al., 2010).
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In the brain, AQP4 localized in perivascular end-feet of astrocytes allows the formation of low-resistance pathways for fluid movement between paravascular spaces and interstitium.
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Indeed, although AQP4 is located in astrocytes around vessels in the brain, it does not allow water to enter the brain vessels, creating a paravascular flow, recognized as the “glymphatic pathway” since like the lymphatic system it constitutes a waste clearance pathway for interstitial solutes and proteins (Iliff et al., 2012; Nakada et al., 2017). In the rodent retina, AQP4 is highly expressed in RMG cells, specifically located at the RMG endfeet, at the ILM and around vessels, and at the optic nerve head (Amann et al., 2016). We have analyzed the distribution of AQP4 and RMG in healthy post-mortem human retinas including the maculae (Figure 11) on sections and on flat-mounted specimen. As expected,
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ACCEPTED MANUSCRIPT AQP4 is located around all retinal vessels in all plexuses (Figure 11A), and in astrocytes and RMG endfeet in the superficial inner retina. However, AQP4 co-localizes with glutamine synthetase only at the level of the deep capillary plexus, where no astrocytes but only RMG cells are present (Figure 2 A and E, and Figure 11F).
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At the fovea, where no vessels are present, AQP4 is highly expressed along central RMG cells (Figure 11 A, B-D) that poorly express GS (Figure 11 C). In the Henle fiber layer, Zshaped RMG cells highly express AQP4 (Figure 11A, dotted contour, and 11D inset). In flatmounted macula from non-human primate and from human, RMG cells (labeled with
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glutamine synthetase and with CRALBP respectively) follow the axon organization towards the optic disc (Figure 12A and Supplementary Figure 1). AQP4 is concentrated in the fovea
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(Figure 12A) and form patches along RMG cells towards the optic nerve head (Figure 12C and D). Higher magnification allows the identification of “channel-like” structures surrounded by AQP4 (Figure 12E and inset).
We raise the hypothesis that like in the brain, AQP4 could create a permanent solute flow
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between RMG cells and a hydraulic path, around the vessels towards the optic nerve head (Figure 13). A glymphatic pathway could thus exist also in the retina and particularly the macula for the clearance of waste solutes and proteins. At the optic nerve head, previous
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studies had identified a defect in blood-retinal barrier integrity, allowing proteins to be drained from the retina (Flage, 1977). More recently, a glymphatic pathway, driven by AQ4 has been
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described at the optic nerve connected with the cerebrospinal fluid, allowing transport of protein below 70KD (Mathieu et al., 2017). In case of excess of protein leakage and overload of the glymphatic drainage of proteins along the Z shaped Müller cells, proteins accumulation will retain water and cause CME. In retinal diseases such as diabetic retinopathy, AQP4 expression and/or localization is modified (Table 4), which could disrupt this potential “glymphatic pathway” and promote protein accumulation within the neurosensory retina, favoring fluid accumulation according to Starling equation. In a human diabetic retina, AQP4 expression is reduced in the fovea (Figure 14 A-C) and increased outside the macula (Figure 14 D-F). Further studies should
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ACCEPTED MANUSCRIPT explore this hypothesis that still remains speculative.
4.3
Increased interstitial pressure: vascular consequences
Increase in protein concentration inside the neuroretina subsequently raises the interstitial
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pressure, reducing capillary blood flow. In particular, the downstream and terminal deep retinal capillary plexus may be more susceptible to flow disruption, since its intravascular pressure is lower than in the superficial plexus. Because Kir4.1 channels of RMG cells are
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mislocalized in several animal models of retinal vein occlusion and diabetic retinopathy
(Table 4), and because regulation of the potassium microenvironment at the deep capillary
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plexus only relies on RMG cells, and not astrocytes (as discussed in 2.1.1.4 and Figures 2 and 11), the local decrease in perivascular potassium concentration is expected to reduce the vascular flow (Longden et al., 2016) (Figure 15). Subsequently, drainage by the putative glymphatic system (see 4.2 and Figure 13) along non-perfused vessels may decrease, creating an amplifying mechanism. The recent clinical observation that cystoid ME of
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vascular origin correlates with areas of reduced flow in the deep capillary plexus (Mané et al., 2016; Spaide, 2016) is a possible manifestation of these mechanisms.
The formation of “cysts”
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4.4
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Very few anatomical eyes specimens presenting ME have been studied, and most of them were analyzed with conventional staining of macular sections, without immunohistochemistry. The description of cystoid ME reveals that the empty cavities begin to form in the Henle fiber layer and can extend over time to other layers. In some cases, the central RMG cell cone seemed absent (Wolter, 1981). Three eyes with cystoid macular edema diagnosed on fluorescein angiography were examined using photonic and electron microscopy, showing widespread swelling and necrosis of RMG cells without enlargement of intercellular spaces, suggesting that cysts are caused by degeneration of RMG cells (Fine and Brucker, 1981; Yanoff et al., 1984). Whether retinal cysts result from degenerating RMG
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ACCEPTED MANUSCRIPT cells remains a subject of debate, but the role of RMG cells in the formation of cystoid macular edema is certain. If a “glymphatic” system exists in the retina, the loss of central RMG cells would prevent the central drainage system to function and permanent recurrence
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of edema.
Depending on the primary mechanism and etiology of ME, different fluid topography and associated signs may be observed on OCT, which may differentiate the causative diagnoses (Munk et al., 2014). For example, in purely retinal vascular disorders such as aneurysmal
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telangiectasia (macular telangiectasia type 1), focal ME tends to localize in the vicinity of vascular lesions (Figure 16). In diabetic ME, leakage from the deep capillary plexus induces
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fluid migration in the outer plexiform layer whereas more superficial plexus leakage induces fluid to accumulate in the inner nuclear layer (Byeon et al., 2012). Interestingly, subretinal fluid accumulation is frequently associated with ME due to diabetes or branch retinal vein occlusion (Celık et al., 2016; Noma et al., 2011b; Ota et al., 2013; Shereef et al., 2014), as
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discussed in 3.1.2.1. This observation suggests that early alteration of outer retinal barriers occurs, as observed in animal models of diabetic retinopathy (Desjardins et al., 2016; Omri et al., 2013; Xu et al., 2011).
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Intracellular edema cannot be detected clinically but has been observed on pathological analyses of eyes with ME (Kohno et al., 1983; Yanoff et al., 1984). It may localize within RPE
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cells, RMG cells, and in neurons, as a consequence of metabolic disturbances. How does intracellular edema contribute to the clinically significant ME remains unclear, but it makes no doubt that intracellular edema contributes per se to neuronal toxicity and to extracellular fluid volume increase.
5 Why does macular edema alter vision? ME is a severe complication of numerous retinal disorders because it affects the central vision. This visual impairment can be variable, and results from multiple mechanisms.
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ACCEPTED MANUSCRIPT Moreover, vision loss may be reversed by ocular therapy or control of favoring systemic factors in recent ME of short-duration, but may become irreversible in long-standing cases.
5.1
Evaluation of visual function in macular edema
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Additionally to central vision loss, usually experienced as a relative central scotoma affecting far- and near-vision, patients with ME often also complain of metamorphopsia (Achiron et al., 2015), reading difficulties (Munk et al., 2013), impaired stereopsis, or disturbed color vision. Visual acuity testing, a subjective method based on letter recognition assessing the
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resolution ability of the fovea, is the most widely employed test, both in the clinical setting
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and as validated clinical research endpoint. However, visual acuity does not reflect all alterations affecting central vision, such as perifoveal scotoma or reading speed reduction. Microperimetry is a subjective test that estimates the contrast sensitivity of the macular area, and can detect perceived or non-perceived micro-scotoma. The contrast sensitivity is altered in the presence of ME, correlates with retinal thickness and visual acuity (Okada et al., 2006;
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Vingolo et al., 2016), and shows a spatial correlation with macular lesions such as outer or inner retinal cysts, serous retinal detachments and hard exudates (Deák et al., 2010). Microperimetry testing also assesses retinal fixation, impaired in diabetic ME (Carpineto et
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al., 2007), and aggravated when hard exudates are present (Vujosevic et al., 2008). Reading speed, another subjective test evaluating near vision, correlates with central macular
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thickness (Kiss et al., 2006; Suñer et al., 2013), and central microperimetry (Edington et al., 2017).
The influence of structural changes induced by ME on visual function has been comprehensively reviewed (Tomkins-Netzer et al., 2015). Retinal contrast sensitivity on microperimetry, as well as reading speed testing, are useful adjuncts to visual acuity testing to evaluate visual impairment caused by diabetic, vascular or uveitic ME. The function of the macula can also be assessed objectively by multifocal electroretinography, recording the retinal electric activity upon focal stimulation at multiple macular locations. Multifocal electroretinography studies showed that this activity is altered in diabetic ME, and that the 48
ACCEPTED MANUSCRIPT degree of signal alteration correlates with retinal thickness and visual acuity (Tehrani et al., 2015; Yamamoto et al., 2001). Interestingly, electroretinography also showed that alterations of neuronal activity affecting all retinal layers preceded retinal thickening (Nagesh et al., 2016), in line with the notion that ME
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results from severe physiological changes disturbing the normal retinal metabolism, before the development of clinically-patent ME. Noteworthy, certain presentations of subretinal or intraretinal fluid may not - or very mildly - alter vision, such as early diabetic ME or acute
subretinal fluid is not the sole cause of visual loss in ME.
Acute effects of macular edema on visual function
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5.2
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central serous chorioretinopathy. This supports the concept that the presence of cysts or
Several mechanisms at the subcellular level, and other related to the architecture and vascular supply of the retina, explain why fluid accumulation disturbs visual function. Several acute effects of ME are reversible, when fluid resorbs spontaneously during disease course
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or as response to therapy, as evidenced by the more favorable functional outcome after earlier therapeutic intervention on ME of various causes in clinical practice. ME may disturb the normal path of light from the inner retinal surface to the photoreceptor outer segments,
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where the light signal is captured, and triggers the phototransduction cascade. First, retinal thickening induces a loss in normal retinal transparency. Second, a change in the refractive
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index of retinal layers, as well as an increased light diffraction may occur secondary to hydroionic deregulation, and protein accumulation in the retinal interstitium. Finally, disruption of the normal RMG cell architecture in the central macula, due to intracellular swelling or extracellular fluid excess, may alter light transmission. The role of RMG cells in light guidance having been established for peripheral cells but not for the foveal glial cells, whether this mechanism play a major role in vision alteration remains questionable (Agte et al., 2011; Reichenbach and Bringmann, 2013). These physical effects of ME are probably reversed after ME resolution and explain the good visual recovery in the absence of major alterations of retinal structure. 49
ACCEPTED MANUSCRIPT ME is also accompanied by vascular changes, either causative or secondary, that have also been linked to the degree of visual impairment. The recent advent of OCT angiography allows to image non-invasively details of the perifoveal vasculature. Foveal avascular zone enlargement, frequently observed in diabetic ME, has been identified as an independent
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factor of visual acuity loss (Balaratnasingam et al., 2016). Capillary density of the superficial and deep capillary plexuses is correlated to visual acuity levels in many retinal vascular
disorders manifesting with ME, such as diabetic retinopathy (Samara et al., 2017), central and branch retinal vein occlusion (Samara et al., 2016), or type 1 idiopathic macular
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telangiectasia (Matet et al., 2016). In particular, there is increasing evidence from clinical imaging studies that alterations of the deep retinal capillary plexus are more pronounced
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than those affecting the superficial plexus, as mentioned in 4.2.3. In addition, areas of deep capillary plexus dropout co-localize with cystoid cavities in diabetic ME (Mané et al., 2016) and vascular ME from other causes such as vein occlusion, macular irradiation, and macular telangiectasia type 1 (Spaide, 2016). Moreover, there is no reconstitution of the disrupted
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deep capillary plexus when ME resolves, and in case of recurrence edema tends to recur in the same locations (Spaide, 2016). The retinal vasculature, and particularly the deep plexus, is essential for the metabolic support of the high energy-demanding inner retinal neural
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network. Therefore, disruption of this vasculature provides a plausible explanation for the impaired retinal neural activity present in ME, as evidenced by multifocal electroretinography.
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Acute visual decrease may also occur indirectly when ME is localized nasal to the fovea and disrupts the normal neural signal transmission in the papillomacular nerve fiber bundle (Konieczka et al., 2016).
5.3
Long-term effects of macular edema on visual function
Long standing ME induces irreversible neural retinal damage and glial reactions, as detailed in 3.2.1, which persist even after ME resolution. Definitive photoreceptor alterations manifest clinically with outer nuclear layer thinning, outer segment atrophy and disruption of the ellipsoid zone signal on OCT. Alterations of this hyperreflective band have been correlated 50
ACCEPTED MANUSCRIPT with the degree of visual dysfunction in ME of various causes, such as diabetes (Maheshwary et al., 2010; Otani et al., 2010), branch (Akagi-Kurashige et al., 2014; Ota et al., 2007, 2008b) or central retinal vein occlusion (Ota et al., 2008a), posterior uveitis (Tortorella et al., 2015), Irvine-Gass syndrome (Hunter et al., 2014), epiretinal membranes
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(Fang et al., 2016) and retinitis pigmentosa (Oishi et al., 2009). Alterations of cone arrangement regularity, potentially linked to irreversible photoreceptor damage, have been observed in diabetic ME using adaptive optics imaging (Lammer et al., 2016; Nesper et al., 2017). Intraretinal ME is variably associated with subretinal detachment, depending on its
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etiology and on the time course of ME. Subretinal detachment may occur early, for instance in retinal vein occlusions, or later, for instance in diabetic ME, as intraretinal fluid is
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progressively drained by the RPE or as RPE barrier dysfunction occurs (Omri et al., 2011, 2013; Xia and Rizzolo, 2017). While short-duration detachment of the neurosensory retina from the RPE may not have functional consequences after resolution, long-standing detachments may lead to photoreceptor damage, as exemplified by the more severe visual
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sequelae in chronic versus acute central serous chorioretinopathy (Daruich et al., 2015b). Hard exudate deposition within the neuroretina have long been recognized as a marker of worse visual prognosis (Coscas and Gaudric, 1984), and studies combining OCT and
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microperimetry have confirmed that they co-localize with areas of decreased retinal sensitivity (Deák et al., 2010).
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In diabetic ME, long-standing enlargement of the foveal avascular zone can lead to irreversible foveal ischemia, clinically described as “ischemic maculopathy” on fluorescein, and recently OCT angiography. This complication of advanced ME leads to irreversible foveal thinning affecting all layers on OCT. In particular, thinning of photoreceptor outer segment, ellipsoid zone (Lee et al., 2013) and ganglion cell layer (Byeon et al., 2009) has been identified following ischemic diabetic ME. Finally, chronic ME can also induce permanent visual loss, due to structural disorganization of retinal inner layers, or “DRIL”, persisting after ME resolution. DRIL is defined as the absence of detectable boundaries on OCT between the ganglion cell-inner plexiform layer
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ACCEPTED MANUSCRIPT complex, the inner nuclear layer, and the outer nuclear layer. DRIL reflects the degree of cellular damage accompanying ME, and its morphological extension has been correlated to the degree of visual loss (Radwan et al., 2015; Sun et al., 2014).
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6 What can we learn from pure phenotypes? Besides the most frequent causes of ME extensively investigated in the literature, such as diabetes, retinal vein occlusions or uveitis, there are specific conditions manifesting with
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intraretinal fluid accumulation, in which one single cellular mechanism predominates. From a translational perspective, these presentations contribute to our understanding of the
6.1
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underlying mechanisms and their clinical expression.
Irvine-Gass postoperative macular edema
Pseudophakic macular edema, also referred to as Irvine-Gass syndrome, is a complication of
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cataract removal or other intraocular procedures (Figure 6 C-D). It is characterized by the release of pro-inflammatory mediators triggered by the surgical “trauma”, that induces bloodocular barrier breakdown (Grzybowski et al., 2016). Although its exact pathogenesis is not
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fully elucidated, it clearly occurs after barrier alteration, and can be therefore considered a pure phenotype. Amongst associated factors suspected to predispose for postoperative
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macular edema, including vitreous traction and inflammation, authors have suggested a role of choroidal blood flow changes and instability (Nicholls, 1954; Welch and Cooper, 1958). Reduction in choroidal blood flow velocity and volume was indeed shown to occur in pseudophakic macular edema (Miyake et al., 2007). Interestingly Irvine-Gass macular edema frequently presents with subclinical optic disc swelling, manifesting on fluorescein angiography as late disc hyperfluorescence (Figure 6C). Whether impaired glymphatic pathway drainage, discussed in 4.2, could be involved in the pathogenesis of pseudophakic macular edema should therefore be evaluated.
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ACCEPTED MANUSCRIPT 6.2
Idiopathic macular telangiectasia type 1
Macular telangiectasia type 1, also termed “aneurysmal macular telangiectasia”, is a retinal vascular disorder of congenital or developmental origin that affects mostly men. It manifests
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during early adulthood or later with multiple exudative dilations of perifoveal capillaries, termed telangiectasia, that cause macular edema. It may be associated with areas of
perifoveal non-perfusion, and in some cases with localized peripheral non-perfusion (Gass
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and Blodi, 1993; Yannuzzi et al., 2006). Since it is unilateral in most cases, and may extend beyond the macula, it has been linked to the spectrum of Coats disease (Cahill et al., 2001).
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Macular telangiectasia type 1 is thought to originate from a vascular abnormality affecting retinal and macular capillaries, without other structural alterations than the presence of cystoid cavities. In particular, there is no metabolic or inflammatory precipitating factors, no RPE or OLM alteration, and good visual function is maintained, except at sites of photoreceptor damage associated with long-standing ME (Takayama et al., 2012). MacTel 1
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can therefore be considered a “pure” phenotype. The molecular basis of this disorder has not been elucidated, but we recently proposed that vessel abnormalities might be a consequence of non-perfusion foci (Matet et al., 2016), and that this effect may be mediated
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by the PGF/VEGFR-1 pathway (Kowalczuk et al., 2016). Leakage from perifoveal telangiectasia is the main feature visible on fluorescein angiography, where it appears more
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intense than in idiopathic macular telangiectasia type 2 (MacTel 2) (Figure 16), and usually correlates with areas of retinal thickening and intraretinal cysts. This leakage is responsible for chronic intraretinal exudation of fluid and proteins. We hypothesize that this protein accumulation over time is responsible for ME, due to focal alteration of the normal protein gradient in the retina.
6.3
Idiopathic macular telangiectasia type 2
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ACCEPTED MANUSCRIPT MacTel 2 is a rare macular disease associating subtle leakage from perifoveal retinal telangiectasia, macular pigment loss, intraretinal cavitations, crystalline deposits, focal disruption of the retinal layers, and photoreceptor loss (Charbel Issa et al., 2013). Intraretinal cavitations have a particular appearance on OCT. They harbor square edges, a thin remnant
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of inner limiting membrane when affecting the inner retina, and are not associated with retinal thickening. This latter feature suggests that they result from loss of tissue replaced by fluid, rather than from true exudation (Oh et al., 2014) (Figure 16). The disease manifests during the fourth-to-sixth decades, and affected subjects often present diabetes mellitus or
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hypertension (Clemons et al., 2013).
The molecular origin of the disorder is not known, and some familial cases have been
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identified, suggesting possible genetic factors (Gillies et al., 2009). Regarding the involved cell type, a defect in RMG function or loss of RMG cells is thought to be the primary pathogenic factor leading to the MacTel 2 phenotype. RMG cell loss has been observed in MacTel 2 eyes (Powner et al., 2010, 2013), and in two proposed rodent models
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recapitulating part of MacTel type 2 phenotype (Shen et al., 2012; Zhao et al., 2015). Recent data obtained with OCT angiography showed advanced deep capillary plexus abnormalities in Mactel 2, with outer retinal invasion and possible capillary proliferation from the deep
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capillary plexus to the outer retina (Gaudric et al., 2015; Spaide et al., 2015, 2016). These manifestations might result from a deregulation of endothelial growth exerted by RMG cells.
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As discussed above (see 2.2.1), only RMG cells interact and control the deep capillary plexus, explaining why loss of normal RMG cell function would primarily affect this plexus. Finally, the absence of intraretinal cystoid cavities and retinal thickening, in the absence of choroidal neovascularization that may complicate the course of Mactel 2, may be explained by the low leakage intensity from altered endothelial junctions in the deep plexus, that would not allow the massive passage of large proteins.
6.4
Paclitaxel-induced maculopathy
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ACCEPTED MANUSCRIPT Patients receiving paclitaxel, a chemotherapy drug of the taxane family, preventing microtubule assembly, may develop bilateral cystoid ME (Ehlers et al., 2013). This adverse event presents with intraretinal cystoid cavities on OCT, and as silent ME on fluorescein angiography, where no leakage is observed, either from the retinal vessels or the RPE
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(Georgakopoulos et al., 2012; Joshi and Garretson, 2007). Although its exact mechanism is not understood, RMG cell dysfunction has been hypothesized, by analogy with MacTel 2, and based on electrophysiological observations (Nakao et al., 2016). Therefore, paclitaxel maculopathy belongs to the “pure” phenotypes listed in this chapter. We suggest that
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inhibition of microtubule polymerization and intracellular RMG transport could cause fluid to accumulate in RMG cells. If confirmed, this unique clinical presentation would demonstrate
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that intracellular fluid accumulation in the retina can cause cystoid ME. Consistently, antiVEGF agents were not efficient in treatment of paclitaxel-induced ME, and the only therapeutic option to date is discontinuation of paclitaxel therapy.
MEK-inhibitor-associated maculopathy
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6.5
Serous macular detachments and cystoid macular edema have been reported during treatment by mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors (e.g.
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Binimetinib, Trametinib) for cutaneous melanoma (Duncan et al., 2015; McCannel et al., 2014; Niro et al., 2015; van Dijk et al., 2015) (Figure 16, E-F). This effect is reversible and
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may originate from either acute RPE toxicity by inhibition of the MAPK pathway that lies downstream of the fibroblast growth factor receptor, a key actor for the maintenance of RPE integrity (van der Noll et al., 2013). But the exact mechanisms might be more complex since ERK activation takes place in the mature neurosensory retina (van Dijk et al., 2016). The upregulation of AQP1 in RPE induced by MEK inhibitor suggest a compensatory mechanism to hyperosmotic pressure in the subretinal space (Stamer et al., 2003).
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ACCEPTED MANUSCRIPT 6.6
Hypoproteinemia
ME can also develop in patients with pronounced hypoproteinemia, due to severe renal failure (Williams et al., 2006), deficient protein intake or other causes. This mechanism may also contribute to manifestations of advanced diabetic ME, such as hard exudate deposits,
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that may improve after initiation of hemodialysis (Matsuo, 2006). Several observations of serous macular detachment due to hypoproteinemia, for instance during the course of
nephrotic syndrome of various origins, have been reported (Bilge et al., 2016; Izzedine et al.,
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2014) (Figure 16, I-J). We suggest that disruption of the normal protein gradients between the vascular and the retinal compartments induced by low proteinemia, leads to an
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impairment of water exit mechanisms, and to the development of ME or subretinal fluid accumulation. Furthermore, this “pure” mechanism is a supportive example for the putative critical role of proteins in retinal hydro-ionic homeostasis.
Paracentral acute middle maculopathy
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6.7
Paracentral acute middle maculopathy is a recently described entity consisting in focal hyperreflective intraretinal lesions at the level of the inner nuclear layer, visible on OCT
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(Figure 16, K-L). These lesions are usually associated to a relative thickening of the inner nuclear layer, and originate from focal retinal ischemia (Sarraf et al., 2013). They develop in
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the context of retinal vascular occlusions, either arterial (Bonini Filho et al., 2015; Christenbury et al., 2015) or venous (Ghasemi Falavarjani et al., 2017; Sarraf et al., 2013). OCT angiography demonstrated that focal decrease in perfusion of the deep capillary network, located at the outer border of the inner nuclear layer, is the probable cause of this phenomenon (Nemiroff et al., 2016). This focal deregulation of fluid homeostasis in the retina results purely from cellular hypoxia in an ischemic microenvironment secondary to vascular occlusive disease and therefore represents a purely ischemic mechanism of ME.
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7 Major pathways identified as causative of macular edema 7.1
VEGF/PGF pathway
The VEGF family includes VEGF-A, -B, -C, -D, -E and PGF. Among all factors, the
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overexpression of PGF and VEGF-A causes increased vascular permeability (Witmer et al., 2003). VEGF-A binds to two high-affinity tyrosine kinase receptors, VEGFR-1 (also known as flt-1) and VEGFR-2 (also known as kinase insert domain-containing receptor or KDR), while
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PGF binds with high affinity only to VEGFR-1. Whether VEGF-A influences vascular permeability via VEGFR-1 or 2 remains controversial. It has been shown in human
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endothelial cells that VEGF-A mutants binding selectively to VEGFR-2 are able to induce vascular permeability in vivo, whereas VEGF-A mutants selectively binding to VEGFR-1 do not exert these effects (Gille et al., 2001; Keyt et al., 1996). However, activation of the VEGFR-1 pathway by PGF/VEGF-A signaling may be involved in other retinal vascular cell types such as pericytes and vascular smooth muscle cells (Luttun et al., 2002). VEGF-A
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mediates vascular hyper-permeability by increasing both paracellular and transcellular permeability. VEGF-A regulates endothelial junction proteins through VEGFR-2 activation (Kim et al., 2009). For instance, in endothelial vascular cells, VEGF-A induces occludin
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phosphorylation via activation of PKCβ (Harhaj et al., 2006; Murakami et al., 2012) and
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occludin down-regulation via an increase of free cytosolic ß-catenin and up-regulation of uPAR (Behzadian et al., 2003). Loss of VE-cadherin by endocytosis has been also implicated in paracellular hyperpermeability via VEGFR-2 (Gavard and Gutkind, 2006). The VEGF/VEGFR-2 pathway also increases transcellular permeability in retinal endothelial cell by transcytosis involving caveolae and a NOS-dependent mechanism (Feng et al., 1999; Hofman et al., 2000). Clinically, aqueous humor and vitreous levels of VEGF-A, PGF, and VEGFR-1 and -2 are elevated in eyes with ME due to diabetes (Table 1) and retinal vein occlusion (Tables 2 and 3) (Funatsu et al., 2003; Noma et al., 2015).
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7.2
Kinin/kallikrein system
Kinins, including bradykinin and kallidin are polypeptides with natural vasodilator and endothelial protector properties. They are produced by the action of kallikreins on kininogens,
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produced by the liver and released into the plasma. There are two types of kallikrein, the tissue and plasma kallikreins. The angiotensin converting enzyme (ACE) belongs to the kinin inactivating enzymes that regulate kinin activity, linking the renin-angiotensin system (RAS) to the kinin/kallikrein system (KKS) (Regoli and Gobeil, 2017; Wilkinson-Berka and Fletcher,
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2004). Effects of kinins are mediated by two related G-protein-coupled receptors, the
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bradykinin receptors, B1R and B2R. Both receptors are expressed in the normal retina and choroid (Ma et al., 1996), but vasodilation may be mediated by B2R that is predominantly expressed in the vascular endothelium, GCL, INL and ONL (Yasuyoshi et al., 2000). However, B1R is over-expressed in response to oxidative stress in the central nervous system, in a type-2-diabetes rat model (Dias et al., 2015), and in post-mortem eyes of
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patients with type 1 or 2 diabetes (Bhat et al., 2014). Cumulative evidence has emerged that the KKS system is activated and exerts pro-edematous and pro-inflammatory effects in diabetic retinopathy. Retinal vascular permeability was increased in animal models using
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intravitreal injection of plasma kallikrein or bradykinin and was reduced after KKS inhibition using either transgenic animals or antagonists, as exhaustively reviewed (Bhat et al., 2014).
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Specific B1R antagonists have been developed and were found efficient in reducing retinal vascular permeability and thickening (Lawson et al., 2005). However, differential and paradoxical effects of bradykinins might be observed, mediated by either B1R or B2R, depending on the pathologic conditions (Sang et al., 2016; Shi et al., 2016). At the cellular level, bradykinin induces vasodilation through the activation of calcium-dependent eNOS and prostacycline via B2R, and induces vasodilation and permeability through the up-regulation of iNOS via B1R. B2R activation can also induce permeability through Src activation pathway (Liu and Feener, 2013). Ultimately, hyper-permeability is, at least in part, mediated by the down-regulation of claudin-5 through a calcium-induced calcium-release mechanism (Zhou 58
ACCEPTED MANUSCRIPT et al., 2014) and by the phosphorylation of cadherin (Liu and Feener, 2013). Finally, the KKS interacts with other pathways involved in the pathogenesis of ME, such as the complement factor system, as illustrated by the occurrence of angioedema due to over-
7.3
Angiopoietin-2 (Ang2)/Tie2/α3β1 pathway
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bradykinin activity and mutations in C1-inhibitor coding genes (Reshef et al., 2016).
The Ang2/Tie-2 pathway is mostly involved in vessel stabilization and proliferation. But it also intervenes in retinal vascular permeability through the neuro-glio-vascular cross-talk. As
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detailed above (3.1.1.3), Ang2 induces pericyte apoptosis through Tie2 and α3β1 binding.
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Ang2 also induces apoptosis of retinal astrocytes through avβ5 binding (Yun et al., 2016a). Novel therapeutic strategies are developed to regulate the angiopioetin-2/Tie2 pathway (Saharinen et al., 2017).
7.4
Renin-angiotensin system and angiotensin-converting enzyme
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The renin-angiotensin system (RAS), particularly via the angiotensin-converting enzyme (ACE), is involved in the progression of diabetic retinopathy (Fletcher et al., 2010). In a recent meta-analysis collecting 21 randomized clinical trials with 13,823 participants, RAS
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inhibition was associated to a reduced risk of progression and an increased regression rate of diabetic retinopathy. Moreover, a polymorphism in the gene encoding ACE is associated
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with the risk of developing diabetic retinopathy, particularly in the type 2 diabetic Asian population (Fletcher et al., 2010). Angiotensin-2 exerts its action via the angiotensin-2 receptors type 1 (AT1) and 2 (AT2) that are differentially expressed in endothelial cells or glial cells. Angiotensin exerts vasoconstriction and pro-angiogenic effects but no direct effect is known on vascular permeability (Fletcher et al., 2010).
7.5
The mineralocorticoid pathway
The mineralocorticoid receptor (MR) is expressed in the retina and particularly in RMG cells
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ACCEPTED MANUSCRIPT and in endothelial cells of the retinal and choroidal vasculature. Aldosterone, the specific MR ligand, regulates the expression and distribution of several ion and water channels in RMG cells (such as AQP4, Kir4.1, αENac) and induces retinal thickening through alteration of glial drainage mechanisms (Zhao et al., 2010). On the other hand, MR activation in the choroidal
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vasculature induces vasodilation and permeability through up-regulation of the endothelial vasodilatory K+ channel KCa2.3, expressed in choroidal but not in retinal vessels (Zhao et al., 2012). In vascular endothelial cells, activation of the MR pathway induced an increased paracellular permeability through activation of the Rho kinase and subsequent actin cytoskeleton
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stress (Kirsch et al., 2013). MR over-activation is the putative pathogenic mechanism accounting for the efficacy of oral MR antagonist therapy in patients with central serous
7.6
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chorioretinopathy (Daruich et al., 2015b).
Inflammation and inflammatory mediators with pro-edematous effects
Inflammation is a protective response of the innate immune system programmed to neutralize harmful stimuli, and initiate a healing process restoring tissue homeostasis. The
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retina is an immune-privileged organ due to the blood-retinal barriers, and the absence of lymphatic vessels or dendritic cells. Yet, local activation of immune competent resident cells such as microglia/macrophages, choroidal mast cells but also astrocytes, RMG and RPE
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cells occurs in response to any injury. When immune resident cells are activated locally without barrier rupture, no sign of inflammation is clinically detected, a silent process termed
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“para-inflammation”. Histamine, VEGF and permeating cytokines, produced by resident immune cells, together with reduced protective mechanisms, contribute to blood-retinal barriers breakdown. ME is one of the cardinal signs of retinal inflammation and should be considered as an “inflammatory sign”. Numerous cytokines, chemokines and permeating factors have been repeatedly found elevated in ocular fluids of eyes with ME, some studies identifying correlations with retinal thickness. Tables 1-3 recapitulate the factors measured in diabetic ME, central and branch vein occlusions, respectively. However, the increased level of a given cytokine is not
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ACCEPTED MANUSCRIPT sufficient to establish a causative pathogenic link. Indeed, many endogenous neutralizing mechanisms are also involved, for instance via soluble receptors which have not been systematically measured in these studies. Moreover, secretion of these inflammatory mediators could also result from ME. Whether inflammation causes ME in a priori non-
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inflammatory retinal diseases remains a subject of debate, but inflammatory pathways are clearly involved, as evidenced by the clinical efficacy of intravitreal corticosteroid agents in ME secondary to diabetes (Boyer et al., 2014; Daruich et al., 2015a), or vein occlusions
(Haller et al., 2011). On the other hand, subclinical retinal chronic inflammation can certainly
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create environmental conditions for ME to develop. Submitted to chronic oxidative,
metabolic, degenerative stress, retinal cells activate pathways that can each favor the
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occurrence of blood barrier function alterations and/or alter physiologic drainage mechanisms. In all these processes, ageing acts as a favoring factor. It precipitates homeostasis disruption, since ocular tissue are modified with advancing age, as illustrated by the increasing deposits in Bruch’s membrane, the basal membrane of the RPE, and the
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decreased density of capillary endothelial cell and pericytes. A list of growth factors and inflammatory mediators potentially involved in retinal or choroidal vascular permeabilization
7.6.1
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is provided in Table 5.
Resident immune-competent cells
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7.6.1.1 Microglia/macrophages Microglia recruitment and activation occurs in response to any retinal stress or insult. Microglia migrate from their physiologic perivascular location to the outer retina causing alteration of RPE functions, release of several pro-permeabilizing factors (particularly TNF, IL-1, NO and VEGF), and reducing the production of anti-inflammatory mediators at the proximity of the retinal vasculature (Grigsby et al., 2014; Vecino et al., 2016; Wang and Wong, 2014). The chemokine, CCL2 (also referred to as MCP-1), is elevated in the ocular
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ACCEPTED MANUSCRIPT media of patients with ME. CCL2 is produced by inflammatory cells, resident microglia, but also by RMG and RPE cells under IL1-β produced by microglia (Natoli et al., 2017).
7.6.1.2 Retinal Müller glial cells
include, among others:
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RMG cells, in response to metabolic or ischemic stress show multiple responses. These
up-regulation of the intermediate filament protein, GFAP,
•
reduced potassium conductance (reduced drainage mechanisms and capillary
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•
dilation)
modified location and expression of AQPs, including AQP4 which reduces drainage
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•
mechanisms (Nicchia et al., 2016) •
production of pro-permeabilizing cytokines such as VEGF, IL-6 and MCP-1 (Eastlake et al., 2016; Liu et al., 2015) and other chemokines (Rutar et al., 2015) activation of the renin-angiotensin pathway (Wilkinson-Berka et al., 2010)
•
activation of the small GTPase Rho (Tura et al., 2009)
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•
In addition, RMG swelling has been reported in response to metabolic stress (Bringmann et
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al., 2004, 2006; Bringmann and Wiedemann, 2012).
7.6.1.3 Astrocytes
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High glucose conditions lead to the production of oxidative stress and induced the production of TNF-α and IL1-β and iNOS through activation of NF-κB. Enhanced Nuclear factor erythroid 2-related factor 2 (Nrf2) translocation and the production of anti-oxidant enzymes was associated with protection against oxidative-stress induced apoptosis (Shin et al., 2014).
7.6.1.4 Retinal pigment epithelial cells RPE cells are posterior uveal cells that express Toll-like receptors (TLR), components of the innate immunity (Kumar et al., 2004) and, P2X7 receptor (Guha et al., 2013), activated by
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ACCEPTED MANUSCRIPT mast cells degranulation and inflammation. RPE can produce a large number of cytokines and chemokines and closely interact with choroidal macrophages/microglia and mast cells. Activation of P2X7 receptor and TLR receptors expressed in RPE cells induces differential responses. Photo-oxidative stress, aging, oxidization of lipids, impairment of autophagy,
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accumulation of β-amyloid, iron loading or Alu RNA, activation of inflammasome through P2X7 receptor (Guha et al., 2013), lead to several metabolic changes contributing to AMD pathogenesis. These changes include the release of pro-inflammatory cytokines, with polarized apical secretion of IL-1β and IL-18, and the basal down-regulation of VEGF
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(Brandstetter et al., 2015; Gelfand et al., 2015; Gnanaguru et al., 2016; J. Liu et al., 2016;
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Mohr et al., 2015; Tarallo et al., 2012). Via TLR-4 activation, LPS induces NF-κB activation, subsequent inflammatory cascade (Wang et al., 2017) and disruption of the epithelial barrier (Zech et al., 1998). The expression of TLR-4 in RPE cells is enhanced by C5a, which induces the production of IL-6 and IL-8 (Zhu et al., 2015). The sub-retinal microglial accumulation contributes to the pro-inflammatory mediator production by RPE cells. We have
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shown that transcytosis of activated microglia/ macrophages through RPE cells contributes to cell trafficking without alteration RPE tight junction (Omri et al., 2011) (Video 3). Other mechanisms of macrophage/microglia elimination by apoptosis regulated by interaction of
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CFH with CD47 have been demonstrated (Calippe et al., 2017). Their impairment could play important role of pathogenic sub-retinal inflammation that takes place in numerous “a priori”
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non-inflammatory retinal diseases.
7.6.2
Inflammatory molecules
7.6.2.1 Complement components The complement system plays important roles in macular edema through mechanisms related to its state of activation. In example, C1 inhibitor is an important regulator of the kinininduced permeability. The anaphylatoxins C3a and C5a induce hyper-permeability through degranulation of mast cells, present in the choroid. In addition, C5a but not C3a induces
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ACCEPTED MANUSCRIPT direct endothelial cell retraction and increases paracellular permeability on human endothelial cells in vitro through PI3K and Src kinase pathway (Schraufstatter et al., 2002). Preclinical experimental studies suggest that mostly TNF and IL-1ß may have direct effect on
7.6.2.2 Tumor Necrosis Factor-α (TNF-α)
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retinal barrier integrity.
TNF-α is one of earliest cytokines with permeating properties being released at the site of inflammation as it is the only cytokine stored in mast cells granules that can be immediately
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released by mast cells degranulation (Gu et al., 2015). The role of TNF-α on vascular
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permeability has been demonstrated by in vitro studies on various endothelial cell types, including brain and retinal microvascular cells, as well as ex vivo and in vivo. Depending on the micro-environment and on its concentration, TNF-α can induce vascular permeability but also endothelial cell death as detailed in 3.1.1.3. Herein, the main mechanisms of TNF-αinduced permeability will be summarized, with an emphasis on its effects on brain and retinal
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endothelial cells.
On cultured human umbilical vein endothelial cell line cells (HUVECs), TNF-α induced permeability through activation of RhoA/ROCK pathway, contraction of actin cytoskeleton
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and reduced VE-Cadherin (J. Peng et al., 2011; Wei et al., 2017). Depending on the applied concentration, TNF-α induced either junction disruption at low dose (identified by claudin-5
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delocalization), or apoptosis at higher concentrations (Miyazaki et al., 2017). On mouse brain endothelial cells, TNF-α repressed claudin-5 expression through NF-kB signaling (Aslam et al., 2012). Using human brain microvascular endothelial cells (HBMvECs), both TNF-α and IL-6 induced permeability through decreased expression, phosphorylation and cytoplasmic translocation of ZO-1 (Rochfort and Cummins, 2015). In bovine retinal endothelial cells, the down-regulation and translocation of ZO-1 and claudin-5 was preceded by PKCζ phosphorylation (Aveleira et al., 2010). On tridimensional chips modelling the multicellular blood-brain barrier component, TNF-α induced the production of G-CSF in presence of
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ACCEPTED MANUSCRIPT endothelial cells and pericytes, and the increase of IL-6 and IL8 in the presence of astrocytes or pericytes. However, this was not observed in endothelial cells cultured alone, suggesting that TNF-α initiates the production of other cytokines at the glio-vascular interface. These results are consistent with observations that TNF-α does not directly disrupt brain endothelial
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cell junctions when co-cultured with astrocytes, but that junctions were disrupted when cocultured astrocytes were pre-exposed to TNF-α-conditioned media (Chaitanya et al., 2011). In addition, TNF-α leads to endothelial barrier disruption by exacerbating several other mechanisms. For instance, polymorphonuclear endothelial transcytosis, increasing
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intercellular permeability, occurs through ICAM-1 and is enhanced by TNF-α (Wong et al., 2007). TNF-α contributes to ischemic VEGF-induced leukostasis but does not seem to
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intervene directly in VEGF-induced retinal permeability, suggesting that inhibition of both pathways could be synergistic (Vinores et al., 2007). Different observations were made in lipopolysaccharide (LPS)-induced uveitis, where anti-TNF-α suppressed leukostasis, retinal vascular leakage, and apoptosis (Koizumi et al., 2003). In addition, TNF-α produced by LPS-
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stimulated microglia was also able to disrupt brain microvascular cells barrier, strengthening the notion that the glio-vascular cross-talk regulates TNF-α-induced permeability (Nishioku et al., 2010).
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TNF-α not only disrupts the inner blood-retinal barrier but also increases RPE permeability. In rodent RPE cells, TNF-α directly influences ZO-1 distribution (Shirasawa et al., 2013; Zech
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et al., 1998), while in human embryonic stem cell (hESC)-derived RPE cells, TNF-α-induced permeability is associated with increased expression of MMP1, MMP2 (Juuti-Uusitalo et al., 2015) and MMP9 (Wang et al., 2012). In contact with RPE, activated macrophages and microglia produce TNF-α in models of AMD, such as the Cxcr5-knock-out mouse (Huang et al., 2017, p. 5; Müller et al., 2003). TNF-α is involved in the close interaction between RPE and macrophages. Indeed, co-cultures of macrophages and RPE produce more MCP-1, IL-6, and VEGF, as compared to separate cultures. This effect is mediated by the production of TNF-α by macrophages, negatively regulated by contact with RPE cells (Yamawaki et al.,
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ACCEPTED MANUSCRIPT 2016). On the other hand, TNF-α induces ICAM-1 expression and promotes monocyte adhesion through TNF receptor-1 (TNFR1) activation and the PKCδ/JNK1/2/c-Jun pathway (Lee et al., 2015). Finally, the interaction of TNF-α and VEGF is complex and may depend on the inflammatory
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status of the RPE. TNF-α decreases VEGF secretion in highly polarized RPE cells but increases it in non-polarized RPE cells, related to the cross-talk between the c-Jun N-
terminal kinase (JNK) and NF-κB pathways (Terasaki et al., 2013). TNF-α also enhances VEGF expression in a model of induced choroidal neovascularization (Wang et al., 2016).
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To summarize, the role of TNF-α in RPE permeability is complex. It implies direct effects through MMP activation, and indirect effects through a controlled cross-talk with
7.6.2.3 Interleukin-1β (IL-1β)
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macrophage/microglia, suggesting differential effects in the normal and pathologic retina.
The intravitreal administration of IL-1β induces a reversible retinal inflammatory response. It
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is accompanied by inner blood-retinal barrier breakdown and leukocyte recruitment, through retinal endothelial transcytosis. This effect is probably mediated by histamine in a biphasic time-dependent pattern, with barrier opening observed 4-8 hours and 24-48 hours post-
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injection (Bamforth et al., 1997). In the rabbit retina, intravitreal injection of IL-1β disrupted endothelial cell junctions (Claudio et al., 1994; Rosenbaum et al., 1987). VEGF can
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contribute to the effect of IL-1β on vessels, as shown in transgenic mice overexpressing IL1β in the lens, in which an early peak of retinal VEGF expression was concomitant to bloodretinal barrier breakdown and inflammatory cell infiltration (Vinores et al., 2003). Similarly, in human brain astrocytes, IL-1β induces the expression of HIF-1α and VEGF-A (Argaw et al., 2006). However, several in vitro experiments have demonstrated direct effects of IL-1β on endothelial cells. After endothelial cells stimulation by IL-1β, over 2500 genes were differentially expressed, including genes associated with apoptosis, cell cycle, NF-kB cascade, chemotaxis, and immune response. Interestingly, claudin-1 was up-regulated, but
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ACCEPTED MANUSCRIPT claudin-5 and occludin were down-regulated (Williams et al., 2008). In brain microvascular endothelial cells, IL-1β down-regulated the expression occludin, which increased permeability, when submitted to hypoxic, but not normoxic conditions (Yamagata et al., 2004). This suggests that the mechanisms of IL-1β on permeability may differ according to
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the pathologic conditions. IL-1β-induced permeability could be driven by: •
PKC-θ-dependent phosphorylation of ZO-1 (Rigor et al., 2012)
•
alterations in the f-actin cytoskeleton due to the phosphorylation of MLCs by ROCK
•
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activation, counter-regulated by neuregulin-1β (Wu et al., 2016)
decreased VE-cadherin expression, counter-regulated by BDNF (Matsuda et al.,
•
melatonin (Yuan et al., 2011).
7.6.2.4 Interleukin-6 (IL-6)
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2015)
Aqueous humor and vitreous levels of IL-6 are elevated in eyes with ME associated to retinal vein occlusion or diabetes (Koss et al., 2012; Noma et al., 2009, 2015; Owen and Hartnett,
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2013). Recently, it has been found that IL-6 produced by microglia induces STAT3 activation in retinal endothelial cells under high glucose conditions, down-regulating occludin and ZO-1, and producing VEGF (Yun et al., 2016b). In human brain microvascular endothelial cells, IL-6
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decreases the expression of VE-cadherin, occludin and claudin-5 (Rochfort et al., 2014).
7.6.2.5 Interleukin-8 (IL-8) Aqueous humor and vitreous levels of IL-8 are also elevated in patients with ME associated to retinal vein occlusion or diabetes (Fonollosa et al., 2010; Noma et al., 2015; Owen and Hartnett, 2013; Sonoda et al., 2014). Human endothelial vascular cells treated with increased concentration of IL-8 showed a down-regulation of tight junction proteins, including occludin, claudin-5 and ZO-1 (Yu et al., 2013). In human microvascular endothelial cells, permeability induced by IL-8 required the activation of VEGFR-2 (Petreaca et al., 2007). IL-1β potently
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ACCEPTED MANUSCRIPT stimulates IL-8 expression in human RMG cells, mainly through the p38 MAPK and ERK1/2 pathways (Liu et al., 2015).
7.6.2.6 Intercellular Adhesion Molecule 1 (ICAM-1)
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Vitreous level of ICAM-1 are elevated in patients with ME associated to retinal vein occlusion (Noma et al., 2011a, 2013, 2014) or diabetes (Funatsu et al., 2005, 2009). Aqueous humor level of ICAM-1 have been correlated with the height of subretinal fluid in DME (Zhu et al., 2014). In a rat model, increased leukostasis, vascular permeability and capillary non-
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expression in the retina (Miyamoto et al., 2000).
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perfusion after intravitreal VEGF injections was associated with up-regulation of ICAM-1
7.6.2.7 Transforming growth factor-ß (TGF-β)
TGF-ß secreted by glial cells stimulates the production of MMP-9, which in turn degrades
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occludin, contributing to inner blood-retinal barrier disruption, as detailed in 3.1.1.1.
In summary, any chronic retinal stress induces multiple subclinical molecular and cellular inflammatory responses, creating a favorable environment for ME to develop. In this context,
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ME appears clinically upon an additive acute event (such as hypertension), or by reaching the threshold of compensatory mechanisms. When ME occurs, breakdown of blood retinal
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barrier leads to the infiltration of circulating cells, that together with activated resident cells amplify these inflammatory reactions.
8 General mechanisms contributing to diabetic ME Chronic hyperglycemia activates cardinal pathways, recognized as main contributors to diabetes-induced damages at cell, tissue and organ levels (Figure 17). In hyperglycemic conditions, retinal cells are subjected to nutrient excess, causing mitochondrial and endoplasmic reticulum stress (Wellen and Thompson, 2010). When early compensatory
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ACCEPTED MANUSCRIPT mechanisms are overwhelmed cellular damage may occur. We will briefly review the mechanisms involved, most of which were previously mentioned in specific paragraphs. Increased fluid entry and impaired fluid drainage are both involved in diabetic ME (Figure 17). The main cellular mechanisms dysregulated in diabetic conditions and leading to ME are
8.1
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summarized in the Video 2.
The Polyol pathway
Upon long-lasting exposure to glucose excess, the hexokinase enzyme becomes saturated
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and glucose is derived toward an alternative glycolysis pathway through sorbitol and fructose
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production. The level of cellular NADPH is lowered, leading to oxidative stress. Sorbitol does not enter the cell membrane creating creating osmotic stress, and it induces nitrogen glycation of proteins, contributing to the accumulation of AGEs.
8.2
The AGE pathway
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Non-enzymatic glucose-protein reactions integrate a heterogeneous group of irreversible molecular adducts called AGEs, whose normal accumulation with ageing is exacerbated by diabetes. AGEs increase fibrosis, and activates inflammation through the formation of
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collagen crosslinks, and by interaction with the AGE-receptor (RAGE), which impairs calcium handling. AGEs have been associated with diabetic microangiopathy (Madonna et al., 2017).
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In experimental diabetic conditions in vitro and in vivo, AGEs were shown to induce retinal pericytes apoptosis (Denis et al., 2002; Yamagishi et al., 1995) and accumulate in RMG cells. These processes cause hydro-ionic channels dysfunction, oxidative stress and GFAP activation (Curtis et al., 2011; Lecleire-Collet et al., 2005), as well as RMG cell inflammatory responses through RAGE activation (Zong et al., 2010). Exposure of RPE cells to AGEs also activated inflammatory response via the NF-kB and JAK-STAT pathways (Lin et al., 2013).
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Protein kinase C (PKC) activation
There are multiple isoforms of PKC. The conventional PKCs (PKC-α, -β1, -β2, and -γ) are activated by phosphatidylserine, calcium, and diacyglycerol (DAG). Novel PKCs (PKC-δ, -ε, θ, and -η) are activated by phosphatidylserine and DAG but not by calcium, and atypical
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PKCs (such as PKC-ζ) are not activated by calcium or diacylglycerol (DAG) but by phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP3), which is mainly produced by the
phosphoinositide 3-kinase (PI-3 kinase) (Idris et al., 2001). As a consequence of impaired glycolysis and glucose overload, DAG synthesis increases in cells and activates conventional
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and novel PKCs, particularly isoforms β δ, ε, and γ. Activation of the DAG/PKC pathway has
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been associated with endothelial permeability and dysfunction, vasoconstriction, angiogenesis, leukostasis and inflammation (Aiello, 2002; Das Evcimen and King, 2007; Geraldes and King, 2010). Under hyperglycemic conditions, the activation of PKC-δ induces retinal vascular cell death (Geraldes et al., 2009). Atypical PKCζ, activated in response to TNF-α in endothelial and RPE cells in diabetic conditions, contributes to tight-junction
al., 2017).
Local renin-angiotensin system (RAS)
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destabilization and cell polarity alteration (Aveleira et al., 2010; Omri et al., 2013; Sewduth et
In addition to the systemic RAS regulating vascular and extravascular concentrations of Na+
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and K+, and maintaining blood volume (Patel et al., 2017), local RAS pathways participate in the homeostasis of vascularized organs such as kidney and retina, and are activated in diabetic conditions (Giese and Speth, 2014). For instance, in the diabetic retina, activation of the local RAS and increased retinal levels of angiotensin-2 can contribute to ME via its receptor AT1, and the downstream pathological chain of events, as detailed in 7.4. This pathway has also indirect effects on other mechanisms involved in diabetic ME such as oxidative stress, production of AGEs, polyol pathway, and PKCs (Steckelings et al., 2009).
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Inflammation and oxidative stress
Recent investigations have shown that chronic hyperglycemia induces oxidative stress and inflammation in the retina, which constitute early processes in the development of diabetic ME (Arroba and Valverde, 2017; Roy et al., 2017; Yu et al., 2015). As detailed in 7.6, local
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inflammation is a major and permanent pathogenic mechanism contributing to diabetic ME. Involved pathways are summarized in Figure 18. Inflammation is not only a consequence of barrier dysfunction, but also an early local mechanism contributing to barrier alteration and leukostasis. As a consequence of the activation of pathways hereby listed from paragraphs
8.6
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8.1 to 8.5, all retinal functions preventing the formation of edema in the retina are altered.
Blood-retinal barrier dysfunction
Hyperglycemia induces a degradation of junctional complex partners, including occludin, claudin, JAM-A, ZO-1 (Saker et al., 2014; Stewart et al., 2016; Tien et al., 2013; Wang et al., 2015) and VE-cadherin (Navaratna et al., 2007; Rangasamy et al., 2011). VEGF contributes
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to diabetes-induced vascular permeability by the rapid phosphorylation of occludin and ZO-1, and by reducing occludin expression in endothelial cells (Antonetti et al., 1998, 1999). VEGF could also stimulate stress fiber formation and VE-cadherin disruption in the diabetic retina
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(Lee et al., 2016). The endothelial barrier is further disrupted by pro-inflammatory cytokines, such as TGF-ß released by glial cells (Behzadian et al., 2001), and interleukin-6 by activated
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microglia (Yun et al., 2016b). Leukostasis induced by hyperglycemia, nitrosative/oxidative stress, VEGF and TNF-α, favors endothelial permeability (Huang et al., 2015; Joussen et al., 2002; Leal et al., 2007; Vinores et al., 2007). It also potentially leads to retinal capillary endothelial cell apoptosis (Joussen et al., 2001). Among hallmarks of diabetic ME, the inner blood-retinal barrier rupture is characterized by important pericyte loss. As mentioned in section 3.1.1.3, several pathways contributing to pericyte loss have been identified. These pathways include Ang-2 via integrin-α3 and -β1 (Cai et al., 2008; Pfister et al., 2008), PEDF signaling (Geraldes et al., 2009),
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oxidative and nitrosative stress (Cao et al., 2013; Giebel et al., 2005, 2005; Kowluru et al., 2012; Navaratna et al., 2007). The OLM, that contributes to the outer blood/retinal barrier, is also damaged in diabetic ME, because of tight junction disruption due to the activation of PKC-ζ by hyperglycemia (Omri et al., 2013).
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More advanced events in the pathophysiology of diabetic ME, such as microaneurysms or neovascularization, contribute to its persistence and are controlled by angiogenic factors,
8.7
Drainage functions
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such as VEGF (Witmer et al., 2003) and PGF (Kowalczuk et al., 2011).
In RMG cells, diabetes alters the cellular distribution and expression of aquaporins (Fukuda
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et al., 2010), and reduces potassium conductance (Pannicke et al., 2006). Alterations in the expression pattern of aquaporins has also been described in the RPE of diabetic animal
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models (Hollborn et al., 2011).
Kinetic of events in the diabetic GK rat model
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The Goto Kakizaki (GK) rat is a spontaneous non-obese type 2 diabetic rat model, recognized as one of the most appropriate to study complication of type 2 diabetes mellitus (Akash et al., 2013). GK rats spontaneously develop hyperglycemia at about 3 months after birth, that persists for lifetime. Retinopathy progressively develops in the GK rat (Gong et al., 2016) and clear signs of microangiopathy can be detected after one year (Figure 19E). Vascular permeability, the hallmark of blood-retinal barrier breakdown was correlated to iNOS expression and activity in the GK rat retina (Carmo et al., 2000) and associated with decreased pericytes coverage, beginning at 8 months (Agardh et al., 1997). Accordingly,
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ACCEPTED MANUSCRIPT VEGF levels increased in the retina of GK rats at 7 months (Sone et al., 1997). We have previously characterized the early retinal changes occurring in GK rats from the onset of the hyperglycemia to the development of microangiopathy (Omri et al., 2013). Note that the GK rat is a spontaneously mutant albino Wistar rat. Unfortunately, fluorescein angiography
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cannot be performed very well in the GK rats. Thus, vascular abnormalities were evaluated by vessel labeling on flat-mounted retina after animal sacrifice and could not be followed longitudinally in vivo. The first clinical sign detectable on OCT and on histologically observed on retinal sections is an increase in choroidal thickness due to choroidal vessels dilation,
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including capillaries, observed as early as 4 months of age (Figure 19 A-D). In the
neurosensory retina, at the onset of hyperglycemia (at 3 months), a significant increase in
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the expression of Kir4.1 and AQP9 was detected. In the Wistar normoglycemic rat, Kir4.1 and AQP4 expression increase between 3 and 6 months and then remain stable. But in the GK diabetic rats, following increase in AQP4 expression at 3 months, the levels of expression of Kir4.1 and AQP4 remain significantly lower than in the Wistar control rat
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(Figure 20 B and D). Immunolocalization confirmed the decreased expression of Kir4.1 and AQP4 in the diabetic GK rat with a more pronounced reduced distribution in the INL and around the vessels (Figure 20 A and C). The aquaglyceroporin AQP9 remains highly up-
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regulated at all time points (Figure 20 E). The retinal thickness increases particularly in the outer retinal layers as early as 8 months and with progresses with longer diabetes duration
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(18 months), demonstrating that retinal edema occurs in GK rats after long-term hyperglycemia.
Alterations of the outer blood-retinal barrier, both at the RPE and OLM, occur early at the onset of hyperglycemia and progress between 6 and 12 months. The earlier changes in the RPE is the membrane translocation and activation of ROCK-1, which phosphorylates components of the actin cytoskeleton, inducing apical constrictions and stress fibers, and that can eventually lead to later non-apoptotic membrane cell blebbing in RPE cells (Figure 21 E, G and H). Polydispersity of RPE cell size and multinucleated syncytium are observed (Figure 21 F, H and I). The atypical PKCζ, which is associated to junction proteins at the cell
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ACCEPTED MANUSCRIPT membrane, translocates in the cytoplasma together with occludin (Figure 22), due to the down-regulation of the regulating N-terminal part of the kinase. This process induces its overactivation and the subsequent deregulation of junction stability (Omri et al., 2013). With prolonged hyperglycemia, PKCζ activity and junction proteins expression decrease. In
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addition, there is a reduction in the transcytosis mechanism through RPE cells. This reduces the elimination of activated microglia from the subretinal space towards the choroid, and subsequent production of pro-inflammatory mediators (Figure 23 and Video 3) (Omri et al., 2011). At 12 months, focal leakage from the choroid to the subretinal space demonstrates
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the functional alteration of the RPE barrier (Rothschild et al., 2017). At the OLM, similar changes are observed leading to alteration of the tight-like junction barrier between
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photoreceptor segments and RMG cell apices. Occludin and ZO-1 are removed from the OLM junction plaques and are internalized into RMG cells (Figure 24), suggesting that OLM alterations may occur earlier at the molecular level, than detected clinically by the loss of the OLM hyperreflective band on OCT (X. Chen et al., 2012). At 18-24 months of age, retinal
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vascular occlusions and enhanced permeability are associated with vascular endothelial cell blebbing (Rothschild PR et al Sci Report 2017). In summary, although no rodent animal model can recapitulate all the features of a human
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retinal disease, the GK diabetic rat develops many features of human diabetic retinopathy. The kinetics of events in this model show that alterations in the choroid may precede
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clinically detectable retinopathy, as in humans (Gupta et al., 2017; Tavares Ferreira et al., 2017). Microangiopathy can be detected in the GK rat but appears as a late phenomenon after alterations occur in retinal neurons, RPE and RMG cells. Importantly, early and continuous modifications of the RPE polarity and cytoskeleton, can have important consequences on transport and on the sub-retinal micro-environment (Philp et al., 2003; Shirasawa et al., 2013). In addition, microglial activation and sub-retinal accumulation of microglia contribute to photoreceptor inflammatory injury (Grigsby et al., 2014; Omri et al., 2011). These important non-clinically detectable retinal alterations, present long before
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ACCEPTED MANUSCRIPT macular edema is clinically detected, may explain the inconstant visual recovery after resolution of ME in diabetic patients.
9 Concluding remarks
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Homeostasis of a tissue is secured by the proper regulation of its energetic metabolism, its hydro-ionic balance, and intercellular communications. As a consequence of acute or chronic stress, edema may occur and spontaneously resolve through lymphatic drainage as tissue
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homeostasis is restored. In the retina, like in brain parenchyma, there are no lymphatic
vessels, and limited protein and fluid drainage takes place in normal conditions because
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barriers actively maintain the local micro-environment from any uncontrolled entry. The retina and particularly the macula are functionally extremely sensitive to any change in their hydration state to allow the proper transmission of light to photoreceptors. In addition, any disturbance of the local microenvironment subsequently threatens the structure and neural function of the macular region. Thus, regulatory mechanisms specific to the macula should
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be present. We have hypothesized that a “glymphatic system”, closely similar to mechanisms observed in the brain, is an important and unappreciated mechanism of fluid and protein drainage from the macula. While extensive research has been conducted to decipher the
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mechanisms of barrier disruption and to identify molecules inducing or preventing the formation of edema, little attention has been paid to the exact molecular and cellular
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specificity of the macula and the fovea. Yet, no animal or cellular model can equal the knowledge gained by analysing the structure of the human macula. With the development of novel imaging technologies, major improvements of the in vivo structural analysis of the macula should be expected. In parallel, molecular and metabolic analysis of cells composing the macula should be explored in normal and pathologic conditions using more sophisticated omics technologies. The return to basic knowledge revisited by multidisciplinary technological tools will most probably expand our vision on macular edema and help us dive below its surface. With this new knowledge, innovative therapies will emerge.
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ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS This work was supported by the Agence Nationale de la Recherche, France (ANR-15-CE180032 “ROCK SUR MER”), by the Swiss National Science Foundation (#320030_156401), by the Faculty of Biology and Medicine Research Commission Fund, University of Lausanne,
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Switzerland, by the Abraham J. & Phyllis Katz Foundation, Atlanta, GA and by the Lowy Medical Research Institute, San Diego, CA. The authors do not have any conflicting interest related to the content of this work.
The authors wish to acknowledge Michèle Savoldelli for electronic microscopy image, and
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Gunilla Norrgren (Allergan, Dublin, Ireland) for technical support in video production.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Schematic representation of retinal layers and main retinal cell types. Histology section of a human retina at the mid-periphery, stained with hematoxylin-eosin. Color drawings of the different neuronal cell types and the retinal glial Müller cells (RMG) are
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superimposed on the histologic picture.
ILM= Inner Limiting Membrane; GCL= Ganglion Cell Layer; NFL= nerve fiber layer; IPL= Inner Plexiform Layer; INL= Inner Nuclear Layer; OPL= Outer Plexiform Layer; ONL= Outer
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Nuclear Layer; OLM= outer limiting membrane; IS= Inner Segment; OS= Outer Segment;
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RPE= Retinal Pigment Epithelium.
Figure 2. Cellular and molecular components of the inner blood-retinal barrier. A. Human retinal section labeled for AQP4 that surrounds the vessels green), glutamine synthetase (GS, retinal Müller glial cells, red), and Dapi (nuclei, blue), showing that retinal glial Müller cells expressing AQP4, ensheath retinal capillaries at the level of the superficial
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and deep capillary plexuses (superior and inferior insets, respectively). In the deep capillary plexus (low inset) AQP4 and GS co-stain the vessel wall. B. Junctional complex between retinal vascular endothelial cells, forming the inner retinal
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barrier. Tight junctions are formed by occludin, claudins 1,2 and 5, JAM-A and C, and are linked to the actin cytoskeleton by ZO-1. Adherens junctions are formed by VE-cadherin, ß-
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catenin and N-cadherin. Gap junctions are formed by hemi-channels of 6 connexins. The pericyte-endothelial cell junction is composed mainly by N-cadherin and connexin-43. C. Schematic representation of the neuro-glio-vascular unit forming the inner blood-retinal barrier, composed by vascular endothelial cells, pericytes (p), retinal glial Müller (RMG) cells, astrocytes (a), microglia (mc). Retinal glial Müller cell projections are present at the level of all retinal vascular plexuses (superficial, SCP; intermediate, ICP and deep, DCP), while astrocytes are only present at the level of the superficial plexus.
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ACCEPTED MANUSCRIPT D. Astrocyte labelling by GFAP surrounding retinal vessels on a human retinal flat-mount shows that in the superficial vascular layers, astrocytes are the major glial component of the gliovascular unit. E. Human retinal section labelled by GFAP (astrocytes, and endfeet of RMG cells, red) and
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collagen IV (vessels, green) showing astrocyte labelling at the level of the superficial vascular plexus (left inset) and absence of astrocytes labeling at the level of the deep
vascular plexus (right inset). In the deep capillary plexus, the main glial component of the neuro-gliovascular unit is the RMG cell.
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GCL=ganglion cell layer; INL= inner nuclear layer; HFL= Henle fiber layer; ONL= outer
nuclear layer; OLM= outer limiting membrane; RMG= retinal Müller glial cell; a= astrocyte,
intermediate capillary plexus
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mc= microglia; SCP= superficial capillary plexus; DCP= deep capillary plexus; ICP=
Figure 3. Morphology of the macula in physiological conditions and with edema.
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A. Histological retino-choroidal section of a normal human macula. B. Macular optical coherence tomography section of a normal human retina and choroid (subfoveal choroidal thickness, arrow)
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C. Histological retino-choroidal section of the macula from a non-human primate (Macaca fascicularis) showing intraretinal and subretinal fluid.
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D. Montage from the same image, with superimposed representation of the intraretinal course of cone photoreceptors (pink), and elongated retinal Müller glial cells (green) and Muller cone cells (light green) E. Optical coherence tomography of a human macula presenting intraretinal and subretinal fluid. Note the smaller, round cystoid cavities at the level of the inner nuclear layer, and the larger, elongated cavities at the level of the outer plexiform/Henle fiber layer. F. En face optical coherence tomography of the same eye showing the radial distribution of the two types of cystoid hyporeflective cavities. Images E-F: courtesy Bruno Lumbroso.
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Figure 4. Multimodal clinical imaging of diabetic macular edema A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in a 70-year old woman with type 1 diabetes and severe nonproliferative diabetic retinopathy. On
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OCT, multiple intraretinal hyperreflective dots, extended cystoid edema, hard exudates and focal attenuation of the ellipsoid zone suggesting photoreceptor suffering are visible.
C-D: OCT and midphase FA in a 65-year old man with type 2 diabetes and moderate
nonproliferative diabetic retinopathy. OCT shows cystoid cavities and hyperreflective dots.
leakage from exudative microanevrysms.
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FA suggests a possible diffuse leakage from the outer blood retinal barrier, in addition to
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E-F: OCT and midphase FA in a 56-year old woman with type 2 diabetes exhibiting minimal nonproliferative diabetic retinopathy. OCT shows intraretinal cystoid cavities and subretinal detachment.
G-I: OCT and midphase FA in a 42-year old woman with type 1 diabetes and untreated
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hypertension. FA shows moderate proliferative diabetic retinopathy with a neovascular lesion on the superior temporal arcade, a crescent-shape inferior retro-hyaloidal hemorrhage, and panretinal peripheral photocoagulation scars (G). OCT showed macular cystoid edema (H).
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Due to the preserved visual acuity and the untreated hypertension, the initial management consisted in blood pressure control, which resulted in the regression of macular edema 3
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months later (I).
Figure 5. Multimodal clinical imaging of macular edema secondary to retinal vein occlusions
A-B: OCT and midphase FA in a 49-year old woman with inferior-temporal branch retinal vein occlusion. FA shows increased venous tortuosity and dilation with flame-shaped hemorrhages in the affected sector, extending to the capillary network drained by the occluded branch. OCT shows macular cystoid edema with discrete subfoveal detachment.
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ACCEPTED MANUSCRIPT C-D: OCT and midphase FA in a 53-year old woman with superior-temporal branch retinal vein occlusion. Wide-field FA shows increased venous tortuosity and dilation in the corresponding drainage territory, and extended peripheral capillary nonperfusion. Cystoid edema affecting the temporal aspect of the macula is visible on OCT with accumulation of
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subretinal fluid at the fovea. E-F: OCT and midphase FA in a 74-year-old woman with inferior hemi-retinal vein occlusion. FA shows a tortuous, dilated and hyperpermeable inferior hemi-retinal venous network. OCT demonstrates intraretinal cystoid cavities and extended inner retinal hyperreflectivity,
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corresponding to paracentral acute middle maculopathy reflecting the decreased perfusion of the deep retinal plexus.
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G-H: OCT and midphase FA in a 56-year-old man with central retinal vein occlusion. FA shows diffuse dot-shaped hemorrhages and moderate venous tortuosity, and cystoid macular edema is visible on OCT.
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(Images A-D courtesy Aude Ambresin)
Figure 6. Multimodal clinical imaging of macular edema secondary to neovascularization, inflammation or other causes.
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A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in an 83-year-old woman with age-related macular degeneration and type 3 choroidal
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neovascularization, leading to focal retinal thickening and intraretinal edema. C-D: OCT and midphase FA in a 76-year-old woman with chronic postoperative macular edema (Irvine-Gass syndrome) following posterior segment surgery for dislocated intraocular lens. FA shows diffuse outer blood-retinal barrier rupture manifesting as multiple pinpoints, and fluorescein filling of the cystoid cavities. OCT shows intraretinal cystoid edema and subretinal fluid. E-F: OCT and midphase FA in a 29-year-old man with macular edema secondary to posterior uveitis in the context of Behcet disease. FA shows diffuse outer blood-retinal barrier rupture while cystoid macular edema with subfoveal detachment is visible on OCT.
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ACCEPTED MANUSCRIPT G-H: OCT and midphase FA in a 54-year-old man with chronic central serous chorioretinopathy. FA shows an area of retinal pigment epithelium atrophy manifesting as a window defect, and fluorescein filling of a large pigment epithelial detachment. This epithelial lesion is visible on OCT, together with intraretinal cystoid cavities, a classical finding in
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chronic forms of the disease. I-J: OCT and midphase FA in a 40-year-old man with radiation maculopathy following ocular irradiation in childhood for retinoblastoma. FA shows the presence of multiple exudative
telangiectasia and progressive filling of cystoid cavities, clearly visualized on OCT, together
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with an irregular ellipsoid zone at the fovea indicating photoreceptor alteration.
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(Images I-J: courtesy Francis Munier)
Figure 7. Cellular and molecular components of the outer blood-retinal barrier. A. Junctional complex of the retinal pigment epithelium (RPE) formed by tight and adherens junctions. Junctional complex of the outer limiting membrane (OLM) formed by tight and
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adherens junctions between retinal Müller glial cells and photoreceptor inner segments B. Confocal microscopy of non-human primate retinal pigment epithelium showing labeling of occludin (green) and PKC-ζ (red), within occludin loops at the tight junction.
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C. Rat retinal section at the level of the outer limiting membrane showing the coimmunolabeling for zonula occludens-1 (ZO-1, red) and glutamine synthetase (GS, retinal
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Müller glial cells, green), particularly around the cones (nuclei labelled by Dapi, blue). D. Primate retinal section at the level of the outer limiting membrane showing the coimmunolabeling for zonula occludens-1 (ZO-1, red) and peanut agglutinin (PNA, cone photoreceptors, green), showing the expression of ZO-1 in the cones. Nuclei are labelled by dapi (blue). E. Rat retina section microscopy of the outer limiting membrane showing the coimmunolabeling for occludin (green) and glutamine synthetase (GS, retinal Müller glial cells, green). Nuclei are labelled by dapi (blue). A and C-E: adapted with permission from (Omri et al., 2010) 128
ACCEPTED MANUSCRIPT GS= glutamine synthetase; PKC= protein kinase C; ZO-1= zonula occludens-1; PNA= peanut agglutinin
Figure 8. Pathophysiological mechanisms leading to macular edema
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Macular edema results from an imbalance between fluid entry and fluid exit, leading to intraretinal or subretinal fluid accumulation, driven by Starling equation.
Figure 9. Role of choroidal mast cells in outer blood-retinal barrier alteration.
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A. Nomarsky phase-contrast microscopy of rat flat-mounted choroid (left) showing resident
cells (right) with hexagonal shape.
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mast cells (white arrows) along choroidal vessels, and binucleated retinal pigment epithelial
B. Optical coherence tomography showing in vivo serous retinal detachment in a rat eye, 6 hours after subconjunctival administration of compound 48/80 resulting in the degranulation of mast cells.
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C. Histo-resin section showing ex vivo serous retinal detachment in a rodent eye, 6 hours after subconjunctival administration of compound 48/80 resulting in the degranulation of mast cells. Vasodilation of choroidal vessels (red star) and RPE swelling and detachment are
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associated with sub-retinal fluid. Inset: Detached RPE cell. D. Flat-mounted retinal pigment epithelium immunostaining by occludin (green) and Dapi
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(blue) showing enlarged retinal pigment epithelial cells with irregular occludin labeling, cytoplasmic translocation (white stars) and junction disruption (white arrow) as a consequence of mast cell degranulation. Images A, B and D: courtesy Yvonne de Kozak. Image B: adapted with permission from (Bousquet et al., 2015).
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ACCEPTED MANUSCRIPT Figure 10. Macular immunohistochemistry and schematic representation A. Schematic representation of macular Müller glial cells at the macula (blue lines) showing their lateral displacement and characteristic Z shape, from the outer limiting membrane (red) to the inner limiting membrane (green)
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B. Immunohistochemistry at the level of the Henle layer, showing the co-labeling of RMG cells with glutamine synthetase (GS, red) and with zonula occludens-1 (ZO-1, red).
C. Flat-mounted macula with GS (red) and ZO-1 immunostaining (red) showing the radial organization of retinal Müller glial cells around the fovea in the Henle layer.
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D. Schematic representation of the nerve fibers from the macula to the optic nerve head (red).
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E. Glutamine synthetase (GS) labeling (red) of retinal Müller glial cells around the fovea on flat-mounted retina. Around the fovea, retinal Müller glial cells are radially oriented. F. Section of human macula stained with GFAP (red). Astrocytes are distributed at the innermost retinal layers around vessels and in the superficial inner nuclear layer.
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At the fovea, GFAP-positive cells form a roof and send extensions toward the Henle layer (inset and yellow arrows). GS labelling shows an intense labelling at the Henle fiber layer where retinal Müller glial cells are densified and have a Z-shaped orientation and then
(Inset).
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vertically oriented all over the retina. Note that at the fovea, the glial cells poorly express GS
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D: adapted with permission from (Matet et al., 2015) RMG= retinal Müller glial cells; GS= glutamine synthetase; GFAP= glial fibrillary acidic protein; ZO-1= zonula occludens-1; ILM= Inner Limiting Membrane; NFL= nerve fiber layer; GCL= Ganglion Cell layer; INL= Inner Nuclear Layer; OPL= Outer Plexiform Layer; ONL= Outer Nuclear Layer; OLM= outer limiting membrane
Figure 11. AQP4 immunostaining in the normal macula A. Transversal section of a normal human macula immuno-stained with AQP4 (green), glutamine synthetase (GS, red) and Dapi (blue).
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ACCEPTED MANUSCRIPT AQP4 is located is astrocytes and retinal Müller glial cells, surrounding vessels. At the fovea, where no vessels are present (central continuous-contour inset and B-D), AQP4 is highly expressed along the central foveal Müller cone cells (B) that poorly express GS (C). At the Henle layer, retinal Müller glial cells highly express AQP4 along the in the Z-shaped retinal
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Müller glial cells (D and magnified inset). Outside the avascular zone (A, dotted-contour inset and E-G); AQP4 is located in astrocytes and endfeet of retinal Müller glial cells, and along the vertical course of retinal Müller glial cells. AQP4 is highly expressed around the vessels in all capillary plexuses. In the superficial plexus, AQP4 co-localizes partly with GS, but in the
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deep plexus, AQP4 completely co-localizes with GS (F, upper and lower insets, respectively).
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GS= glutamine synthetase
Figure 12. AQP4 immunostaining in retinal Müller glial cells on flat-mounted human macula and the glymphatic hypothesis
A. Flat-mounted human macula with glutamine synthetase (GS, blue) and AQP4 (green) co-
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staining. At low magnification, AQP4 is concentrated in the macula and along RMG cells that follow the interpapillo-macular fibers.
B. Higher magnification (inset in A) showing the concentration of AQP4 around the fovea.
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C-D. Higher magnification (lower inset in B) shows that AQP4 distribution follows a structured pattern around a network composed of small channels running in parallel (C), and
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corresponding to the orientation of glial cells stained by GS (D). E. Identification of a similar channel pattern in the upper inset of B. F. Schematic hypothesis of AQP4-retinal Müller glial cell glymphatic drainage (green dotted line) from the macula towards the optic nerve head (yellow arrows). GS= glutamine synthetase; AQP= aquaporin
Figure 13. Schematic representation of the hypothetical glymphatic system. The distribution of aquaporin channels at retinal glial Müller cell membrane, allows water drainage along vessels, or “paravascular” drainage (A) and results in a radial network of
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ACCEPTED MANUSCRIPT draining channels between retinal glial Müller cells (B), orientated towards the optic nerve head.
Figure 14. Confocal microscopy of a human diabetic retina labelled with AQP4
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A-C: Staining for aquaporin 4 (AQP4, green), glutamine synthetase (GS, red) and Dapi (blue) showing a reduced expression of AQP4 in the perifoveal region together with reduced GS expression of the Z-shaped RMG cells. AQP4 is rather concentrated only around vessels. Outside the macula (Figure A, small inset and D, E), AQP4 is highly co-expressed with GS
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AQP= aquaporin; GS= glutamine synthetase
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along RMG cells, and is particularly concentrated around vessels (F, arrow).
Figure 15. Schematic representation of gliovascular changes in diabetic macular edema and correlation with OCT-A changes
A. In the normal retina, Kir4.1 (orange channels) is located around vessels expressed by
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astrocytes and retinal Müller glial cells. Microglia surrounds vessels. B. In the diabetic retina, Kir4.1 distribution is altered in retinal Müller glial cells, leading to Kir4.1 channel loss around vessels in the deep capillary plexus, since there are no astrocytes
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in the deep capillary plexus. In the more superficial vessels Kir 4.1 is still located around vessels, expressed by astrocytes. Since potassium intervenes in the regulation of blood flow,
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flow decreases in the deep capillary plexus. Retinal Müller glial cells are hypertrophic, and Kir 4.1 is mislocalized at their apices. Activated microglia migrate in the sub-retinal space. C: Optical coherence tomography angiography of a normal retina. D: coherence tomography angiography of diabetic macular edema showing focal defects in the deep capillary network adjacent to hyporeflective cystoid edema spaces. RMG= retinal Müller glial cell; a= astrocyte, mc= microglia; OLM= outer limiting membrane; SCP= superficial capillary plexus; DCP= deep capillary plexus; ICP= intermediate capillary plexus”
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ACCEPTED MANUSCRIPT Figure 16. Multimodal clinical imaging of “pure” macular edema phenotypes. A-B: Optical coherence tomography (OCT) and midphase fluorescein angiography (FA) in a 45-year-old man with idiopathic macular telangiectasia type 1 showing intraretinal cystoid edema and intraretinal exudates of pure vascular origin, related to an intense leakage from
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perifoveal telangiectasia and hyperpermeable capillaries. C-D: OCT and midphase FA in a 72-year-old man with idiopathic macular telangiectasia type 2 showing intraretinal cavitations without retinal thickening, and focal loss of the normal
retinal layer structure, resulting from a cellular neuro-vasculo-glial degenerative process of
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unknown origin. There is a mild fluorescein leakage from fine perifoveal telangiectasia, but intraretinal cavities result from tissue loss and not from exudation.
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E-F: OCT and midphase FA in a 48-year-old man treated by Trametinib, a mitogen-activated protein kinase kinase (MEK) inhibitor, for metastatic melanoma, showing marked cystoid macular edema and retinal thickening, but absence of vascular fluorescein leakage. A moderate filling of intraretinal cavities is visible on FA. Adapted with permission (Duncan et
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al., 2015).
G-H: OCT and midphase FA in a 59-year-old woman with receiving Paclitaxel (formulated as albumin-bound nanoparticles) for metastatic breast cancer, showing cystoid macular edema
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but absence of fluorescein leakage. Adapted with permission (Ehlers et al., 2013). I-J: OCT and midphase FA in a 69-year-old man with hypoproteinemia due to nephrotic
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syndrome, showing subretinal fluid accumulation and absence of fluorescein leakage. K-L: OCT in a 44-year-old woman with inferior hemi-central retinal vein occlusion complicated by paracentral middle acute middle maculopathy, showing a focal hyperreflectivity on OCT localized across several relatively thickened layers, from the inner nuclear to the Henle fiber layers, with a focal hyporeflectivity on infrared reflectance. These changes indicate intraretinal fluid accumulation related to focal cellular ischemia due to a focal perfusion decrease in the deep capillary plexus. E-F: adapted with permission from (Duncan et al., 2015). G-H: adapted with permission from (Ehlers et al., 2013)
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Figure 17. Mechanisms induced by hyperglycemia in the retina leading to diabetic macular edema.
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Figure 18. Implication of inflammation in diabetic macular edema. A. Schematic view of inflammation, aging and oxidative stress as contributors to diabetic macular edema.
B. Pathways activated in hyperglycemic conditions triggering inflammation
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AGE= advanced glycation endproducts; RAGE= receptor for advanced glycation
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endproducts; PKC= protein kinase C; ROS= reactive oxygen species
Figure 19. Kinetics of retinopathy in the Goto-Kakizaki rat model A-D Histologic paraffin sections of rat retina stained with hemalun-eosin A: Retina from a one-year-old Wistar rat, with normoglycemic control
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B-D: Histology of Goto-Kakizaki rat retinas at 4, 8 and 18 months showing progressive retinal thickening with fluid accumulation in the photoreceptor outer segment layer (white arrows) and outer nuclear layer (yellow arrows). Progressive dilation of choroidal vessels is also
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visible (black arrows and blue stars).
E: Lectin-labelled flat-mounted retina of a Goto-Kakizaki rat at 18 months showing clear
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signs of retinal microangiopathy
WS= Wistar rat; GK= Goto Kakizaki rat; INL= inner nuclear layer; ONL= outer nuclear layer
Figure 20. Kir4.1 and aquaporin expression kinetics in the Goto-Kakizaki diabetic rat model A and C: immunostaining on retinal cryosections from Wistar control rats and Goto-Kakizaki diabetic rats with Kir4.1 (A) or AQP4 (C) labeling, showing decreased Kir4.1 and AQP4 fluorescence in the outer retina and in the retinal glial Müller cell processes surrounding the retinal vessels.
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ACCEPTED MANUSCRIPT B, D and E: time-dependent evolution of Kir4.1 (B), AQP4 (D) and aquaglyceroporin AQP9 (E) expression in the retina of Goto-Kakizaki diabetic rats, compared to age-matched control Wistar rats, using quantitative PCR (HPRT1 and 18S serving as internal controls). Data were expressed as mean ± standard error of the mean (n=6).
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*, P<0.05; **, P<0.01, ***, P>0.001. WS= Wistar rat; GK= Goto Kakizaki rat; GCL= ganglion cell layer; INL= inner nuclear layer; ONL= outer nuclear layer; AQP= aquaporin.
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Figure 21. Retinal pigment epithelium changes in the Goto-Kakizaki diabetic rat model In the Wistar non-diabetic rat, phalloidin stains the actin cytoskeleton that borders the inner
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region of tight junctions (B) and shows a regular mosaic pattern of the retinal pigment epithelium. ROCK-1 is localized in the cytoplasma (C). On electron microscopy, RPE cells have a regular morphology.
In the Goto-Kakizaki rat, at 6 months, apical constriction of the cytoskeleton (E, as compared
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to A; and F, dotted circle) is associated with membrane translocation of ROCK-1 (G) and formation of membrane blebs (G inset and H, yellow dotted line). At one year, large syncytium can be observed (H, yellow arrows) together with areas of disrupted intercellular
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junctions in the diabetic Goto-Kakizaki model (J, arrow) as compared to Wistar rat (I). WS= Wistar rat; GK= Goto Kakizaki rat; POS= photoreceptor outer segments; RPE= retinal
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pigment epithelium
Figure 22. Schematic representation of hyperglycemia-induced changes in the PAR3/PAR6/PKCζ complex in retinal pigment epithelial cells of Goto-Kakizaki diabetic rats The PAR-6/PKCζ/PAR-3 scaffold complex is involved in tight junction (TJ) formation and maintenance. Alteration of any of those junctional partners leads to mislocalization of the others and to tight junction disruption. Hyperglycemia induces a biphasic activation of PKCζ. Higher (GK 6 months) and lower panels (GK 12 months): the PKCζ-PT410/ PKCζ ratio, in
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ACCEPTED MANUSCRIPT western blotting, shows that PKCζ activity increases significantly at 6 months (~ +40%) and then decreases at 12 months (~ -60%) after diabetes onset, compared to control Wistar rats. Upper panels: In 6-month-old controls, PKCζ staining (red) appeared as focal spots within loops formed by occludin protrusions (green). During hyperglycemia, 6 months after diabetes
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onset, phosphorylation of PKCζ (PKCζ-P) by PDK1 (Pyruvate Dehydrogenase Kinase 1) increases, inducing ROCK-1 phosphorylation (ROCK1-P). ROCK1 activation induces PAR3 phosphorylation, disrupting the PAR-3 / PKCζ interaction and thereby suppressing the activity of the PAR-6/PKCζ/PAR-3 complex.
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Lower panel: twelve months after diabetes onset, the PKCζ staining associated to tight
junctions was either barely or not detectable. The decrease of PKCζ activity leads to tight
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junction opening and occludin internalization from the cell membrane to the cytoplasm, further contributing to tight junction disruption and outer retinal barrier disruption. Administration of a PKCζ inhibitor induces similar changes.
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GK= Goto-Kakizaki rat; PKC= protein kinase C
Figure 23. Transcytosis of microglia/macrophages through retinal pigment epithelial cells
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A. Semi-thin section of rat retinal pigment epithelium showing the formation of a “pore” resulting from the invagination of the apical towards the basal cell membrane (arrow).
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B and C: Electron microscopy imaging of a retinal pigment epithelial cells showing the formation of a “pore” (B), inducing the lateral displacement of a cell nucleus (C, arrow). D: tridimensional representation of a microglial cell transcytosis (arrow) from the subretinal space through a retinal pigment epithelial cell. A and C: Adapted with permission from (Omri et al., 2011)
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ACCEPTED MANUSCRIPT Figure 24. Changes in the outer limiting membrane in the Goto-Kakizaki diabetic rat model Flat-mounted retinas imaged by confocal microscopy at the level of the outer limiting membrane (A-D) and retinal sections magnified around the outer limiting membrane (E-F).
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A-B: Labeling for glutamine synthetase (GS, red), a marker of retinal Müller glial cells, shows that the outer limiting membrane network formed by retinal Müller glial cells is disrupted in the diabetic model.
C-D: Additional labelling for occludin, that is expressed between retinal Müller glial cells, and
SC
between these cells and photoreceptors, shows a focal disruption of occludin staining in the diabetic model (C, arrow).
limiting membrane (arrows).
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E-F: Staining for PKCζ on retinal sections shows delocalization of PKCζ from the outer
Adapted with permission from (Omri et al., 2010) and (Omri et al., 2013) WS= Wistar rat; GK= Goto Kakizaki rat; CTL= control (WS rat); DIA= diabetic GK rat; PKC=
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protein kinase C; GS= glutamine synthetase
Supplementary Figure 1. Radial distribution of retinal glial Müller cells in a non-human
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primate retina.
A. Flat-mounted retina from a Macaca fascicularis, labelled with glutamine synthetase,
head.
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showing the radial orientation of retinal glial Müller cells from the fovea to the optic nerve
B. Flat-mounted retina from a normal post mortem human donor eye, labelled with CRALBP, showing the radial orientation of retinal glial Müller cells around to fovea and towards the optic nerve head. GS= glutamine synthetase; F= fovea, ONH= optic nerve head
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ACCEPTED MANUSCRIPT VIDEO LEGENDS
Video 1. Mechanisms of hydro-ionic homeostasis in the retina.
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Video 2. Mechanisms of diabetic macular edema
Video 3. Transcytosis across retinal pigment epithelial cells allowing microglia
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migration from the retina to the choroid
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TABLE 1. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with diabetic macular edema and reported in the literature. Patients, No. (Controls, No.)
Correlation with central macular thickness (+/-) _ _
Reference
23 (22) 13 (14)
Fold increase Patients vs Controls 3.6 × NS*
Interleukin 3 (IL-3)
Aqueous Vitreous
Multiplex Multiplex
Interleukin 6 (IL-6)
Aqueous
Multiplex
23 (22)
10.4 ×
_
(Jonas et al., 2012)
Vitreous
ELISA
26 (12)
19.8 ×
_
(Funatsu et al., 2003)
Vitreous Vitreous
ELISA Multiplex
53 (15) 92 (83)
10.2 × 14.4 ×
Yes (+) _
(Funatsu et al., 2009) (Yoshimura et al., 2009)
Aqueous
Multiplex Multiplex
18 (16) 13 (14)
2.3 × NS*
No _
(Lee et al., 2012) (Ghodasra et al., 2016)
Interleukin 8 (IL-8)
Aqueous Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex Multiplex
23 (22) 92 (83) 18 (16) 13 (14)
10.3 × 8.9 × 2.3 × NS*
_ _ Yes (+) _
(Jonas et al., 2012) (Yoshimura et al., 2009) (Lee et al., 2012) (Ghodasra et al., 2016)
Interleukin 13 (IL-13)
Aqueous
Multiplex
18 (16)
No
(Lee et al., 2012)
Epidermal Growth Factor (EGF)
Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex
0.9 × NS*
_ _ _
(Ghodasra et al., 2016) (Jonas et al., 2012) (Yoshimura et al., 2009)
Aqueous Vitreous Aqueous Vitreous Aqueous Vitreous
Multiplex Multiplex Multiplex ELISA Multiplex ELISA
18 (16) 13 (14) 23 (22) 20 (8) 23 (22) 20 (8)
NS NS* 1.9 × 3.6 × 2.4 × 0.025 ×
No _ _ _ _ _
(Lee et al., 2012) (Ghodasra et al., 2016) (Jonas et al., 2012) (Patel et al., 2006) (Jonas et al., 2012) (Patel et al., 2006)
Aqueous
ELISA
42 (23)
1.8 ×
_
(Kocabora et al., 2016)
Transforming Growth Factor ß (TGF-ß)
Tumor Necrosis Factor alpha (TNF-α α)
TE D 13 (14) 23 (22) 92 (83)
EP
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Hepatocyte Growth Factor (HGF)
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Technique
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Ocular media
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Biological factor (acronym)
1.7 × NS
(Jonas et al., 2012) (Ghodasra et al., 2016)
1
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Platelet Derived Growth Factor (PDGFAB) Platelet Derived Growth Factor (PDGFBB) InterCellular Adhesion Molecule 1 (ICAM-1, CD54)
Vascular cell adhesion molecule 1 (VCAM) C-X-C motif chemokine 10 (CXCL-10) (=Interferon gamma-induced protein 10, IP10) Chemokine ligand 2 (CCL2) (=monocyte chimoattractant protein 1, MCP-1)
(Jonas et al., 2012) (Jonas et al., 2012) (Funatsu et al., 2003) (Funatsu et al., 2005) (Funatsu et al., 2005) (Patel et al., 2006) (Funatsu et al., 2009)
Multiplex ELISA Multiplex ELISA ELISA ELISA ELISA Multiplex Multiplex ELISA
92 (83) 15 (15) 18 (16) 36 (6) 20 (8) 53 (15) 15 (15) 18 (16) 13 (14) 15 (15)
2.1 ×
_ _ No Yes (-) _ Yes (-) _ No _ _
(Yoshimura et al., 2009) (Praidou et al., 2011) (Lee et al., 2012) (Funatsu et al., 2005) (Patel et al., 2006) (Funatsu et al., 2009) (Praidou et al., 2011) (Lee et al., 2012) (Ghodasra et al., 2016) (Praidou et al., 2011)
Vitreous
ELISA
15 (15)
0.56 ×
_
(Praidou et al., 2011)
Aqueous Vitreous
Multiplex ELISA
23 (22) 33 (13)
3.2 × 2.5 ×
_ Yes (+)
(Jonas et al., 2012) (Funatsu et al., 2005)
Vitreous
ELISA
53 (15)
2.2 ×
Yes (+)
(Funatsu et al., 2009)
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_ _ _ Yes (+) Yes (+) _ Yes (+)
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Vitreous Vitreous Aqueous Vitreous Vitreous Vitreous Vitreous Aqueous Vitreous Vitreous
7.9 × 2× 46 × 60.5 × 58 × 4.5 × 30.5 × NS 7.6 × 2.8 × 0.15 × NS
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23 (22) 23 (22) 26 (12) 36 (8) 33 (13) 20 (8) 53 (15)
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Platelet Derived Growth Factor (PDGFAA)
Multiplex Multiplex ELISA ELISA ELISA ELISA ELISA
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Pigment Epithelium Derived Factor (PEDF)
Aqueous Aqueous Vitreous Vitreous Vitreous Vitreous Vitreous
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Placental Growth Factor (PGF) Vascular Endothelial Growth Factor (VEGF)
0.13 × 2.4 × 1.5 × NS*
Aqueous
Multiplex
23 (22)
2.7 ×
_
(Jonas et al., 2012)
Aqueous Vitreous
Multiplex Multiplex
23 (22) 13 (14)
1.4 × NS*
_ _
(Jonas et al., 2012) (Ghodasra et al., 2016)
Aqueous Vitreous Vitreous
Multiplex ELISA Multiplex
23 (22) 53 (15) 92 (83)
2.1 × 2.6 × 15.8 ×
_ Yes (+) _
(Jonas et al., 2012) (Funatsu et al., 2009) (Yoshimura et al., 2009)
2
Multiplex Multiplex
18 (16) 23 (22)
2.5 × 1.6 ×
No _
(Lee et al., 2012) (Jonas et al., 2012)
Aqueous Vitreous Aqueous
Multiplex ELISA Multiplex
23 (22) 20 (8) 23 (22)
6.1 × NS
_ _ _
(Jonas et al., 2012) (Patel et al., 2006) (Jonas et al., 2012)
Vitreous
ELISA
20 (8)
0.67 ×
_
(Patel et al., 2006)
Vitreous
Multiplex
92 (83)
NS
_
(Yoshimura et al., 2009)
4.1 ×
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Plasminogen activator inhibitor-1 (PAI1) Soluble fms-like tyrosine kinase-1 (sFlt1) (=soluble vascular endothelial growth factor 1, sVEGFR-1) Tumor Necrosis Factorα α (TNF-α α)
Aqueous Aqueous
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C-X-C motif chemokine ligand 9 (CXCL-9) or Monokine induced by γinterferon (MIG) Matrix metallopeptidase 9 (MMP-9)
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ACCEPTED MANUSCRIPT
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* Multivariate analysis with adjustment on all biological factors assessed NS= non-significant; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
3
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TABLE 2. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with macular edema due to central retinal vein occlusion and reported in the literature. Technique
Patients, No. (Controls, No.)
Interleukin-1ß (IL-1ß)
Aqueous
Multiplex
Interleukin 6 (IL-6)
Vitreous
No
(Feng et al., 2013)
Multiplex
13 (83)
81.4 ×
_
(Yoshimura et al., 2009)
Vitreous
ELISA
27 (16)
47.9 ×
Yes (+)
(Noma et al., 2009)
Aqueous
Multiplex
5 (13)
39.6 ×
_
(Funk et al., 2009)
Vitreous
CBA
35 (14)
10.4 ×
_
(Koss et al., 2012)
Aqueous
Multiplex
18 (10)
2.3 ×
No
(Feng et al., 2013)
Vitreous
Multiplex
13 (83)
35.3 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
5 (13)
63.8 ×
_
(Funk et al., 2009)
Aqueous
Multiplex
18 (10)
1.3 ×
No
(Feng et al., 2013)
Aqueous
Multiplex
18 (10)
2.1 ×
No
(Feng et al., 2013)
Vitreous
Multiplex
13 (83)
14.7 ×
_
(Yoshimura et al., 2009)
Vitreous
ELISA
27 (16)
7×
Yes (+)
(Noma et al., 2009)
Aqueous
Multiplex
5 (13)
23.7 ×
_
(Funk et al., 2009)
CBA
35 (14)
39.8 ×
_
(Koss et al., 2012)
Multiplex
18 (10)
1.6 ×
No
(Feng et al., 2013)
Multiplex
18 (10)
1.9 ×
No
(Feng et al., 2013)
Multiplex
5 (13)
4.4 ×
_
(Funk et al., 2009)
Vitreous Aqueous Basic fibroblast growth factor (bFGF) C-X-C motif chemokine 10 (CXCL-10) or Interferon gamma-
Aqueous Aqueous
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Transforming Growth Factor ß (TGF-ß) Vascular Endothelial Growth Factor (VEGF)
Reference
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Interleukin 8 (IL-8)
Correlation with CMT (+/-)
8 (10)
Fold increase Patients vs Controls 1.4 ×
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Ocular media
SC
Biological factor (acronym)
ACCEPTED MANUSCRIPT
induced protein 10 (IP10) 13 (83)
28.8 ×
Aqueous
Multiplex
5 (13)
5.6 ×
Vitreous
CBA
35 (14)
4×
Aqueous
Multiplex
5 (13)
2.2 ×
Aqueous
Multiplex
18 (10)
2.1 ×
_
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Multiplex
SC
Platelet Derived Growth Factor (PDGFAA) Serum Amyloid A (SAA)
Vitreous
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Chemokine ligand 2 (CCL2) or monocyte chimoattractant protein 1 (MCP-1)
(Yoshimura et al., 2009)
_
(Funk et al., 2009)
_
(Koss et al., 2012)
_
(Funk et al., 2009)
(Feng et al., 2013)
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CBA= cytometric bead array; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
ACCEPTED MANUSCRIPT
Technique
Patients, No. (Controls, No.)
Correlation with CMT (+/-)
Reference
8 (10)
Fold increase Patients vs Controls 2.1 ×
IL-1 beta (Interleukin 1 beta) IL-6 (Interleukin 6) Master inflammatory cytokine
Aqueous
Multiplex
No
(Feng et al., 2013)
Vitreous
Multiplex
30 (83)
5.4 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
38 (18)
1.8 ×
Yes (+)
(Kaneda et al., 2011)
Aqueous
Multiplex
12 (16)
NS
No
(Lee et al., 2012)
Vitreous
CBA
43 (14)
NS
_
(Koss et al., 2012)
Vitreous
CBA
43 (28)
NS
No
(Pfister et al., 2013)
Aqueous
Multiplex
8 (10)
2.3 ×
No
(Feng et al., 2013)
Vitreous
ELISA
28 (17)
9.6 ×
Yes (+)
(Noma et al., 2014)
Vitreous
Multiplex
30 (83)
16.4 ×
_
(Yoshimura et al., 2009)
Aqueous
Multiplex
38 (18)
7.1 ×
Yes (+)
(Kaneda et al., 2011)
Aqueous
Multiplex
12 (16)
2.1 ×
Yes (+)
(Lee et al., 2012)
Aqueous
Multiplex
8 (10)
1.3 ×
No
(Feng et al., 2013)
IL-12 (Interleukin 12)
Aqueous
Multiplex
38 (18)
2.2 ×
No
(Kaneda et al., 2011)
IL-15 (Interleukin 15)
Aqueous
Multiplex
38 (18)
1.6 ×
No
(Kaneda et al., 2011)
IL-17 (Interleukin 17)
Aqueous
Multiplex
38 (18)
1.8 ×
No
(Kaneda et al., 2011)
IL-23 (Interleukin 23) TGF beta (Transforming Growth Factor Beta) VEGF
Aqueous
Multiplex
38 (18)
2.6 ×
No
(Kaneda et al., 2011)
Multiplex
8 (10)
2.1 ×
No
(Feng et al., 2013)
Multiplex
30 (83)
NS
_
(Yoshimura et al., 2009)
Aqueous
Vitreous
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EP
AC C
IL-8 (Interleukin 8)
SC
Ocular media
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Biological factor (acronym)
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TABLE 3. Major biological factors, including cytokines and angiogenic factors, detected in ocular media of eyes with macular edema due to branch retinal vein occlusion and reported in the literature.
2.4 ×
No
(Lee et al., 2012)
Aqueous
Multiplex
38 (18)
NS
No
(Kaneda et al., 2011)
Vitreous
Multiplex
43 (14)
23.1 ×
_
(Koss et al., 2012)
Vitreous
Multiplex
43 (28)
23.1 ×
Aqueous
Multiplex
8 (10)
1.6 ×
Vitreous
Multiplex
28 (17)
48.3 ×
Aqueous
Multiplex
8 (10)
1.7 ×
Vitreous
ELISA
28 (17)
2.4 ×
Vitreous
Multiplex
30 (83)
Aqueous
Multiplex
38 (18)
Aqueous
Multiplex
12 (16)
Vitreous
CBA
43 (14)
Vitreous
CBA
Vitreous
ELISA
Vitreous
ELISA
Aqueous
Multiplex
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12 (16)
(Pfister et al., 2013)
No
(Feng et al., 2013)
Yes (+)
(Noma et al., 2014)
No
(Feng et al., 2013)
Yes (+)
(Noma et al., 2014)
4.4 ×
_
(Yoshimura et al., 2009)
NS
No
(Kaneda et al., 2011)
NS
No
(Lee et al., 2012)
2.4 ×
_
(Koss et al., 2012)
43 (28)
2.4 ×
No
(Pfister et al., 2013)
28 (17)
3×
Yes (+)
(Noma et al., 2014)
28 (17)
1.9 ×
Yes (+)
(Noma et al., 2014)
2.2 ×
No
(Feng et al., 2013)
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No
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sVEGFR2 (soluble Vascular Endothelial Growth Factor Receptor 2) SAA (Serum Amyloid A)
Multiplex
EP
bFGF (Basic fibroblast growth factor) sICAM-1 (soluble InterCellular Adhesion Molecule 1) or CD54 CCL2 (Chemokine ligand 2) or MCP-1 (Monocyte chimoattractant protein 1)
Aqueous
AC C
(Vascular Endothelial Growth Factor)
SC
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8 (10)
CBA= cytometric bead array; NS= non-significant; (+) = positive correlation with central macular thickness; (-) = negative correlation with central macular thickness
ACCEPTED MANUSCRIPT
Identified mechanism(s)
Vascular endothelial growth factor-A (VEGF-A) / VEGF-R2
Junction integrity alteration
Occludin phosphorylation via PKCβ activation
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Biological factor / receptor
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TABLE 5. Major growth factors and inflammatory mediators potentially involved in retinal and choroidal vascular permeabilization
Occludin downregulation by increasing free cytosolic ß-catenin, and upregulating u-PAR
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VE-cadherin endocytosis via Rac activation/ß-arrestin-2
VE-cadherin disruption via increased transglutaminase-2 activity by raising intracellular Ca and ROS concentrations Up-regulation of ICAM-1 expression: leukostasis
(Miyamoto et al., 2000)
Occludin internalization and ubiquitinization via Src-family kinases
(Scheppke et al., 2008)
Transcellular permeability
Transcytosis in caveolae via an eNOS-dependent mechanism
(Feng et al., 1999)0) (Hofman et al., 2000) (Wisniewska-Kruk et al., 2016)
Junction integrity alteration
Changes in occludin distribution in RPE cells MEK signaling pathway
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Degradation of ZO-1 and VE-cadherin
Bradykinin (BK) / B2R and B1R
Tie-2 receptor α3β1-Integrin
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Loss of pericytes
(Lee et al., 2016)
(Miyamoto et al., 2007) (Otrock et al., 2007) (Kowalczuk et al., 2011) (Huang et al., 2015) (Hammes et al., 2004) (Cai et al., 2008) (Pfister et al., 2008) (Park et al., 2014)
Loss of astrocytes
avβ5 binding
Junction integrity alteration Vasodilatation and hyperpermeability Junction integrity alteration
Reduces VE-cadherin by phosphorylation
(Rangasamy et al., 2011)
eNOS-mediated B1R stimulation iNOS-mediated B2R stimulation Src kinase-mediated VE-cadherin phosphorylation
(Kita et al., 2015)
Claudin-5 down-regulation
(Zhou et al., 2014)
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Angiopoietin-2 (Ang-2)
(Antonetti et al., 1999) (Harhaj et al., 2006) (Murakami et al., 2012) (Liu et al., 2016) (Behzadian et al., 2003) (Gavard and Gutkind, 2006)
2+
Induces plasmalemma vesicle-associated protein formation Placental growth factor (PGF) / VEGFR-1
Reference
(Yun et al., 2016a)
(Liu and Feener, 2013)
Angiotensin II
Pericyte migration
-
(Wilkinson-Berka, 2006)
Stimulates VEGF
(Gilbert et al., 2000)
Aldosterone / MR
Junction integrity alteration Choroidal permeability
Up-regulation of the endothelial vasodilatory K channel KCa2.3 in choroidal endothelial cells and
(Zhao et al., 2012)
ACCEPTED MANUSCRIPT
adjacent smooth muscle cell relaxation (Zhao et al., 2010) (Gaudenzio et al., 2016)
Decreased ZO-1 and claudin-5 expression and alteration of their sub-cellaular distribution in retinal endothelial cells Via PKC-mediated NF-kB activation ZO-1 disruption in RPE cells Via p38 MAPK Increased VEGF and ICAM
(Aveleira et al., 2010)
Apoptosis
(Behl et al., 2008)
Retraction of endothelial cells Junction integrity alteration
Endothelial cell death
RI PT
Regulates the expression and distribution of ion and water channels in RMG cells Histamine-mediated vasodilation Release of biogenic amines (e.g. histamine), proteases, angiogenin, cytokines (e.g. TNF-α,) chemokines (e.g. CCL5, IL-8, MCP-1, eotaxin) Activation of PI3K and Src kinase pathways (C5a)
SC
Tumor necrosis factor-α (TNF-α) / TNF-Rs
Decreased drainage Mast cell degranulation
Necrosis
Interleukin-1ß (IL1-ß)
Junction integrity alteration
M AN U
Complement components (C3a and C5a)
(Schraufstatter et al., 2002)
(Shirasawa et al., 2013) (Penfold et al., 2002)
(Claudio et al., 1994)
Decreased cx43-mediated glio-vascular communication
(Muto et al., 2014)
Recruitment of leukocytes Histamine production VEGF-A production
(Bamforth et al., 1997)
Occludin down-regulation under hypoxic conditions
TE D
PKC-θ-dependent phosphorylation of ZO-1
(Vinores et al., 2003) (Yamagata et al., 2004) (Rigor et al., 2012)
Alterations in the f-actin cytoskeleton due to the phosphorylation of MLCs by ROCK activation
(Wu et al., 2016)
Decreased VE-cadherin expression
(Matsuda et al., 2015)
Junction integrity alteration Pericyte loss
Occludin degradation via MMP-9 production
(Behzadian et al., 2001)
-
(Betts-Obregon et al., 2016)
Interleukin-6 (IL-6)
Junction integrity alteration
Down-regulation of occludin and ZO-1 VEGF production by STAT3 activation
(Yun et al., 2016b)
Decreases the expression of VE-cadherin, occludin and claudin-5
(Rochfort et al., 2014)
Junction integrity alteration
AC C
IL-8
EP
Transforming growth factor-ß (TGF-ß)
Down-regulation of tight junction proteins, including occludin, claudin-5 and ZO-1
(Yu et al., 2013)
VEGF= vascular endothelial growth factor; VEGFR-2= kinase insert domain-containing receptor or KDR; PGF= placental growth factor; VEGFR-1 (Flt-1)= VEGF receptor-1 (fms-like tyrosine kinase-1); MAPK= mitogen-activated protein kinase; PACAP= pituitary adenylate cyclase-activating polypeptide; VIP= vasoactive intestinal peptide; B1R= bradykinin B1 receptor; B2R= bradykinin B2 receptor; eNOS= endothelial nitric oxide synthase; iNOS= inducible nitric oxide synthase; pKal= plasma kallikrein; PCK= protein kinase C; ROCK= Rho-associated protein kinase; NO= nitric oxide; MMP= matrix metalloproteinase; IL=interleukin; uPAR= urokinase plasminogen activator receptor; MR= mineralocorticoid receptor; ROS= reactive oxygen species; TNF= tumor necrosis factor; TNF-Rs= TNF receptors; cx43= connexin-43; MLC= myosin light chain
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Kir4.1 expression Immunohistochemistry
Ischemiareperfusion
RT-PCR
Immunohistochemistry
Decreased
Slight reduction in IPL (D1-7)
Decreased
Lewis Rat
Decreased around vessels and at RMG cell endfeet
-
Horse
Absent
Decreased
Wistar rats
Globally decreased. Absent at OLM and perivascular membranes (6 months)
-
Wistar rats
-
Long Evans rats
Decreased. Absent at ILM and around vessels. Mislocalized in IPL
Absent around vessels and at RMG cell endfeet
Reference
Westernblot Decreased
RT-PCR Unchanged
(Liu et al., 2007)
-
Unchanged
(Zhao et al., 2011)
-
Absent at RMG cells in inner layers. Mislocalized at ONL and OLM
-
-
(Eberhardt et al., 2011)
-
-
-
-
(Pannicke et al., 2006)
Absent at superficial capillary plexus. Present in NFL and GCL. Not altered at deep capillary plexus (6 months) Slightly increased at IPL
-
-
(Iandiev et al., 2007)
Slightly decreased (D7)
Unaltered -
(Hollborn et al., 2011) (Pannicke et al., 2004)
TE D
Decreased (D1) Disappeared (D3-7)
-
-
Decreased (D7)
-
EP
Equine recurrent autoimmune uveitis Streptozotocyninduced diabetes
Westernblot Decreased
Wistar rats
AC C
Endotoxininduced uveitis
AQP4 expression
SC
Animal
M AN U
Disease model
RI PT
TABLE 4. Changes in the membrane channels Kir4.1 and Aquaporin-4 expression and localization in animal models of retinal disorders relevant to study cellular mechanisms of macular edema
Long Evans rats
Absent at superficial vascular plexus. Normal at deep vascular plexus (D7)
-
-
-
-
-
(Iandiev et al., 2006a)
Long Evans rats C57BL/6 mice
-
-
-
Decreased (D7)
-
-
Slightly increased at IPL
Decreased D7 -
(Iandiev et al., 2006b)
Decreased around vessels
Decreased (D7) -
(Hirrlinger et al., 2010)
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Wistar rats
-
Decreased (D7-14)
Decreased (D7-14)
-
Decreased (D7-14)
Decreased (D7-14)
(Dibas et al., 2010)
Branch Retinal Vein Occlusion
Long Evans rats
Decreased
-
Decreased (D1-3)
Not altered (except in cysts)
-
Decreased (D1-3)
(Rehak et al., 2009)
Light induce damage
Pigmented C57BL/6SV129 mice
Increased in outer retina
-
-
Increased in outer retina
-
-
(Iandiev et al., 2008)
RI PT
Optic nerve crush
AC C
EP
TE D
M AN U
SC
AQP4= Aquaporin-4; RT-PCR= real-time polymerase chain reaction; D= day; RMG cells= retinal Müller glial cells; ILM= inner limiting membrane; NFL= nerve fiber layer; GCL= ganglion cell layer; IPL= inner plexiform layer; ONL= outer nuclear layer; OLM= outer limiting membrane Time of observations after model induction is indicated between parentheses
ED
M AN U
CE PT ED
M AN US C
R
PT ED
M AN US
AC C
EP TE D
M AN US C
RI P
CC
EP TE D
M AN US C
RI P
AC C
EP
TE D M AN US C
RI P
EP TE D
M AN US C
ED
M AN
EP TE D
M AN US C
R
CE PT ED
M AN US C
RI
PT ED
M AN US
PT ED
M AN US
D
M AN
PT ED
M AN US C
EP TE D
M AN US C
AC C
EP
TE D M AN US C
RI P
PT ED
M AN US
CC
EP TE D
M AN US C
RI
EP TE D
M AN US C
R
EP TE D
M AN US C
R
EP TE D
M AN US C
EP TE D
M AN US C
PT ED
M AN US
ED
M AN
ACCEPTED MANUSCRIPT ARTICLE HIGHLIGHTS •
Cells forming inner and outer blood-retinal barriers maintain retinal homeostasis
•
Macular edema results from an imbalance between fluid entry and drainage mechanisms Intraretinal accumulation of macromolecules osmotically attracts water and solutes
•
The structural organization of the retina explains why edema develops in the macula
•
A glymphatic system may be formed by AQP4 expression along macular Müller cells
AC C
EP
TE D
M AN U
SC
RI PT
•