Brain Pericytes As Mediators of Neuroinflammation

TIPS 1399 No. of Pages 14

Review

Brain Pericytes [6_TD$IF]As Mediators of Neuroinflammation Justin Rustenhoven,1,2 Deidre Jansson,1,2 Leon C. Smyth,1,2 and Mike Dragunow1,2,* Brain pericytes are perivascular cells that regulate capillary function, and this localization puts them in a pivotal position for the regulation of central nervous system (CNS) inflammatory responses at the neurovascular unit. Neuroinflammation, driven by microglia and astrocytes or resulting from peripheral leukocyte infiltration, has both homeostatic and detrimental consequences for brain function and is present in nearly every neurological disorder. More recently, brain pericytes have been shown to have many properties of immune regulating cells, including responding to and expressing a plethora of inflammatory molecules, presenting antigen, and displaying phagocytic ability. In this review we highlight the emerging role of pericytes in neuroinflammation and discuss pericyte-mediated neuroinflammation as a potential therapeutic target for the treatment of a range of devastating brain disorders. What Is a Pericyte? Pericytes are encapsulated within the basement membrane and ensheathe the capillary endothelium throughout the body. Due to their ubiquitous presence, it is likely that these cells serve both generic and organ/tissue-specific functions. In the brain they are a vital component of the blood–brain barrier (BBB)/neurovascular unit and have extensive contacts with endothelial cells lining the capillaries, astrocyte end feet that enclose cerebral vessels, perivascular microglia/ macrophages, and parenchymal neurons (Figure 1) [1]. The importance of pericytes to CNS function is demonstrated by the brain having the highest ratio of pericyte-to-endothelial cell numbers and the severe neurological pathologies caused by pericyte deficiency [2]. Recently, there has been significant debate regarding what defines a true pericyte [3,4]. This difficulty in classification has largely resulted from their plasticity and the lack of definitive markers to distinguish them from closely related cell types, particularly vascular smooth muscle cells and mesenchymal stromal/stem cells. Further, pericytes display heterogeneity in the brain and have distinct morphologies, and likely functions, depending on their positioning within the vasculature. While precapillary pericytes on the arteriolar end of vessels display processes wrapping around the vasculature, and these are likely to modify vascular diameter, true-capillary pericytes display longitudinal processes along the vessel and contribute more to BBB maintenance [5]. Additionally, pericyte subtypes have been isolated from human brain tissue by differential CD90 expression and these cells display differing phenotypes and functional responses [6]. Several studies have also suggested that pericytes are tissue-resident mesenchymal stromal/stem cells, and this ability to adapt to the local microenvironment may explain their diverse range of functions [3,4]. Brain pericytes are uniquely positioned to regulate multiple aspects of CNS functioning. While a role for brain pericytes in BBB formation, non-glial scarring, and the regulation of cerebral blood flow has been well documented [1,7], the contribution of pericytes to neuroinflammation has been underappreciated. The perivascular localization of pericytes makes them ideally situated to control

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

Trends Pericytes in the brain and spinal cord constitute a new class of cell mediating inflammation in the nervous system. Capillary pericytes control the movement of peripheral immune cells into the nervous system and show significant phagocytic activity associated with clearance of toxic proteins. Pericytes may assume a microglia-like phenotype under some conditions. Capillary pericytes regulate the formation and stability of the blood–brain barrier and the blood–spinal cord barrier and their dysfunction is associated with leakage through these barriers. Immune modulators elicit the release of a plethora of inflammatory mediators from pericytes that may be responsible for the disruption of barriers in the brain and spinal cord. Immune modulators such as interferon gamma impair pericyte function by preventing signalling through plateletderived growth factor receptor beta, which is critical for the proliferation and survival of these cells.

1 Department of Pharmacology and Clinical Pharmacology, The University of Auckland, Auckland, New Zealand 2 Centre for Brain Research, The University of Auckland, Auckland, New Zealand

*Correspondence: [email protected] (M. Dragunow).

http://dx.doi.org/10.1016/j.tips.2016.12.001 © 2016 Elsevier Ltd. All rights reserved.

1

TIPS 1399 No. of Pages 14

Neuron Astrocyte endfoot

Arteriole Artery

Pericyte Endothelial cell Capillary

Basement membrane Microglia

Figure 1. Composition of the Blood–Brain Barrier/Neurovascular Unit. Brain pericytes are embedded in the basement membrane and ensheathe the endothelial cells lining cerebral capillaries. Together with astrocyte end feet, perivascular microglia/macrophages, and neurons, these cells form the neurovascular unit.

several aspects of the CNS immune response, including leukocyte extravasation, inflammationinduced BBB disruption, clearance of waste products, propagation of peripheral and central inflammation, polarization of inflammatory cells in the BBB or brain parenchyma, and adaptive immunity. Furthermore, pericytes express the appropriate receptors to respond to numerous forms of inflammatory insults and are likely to propagate cues derived from the periphery or the brain itself to further worsen CNS outcomes (Figure 2, Key Figure). Here we review the emerging role of pericytes in neuroinflammation, discuss the importance of species differences, describe in vitro models to study these cells, and propose pericyte-mediated neuroinflammation as a potential therapeutic target for the treatment of a range of devastating brain disorders.

Immunological Functions of Brain Pericytes Recruitment of Leukocytes The human brain is typically considered an immune-privileged site with restricted leukocyte access. This is achieved largely by the specialized BBB, which prevents leukocyte surveillance under homeostatic conditions. However, this is not preserved during inflammatory states, neurological disease, or old age. Pericytes express several mediators that can enhance leukocyte extravasation and contribute to an inflammatory cerebral phenotype. During both basal and inflammation-stimulated states [e.g., IL-1b, tumor necrosis factor alpha (TNF/), interferon gamma (IFNg), lipopolysaccharide (LPS), transforming growth factor beta 1 (TGFb1)] pericytes secrete numerous chemokines that function to attract circulating leukocytes to the brain by a concentration-directed gradient [8–16]. Chemokines can be classified into four subfamilies (CXC, CC, CX3C, and XC) that display some degree of leukocyte subtype specificity. Pericytes express members of the CC chemokines [(C-C motif) chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1)], the CX3 chemokines [(C-X-C motif) ligand 1 (CXCL1)], and the CX3C chemokines [(C-X3-C motif) ligand 1 (CX3CL1)] (for the full secretome, see Table 1), allowing them to recruit monocytes, T cells, eosinophils, and neutrophils [8–16]. With respect to human brain pericytes, chemokines represent the predominant component of their secretome. In accordance with the immune privilege observed in the brain, these cells demonstrate a preference for inflammatory (inducible) chemokines as opposed to those involved in homeostatic leukocyte surveillance [8].

2

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

Key Figure

Brain Pericytes As Mediators of Neuroinflammation (A)

(D)

Recruitment of leukocytes

Response to inflammatory smuli

Proinflammatory smulus ICAM1 and VCAM1 mediated leukocyte crawling PAMPs

DAMPs

Chemokine secreon

IL-1β TNFα TGFβ1

NF-κB SMAD2/3

IFNγ

STAT1

Inflammatory response

(B)

(E)

BBB Disrupon

“Resng” microglia

Proinflammatory smulus

Infiltraon of leukocytes

Polarisaon of microglia An-inflammatory phenotype

Entry of plasma proteins Breakdown of basal lamina

ROS/RNS

Pro-inflammatory phenotype

CX3CL1 IL-33

IL-6

MMPs VEGFs increase permeability

(C)

(F)

Endocytosis

Re-acvated T-cell

Degradaon Receptor-mediated endocytosis

Adapve immunity

Phagocytosis of insoluble material Primed CD4+ T-cell TCR MHC-II

Anergy

Internalisaon of angen

T-reg

Naïve CD4+ T-cell

Figure 2. Chemokine secretion by pericytes acts to recruit circulating leukocytes to an inflamed site. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) guidance aids the leukocytes in crawling to gaps in the pericyte coverage, allowing entrance to the brain parenchyma (A). Matrix metalloproteinase (MMP) expression, reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, and vascular endothelial growth factor (VEGF) secretion promote breakdown of the BBB through basement membrane degradation and tight junction disruption (B). Phagocytic and endocytic receptors facilitate uptake of waste products, aggregated proteins, or dying cells, promoting central nervous system clearance (C). Pericytes express receptors for damage-associated molecular patterns (DAMPS), pathogen-associated molecular patterns (PAMPS), and endogenous cytokines, allowing them to mount an immune response or propagate inflammatory responses generated in the brain or periphery (D). Pericytes secrete inflammatory mediators that can polarize parenchymal microglia to a pro- or antiinflammatory phenotype (E). Pericytes internalize antigens and present them to naïve or primed CD4+ T cells to promote T cell anergy, reactivation[8_TD$IF], or T-reg formation (F). NF-kB, nuclear factor kappa light chain enhancer of activated B cells; IFNg, interferon gamma; TNF/, tumor necrosis factor alpha; TGFb1, transforming growth factor beta 1; TCR, T cell receptor; MHCII, MHC class II; CX3CL1, chemokine (C-X3-C motif) ligand 1.

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

3

TIPS 1399 No. of Pages 14

Table 1. Summary of Immune Mediators Expressed by Pericytes Immune family

Immune mediator

Refs

Adhesion molecules

ICAM-1, VCAM-1, MCAM

[6,11,13,18,21]

Chemokines

CCL2 (MCP-1), CCL3, CCL4, CCL5, CCL11, CXCL1, CXCL8 (IL-8), CXCL10 (IP-10), CX3CL1

[8,10–15,80]

Cytokines

IL-1/, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IL-18, IL-33, IFNg, TNF/, G-CSF, GM-CSF

[8,11,13–15,54,83]

Antigen presentation

MHCII/HLA-DR

[9,16,18]

ROS/RNS

iNOS/NO, NOX4/O2 [7_TD$IF]

[13,15,48,49]

Transcription factors

NF-kB, C/EBPd, STAT1, SMAD2/3

[8,11,13,54,81]

Phagocytic/endocytic receptors

Fc receptor, CR3, CD36, CD47, CD68, LRP-1

[13,15,65,66,68]

MMPs

MMP2, MMP9

[13,50,51]

IP-10, IFNg-induced protein 10; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colonystimulating factor; CR3, complement receptor 3.

As well as enhancing chemoattraction to the neurovasculature, chemotactic recognition stimulates conformational changes in leukocyte integrins allowing them to appropriately bind endothelial adhesion molecules. Leukocytes can then undergo rolling adhesion, tight adhesion, and subsequent transmigration through endothelial cells to the perivascular space. To gain access to the brain parenchyma, leukocytes must also traverse pericytes. Pericytes express several adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which enable leukocyte adhesion and help direct their crawling along pericyte processes to gaps in endothelial coverage, preferentially at sites with less basement membrane [17]. Numerous inflammatory stimuli including cytokines (IL-1b and TNF/), pathogen-associated molecular patterns (PAMPS) (e.g., LPS), and damage-associated molecular patterns (DAMPS) [e.g., high-mobility group Box 1 (HMGB1), ATP] enhance pericyte ICAM-1 and VCAM-1 expression [8,10,11,13,17–21]. Blocking ICAM-1/VCAM-1 interactions inhibits neutrophil, monocyte, and T cell adhesion in vitro while ICAM-1 blockade reduces pericyte-mediated leukocyte migration and crawling in vivo [17,19,21]. Pericytes also display prominent expression of melanoma cell adhesion molecule (MCAM/CD146), which has been implicated in endothelial leukocyte adhesion and may be another putative mechanism by which pericytes aid leukocyte attachment [6]. Interestingly, leukocytes appear to congregate in perivascular regions following extravasation and, without appropriate adhesion molecule-mediated guidance by pericytes, may be cleared via perivascular drainage pathways [22]. This could therefore represent a rate-limiting step in CNS leukocyte entrance. However, there are also microvascular regions lacking pericyte coverage and associated basement membrane expression through which leukocytes can more efficiently migrate [23–25]. Proinflammatory cytokines can induce remodelling of these basement membrane components, particularly collagen-IV and laminin-511, enhancing the size of these low-expression regions [24,25]. Similarly, proinflammatory cytokines promote an elongated pericyte morphology in vitro [26] corresponding with enlarged gaps between adjacent processes in vivo [17]. Together these pericyte alterations could contribute to elevated leukocyte breaching of the perivascular space following extravasation. For leukocytes to breach the BBB, they first need to reach the inflamed site. Vasodilation is a typical feature of inflammation and allows elevated blood flow and associated immune cell trafficking. In both the healthy and the diseased brain, pericytes are able to regulate CNS blood

4

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

Box 1. Involvement of Pericytes in Brain Disorders Pericyte dysfunction and inflammation are present in many brain diseases [1]. In AD and MND, loss of capillary pericyte coverage has been related to compromised BBB [68] and blood–spinal cord barrier [107] leakage, respectively. The mechanism underlying this loss of pericyte coverage is unclear, although Ab may be toxic to pericytes [68,108] and this may be the causative agent in AD. Furthermore, TGFb1, which accumulates in AD vessels, induces pericyte expression of several molecules that can damage endothelial cells and the BBB [13]. Less is known about the causative agents in MND but perhaps inflammatory mediators such as IFNg, IL-1b, and TNF/, which are known to compromise pericyte function [57,58], are involved. Leakage through the BBB and the subsequent infiltration of activated leukocytes into the brain parenchyma is considered one of the crucial components of MS development. Stabilization/promotion of pericyte BBB coverage would be an appealing target for combination treatment and has shown promise for the repair of BBB leakage in a model of MS [109]. Pericyte loss also correlates with inflammation-mediated disruption of both the BBB and the blood–retina barrier in diabetes and contributes to tissue pathology [110]. Pericyte dysfunction is also likely to be involved in seizure disorders, where loss of the BBB has been studied as both a cause of epileptogenesis and a consequence of seizures, especially status epilepticus [111]. There is also evidence for pericyte involvement in cerebrovascular disorders, particularly stroke [112]. Traumatic brain and spinal cord disorders also involve inflammation and BBB and spinal barrier leakage [113,114] and targeting pericytes may provide opportunities to understand and treat these traumatic disorders. Finally, there is ongoing research into the role of pericytes in glioblastoma multiforme, which may involve an immunosuppressive pericyte phenotype [115]. Augmenting rather than ablating pericyte inflammatory signaling might be therapeutic in this context. Other research indicates that glioma stem cells may convert into a pericyte phenotype to promote the formation and stabilization of tumor vasculature [116]. Overall, although it remains in its infancy the results of studies of pericyte biology in various brain diseases suggests that there are disease-specific alterations. Loss of pericytes has been demonstrated in AD, MND, and diabetic retinopathy, although the inducers and probably the mechanisms are likely to be varied. Less is known about the role of pericytes in other brain disorders, but pericytes are also likely to be involved in brain scar formation [114].

flow through prostaglandin E2 (or other EP4 agonists) and nitric oxide (NO) production in response to the neurotransmitters noradrenaline and glutamate [27]. Importantly, both NO and prostaglandin E2 are elevated during neuroinflammatory states by inducible NO synthase (iNOS) and COX-2, respectively, and these could potentially contribute to pericyte-mediated vascular dilation [28,29]. During proinflammatory states pericyte-mediated vasodilation and associated elevations in blood flow could contribute to enhanced leukocyte delivery and subsequent entrance into the brain, further worsening CNS outcomes. BBB Disruption The BBB is a highly selective permeability barrier separating the brain from the blood. Unlike the peripheral vasculature, cerebral vessels comprise endothelial cells connected by tight junctions preventing the paracellular transport of large or hydrophilic molecules while allowing the passage of water, gas, and certain lipid-soluble molecules via passive diffusion [30,31]. In the absence of paracellular transport pathways, the BBB contains several active transport mechanisms to facilitate molecule uptake as well as numerous methods for peptide transport [32–34]. This unique arrangement of the neurovasculature also functions to largely restrict the passage of leukocytes, neurotoxins, and microbes to the CNS under normal conditions, thereby preventing extensive immune surveillance of the brain parenchyma and the entrance of pathological mediators [35]. In the non-inflammatory/non-diseased brain, pericytes maintain BBB integrity and their loss drastically enhances vascular permeability [7]. BBB impairment and microvascular pathology has been observed during normal aging, mild cognitive impairment, and numerous neurological disorders including Alzheimer's disease (AD), motor neurone disease (MND), multiple sclerosis (MS), and stroke, by both MRI studies and

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

5

TIPS 1399 No. of Pages 14

post-mortem analysis of human brain tissue [36–40]. More recently, BBB leakage has been linked to pericyte dysfunction/loss in human disease, highlighting the importance of these cells in maintaining barrier function [41–43]. Pericyte loss or dysfunction in numerous diseases promotes a leaky BBB allowing the extravasation of toxic-blood derived products such as fibrinogen, which accumulates around the neurovasculature as insoluble fibrin [44]. Fibrinogen/fibrin infiltration results in perivascular clustering and activation of microglia as well as chemokine- and antigen presentation-mediated recruitment and activation of T cells precipitating axonal degeneration [45,46]. Furthermore, while fibrin may enter the brain only through a compromised BBB, fibrinogen depletion attenuated neuroinflammation and vascular damage in a transgenic AD mouse model, suggesting that these deposits further contribute to neurovascular dysfunction following BBB compromise [47]. While pericyte loss clearly promotes BBB disruption, viable pericytes can also produce numerous proinflammatory mediators detrimental to BBB function. Reactive oxygen/nitrogen species (ROS/RNS) precipitate oxidative/nitrosative stress-induced apoptosis and pericytes express several forms, particularly iNOS-derived NO and NADPH oxidase 4 (NOX4)-derived superoxide (O2 [10_TD$IF]). Pericyte-derived NO and nitrosative stress are induced in vitro in response to the bacterial cell membrane endotoxin LPS [15]. Furthermore, NOX4 is expressed in human pericytes under basal conditions and is inducible in vitro by hypoxia and TGFb1 or by permanent/transient middle cerebral artery occlusion (pMCAO/tMCAO)-induced cerebral hypoxia in mice [13,48,49]. Interestingly, NOX4 overexpression in a pMCAO stroke model exacerbated BBB breakdown and enhanced nuclear factor kappa light chain enhancer of activated B cells (NF-kB) phosphorylation and proinflammatory signaling including matrix metalloproteinase 9 (MMP9) induction [49]. MMPs represent another source of pericyte-derived products implicated in BBB breakdown. Pericytes express two main family members, MMP2 and MMP9, which cleave basement membrane substrates including collagen-IV, laminin, and fibronectin. MMP9 but not MMP2 is induced by TNF/ [50] while TGFb1 elevated both MMP9 and MMP2 production in human brain pericytes [13,51]. Basement membrane cleavage precipitates a weakened BBB and enhanced endothelial permeability. Additionally, MMP2/9 promotes the activation of vascular endothelial growth factor (VEGF), a growth factor that is inducible by MCAO in vivo and sodium cyanide-induced chemical hypoxia in vitro and enhances BBB permeability [52–54]. Furthermore, MMPs contribute to BBB breakdown and enhanced permeability through tight junction [zona occludens-1 (ZO-1) and occludin] rearrangement [55]. Interestingly, MMP9 expression is elevated in the cerebrospinal fluid of patients with the apolipoprotein E4 allele and correlates with BBB breakdown [56]. Similarly, both pericyte expression of MMP9 and BBB breakdown are enhanced in post-mortem AD brains of apolipoprotein E4 carriers [43], suggesting that this may contribute to pericyte MMP9-induced BBB disruption. Furthermore, inflammatory mediators can alter the function of brain pericytes. For example, chronic treatment of brain pericytes with the proinflammatory cytokines IL-1b and TNF/ decreased mediators promoting BBB formation (angiopoietin 1), basement membrane deposition (fibronectin 1), pericyte function [platelet-derived growth factor receptor beta (PDGFRb)], and gap junction formation (connexin 43) [57]. Similarly, chronic treatment of primary adult human brain pericytes with the proinflammatory cytokine IFNg blocks PDGFRb signaling in brain pericytes, suggesting that a proinflammatory cerebral environment deregulates critical mediators of BBB function [58]. The effects of IFNg on the PDGFRb signaling cascade were particularly dramatic as this ultimately stopped platelet-derived growth factor subunit B (PDGF-BB) from increasing pericyte proliferation and migration. Given that the PDGF-BB–PDGFRb signaling pathway in pericytes is crucial for brain capillary development and function, the implications of this action are widespread. Furthermore, peripheral inflammation (e.g., sepsis) as well as

6

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

ischemic stroke may promote either detachment of pericytes from the BBB or pericyte death, attenuating endothelial coverage and further worsening vessel stability [59,60]. Interestingly, hypoxia and amyloid beta 1–40/42 (Ab1–40/42) also promotes membrane shedding of critical pericyte proteins, including NG2 and PDGFRb, impairing their normal functions and likely contributing to pericyte dysfunction [61,62]. Endocytosis Correct cerebral functioning requires strict regulation of factors entering the CNS microenvironment. While the BBB normally limits cerebral access, its disruption allows the penetrance of neurotoxic blood-derived products precipitating neuroinflammation and neurodegeneration [7]. Pericytes can internalize a range of matter entering a breached BBB, including small molecules (dextran and horseradish peroxidase) and neurotoxic blood-derived products (fibrin and immunoglobulins), through receptor-mediated endocytosis or nonspecific pinocytosis and possess numerous lysosomal granules, suggesting that they are structurally equipped to degrade internalized material [44,63,64]. Pericytes can also internalize large solid matter through phagocytic pathways and display IgG, complement, and scavenger receptors to coordinate recognition of a diverse range of substrates [65]. In situ, pericytes have been found to accumulate whole erythrocytes of the order of 9 mm in size, while in vitro studies demonstrate phagocytosis of mock waste products in the form of polystyrene beads (0.1–9 mm diameter) [9,13,66,67]. While the proinflammatory cytokines TNF/ and IFNg enhance phagocytic uptake by pericytes, the anti-inflammatory mediator TGFb1 attenuates phagocytic uptake, possibly by reducing scavenger receptor expression [9,13]. Pericytes may also clear matter derived from the brain parenchyma, particularly disease-related proteins, and their perivascular location makes them ideally positioned to do so. Through receptor-mediated endocytic pathways, particularly low-density lipoprotein receptor-related protein 1 (LRP-1), pericytes clear soluble Ab from the brain preventing parenchymal and perivascular plaque deposition [68–72]. Recently, pericytes have also been shown to internalize preparations enriched for amylin oligomers in vitro and demonstrate amylin inclusions in situ [64]. Importantly, accumulation of both Ab1–40 monomers and amylin oligomers promoted pericyte toxicity. Furthermore, vascular smooth muscle cells, which appear phenotypically similar to brain pericytes, can internalize aggregates of Ab1–42 and, unlike monomer or oligomers, aggregation negates Ab-induced toxicity; it is tempting to postulate that pericytes could also phagocytose aggregated forms of these proteins [73,74]. While parenchymal plaques of Ab1–42 are increasingly considered to be inert, perivascular Ab1–40/1–42 depositions present in AD and cerebral amyloid angiopathy (CAA) are highly detrimental to perivascular and glymphatic drainage and neurovasculature viability and their clearance is likely to be beneficial [75–77]. Pericyte loss enhances vascular Ab deposition, supporting a role for these cells in Ab clearance [68]. During cerebral ischemia or oxygen–glucose deprivation, brain pericytes display a reduction of pericyte markers [CD13, alpha smooth muscle actin (/SMA), and PDGFRb] and enhanced expression of microglial markers [ionized calcium-binding adapter molecule 1 (IBA1), MHC class II (MHCII), CD11b, and CD68] corresponding with phagocytic uptake of polystyrene beads [78,79]. As pericytes also display phagocytic capacity, it would be interesting to determine whether this function is induced in these microglia-like pericytes. Following ischemic stroke, enhanced phagocytic uptake precipitated by a microglia-like phenotype could be beneficial in clearing compromised cells or neurotoxic matter breaching an impaired BBB. Augmenting this function in pericytes may be beneficial in promoting the clearance of other disease-related proteins. Pericyte-Mediated Propagation of Peripheral or CNS Inflammation As well as contributing directly to CNS immunity, pericytes can propagate inflammatory responses generated elsewhere. Pericytes mount an inflammatory response following

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

7

TIPS 1399 No. of Pages 14

recognition of endogenous cytokines (e.g., TNF/, IL-1b, IFNg, TGFb1) and the release of intracellular host-derived DAMPs (e.g., HMGB1, ATP), suggesting innate immune receptor expression [8–10,12–14,21]. Additionally, pericytes contain receptors for PAMPs, including Toll-like receptor 4 (TLR4) and TLR2, which recognize Gram-negative bacterial membrane components, fungi, bacterial peptidoglycans, and certain viruses to induce an inflammatory response [8,9,15,21]. Recently, pericytes have also been shown to express non-TLR pattern recognition receptors (PRRs) including nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which recognizes peptidoglycan components of Gram-negative bacteria to mount an immune response [80]. Together these findings demonstrate that pericytes can respond to numerous forms of insult, including circulating proinflammatory cytokines, and compromised cells present in peripheral sterile inflammation or bacterial or viral infection to enhance vascular inflammation. Additionally, pericytes could propagate inflammation derived from local CNS inflammation, including DAMPS present in acute brain injuries (stroke and traumatic brain injuries) and inflammatory cytokines generated by reactive microglia or invading leukocytes in neurodegenerative diseases, as well as bacterial or viral infection to further enhance the cerebral immune response. The induction of pericyte inflammatory mediators following immunogenic recognition requires transcriptional regulation. NF-kB-mediated gene transcription is implicated in immunogenic recognition of the majority of immunogenic stimulus responses (LPS, IL-1b, TNF/, C12-iE-DAP) [8,12,80]. Additionally, IFNg and TGFb1 signal through the signal transducer and activator of transcription 1 (STAT1) and mothers against decapentaplegic homolog 2/3 (SMAD2/3) transcription factors respectively and both can modify pericyte-mediated inflammation [13,81]. Furthermore, a member of the CCAAT/enhancing binding protein (C/EBP) family, C/EBPd, dampens adhesion molecule and chemokine secretion in pericytes, probably as a secondary consequence of NF-kB-mediated induction [11]. The investigation of pericyte-specific transcription factors is of significant value for our understanding and selective regulation of pericyte immune responses. Polarization of Parenchymal Immune Cells Pericytes secrete numerous immunomodulatory factors that can impact the polarization state adopted by neighboring cells of the neurovascular unit. In particular, pericytes secrete several proinflammatory mediators following immunological activation, including IL-1b, TNF/, IFNg, and IL-6, each of which can induce a proinflammatory state in astrocytes, microglia, and endothelial cells and precipitate apoptotic neuronal death [14,15,82]. Conversely, pericytes can also secrete several factors implicated in anti-inflammatory roles, including CX3CL1 and IL-33 [13,83]. IL-33 has been shown to be beneficial in preventing microglial activation in AD and can be specifically enhanced in pericytes through PDGF-BB stimulation [83,84]. Similarly, CX3CL1 promotes a strong anti-inflammatory microglial phenotype and appears to be neuroprotective [85]. The exact contribution of the pericyte secretome is likely to depend on the specific context, but coculture systems will be highly useful in elucidating the polarization state following pericyte inflammation. Adaptive Immunity Besides contributing to innate immunity, pericytes may also modulate adaptive CNS immune functions. Pericytes display PRRs facilitating the uptake of foreign or self-antigens and IFNginducible MHCII/human leukocyte antigen (HLA)-antigen D related (HLA-DR), -DP, and -DQ complexes, suggesting that they can present antigens to CD4+ T lymphocytes [9,16,18]. However, MHCII–T cell receptor binding is not sufficient alone for activation of naïve T cells but requires further co-stimulatory molecules (e.g., CD86, CD80) and instructive cytokines (e.g., IL-12) that appear to be lacking in pericytes [86]. In the absence of these secondary signals, antigen presentation promotes T cell anergy, a phenomenon in which T cells

8

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

remain viable but are functionally inactivated; this was observed utilizing in vitro co-culture models of brain pericytes and naïve CD4+[1_TD$IF] T cells [86,87]. Interestingly, despite their lack of co-stimulatory molecules, pericytes were found to specifically promote the formation of functionally immunosuppressive T-reg populations promoting an anti-inflammatory phenotype [87]. While a very small number of circulating naïve T cells can be found in the brain [88], these are predominantly situated in secondary lymphoid organs. While perivascular and glymphatic drainage pathways terminate in cervical lymph nodes and pericytes have been shown to detach from the cerebral vasculature and enter the peripheral circulation during pathological conditions (e.g., sepsis, stroke), there is currently no evidence that pericytes can home to these sites to present antigens to naïve T cells [59,60,89]. Instead, pericytes are likely [12_TD$IF]involved [13_TD$IF]in [14_TD$IF]the reactivation of extravasated T cells previously primed by professional antigen-presenting cells (APCs) [90]. Pericytes stimulate the expansion of and cytokine secretion by primed T cells [18,86], with a preference for Th1 lymphocytes over Th2 [91]. While T cells are typically excluded from the brain under normal conditions by the BBB, extensive infiltration occurs in MS and to a lesser extent in other neurological diseases [35]. Prevention of pericyte-mediated re-priming could attenuate the detrimental aspects of these cells in such disorders.

Species Differences Recent data suggest that rodent models poorly reflect human immune responses and, more importantly, human neuroinflammation (reviewed in [92]). This could partially explain why drugs developed in rodent models to treat brain inflammation have not worked in humans. Like parenchymal brain immune cells such as microglia, this discrepancy appears to also hold true for brain pericytes. Nonhuman-derived pericytes (murine and porcine) express numerous proand anti-inflammatory mediators that appear to be absent, significantly attenuated, or unconfirmed in human pericytes. In particular, human pericytes appear to secrete large amounts of MCP-1, IL-6, IL-8, and CXCL1 but significantly lower or non-detectable levels of other mediators, particularly the proinflammatory cytokines IL-1b, IFNg, and TNF/, found in rodent or porcine in vitro models [8–14,54]. These findings emphasize the importance of species considerations in the study of basic pericyte biology and pharmacology. Such differences could drastically alter the in vivo function of brain pericytes and indicate that in vitro studies of pericyte biology and pharmacology are best performed on human brain-derived cells. To facilitate such studies, commercial human brain pericytes are available. Our studies have focused on the use of pericytes derived from adult human brain autopsy and biopsy specimens and we have published these standardized protocols [11–13,58]. These isolated pericytes can be utilized to explore inflammatory contributions in response to numerous immunogenic stimuli to better understand pericyte inflammatory biology as well as provide a valuable tool for the investigation of antiinflammatory interventions. In particular, the ability to culture, study, and test pericytes derived from donors with neurological disorders has enormous potential for translational drug development. While in vivo animal models of pericyte function are likely to have significant species differences, it is currently unclear whether in vitro models of human pericytes accurately recapitulate in vivo phenotypes. Ideally, future studies will investigate neuroinflammation in live human patients. Functional imaging of neuroinflammation in live human patients is currently possible through the use of positron emission tomography and single-photon emission CT combined with various radio tracers that specifically bind activated microglia or disease-related proteins including Ab and tau [93]. Future development of pericyte-specific tracers, particularly inflammation-activated pericytes, would enable similar observations in humans. Combining such studies with dynamic contrast-enhanced MRI to detect BBB permeability and with biomarkers for pericyte-specific dysfunction such as soluble PDGFRb [38,42,61] could be highly beneficial in furthering our understanding of pericyte (dys)function in humans.

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

9

TIPS 1399 No. of Pages 14

In Vitro Co-culture Systems to Study Pericyte Biology While isolated cultures of pericytes allow the discrimination of cell-specific effects, particularly inflammatory secretions or gene expression data, they fail to recapitulate the multicellular microenvironment of the BBB. To address these issues, several co-culture models have been developed, typically with endothelial cells. Simple same-well 2D co-cultures [94] can enable paracrine signaling between pericytes and endothelial cells, which is lacking in monoculture systems, and be used to investigate how each cell type influences the inflammatory state of the other. Multitiered [9_TD$IF]transwell cultures incorporating well inserts [95] allow detailed investigations of how pericytes can modify endothelial barrier formation and immune cell trafficking between two semi-isolated compartments. Endothelial pericyte cultures in 3D extracellular matrices spontaneously form vessel-like structures [95] and can be utilized to investigate how inflammatory phenotypes alter angiogenesis. Last, complicated dynamic models on microfluidic devices incorporating both 3D structures and aspects of sheer stress [96] can more reliably model BBB extravasation in the presence of physiological blood flow (Figure 3). These co-culture models allow more biologically relevant interactions with cells of the neurovasculature – typically, pericyte–endothelial cell interactions, but often also incorporating astrocytes. Furthermore, these systems can also be manipulated to study the effects of other cell types; for example, whether pericytes alter the microglial inflammatory state through the release of CX3CL1 [13] or contribute to neuronal death by ROS/RNS-mediated mechanisms [15,48]

Pericyte-Mediated Inflammation [6_TD$IF]As a CNS Drug Target Neuroinflammation is present in almost all neurological diseases and its attenuation is likely to be beneficial in promoting neuronal survival. Targeting various aspects of pericyte immune functions therefore has promise in the treatment of a diverse range of neurological disorders (Box 1). Due to their close association with the cerebral vasculature, pericytes represent an attractive drug target. Unlike cells in the brain parenchyma, their perivascular positioning means that they do not require a significant amount of drug diffusion once it has passed the endothelial cells. The crowded cellular composition of the brain significantly limits passive drug diffusion, often creating

Monolayer

Mullayer

Transwell

Three-dimensional

Dynamic

Key: Astrocyte Pericyte

Pericytes and endothelia

Endothelia

Pericytes, astrocytes and endothelia

Figure 3. In Vitro Models for the Study of Pericyte Biology. Primary pericytes isolated from brain tissue or purchased cell lines can be utilized in isolation or in coculture systems with other cells of the blood–brain barrier (BBB). In order of increasing complexity and physiological relevance, pericytes can be cultured in a mixed adherent monolayer system with endothelial cells or in a multilayer system to better enable endothelial barrier formation and pericyte coverage. To enable permeability and migration studies, these multilayer systems can be modified to utilize [9_TD$IF]transwell inserts, where cells can be cultured on either side of the inserts or in other combinations allowing paracrine signaling without physical contact. Utilizing various extracellular matrices rather than adherent culture dishes, 3D co-cultures of pericytes and endothelial cells spontaneously organize themselves into tube-like structures with pericyte coverage resembling blood vessels and such systems can be utilized to study angiogenesis. Last, dynamic microfluidic models incorporating shear forces on endothelial cells provided by fluid movement model physiological blood flow and can enable transmigration studies that closely resemble the in vivo phenotype. To further enhance the physiological relevance of these co-culture systems, astrocytes can be incorporated into the above models to model the physical contact or barrier-enhancing properties provided by astrocyte end feet at the BBB.

10

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

concentration gradients above and below therapeutic ranges [97]. Furthermore, in several neurological diseases a disrupted BBB allows the passage of drugs that would not cross an intact barrier. In various forms of brain cancer, this barrier is selectively disrupted in the region of the tumor allowing selective drug entrance. These features make the targeting of pericytes an attractive candidate for drug development. Importantly, pericytes are not specific to the brain but are present in all of the peripheral vasculature. In contrast to the BBB, endothelial barriers in most other tissues lack tight junctions, allowing paracellular transport and greater drug penetration [31]. Like brain pericytes, peripheral pericytes respond to proinflammatory cytokines and DAMPS to mount a functional immune response (chemokine/cytokine secretion and adhesion molecule expression) but whether this differs from the brain is currently unclear [17,98]. Such matters complicate the ability to selectively target brain pericytes for neurological disorders. However, pericytes in the forebrain are unique in that they arise from the neuroectoderm [1], and have differentiation potential for cells of the CNS (e.g., neurons, astrocytes, microglia) [79,99]. This suggests that brain pericytes may differ intrinsically from the mesoderm-derived peripheral pericytes, which holds promise for the ability to selectively target such cells. Transcriptome studies of other CNS immune cells, namely microglia, have enabled the identification of specific genes [100] or cytokine-response patterns [101] distinguishing them from peripheral macrophages and future studies should seek to isolate brain pericyte-specific signatures for pharmacological manipulation. Several attempts have been made to specifically target pericytes for therapeutic purposes. In particular, the elimination of tumors through vascular disruption by antibody-mediated blockade of PDGFRb or the addition of drug-carrying liposomes with pericyte-specific motif (e.g., NG2)conjugated peptides has been examined [102,103]. Furthermore, exogenous application of PDGF-BB, the ligand for PDGFRb, is found to be neuroprotective in a mouse model of Parkinson's disease, and restoring BBB function could be beneficial in numerous other diseases with BBB breakdown [104]. Translation of these strategies could be beneficial for the specific targeting of pericyte-mediated inflammation and other aspects of pericyte biology. Furthermore, augmenting the anti-inflammatory functions of pericytes, particularly the release of CX3CL1 and IL-33, and enhancing their phagocytic phenotype might be beneficial in maintaining an appropriate microglial phenotype and attenuating global inflammation. A probable weakness in many anti-inflammatory drug interventions is the targeting of specific factors; for example, inhibitors of MCP-1. Such strategies overlook the complexity of inflammatory responses involving chemokine, cytokine, adhesion molecule, ROS, and MMP responses. While multidrug-target approaches may be valid, another interventional strategy is the targeting of transcription factors that regulate these immune functions following inflammatory insult. Due to the conserved promoter/enhancer regions, a single transcription factor can regulate the expression of a range of inflammatory mediators, making them attractive targets. While pericytespecific [15_TD$IF]transcription factors have not been demonstrated, their identification could prove useful. Furthermore, pericytes express differential receptors for inflammatory cytokines compared with other cell types in the BBB. For example, pericytes mount a proinflammatory response to IL-17 while endothelial cells do not [105]. Identifying pericyte-specific receptors would be useful for their selective targeting. Recently, the transcriptome of brain pericytes has been examined, allowing the investigation of pericyte-specific immune functions including secreted cytokines, receptors, and transcription factors that can contribute to neuroinflammation [106].

Outstanding Questions How relevant are in vitro studies to the whole human? Given that a large proportion of pericyte studies are undertaken using in vitro models (largely rodent with a few human), a key issue in the study of pericyte biology and pharmacology is the relevance of this work to the human brain and spinal cord. This is a critical question. Central to this question is the culture and identification of pericytes in vitro. Different culture conditions can generate pericytes with varied phenotype – which conditions match in vivo reality? How do capillary-based pericytes differ from arteriole-based smooth muscle cells phenotypically and functionally? Although /SMA is used to identify pericytes, when the expression of this protein is studied in the human brain it labels predominantly larger vessels and not capillaries. By contrast, PDGFRb labels both capillaries and larger vessels. Do the two respond similarly to inflammatory cues? Do pericytes from neurodegenerative disorders such as AD and MND display a disease-related phenotype? If so, can these be targeted for disease-specific pericyte-mediated therapies? Finally, perhaps the most important question: will enhancing pericyte ‘health’ and/or proliferation/number and/or reducing pericyte inflammation repair a damaged BBB or spinal cord–blood barrier and provide a therapeutic outcome in brain and spinal cord disorders?

Concluding Remarks Brain pericytes are gaining widespread attention in neuropharmacology as critical mediators of neuroinflammation and as regulators of barrier functions in the brain and spinal cord. In this review we have summarized the current research surrounding the significant role of pericytes in

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

11

TIPS 1399 No. of Pages 14

neuroinflammation, which is tantalizing for those interested in the brain's immune functions. Pericytes occupy a pivotal perivascular niche, making them important regulators of the neurovascular unit interfacing between the peripheral immune system and the brain and spinal cord. Understanding this interaction and how pericytes contribute to brain inflammation and BBB and blood–spinal cord barrier dysfunction will provide novel insights into the pharmacological management of a range of CNS and spinal cord inflammatory disorders and injuries (see Outstanding Questions). However, much research is still required to understand the roles of pericytes in neuroinflammation and neurodegeneration. For example, although there are compelling data showing that pericytes secrete a plethora of inflammatory mediators, these are mainly derived from in vitro studies. We still need to demonstrate that pericytes in vivo secrete inflammatory mediators that contribute to neuroinflammation, BBB leakage, and neurodegeneration. Furthermore, we still do not have direct evidence that brain pericytes secrete inflammatory mediators in human neurodegenerative disorders. It will be especially interesting to determine whether there are neuroinflammatory pericyte signatures associated with specific neurodegenerative disorders. We also have no direct evidence that inhibiting pericyte-mediated inflammation has any protective effect for the brain or the BBB. These studies will be vital in translating this laboratory research into clinical outcomes. Overall, despite these caveats, pericytes hold great promise for clinical neuropharmacology: identifying molecules that promote pericyte health, especially in the injured, diseased, or aging human brain, may provide a novel strategy for the promotion of brain health. References 1.

Winkler, E.A. et al. (2011) Central nervous system pericytes in health and disease. Nat. Neurosci. 14, 1398–1405

2.

Winkler, E.A. et al. (2014) The pericyte: a forgotten cell type with important implications for Alzheimer's disease? Brain Pathol. 24, 371–386

3.

de Souza, L.E. et al. (2016) Mesenchymal stem cells and pericytes: to what extent are they related? Stem Cells Dev. Published online November 3, 2016. http://dx.doi.org/10.1089/ scd.2016.0109

4.

5.

Attwell, D. et al. (2015) What is a pericyte? J. Cereb. Blood Flow Metab. Published online September 10, 2015. http://dx.doi.org/ 10.1177/0271678X15610340 Armulik, A. et al. (2011) Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215

15. Kovac, A. et al. (2011) Brain microvascular pericytes are immunoactive in culture: cytokine, chemokine, nitric oxide, and LRP-1 expression in response to lipopolysaccharide. J. Neuroinflammation 8, 139 16. Smith, A.M. et al. (2013) Adult human glia, pericytes and meningeal fibroblasts respond similarly to IFNy but not to TGFb1 or MCSF. PLoS One 8, e80463 17. Proebstl, D. et al. (2012) Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. J. Exp. Med. 209, 1219–1234 18. Balabanov, R. et al. (1999) Role of central nervous system microvascular pericytes in activation of antigen-primed splenic T-lymphocytes. J. Neurosci. Res. 55, 578–587 19. Verbeek, M.M. et al. (1995) T lymphocyte adhesion to human brain pericytes is mediated via very late antigen-4/vascular cell adhesion molecule-1 interactions. J. Immunol. 154, 5876– 5884

6.

Park, T.I. et al. (2016) Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression. Sci. Rep. 6, 26587

7.

Armulik, A. et al. (2010) Pericytes regulate the blood–brain barrier. Nature 468, 557–561

20. Ayres-Sander, C.E. et al. (2013) Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes. PLoS One 8, e60025

8.

Guijarro-Munoz, I. et al. (2014) Lipopolysaccharide activates TLR4-mediated NF-kB signaling pathway and proinflammatory response in human pericytes. J. Biol. Chem. 289, 2457– 2468

21. Stark, K. et al. (2013) Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and ‘instruct’ them with pattern-recognition and motility programs. Nat. Immunol. 14, 41–51

9.

Pieper, C. et al. (2014) Brain capillary pericytes contribute to the immune defense in response to cytokines or LPS in vitro. Brain Res. 1550, 1–8

22. Owens, T. et al. (2008) Perivascular spaces and the two steps to neuroinflammation. J. Neuropathol. Exp. Neurol. 67, 1113–1121

10. Pieper, C. et al. (2013) Pericytes support neutrophil transmigration via interleukin-8 across a porcine co-culture model of the blood–brain barrier. Brain Res. 1524, 1–11 11. Rustenhoven, J. et al. (2015) An anti-inflammatory role for C/EBPd in human brain pericytes. Sci. Rep. 5, 12132 12. Jansson, D. et al. (2014) A role for human brain pericytes in neuroinflammation. J. Neuroinflammation 11, 104 13. Rustenhoven, J. et al. (2016) TGF-b1 regulates human brain pericyte inflammatory processes involved in neurovasculature function. J. Neuroinflammation 13, 1–15 14. Matsumoto, J. et al. (2014) Tumor necrosis factor-alpha-stimulated brain pericytes possess a unique cytokine and chemokine release profile and enhance microglial activation. Neurosci. Lett. 578, 133–138

12

23. Sweeney, M.D. et al. (2016) Pericytes of the neurovascular unit: key functions and signaling pathways. Nat. Neurosci. 19, 771–783 24. Voisin, M-B. et al. (2010) Venular basement membranes ubiquitously express matrix protein low-expression regions: characterization in multiple tissues and remodeling during inflammation. Am. J. Pathol. 176, 482–495 25. Wang, S. et al. (2006) Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils. J. Exp. Med. 203, 1519–1532 26. Tigges, U. et al. (2013) TNF-/ promotes cerebral pericyte remodeling in vitro, via a switch from /1 to /2 integrins. J. Neuroinflammation 1, 331–310 27. Hall, C.N. et al. (2014) Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1399 No. of Pages 14

28. Minghetti, L. (2004) Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J. Neuropathol. Exp. Neurol. 63, 901–910 29. Zhao, M-L. et al. (1998) Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain. Brain Res. 813, 402–405 30. Wolburg, H. and Lippoldt, A. (2002) Tight junctions of the blood– brain barrier: development, composition and regulation. Vascul. Pharmacol. 38, 323–337 31. Mann, G. et al. (1985) Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon. BBA Biomembranes 819, 241–248 32. Zlokovic, B.V. (1995) Cerebrovascular permeability to peptides: manipulations of transport systems at the blood–brain barrier. Pharm. Res. 12, 1395–1406 33. Zlokovic, B.V. et al. (1990) Kinetics of arginine-vasopressin uptake at the blood–brain barrier. BBA Biomembranes 1025, 191–198 , B.V. et al. (1987) Transport of leucine-enkephalin 34. Zlokovic across the blood–brain barrier in the perfused guinea pig brain. J. Neurochem. 49, 310–315 35. Ransohoff, R.M. et al. (2003) Three or more routes for leukocyte migration into the central nervous system. Nat. Rev. Immunol. 3, 569–581 36. van de Haar, H.J. et al. (2016) Blood–brain barrier leakage in patients with early Alzheimer disease. Radiology 281, 527–535 37. Zlokovic, B.V. (2011) Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders. Nat. Rev. Neurosci. 12, 723–738 38. van de Haar, H.J. et al. (2016) Neurovascular unit impairment in early Alzheimer's disease measured with magnetic resonance imaging. Neurobiol. Aging 45, 190–196 39. Zipser, B. et al. (2007) Microvascular injury and blood–brain barrier leakage in Alzheimer's disease. Neurobiol. Aging 28, 977–986 40. Farkas, E. and Luiten, P.G. (2001) Cerebral microvascular pathology in aging and Alzheimer's disease. Prog. Neurobiol. 64, 575– 611 41. Sengillo, J.D. et al. (2013) Deficiency in mural vascular cells coincides with blood–brain barrier disruption in Alzheimer's disease. Brain Pathol. 23, 303–310 42. Montagne, A. et al. (2015) Blood–brain barrier breakdown in the aging human hippocampus. Neuron 85, 296–302

52. Thanabalasundaram, G. et al. (2010) Regulation of the blood– brain barrier integrity by pericytes via matrix metalloproteinases mediated activation of vascular endothelial growth factor in vitro. Brain Res. 1347, 1–10 53. Jiang, S. et al. (2014) Vascular endothelial growth factors enhance the permeability of the mouse blood–brain barrier. PLoS One 9, e86407 54. Bai, Y. et al. (2015) Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: implications for stroke. PLoS One 10, e0124362 55. Bauer, A.T. et al. (2010) Matrix metalloproteinase-9 mediates hypoxia-induced vascular leakage in the brain via tight junction rearrangement. J. Cereb. Blood Flow Metab. 30, 837–848 56. Halliday, M.R. et al. (2013) Relationship between cyclophilin a levels and matrix metalloproteinase 9 activity in cerebrospinal fluid of cognitively normal apolipoprotein E4 carriers and blood– brain barrier breakdown. JAMA Neurol. 70, 1198–1200 57. Persidsky, Y. et al. (2015) Dysfunction of brain pericytes in chronic neuroinflammation. J. Cereb. Blood Flow Metab. 36, 794–807 58. Jansson, D. et al. (2016) Interferon-g blocks signalling through PDGFRb in human brain pericytes. J. Neuroinflammation 13, 1–19 59. Nishioku, T. et al. (2009) Detachment of brain pericytes from the basal lamina is involved in disruption of the blood–brain barrier caused by lipopolysaccharide-induced sepsis in mice. Cell. Mol. Neurobiol. 29, 309–316 60. Jung, K-H. et al. (2011) Multipotent PDGFRb-expressing cells in the circulation of stroke patients. Neurobiol. Dis. 41, 489–497 61. Sagare, A.P. et al. (2015) Shedding of soluble platelet-derived growth factor receptor-b from human brain pericytes. Neurosci. Lett. 607, 97–101 62. Schultz, N. et al. (2014) Involvement of matrix metalloproteinase9 in amyloid-beta 1-42-induced shedding of the pericyte proteoglycan NG2. J. Neuropathol. Exp. Neurol. 73, 684–692 63. Kristensson, K. and Olsson, Y. (1973) Accumulation of protein tracers in pericytes of the central nervous system following systemic injection in immature mice. Acta Neurol. Scand. 49, 189–194 64. Schultz, N. et al. (2016) Amylin alters human brain pericyte viability and NG2 expression. J. Cereb. Blood Flow Metab. Published online June 28, 2016. http://dx.doi.org/10.1177/ 0271678X16657093 65. Thomas, W.E. (1999) Brain macrophages: on the role of pericytes and perivascular cells. Brain Res. Rev. 31, 42–57

43. Halliday, M.R. et al. (2016) Accelerated pericyte degeneration and blood–brain barrier breakdown in apolipoprotein E4 carriers with Alzheimer's disease. J. Cereb. Blood Flow Metab. 36, 216– 227

66. Balabanov, R. et al. (1996) CNS microvascular pericytes express macrophage-like function, cell surface integrin alpha M, and macrophage marker ED-2. Microvasc. Res. 52, 127–142

44. Bell, R.D. et al. (2010) Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron 68, 409–427

67. Castejón, O. (1984) Submicroscopic changes of cortical capillary pericytes in human perifocal brain edema. J. Submicrosc. Cytol. 16, 601–618

45. Davalos, D. et al. (2012) Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation. Nat. Commun. 3, 1227

68. Sagare, A.P. et al. (2013) Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat. Commun. 4, 2932

46. Ryu, J.K. et al. (2015) Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation. Nat. Commun. 6, 8164

69. Zlokovic, B.V. et al. (2010) Low-density lipoprotein receptorrelated protein-1: a serial clearance homeostatic mechanism controlling Alzheimer's amyloid b-peptide elimination from the brain. J. Neurochem. 115, 1077–1089

47. Paul, J. et al. (2007) Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease. J. Exp. Med. 204, 1999–2008

70. Deane, R. et al. (2008) ApoE isoform-specific disruption of amyloid b peptide clearance from mouse brain. J. Clin. Invest. 118, 4002–4013

48. Kuroda, J. et al. (2014) Nox4 is a major source of superoxide production in human brain pericytes. J. Vasc. Res. 51, 429–438

71. Deane, R. et al. (2004) LRP/amyloid b-peptide interaction mediates differential brain efflux of Ab isoforms. Neuron 43, 333–344

49. Nishimura, A. et al. (2016) Detrimental role of pericyte Nox4 in the acute phase of brain ischemia. J. Cereb. Blood Flow Metab. 36, 1143–1154

72. Shibata, M. et al. (2000) Clearance of Alzheimer's amyloid-b 1-40 peptide from brain by LDL receptor-related protein-1 at the blood–brain barrier. J. Clin. Invest. 106, 1489–1499

50. Takata, F. et al. (2011) Brain pericytes among cells constituting the blood–brain barrier are highly sensitive to tumor necrosis factor-alpha, releasing matrix metalloproteinase-9 and migrating in vitro. J. Neuroinflammation 8, 106

73. Bell, R.D. et al. (2009) SRF and myocardin regulate LRP-mediated amyloid-b clearance in brain vascular cells. Nat. Cell Biol. 11, 143–153

51. Takahashi, Y. et al. (2014) p38 MAP kinase mediates transforming-growth factor-1-induced upregulation of matrix metalloproteinase-9 but not -2 in human brain pericytes. Brain Res. 1593, 1–8

74. Davis-Salinas, J. and Van Nostrand, W.E. (1995) Amyloid bprotein aggregation nullifies its pathologic properties in cultured cerebrovascular smooth muscle cells. J. Biol. Chem. 270, 20887–20890

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

13

TIPS 1399 No. of Pages 14

75. Dorr, A. et al. (2012) Amyloid-b-dependent compromise of microvascular structure and function in a model of Alzheimer's disease. Brain 135, 3039–3050

96. Bersini, S. and Moretti, M. (2015) 3D functional and perfusable microvascular networks for organotypic microfluidic models. J. Mater. Sci. Mater. Med. 26, 180

76. Iliff, J.J. et al. (2013) Cerebral arterial pulsation drives paravascular CSF–interstitial fluid exchange in the murine brain. J. Neurosci. 33, 18190–18199

97. Wolak, D.J. and Thorne, R.G. (2013) Diffusion of macromolecules in the brain: implications for drug delivery. Mol. Pharm. 10, 1492–1504

77. Carare, R. et al. (2008) Solutes, but not cells, drain from the brain parenchyma along basement membranes of capillaries and arteries: significance for cerebral amyloid angiopathy and neuroimmunology. Neuropathol. Appl. Neurobiol. 34, 131–144

98. Leaf, I.A. et al. (2016) Pericyte MyD88 and IRAK4 control inflammatory and fibrotic responses to tissue injury. J. Clin. Invest. Published online November 21, 2016. http://dx.doi.org/ 10.1172/JCI87532

78. Ozen, I. et al. (2014) Brain pericytes acquire a microglial phenotype after stroke. Acta Neuropathol. 128, 381–396

99. Nakagomi, T. et al. (2015) Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells. Stem Cells 33, 1962–1974

79. Sakuma, R. et al. (2016) Brain pericytes serve as microgliagenerating multipotent vascular stem cells following ischemic stroke. J. Neuroinflammation 13, 1 80. Navarro, R. et al. (2016) Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation. J. Cell. Mol. Med. 20, 980–986 81. Bansal, R. et al. (2012) Selective targeting of interferon g to stromal fibroblasts and pericytes as a novel therapeutic approach to inhibit angiogenesis and tumor growth. Mol. Cancer Ther. 11, 2419–2428 82. Brown, G.C. and Vilalta, A. (2015) How microglia kill neurons. Brain Res. 1628, 288–297 83. Yang, Y. et al. (2016) The PDGF-BB–SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages. Nat. Commun. 7, 11385 84. Fu, A.K. et al. (2016) IL-33 ameliorates Alzheimer's disease-like pathology and cognitive decline. Proc. Natl Acad. Sci. U. S. A. 113, E2705–E2713 85. Cardona, A.E. et al. (2006) Control of microglial neurotoxicity by the fractalkine receptor. Nat. Neurosci. 9, 917–924 86. Maier, C.L. and Pober, J.S. (2011) Human placental pericytes poorly stimulate and actively regulate allogeneic CD4 T cell responses. Arterioscler. Thromb. Vasc. Biol. 31, 183–189 87. Domev, H. et al. (2014) Immunoevasive pericytes from human pluripotent stem cells preferentially modulate induction of allogeneic regulatory T cells. Stem Cells Transl. Med. 3, 1169–1181 88. Owens, T. (2000) Naive T lymphocytes traffic to inflamed central nervous system, but require antigen recognition for activation. Eur. J. Immunol. 30, 1002–1009 89. Melgar, M.A. et al. (2005) Postischemic reperfusion: ultrastructural blood–brain barrier and hemodynamic correlative changes in an awake model of transient forebrain ischemia. Neurosurgery 56, 571–581

100. Buttgereit, A. et al. (2016) Sall1 is a transcriptional regulator defining microglia identity and function. Nat. Immunol. 17, 1397–1406 101. Butovsky, O. et al. (2014) Identification of a unique TGF-b-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131–143 102. Raica, M. and Cimpean, A.M. (2010) Platelet-derived growth factor (PDGF)/PDGF receptors (PDGFR) axis as target for antitumor and antiangiogenic therapy. Pharmaceuticals 3, 572–599 103. Kang, E. and Shin, J.W. (2016) Pericyte-targeting drug delivery and tissue engineering. Int. J. Nanomedicine 11, 2397 104. Padel, T. et al. (2016) Platelet-derived growth factor-BB has neurorestorative effects and modulates the pericyte response in a partial 6-hydroxydopamine lesion mouse model of Parkinson's disease. Neurobiol. Dis. 94, 95–105 105. Liu, R. et al. (2016) IL-17 promotes neutrophil-mediated immunity by activating microvascular pericytes and not endothelium. J. Immunol. 197, 2400–2408 106. He, L. et al. (2016) Analysis of the brain mural cell transcriptome. Sci. Rep. 6, 35108 107. Winkler, E.A. et al. (2013) Blood–spinal cord barrier breakdown and pericyte reductions in amyotrophic lateral sclerosis. Acta Neuropathol. 125, 111–120 108. Verbeek, M.M. et al. (1997) Rapid degeneration of cultured human brain pericytes by amyloid b protein. J. Neurochem. 68, 1135–1141 109. Muramatsu, R. et al. (2015) Prostacyclin prevents pericyte loss and demyelination induced by lysophosphatidylcholine in the central nervous system. J. Biol. Chem. 290, 11515–11525 110. Warmke, N. et al. (2016) Pericytes in diabetes-associated vascular disease. J. Diabetes Complications 30, 1643–1650

90. MacLeod, M.K. et al. (2010) Memory CD4 T cells: generation, reactivation and re-assignment. Immunology 130, 10–15

111. Marchi, N. and Lerner-Natoli, M. (2013) Cerebrovascular remodeling and epilepsy. Neuroscientist 19, 304–312

91. Fabry, Z. et al. (1993) Differential activation of Th1 and Th2 CD4+ cells by murine brain microvessel endothelial cells and smooth muscle/pericytes. J. Immunol. 151, 38–47

112. Liu, S. et al. (2012) The role of pericytes in blood–brain barrier function and stroke. Curr. Pharm. Des. 18, 3653–3662

92. Smith, A.M. and Dragunow, M. (2014) The human side of microglia. Trends Neurosci. 37, 125–135

113. Zehendner, C.M. et al. (2015) Traumatic brain injury results in rapid pericyte loss followed by reactive pericytosis in the cerebral cortex. Sci. Rep. 5, 13497

93. Arora, A. and Bhagat, N. (2016) Insight into the molecular imaging of Alzheimer's disease. Int. J. Biomed. Imaging 2016, 7462014

114. Göritz, C. et al. (2011) A pericyte origin of spinal cord scar tissue. Science 333, 238–242

94. Jennewein, M. et al. (2016) Two-and three-dimensional co-culture models of soft tissue healing: pericyte–endothelial cell interaction. Cell Tissue Res. 365, 279–293

115. Ochs, K. et al. (2013) Immature mesenchymal stem cell-like pericytes as mediators of immunosuppression in human malignant glioma. J. Neuroimmunol. 265, 106–116

95. Tarallo, S. et al. (2012) Human pericyte–endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment. Acta Diabetol. 49, 141–151

116. Cheng, L. et al. (2013) Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth. Cell 153, 139–152

14

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy