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Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways
CHAPTER EIGHTEEN
Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways Ernst R. Tamm1, Barbara M. Braunger, Rudolf Fuchshofer Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany 1 Corresponding author: e-mail address: [email protected]
Contents 1. Intraocular Pressure and Aqueous Humor Outflow 2. Trabecular Meshwork 3. Schlemm's Canal 4. Outflow Resistance 5. Contractile Mechanisms in the Trabecular Outflow Pathways 6. Resistance of the Trabecular Outflow Pathways in Primary Open-Angle Glaucoma References
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Abstract Intraocular pressure (IOP), the critical risk factor for glaucoma, is generated and maintained by the aqueous humor circulation system. Aqueous humor is secreted from the epithelial layers of the ciliary body and exits the eye through the trabecular meshwork or the uveoscleral outflow pathways. IOP builds up in response to a resistance to aqueous humor flow in the trabecular outflow pathways. The trabecular outflow resistance is localized in the inner wall region, which comprises the juxtacanalicular connective tissue (JCT) and the inner wall endothelium of Schlemm's canal (SC). Outflow resistance in this region is lowered through the relaxation of contractile myofibroblast-like cells in trabecular meshwork and the adjacent scleral spur, or the contraction of the ciliary muscle. In primary open-angle glaucoma, the most frequent form of glaucoma, outflow resistance of the inner wall region is typically higher than normal. There is evidence that the increase in resistance is related to characteristic biological changes in the resident cells of the JCT, which more and more acquire the structural and functional characteristics of contractile myofibroblasts. The changes involve an augmentation of their actin cytoskeleton and of their surrounding fibrillary extracellular matrix, which connects to JCT cells via integrins. This scenario leads to an overall stiffening of the inner wall region, and is modulated by transforming growth factor-β/ Progress in Molecular Biology and Translational Science, Volume 134 ISSN 1877-1173 http://dx.doi.org/10.1016/bs.pmbts.2015.06.007
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connective tissue growth factor signaling. Essentially comparable changes appear to occur in SC endothelial cells. Stiffening of JCT and SC cells is very likely a critical causative factor for the increase in trabecular outflow resistance in POAG.
1. INTRAOCULAR PRESSURE AND AQUEOUS HUMOR OUTFLOW Glaucoma, the leading cause of irreversible blindness throughout the world,1 is a chronic, progressive optic nerve neuropathy, in which optic nerve axons are damaged at the optic disc, the site of their exit from the eye. In several prospective randomized multi-center studies, intraocular pressure (IOP) has been identified as the critical causative risk factor for glaucoma and, consequently, the reduction of IOP delays or prevents the damage of optic nerve axons.2–8 This chapter will review the mechanisms that are thought to generate IOP in the normal eye and in eyes affected with the most common form of glaucoma, primary open-angle glaucoma (POAG). In addition, we will outline a unifying concept to explain the molecular changes that change the biology of the aqueous humor outflow pathways to affect IOP in POAG.9 IOP depends on the aqueous humor circulation system, in which aqueous humor is secreted from the epithelial layers of the ciliary body into the posterior chamber10 and exits the eye in the chamber angle via the conventional or trabecular outflow pathway, or the unconventional or uveoscleral outflow pathway (Fig. 1).11 The trabecular outflow pathway comprises the trabecular meshwork (made up by the uveal and corneoscleral meshworks), the juxtacanalicular connective tissue, the endothelial lining of Schlemm’s canal, the collecting channels, and the aqueous veins (Fig. 1).11,12 After having passed through the trabecular outflow pathways, aqueous humor drains into the episcleral venous system.13 In the unconventional or uveoscleral outflow pathways, aqueous humor diffuses through the interstitial spaces between the ciliary muscle bundles, and through the supraciliary and suprachoroidal spaces to reach the capillaries of the ciliary body or the lymphatic vessels of the orbit.14 Accurate measurements of the rate of uveoscleral outflow are difficult to perform in the living eye.14 In human cadaver eyes, the rate of uveoscleral outflow was measured to constitute 4–14% of total outflow.15 Under physiological conditions, only the trabecular outflow pathways are relevant for the generation and maintenance of IOP.16 The trabecular outflow pathways provide resistance against the flow of the aqueous humor and consequently lead to the generation of
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Figure 1 (A) Light micrograph of a meridional section through the trabecular meshwork. (B) is a magnification of (A). In the plane of this section, Schlemm's canal (SC) has two lumens that are separated by a septum. TM, trabecular meshwork; SS, scleral spur; CM, ciliary muscle; AC, anterior chamber; JCT, juxtacanalicular tissue; CTM, corneoscleral trabecular meshwork; UTM, uveal trabecular meshwork. Arrows in (B) point to giant vacuoles in the inner wall endothelium of SC. Magnification bars: 20 μm (A), 5 μm (B). From Ref. 11.
IOP. In eyes with POAG, outflow resistance in the trabecular outflow pathways is usually higher than in age-matched normal control eyes.16 Passage of aqueous humor in the trabecular outflow pathways occurs as bulk flow driven by pressure gradient only and if IOP is in a steady state, the outflow of the aqueous humor equals its secretion from the epithelium of the ciliary body.16
2. TRABECULAR MESHWORK The trabecular meshwork comprises connective tissue lamellae that are covered by flat, epithelial-like trabecular meshwork cells, which rest on a basal lamina and are of neural crest origin.11,17 Anteriorly, the trabecular
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lamellae attach to the peripheral cornea in a region termed Schwalbe’s line. Posteriorly, the trabecular lamellae are connected to the stroma of the ciliary body and iris at their junction, and to the scleral spur (Fig. 1). There is considerable evidence that the region close to Schwalbe’s line serves as a niche for adult stem/progenitor cells that are capable of dividing and repopulating the trabecular meshwork after injury.18 The outermost part of the trabecular meshwork, the juxtacanalicular tissue, lies immediately adjacent to the endothelium of Schlemm’s canal. The juxtacanalicular tissue is not built up by lamellae, but largely consists of loose connective tissue with two to five layers of cells showing no epithelial, but rather a mesenchymal morphology, quite comparable to that of fibroblasts. The cells of the juxtacanalicular tissue are surrounded by an extracellular matrix that consists of fibrillar components and of an amorphous ground substance of hyaluronan and proteoglycans.19 Throughout the juxtacanalicular tissue, a network of elastic fibers spreads, the cribriform plexus, which spans tangentially to the endothelium of Schlemm’s canal. 20,21 The elastic fibers of the cribriform plexus consist of an elastin-containing core and of banded sheath material with a periodicity of about 50 nm. The sheath material contains collagen type VI and fibronectin.20,22 The fibers of the cribriform plexus are connected to the endothelial cells of Schlemm’s canal by fine fibrils that emerge from the sheath material (Fig. 2).
3. SCHLEMM'S CANAL Schlemm’s canal is a modified capillary blood vessel that transiently forms intra- and intercellular pores (Fig. 2), which can acquire unusually large diameters of up to 1 μm.16 As result, the hydraulic conductivity of Schlemm’s canal is the highest in the body and even higher than that in the fenestrated capillaries of the glomeruli in the kidney or the sinusoids in the liver.16 Aqueous humor passes Schlemm’s canal endothelial cells in a basal-luminal direction, which results in the formation of cellular outpouchings of Schlemm’s canal cells (so-called giant vacuoles) in response to the pressure gradient associated with aqueous humor flow. The contacts of Schlemm’s canal endothelial cells with the connecting fibrils of the cribriform plexus prevent the detachment of the endothelium in response to aqueous humor flow. Schlemm’s canal pores likely develop from 60 nm mini-pores that are covered by a diaphragm containing plasmalemmavesicle associated protein (PLVAP).23,24 The molecular processes of pore formation are ill understood, but PLVAP is likely involved, as the formation of vascular pores is largely impaired in PLVAP-deficient mice.23,25
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Figure 2 Transmission electron microscopy of the SC inner wall region. (A) Connecting fibers (CF) in the juxtacanalicular tissue, which emerge from the cribriform elastic plexus, connect the plexus with the inner wall endothelium of Schlemm's canal (SC). The connection with the inner wall endothelium is made via the banded sheath material of the fibers or via fine fibrils that emerge from it (arrows). (B) A giant vacuole (GV) in the inner wall of SC forms an intracellular pore (arrow). Magnification bars: 1 μm. From Ref. 11.
4. OUTFLOW RESISTANCE It is generally accepted that the outflow resistance of the trabecular outflow pathways is localized in the inner wall region comprising the juxtacanalicular tissue and the inner wall endothelium of Schlemm’s canal.16 There are three potential mechanisms, which may be involved in the
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generation of trabecular outflow resistance and which will be discussed here. (1) Outflow resistance largely depends on the quality and quantity of the extracellular matrix in the extracellular spaces of the juxtacanalicular tissue. (2) Outflow resistance depends on the hydraulic conductivity of the inner endothelial layer of Schlemm’s canal. (3) Outflow resistance is generated through a synergistic interaction between the extracellular outflow pathways of the juxtacanalicular tissue and the pores of Schlemm’s canal endothelium. Support for a critical role of the juxtacanalicular tissue extracellular matrix in generating outflow resistance and for mechanism (1) comes from experiments, which indicate that the perfusion of anterior segment organ cultures with enzymes that degrade extracellular matrix compounds, lowers outflow resistance. 26 Arguments against this mechanism are based on studies using morphometry in conjunction with conventional transmission electron microscopy.27,28 The studies show that the area in the juxtacanalicular outflow pathways occupied by electron-dense extracellular matrix is much too small to generate the physiological outflow resistance. Still, the procedures that are applied to process tissue for transmission electron microscopy cause a substantial loss of nonfibrillar extracellular matrix components such as hyaluronan and proteoglycans. In fact, by quick-freeze deep-etch electron microscopy considerable higher amounts of extracellular matrix in the juxtacanalicular tissue were observed than with conventional electron microscopy. 29 Results from studies in which the endothelial pores were visualized by scanning electron microscopy (SEM) and their sizes quantitatively evaluated argue against a critical role of Schlemm’s canal endothelium and against mechanism (2) in generating outflow resistance.30 According to the data of those studies, only 10% of the trabecular outflow resistance would be generated in Schlemm’s canal endothelium. In contrast, more recent observations show that the number of pores in Schlemm’s canal endothelium is influenced by the duration of chemical fixation,31,32 indicating that the number of pores in vivo might actually be significantly smaller than seen with SEM. The idea behind mechanism (3) and the concept of a synergistic functional interplay between juxtacanalicular tissue and Schlemm’s canal endothelium is based on the hypothesis that the extracellular matrix of the juxtacanalicular tissue funnels the aqueous humor right to the pores in Schlemm’s canal endothelium (funneling hypothesis).33 Funneling results in a restriction of aqueous humor flow and thereby generates outflow resistance. Outflow resistance is reduced if either the number of pores in
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Schlemm’s canal endothelium or the flow pathways in the juxtacanalicular tissue increase and attenuate the funneling effect. Currently, the funneling hypothesis appears to be in agreement with the available experimental data.
5. CONTRACTILE MECHANISMS IN THE TRABECULAR OUTFLOW PATHWAYS There are two contractile systems that influence the architecture of the trabecular outflow pathways to modify outflow resistance in the inner wall region. The ciliary muscle is attached to the scleral spur, the posterior attachment of the trabecular meshwork, via tendon-like structures.34,35 Consequently, contraction of the ciliary muscle results in a changed geometry of the trabecular outflow pathways and reduces outflow resistance in the inner wall region. Experimental disinsertion of ciliary muscle’s anterior insertion to the scleral spur completely abolishes its effects on outflow resistance and IOP in primates.36 The second contractile system comprises cells in the trabecular meshwork and the juxtacanalicular tissue, which show properties of contractile myofibroblasts.37 The cells are particularly numerous in the posterior part of both regions and close to their attachment to the scleral spur (scleral spur cells) (Fig. 3).34 The cells in this region show all the structural characteristics of myofibroblasts, like a cytoplasm that contains numerous myofilaments or cellular processes that form tendon-like contacts with the neighboring extracellular matrix fibers. Moreover, the cells contain high amounts of α-smooth muscle actin, the characteristic actin isoform of smooth muscle cells. A major difference between the myofibroblast-like cells in the trabecular outflow pathways and the cells of the neighboring ciliary muscle is the fact that the myofibroblasts are orientated in a circumferential direction and thus right perpendicularly to the longitudinally orientated bundles of the ciliary muscle (Fig. 3B). Mechanosensitive neuronal endings are frequently observed in this region and are most likely part of a proprioceptive system controlling the tone of both contractile systems.39 Experimental data obtained in the trabecular outflow pathways of bovine eyes, where myofibroblast-like cells are especially numerous,40 indicate that an elevated tone of contractile cells in the trabecular outflow pathways results in an elevated outflow resistance in the trabecular meshwork while their relaxation leads to a reduction of outflow resistance.41 The same appears to be true for the trabecular outflow resistance in the primate eye.42 Apparently, ciliary muscle and myofibroblast-like cells of the trabecular outflow system act as
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Figure 3 Meridional (A) and tangential section (B) through ciliary muscle (CM), scleral spur (SS), and trabecular meshwork (TM) stained with antibodies against α-smooth muscle actin. (A) Ciliary muscle cells and vascular smooth muscle cells stain positively with antibodies against α-smooth muscle actin. Arrows indicate the scleral spur, where all cells show intense immunoreactivity for α-smooth muscle actin. (B) Tangential section of scleral spur, trabecular meshwork, and ciliary muscle. Positively stained cells oriented in a circular direction are seen throughout the entire spur tissue. While ciliary muscle cells also stain positive, no staining is seen in the trabecular meshwork. (C) Schematic drawing (orientation analogue to B) depicting the different orientation of the contractile systems that influence the geometry of the trabecular outflow pathways. The anterior longitudinal or meridional ciliary muscle bundles, which attach to scleral spur and trabecular meshwork are oriented perpendicularly to the circumferential or equatorially oriented contractile myofibroblasts of posterior trabecular meshwork (TM) and scleral spur. Arrows indicate movements following contraction or relaxation. Panels (A) and (B): From Ref. 34; Panel (C): From Ref. 38.
functional antagonists while modulating outflow resistance. The likely explanation for their different function is their different orientation at an angle of 90° to each, which, upon contraction, leads to a modification of trabecular outflow pathway geometry in an antagonistic manner (Fig. 3C). Studies in genetically modified mice provided evidence for a critical autoregulative role of nitric oxide that is released from the resident cells
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of the trabecular meshwork outflow pathways as part of an intrinsic mechano-regulatory system. Accordingly, an increasing mechanical load of the trabecular meshwork, e.g., caused by increased IOP increases the trabecular release of nitric oxide and, subsequently, decreases outflow resistance and IOP due to the nitric oxide-mediated relaxation of the cells of the trabecular outflow system.43
6. RESISTANCE OF THE TRABECULAR OUTFLOW PATHWAYS IN PRIMARY OPEN-ANGLE GLAUCOMA The juxtacanalicular tissue of patients with POAG develops very characteristic structural changes in the course of the disease.44 The quality and quantity of the elastic fibers sheath material in the cribriform plexus changes, and there is an increase of material originally described as “sheath-derived plaques.”45,46 The increase in the amounts of sheath-derived plaque material in the juxtacanalicular tissue correlates with the severity of optic nerve damage in POAG.47 However, the amount of sheath-derived plaque material does not correlate with the level of IOP in individual eyes of patients with POAG,47 a finding that strongly indicates that the increase in sheath-derived material is rather a symptom, but not the cause for the increase in trabecular outflow resistance in POAG. The increase in extracellular matrix in the juxtacanalicular tissue occurs in parallel to an increase in the levels of transforming growth factor (TGF)-β2 in the aqueous humor of most patients with POAG.48 Cultured trabecular meshwork increase their extracellular matrix synthesis upon treatment with TGF-βs,48 and may well do so in vivo in response to higher levels of TGF-β2. In addition, treatment with TGF-βs increases the contractile properties of the trabecular meshwork actin cytoskeleton.48 Overall, the effects of TGF-β are modulated by a complex homeostatic signaling system of molecules that stimulate or inhibit its activity in the trabecular outflow pathways.49 Connective tissue growth factor (CTGF) appears to be the downstream molecule that mediates the effects of TGF-βs on trabecular meshwork extracellular matrix synthesis and the actin cytoskeleton of trabecular meshwork cells.50,51 CTGF is critically required for the effects of TGF-β on extracellular matrix synthesis and for increasing the contractile properties of mesenchymal cells including that of the trabecular meshwork.51,52 In general, the force generated by the actin cytoskeleton of mesenchymal cells is transmitted to the surrounding fibrillar extracellular matrix components via integrin-based cell-matrix contacts. A switch to a myofibroblast-like phenotype, a scenario
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that includes the augmentation of the actin cytoskeleton and the directly associated fibrillar extracellular matrix, is apparently caused by high activity of TGF-β signaling and mediated via the downstream mediator CTGF. This concept of a TGF-β/CTGF-mediated switch as the critical causative factor for the increase in outflow resistance and IOP in eyes with POAG is directly supported by results from recent studies in genetically engineered rodents. Transgenic mice with ectopic overexpression of CTGF in their eyes develop POAG characterized by an increase in IOP that correlates with the loss of optic nerve axons.51 The trabecular meshwork outflow pathways of the mice accumulate fibrillar extracellular matrix components and α-smooth muscle actin positive cells as sign of an increase in actin-based contractility. If the mice are treated with a Rho-kinase inhibitor that interferes with actin contractility, IOP returns within hours to levels seen in normal animals. The findings clearly indicate that the effects of CTGF on IOP are mediated through the modification of the trabecular meshwork actin cytoskeleton. We hypothesize that high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a similar change in the phenotype of trabecular outflow cells in human patients as seen in CTGF-overexpressing mice with glaucoma. In response to high activity of TGF-β signaling, the cells switch to a myofibroblast-like phenotype, a scenario, which strengthens simultaneously both their actin cytoskeleton and their directly associated extracellular matrix fibrils (Fig. 4). Because of the increased tone and the accumulation of extracellular matrix material, the trabecular meshwork becomes stiffer and loses its capability to respond to endogenous signals that cause its relaxation and consequently decrease outflow resistance under physiologic conditions. Experiments using atomic force microscopy support this concept as the trabecular meshwork of patients with POAG is stiffer than that of healthy age-matched controls.53 Additional support for the hypothesis comes from a recent study in rats with a vector-based expression of a constitutively active RhoAGTPase (RhoAV14) in the trabecular outflow pathways.54 The rats develop high IOP that is associated with an increase in extracellular matrix and F-actin in the trabecular meshwork outflow pathways. The changes are ameliorated following topical application of a Rho-kinase inhibitor.54 Quite intriguingly, the switch to a stiffer cellular phenotype in POAG is apparently not restricted to the cells of the juxtacanalicular tissue, but happens simultaneously in Schlemm’s canal endothelial cells, the other cell type in the trabecular meshwork outflow pathways that is very likely involved in the generation of outflow resistance. Schlemm’s canal cells of glaucomatous eyes
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Figure 4 Schematic drawing of the actin cytoskeleton and the fibrillar extracellular matrix of cells in the juxtacanalicular tissue. The force generated by the actin cytoskeleton (red) is transmitted to the fibrillar extracellular matrix (green) by integrin-based cell-matrix contacts (yellow). The high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a switch to a myofibroblast-like phenotype including an augmentation of the actin cytoskeleton and its directly associated extracellular fibrillar matrix. Overall, the changes cause an increase in TM rigidity and aqueous humor outflow resistance. From Ref. 38.
were recently shown to have an increased cytoskeletal stiffness that leads to reduced pore formation in the cells, changes that likely contribute to an increase in outflow resistance.55 The stiffness positively correlates with elevated altered expression of several key genes, particularly of CTGF.55 Increase in cellular stiffness may be the common causative theme for the increase in outflow resistance in the trabecular outflow pathways.
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42. Tian B, Gabelt BT, Geiger B, Kaufman PL. The role of the actomyosin system in regulating trabecular fluid outflow. Exp Eye Res. 2009;88(4):713–717. 43. Stamer WD, Lei Y, Boussommier-Calleja A, Overby DR, Ethier CR. eNOS, a pressure-dependent regulator of intraocular pressure. Invest Ophthalmol Vis Sci. 2011;52(13):9438–9444. 44. Tektas OY, Lu¨tjen-Drecoll E. Structural changes of the trabecular meshwork in different kinds of glaucoma. Exp Eye Res. 2009;88(4):769–775. 45. Rohen JW, Futa R, Lu¨tjen-Drecoll E. The fine structure of the cribriform meshwork in normal and glaucomatous eyes as seen in tangential sections. Invest Ophthalmol Vis Sci. 1981;21:574–585. 46. Rohen JW, Witmer R. Electron microscopic studies on the trabecular meshwork in glaucoma simplex. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1972;183:251–266. 47. Gottanka J, Johnson DH, Martus P, Lutjen-Drecoll E. Severity of optic nerve damage in eyes with POAG is correlated with changes in the trabecular meshwork. J Glaucoma. 1997;6(2):123–132. 48. Fuchshofer R, Tamm ER. The role of TGF-beta in the pathogenesis of primary openangle glaucoma. Cell Tissue Res. 2012;347(1):279–290. 49. Fuchshofer R, Tamm ER. Modulation of extracellular matrix turnover in the trabecular meshwork. Exp Eye Res. 2009;88(4):683–688. 50. Junglas B, Yu AH, Welge-Lussen U, Tamm ER, Fuchshofer R. Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells. Exp Eye Res. 2009;88(6):1065–1075. 51. Junglas B, Kuespert S, Seleem AA, et al. Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork. Am J Pathol. 2012;180(6):2386–2403. 52. Fuchshofer R, Ullmann S, Zeilbeck LF, Baumann M, Junglas B, Tamm ER. Connective tissue growth factor modulates podocyte actin cytoskeleton and extracellular matrix synthesis and is induced in podocytes upon injury. Histochem Cell Biol. 2011;136(3): 301–319. 53. Last JA, Pan T, Ding Y, et al. Elastic modulus determination of normal and glaucomatous human trabecular meshwork. Invest Ophthalmol Vis Sci. 2011;52(5):2147–2152. 54. Pattabiraman PP, Rinkoski T, Poeschla E, Proia A, Challa P, Rao PV. RhoA GTPaseinduced ocular hypertension in a rodent model is associated with increased fibrogenic activity in the trabecular meshwork. Am J Pathol. 2015;185(2):496–512. 55. Overby DR, Zhou EH, Vargas-Pinto R, et al. Altered mechanobiology of Schlemm’s canal endothelial cells in glaucoma. Proc Natl Acad Sci USA. 2014;111(38):13876–13881.
Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways Ernst R. Tamm1, Barbara M. Braunger, Rudolf Fuchshofer Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany 1 Corresponding author: e-mail address: [email protected]
Contents 1. Intraocular Pressure and Aqueous Humor Outflow 2. Trabecular Meshwork 3. Schlemm's Canal 4. Outflow Resistance 5. Contractile Mechanisms in the Trabecular Outflow Pathways 6. Resistance of the Trabecular Outflow Pathways in Primary Open-Angle Glaucoma References
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Abstract Intraocular pressure (IOP), the critical risk factor for glaucoma, is generated and maintained by the aqueous humor circulation system. Aqueous humor is secreted from the epithelial layers of the ciliary body and exits the eye through the trabecular meshwork or the uveoscleral outflow pathways. IOP builds up in response to a resistance to aqueous humor flow in the trabecular outflow pathways. The trabecular outflow resistance is localized in the inner wall region, which comprises the juxtacanalicular connective tissue (JCT) and the inner wall endothelium of Schlemm's canal (SC). Outflow resistance in this region is lowered through the relaxation of contractile myofibroblast-like cells in trabecular meshwork and the adjacent scleral spur, or the contraction of the ciliary muscle. In primary open-angle glaucoma, the most frequent form of glaucoma, outflow resistance of the inner wall region is typically higher than normal. There is evidence that the increase in resistance is related to characteristic biological changes in the resident cells of the JCT, which more and more acquire the structural and functional characteristics of contractile myofibroblasts. The changes involve an augmentation of their actin cytoskeleton and of their surrounding fibrillary extracellular matrix, which connects to JCT cells via integrins. This scenario leads to an overall stiffening of the inner wall region, and is modulated by transforming growth factor-β/ Progress in Molecular Biology and Translational Science, Volume 134 ISSN 1877-1173 http://dx.doi.org/10.1016/bs.pmbts.2015.06.007
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connective tissue growth factor signaling. Essentially comparable changes appear to occur in SC endothelial cells. Stiffening of JCT and SC cells is very likely a critical causative factor for the increase in trabecular outflow resistance in POAG.
1. INTRAOCULAR PRESSURE AND AQUEOUS HUMOR OUTFLOW Glaucoma, the leading cause of irreversible blindness throughout the world,1 is a chronic, progressive optic nerve neuropathy, in which optic nerve axons are damaged at the optic disc, the site of their exit from the eye. In several prospective randomized multi-center studies, intraocular pressure (IOP) has been identified as the critical causative risk factor for glaucoma and, consequently, the reduction of IOP delays or prevents the damage of optic nerve axons.2–8 This chapter will review the mechanisms that are thought to generate IOP in the normal eye and in eyes affected with the most common form of glaucoma, primary open-angle glaucoma (POAG). In addition, we will outline a unifying concept to explain the molecular changes that change the biology of the aqueous humor outflow pathways to affect IOP in POAG.9 IOP depends on the aqueous humor circulation system, in which aqueous humor is secreted from the epithelial layers of the ciliary body into the posterior chamber10 and exits the eye in the chamber angle via the conventional or trabecular outflow pathway, or the unconventional or uveoscleral outflow pathway (Fig. 1).11 The trabecular outflow pathway comprises the trabecular meshwork (made up by the uveal and corneoscleral meshworks), the juxtacanalicular connective tissue, the endothelial lining of Schlemm’s canal, the collecting channels, and the aqueous veins (Fig. 1).11,12 After having passed through the trabecular outflow pathways, aqueous humor drains into the episcleral venous system.13 In the unconventional or uveoscleral outflow pathways, aqueous humor diffuses through the interstitial spaces between the ciliary muscle bundles, and through the supraciliary and suprachoroidal spaces to reach the capillaries of the ciliary body or the lymphatic vessels of the orbit.14 Accurate measurements of the rate of uveoscleral outflow are difficult to perform in the living eye.14 In human cadaver eyes, the rate of uveoscleral outflow was measured to constitute 4–14% of total outflow.15 Under physiological conditions, only the trabecular outflow pathways are relevant for the generation and maintenance of IOP.16 The trabecular outflow pathways provide resistance against the flow of the aqueous humor and consequently lead to the generation of
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Figure 1 (A) Light micrograph of a meridional section through the trabecular meshwork. (B) is a magnification of (A). In the plane of this section, Schlemm's canal (SC) has two lumens that are separated by a septum. TM, trabecular meshwork; SS, scleral spur; CM, ciliary muscle; AC, anterior chamber; JCT, juxtacanalicular tissue; CTM, corneoscleral trabecular meshwork; UTM, uveal trabecular meshwork. Arrows in (B) point to giant vacuoles in the inner wall endothelium of SC. Magnification bars: 20 μm (A), 5 μm (B). From Ref. 11.
IOP. In eyes with POAG, outflow resistance in the trabecular outflow pathways is usually higher than in age-matched normal control eyes.16 Passage of aqueous humor in the trabecular outflow pathways occurs as bulk flow driven by pressure gradient only and if IOP is in a steady state, the outflow of the aqueous humor equals its secretion from the epithelium of the ciliary body.16
2. TRABECULAR MESHWORK The trabecular meshwork comprises connective tissue lamellae that are covered by flat, epithelial-like trabecular meshwork cells, which rest on a basal lamina and are of neural crest origin.11,17 Anteriorly, the trabecular
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lamellae attach to the peripheral cornea in a region termed Schwalbe’s line. Posteriorly, the trabecular lamellae are connected to the stroma of the ciliary body and iris at their junction, and to the scleral spur (Fig. 1). There is considerable evidence that the region close to Schwalbe’s line serves as a niche for adult stem/progenitor cells that are capable of dividing and repopulating the trabecular meshwork after injury.18 The outermost part of the trabecular meshwork, the juxtacanalicular tissue, lies immediately adjacent to the endothelium of Schlemm’s canal. The juxtacanalicular tissue is not built up by lamellae, but largely consists of loose connective tissue with two to five layers of cells showing no epithelial, but rather a mesenchymal morphology, quite comparable to that of fibroblasts. The cells of the juxtacanalicular tissue are surrounded by an extracellular matrix that consists of fibrillar components and of an amorphous ground substance of hyaluronan and proteoglycans.19 Throughout the juxtacanalicular tissue, a network of elastic fibers spreads, the cribriform plexus, which spans tangentially to the endothelium of Schlemm’s canal. 20,21 The elastic fibers of the cribriform plexus consist of an elastin-containing core and of banded sheath material with a periodicity of about 50 nm. The sheath material contains collagen type VI and fibronectin.20,22 The fibers of the cribriform plexus are connected to the endothelial cells of Schlemm’s canal by fine fibrils that emerge from the sheath material (Fig. 2).
3. SCHLEMM'S CANAL Schlemm’s canal is a modified capillary blood vessel that transiently forms intra- and intercellular pores (Fig. 2), which can acquire unusually large diameters of up to 1 μm.16 As result, the hydraulic conductivity of Schlemm’s canal is the highest in the body and even higher than that in the fenestrated capillaries of the glomeruli in the kidney or the sinusoids in the liver.16 Aqueous humor passes Schlemm’s canal endothelial cells in a basal-luminal direction, which results in the formation of cellular outpouchings of Schlemm’s canal cells (so-called giant vacuoles) in response to the pressure gradient associated with aqueous humor flow. The contacts of Schlemm’s canal endothelial cells with the connecting fibrils of the cribriform plexus prevent the detachment of the endothelium in response to aqueous humor flow. Schlemm’s canal pores likely develop from 60 nm mini-pores that are covered by a diaphragm containing plasmalemmavesicle associated protein (PLVAP).23,24 The molecular processes of pore formation are ill understood, but PLVAP is likely involved, as the formation of vascular pores is largely impaired in PLVAP-deficient mice.23,25
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Figure 2 Transmission electron microscopy of the SC inner wall region. (A) Connecting fibers (CF) in the juxtacanalicular tissue, which emerge from the cribriform elastic plexus, connect the plexus with the inner wall endothelium of Schlemm's canal (SC). The connection with the inner wall endothelium is made via the banded sheath material of the fibers or via fine fibrils that emerge from it (arrows). (B) A giant vacuole (GV) in the inner wall of SC forms an intracellular pore (arrow). Magnification bars: 1 μm. From Ref. 11.
4. OUTFLOW RESISTANCE It is generally accepted that the outflow resistance of the trabecular outflow pathways is localized in the inner wall region comprising the juxtacanalicular tissue and the inner wall endothelium of Schlemm’s canal.16 There are three potential mechanisms, which may be involved in the
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generation of trabecular outflow resistance and which will be discussed here. (1) Outflow resistance largely depends on the quality and quantity of the extracellular matrix in the extracellular spaces of the juxtacanalicular tissue. (2) Outflow resistance depends on the hydraulic conductivity of the inner endothelial layer of Schlemm’s canal. (3) Outflow resistance is generated through a synergistic interaction between the extracellular outflow pathways of the juxtacanalicular tissue and the pores of Schlemm’s canal endothelium. Support for a critical role of the juxtacanalicular tissue extracellular matrix in generating outflow resistance and for mechanism (1) comes from experiments, which indicate that the perfusion of anterior segment organ cultures with enzymes that degrade extracellular matrix compounds, lowers outflow resistance. 26 Arguments against this mechanism are based on studies using morphometry in conjunction with conventional transmission electron microscopy.27,28 The studies show that the area in the juxtacanalicular outflow pathways occupied by electron-dense extracellular matrix is much too small to generate the physiological outflow resistance. Still, the procedures that are applied to process tissue for transmission electron microscopy cause a substantial loss of nonfibrillar extracellular matrix components such as hyaluronan and proteoglycans. In fact, by quick-freeze deep-etch electron microscopy considerable higher amounts of extracellular matrix in the juxtacanalicular tissue were observed than with conventional electron microscopy. 29 Results from studies in which the endothelial pores were visualized by scanning electron microscopy (SEM) and their sizes quantitatively evaluated argue against a critical role of Schlemm’s canal endothelium and against mechanism (2) in generating outflow resistance.30 According to the data of those studies, only 10% of the trabecular outflow resistance would be generated in Schlemm’s canal endothelium. In contrast, more recent observations show that the number of pores in Schlemm’s canal endothelium is influenced by the duration of chemical fixation,31,32 indicating that the number of pores in vivo might actually be significantly smaller than seen with SEM. The idea behind mechanism (3) and the concept of a synergistic functional interplay between juxtacanalicular tissue and Schlemm’s canal endothelium is based on the hypothesis that the extracellular matrix of the juxtacanalicular tissue funnels the aqueous humor right to the pores in Schlemm’s canal endothelium (funneling hypothesis).33 Funneling results in a restriction of aqueous humor flow and thereby generates outflow resistance. Outflow resistance is reduced if either the number of pores in
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Schlemm’s canal endothelium or the flow pathways in the juxtacanalicular tissue increase and attenuate the funneling effect. Currently, the funneling hypothesis appears to be in agreement with the available experimental data.
5. CONTRACTILE MECHANISMS IN THE TRABECULAR OUTFLOW PATHWAYS There are two contractile systems that influence the architecture of the trabecular outflow pathways to modify outflow resistance in the inner wall region. The ciliary muscle is attached to the scleral spur, the posterior attachment of the trabecular meshwork, via tendon-like structures.34,35 Consequently, contraction of the ciliary muscle results in a changed geometry of the trabecular outflow pathways and reduces outflow resistance in the inner wall region. Experimental disinsertion of ciliary muscle’s anterior insertion to the scleral spur completely abolishes its effects on outflow resistance and IOP in primates.36 The second contractile system comprises cells in the trabecular meshwork and the juxtacanalicular tissue, which show properties of contractile myofibroblasts.37 The cells are particularly numerous in the posterior part of both regions and close to their attachment to the scleral spur (scleral spur cells) (Fig. 3).34 The cells in this region show all the structural characteristics of myofibroblasts, like a cytoplasm that contains numerous myofilaments or cellular processes that form tendon-like contacts with the neighboring extracellular matrix fibers. Moreover, the cells contain high amounts of α-smooth muscle actin, the characteristic actin isoform of smooth muscle cells. A major difference between the myofibroblast-like cells in the trabecular outflow pathways and the cells of the neighboring ciliary muscle is the fact that the myofibroblasts are orientated in a circumferential direction and thus right perpendicularly to the longitudinally orientated bundles of the ciliary muscle (Fig. 3B). Mechanosensitive neuronal endings are frequently observed in this region and are most likely part of a proprioceptive system controlling the tone of both contractile systems.39 Experimental data obtained in the trabecular outflow pathways of bovine eyes, where myofibroblast-like cells are especially numerous,40 indicate that an elevated tone of contractile cells in the trabecular outflow pathways results in an elevated outflow resistance in the trabecular meshwork while their relaxation leads to a reduction of outflow resistance.41 The same appears to be true for the trabecular outflow resistance in the primate eye.42 Apparently, ciliary muscle and myofibroblast-like cells of the trabecular outflow system act as
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Figure 3 Meridional (A) and tangential section (B) through ciliary muscle (CM), scleral spur (SS), and trabecular meshwork (TM) stained with antibodies against α-smooth muscle actin. (A) Ciliary muscle cells and vascular smooth muscle cells stain positively with antibodies against α-smooth muscle actin. Arrows indicate the scleral spur, where all cells show intense immunoreactivity for α-smooth muscle actin. (B) Tangential section of scleral spur, trabecular meshwork, and ciliary muscle. Positively stained cells oriented in a circular direction are seen throughout the entire spur tissue. While ciliary muscle cells also stain positive, no staining is seen in the trabecular meshwork. (C) Schematic drawing (orientation analogue to B) depicting the different orientation of the contractile systems that influence the geometry of the trabecular outflow pathways. The anterior longitudinal or meridional ciliary muscle bundles, which attach to scleral spur and trabecular meshwork are oriented perpendicularly to the circumferential or equatorially oriented contractile myofibroblasts of posterior trabecular meshwork (TM) and scleral spur. Arrows indicate movements following contraction or relaxation. Panels (A) and (B): From Ref. 34; Panel (C): From Ref. 38.
functional antagonists while modulating outflow resistance. The likely explanation for their different function is their different orientation at an angle of 90° to each, which, upon contraction, leads to a modification of trabecular outflow pathway geometry in an antagonistic manner (Fig. 3C). Studies in genetically modified mice provided evidence for a critical autoregulative role of nitric oxide that is released from the resident cells
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of the trabecular meshwork outflow pathways as part of an intrinsic mechano-regulatory system. Accordingly, an increasing mechanical load of the trabecular meshwork, e.g., caused by increased IOP increases the trabecular release of nitric oxide and, subsequently, decreases outflow resistance and IOP due to the nitric oxide-mediated relaxation of the cells of the trabecular outflow system.43
6. RESISTANCE OF THE TRABECULAR OUTFLOW PATHWAYS IN PRIMARY OPEN-ANGLE GLAUCOMA The juxtacanalicular tissue of patients with POAG develops very characteristic structural changes in the course of the disease.44 The quality and quantity of the elastic fibers sheath material in the cribriform plexus changes, and there is an increase of material originally described as “sheath-derived plaques.”45,46 The increase in the amounts of sheath-derived plaque material in the juxtacanalicular tissue correlates with the severity of optic nerve damage in POAG.47 However, the amount of sheath-derived plaque material does not correlate with the level of IOP in individual eyes of patients with POAG,47 a finding that strongly indicates that the increase in sheath-derived material is rather a symptom, but not the cause for the increase in trabecular outflow resistance in POAG. The increase in extracellular matrix in the juxtacanalicular tissue occurs in parallel to an increase in the levels of transforming growth factor (TGF)-β2 in the aqueous humor of most patients with POAG.48 Cultured trabecular meshwork increase their extracellular matrix synthesis upon treatment with TGF-βs,48 and may well do so in vivo in response to higher levels of TGF-β2. In addition, treatment with TGF-βs increases the contractile properties of the trabecular meshwork actin cytoskeleton.48 Overall, the effects of TGF-β are modulated by a complex homeostatic signaling system of molecules that stimulate or inhibit its activity in the trabecular outflow pathways.49 Connective tissue growth factor (CTGF) appears to be the downstream molecule that mediates the effects of TGF-βs on trabecular meshwork extracellular matrix synthesis and the actin cytoskeleton of trabecular meshwork cells.50,51 CTGF is critically required for the effects of TGF-β on extracellular matrix synthesis and for increasing the contractile properties of mesenchymal cells including that of the trabecular meshwork.51,52 In general, the force generated by the actin cytoskeleton of mesenchymal cells is transmitted to the surrounding fibrillar extracellular matrix components via integrin-based cell-matrix contacts. A switch to a myofibroblast-like phenotype, a scenario
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that includes the augmentation of the actin cytoskeleton and the directly associated fibrillar extracellular matrix, is apparently caused by high activity of TGF-β signaling and mediated via the downstream mediator CTGF. This concept of a TGF-β/CTGF-mediated switch as the critical causative factor for the increase in outflow resistance and IOP in eyes with POAG is directly supported by results from recent studies in genetically engineered rodents. Transgenic mice with ectopic overexpression of CTGF in their eyes develop POAG characterized by an increase in IOP that correlates with the loss of optic nerve axons.51 The trabecular meshwork outflow pathways of the mice accumulate fibrillar extracellular matrix components and α-smooth muscle actin positive cells as sign of an increase in actin-based contractility. If the mice are treated with a Rho-kinase inhibitor that interferes with actin contractility, IOP returns within hours to levels seen in normal animals. The findings clearly indicate that the effects of CTGF on IOP are mediated through the modification of the trabecular meshwork actin cytoskeleton. We hypothesize that high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a similar change in the phenotype of trabecular outflow cells in human patients as seen in CTGF-overexpressing mice with glaucoma. In response to high activity of TGF-β signaling, the cells switch to a myofibroblast-like phenotype, a scenario, which strengthens simultaneously both their actin cytoskeleton and their directly associated extracellular matrix fibrils (Fig. 4). Because of the increased tone and the accumulation of extracellular matrix material, the trabecular meshwork becomes stiffer and loses its capability to respond to endogenous signals that cause its relaxation and consequently decrease outflow resistance under physiologic conditions. Experiments using atomic force microscopy support this concept as the trabecular meshwork of patients with POAG is stiffer than that of healthy age-matched controls.53 Additional support for the hypothesis comes from a recent study in rats with a vector-based expression of a constitutively active RhoAGTPase (RhoAV14) in the trabecular outflow pathways.54 The rats develop high IOP that is associated with an increase in extracellular matrix and F-actin in the trabecular meshwork outflow pathways. The changes are ameliorated following topical application of a Rho-kinase inhibitor.54 Quite intriguingly, the switch to a stiffer cellular phenotype in POAG is apparently not restricted to the cells of the juxtacanalicular tissue, but happens simultaneously in Schlemm’s canal endothelial cells, the other cell type in the trabecular meshwork outflow pathways that is very likely involved in the generation of outflow resistance. Schlemm’s canal cells of glaucomatous eyes
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Figure 4 Schematic drawing of the actin cytoskeleton and the fibrillar extracellular matrix of cells in the juxtacanalicular tissue. The force generated by the actin cytoskeleton (red) is transmitted to the fibrillar extracellular matrix (green) by integrin-based cell-matrix contacts (yellow). The high activity of TGF-β signaling in POAG causes, via the downstream mediator CTGF, a switch to a myofibroblast-like phenotype including an augmentation of the actin cytoskeleton and its directly associated extracellular fibrillar matrix. Overall, the changes cause an increase in TM rigidity and aqueous humor outflow resistance. From Ref. 38.
were recently shown to have an increased cytoskeletal stiffness that leads to reduced pore formation in the cells, changes that likely contribute to an increase in outflow resistance.55 The stiffness positively correlates with elevated altered expression of several key genes, particularly of CTGF.55 Increase in cellular stiffness may be the common causative theme for the increase in outflow resistance in the trabecular outflow pathways.
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