MATRICELLULAR SPECIAL ISSUE

JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 31, Number 7, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/jop.2014.0163

The Function of Matricellular Proteins in the Lamina Cribrosa and Trabecular Meshwork in Glaucoma Deborah M. Wallace,1,2 Olya Pokrovskaya,1,2 and Colm J. O’Brien1,2

Abstract

Purpose: To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM). Methods: A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma. Results: In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility. Conclusion: This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.

open-angle glaucoma (POAG) due to increased outflow resistance.9,10 In POAG, the resistance to outflow increases11 in the TM, particularly in the juxtacanalicular connective tissue ( JCT) region, culminating in elevated IOP.12 Several factors are implicated in this process, including the altered cytoskeletal morphology of TM cells in glaucoma, as well as the deposition of ECM. Changes in the ECM alone are insufficient to account for the increased resistance to aqueous outflow observed in glaucoma.13,14 Cellular contraction and relaxation of TM and SC cells is central to the maintenance of normal aqueous humour outflow facility and agents that can alter contraction can change outflow rates.15–21 Furthermore, various studies have established the endothelial cell of the SC as a reactive but variable mechanical component of the aqueous humor outflow pathway.12,22,23 Activation of molecular pathways in glaucoma, such as the Rho kinase pathway, leads to cytoskeletal changes, including increased actin stress fiber formation and increased cellular contractility—which can impair aqueous outflow.24 Regulatory feedback mechanisms exist between the cytoskeletal changes and ECM production—suggesting that both these processes together contribute to raised IOP in glaucoma.

Glaucoma

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laucoma is the second leading cause of irreversible blindness worldwide, affecting 1%–2% of the population older than 40 years and the prevalence rises to 5% of those aged 70 years and older in the western world and approximately thought to affect 60 million people worldwide.1,2 There are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the optic nerve head (ONH) and subsequent visual field defects. The primary risk factor for onset and progression of glaucoma is raised intraocular pressure (IOP).3–7 IOP is defined as the rate of aqueous humor production by the ciliary body and the aqueous humor resistance through the iridocorneal tissues, trabecular meshwork (TM), and Schlemm’s canal (SC) and to a lesser extent the uveoscleral pathway. IOP is generated in the anterior eye through the aqueous humor circulation system, secreted by the ciliary epithelium, and flows to the anterior chamber to leave through the TM outflow pathways.8 The TM and SC provide the major route for outflow of the aqueous humor from the eye and are responsible for the increased IOP associated with primary 1 2

School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. Department of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.

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Inducing a more relaxed cellular morphology through inhibition of the Rho pathway causes a reduction of IOP and an increase in outflow facility.25 Chronic elevation in IOP causes a deformation at the ONH, specifically at the lamina cribrosa (LC) region in the ONH. The LC undergoes thickening26 and posterior migration27 in the early stages of the disease process and later undergoes shearing and collapse of the LC plates, finally leading to a thin fibrotic connective tissue structure/scar28 where there is disturbed ECM metabolism29 and increased deposition of collagens and elastin.30 Hernandez et al. showed that it is the LC that undergoes fibrosis and mechanical failure in POAG.31 The connective tissue changes in POAG affect the TM and the LC and may result from a common defect in these cells as it has been suggested that the TM and LC are biochemically similar tissues; in addition, the cells cultured from the 2 are very similar.32–39 The fibrotic phenotype associated with glaucoma in the LC and TM regions has been widely reported.33,39,40 In the TM of patients with POAG, there is an increase in outflow resistance with subsequent elevated IOP due to the accumulation of ECM material.41 The disturbed ECM remodeling at the ONH is particularly evident in the LC region.29 The LC region of the ONH consists of perforated fibroelastic plates through which the unmyelinated retinal ganglion cell axons pass through before they converge as the optic nerve.42 ONH astrocytes and LC cells are members of the glial cell population of the ONH and attach to the basement membrane. LC cells can be differentiated from astrocytes by their nonexpression of glial fibrillary acid protein.43 These laminar plates contain ECM such as elastin and collagen I, III, V, and VI and it is here that the nerve axons degenerate in parallel with the apoptotic cell death of retinal ganglion cells and result in progressive visual field loss. Axonal degeneration may be caused by blockade of the anterograde and retrograde axonal transport systems at the level of the LC leading to deprivation of neurotrophic signals44 and is accompanied by local remodeling of the ECM in the ONH. The ECM is a key component of multicellular organisms forming an intricate proteinaceous network that fills the extracellular spaces and provides structural support and tissue organization.45 Maintaining the integrity of the ECM is necessary for the normal structure and function of connective tissue. Not only do we observe a change in ECM production in POAG but also a change in the expression of enzymes that regulate matrix homeostasis. Matrix metalloproteinases (MMPs) are a group of proteolytic enzymes that degrade components of ECM such as collagen and fibronectin. MMPs are important modulators of aqueous humor outflow through their ability to remodel the TM ECM and maintain a constant outflow resistance and ensuing IOP.46 Numerous pathological processes involving ECM remodeling have been associated with increased secretion of MMPs by activated glial cells.47–50 Alterations in the levels of modulators of ECM homeostasis such as MMP-2, - 3, and - 14 occur in the POAG LC.51,52 Yan et al. have shown that there is increased immunostaining for MMP-1, - 2, and - 3 in glaucomatous ONH sections when compared to agematched controls.51 Yuan and Neufeld have also shown that expression of MMP-1, - 2, - 3, and - 14 is more abundant in glaucomatous tissue.52 Work from our laboratory has also shown that both cyclical stretch and exposure to exogenous transforming growth factor (TGF)-b1 significantly increased

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the activity of MMP-2 in LC cells from the ONH and may therefore play a part in matrix remodeling in the glaucomatous ONH.53 MMPs have been described as important modulators of aqueous humor outflow through their ability to remodel the TM ECM, maintain a constant outflow resistance, and ensuing IOP. The TM is constantly being remodeled by MMPs, which in turn generates outflow resistance. MMP-1, - 2, - 3, - 9, and - 14 are constitutively secreted by TM cells54–56 and are upregulated during mechanical stretch while reducing the endogenous inhibitor TIMP-2.56–63

Matricellular Proteins in Glaucoma Paul Bornstein first coined the term ‘‘matricellular proteins’’ to describe a group of proteins that serve as links between cells and the ECM.64 They are nonstructural secreted glycoproteins that enable cells to communicate with and control their surrounding ECM and include secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP1) and TSP-2, osteonectin, connective tissue growth factor (CTGF) or CCN2, periostin, tenascins C and X, SC1/hevin. As previously described, there is a significant role for both ECM deposition and turnover in the TM and LC regions in glaucoma pathology. Therefore, it is reasonable to predict that there is an essential role for matricellular proteins in this neuropathy as a number of these proteins have been shown to be widely expressed in the cornea, lens, retina, vitreous, aqueous, and TM playing a specific defined role in each.65–72

Thrombospondin The aqueous humor of POAG patients contains significantly elevated levels of TGFb273,74; however, most of this is present in the latent inactive form and is unable to interact with its cellular receptors.75,76 This latent form can be activated in vitro and in vivo by TSP1.77,78 TSP1 is a potent activator of TGFb1 and binds to and activates TGFb under physiological conditions.79 TSP1 belongs to a small family (TSP1–5)80 of secreted glycoproteins.81 TGFb2 is activated by TSP1 through its binding to specific sequences in the type 1 repeats or thrombospondin repeats, which in turn induce conformational changes in the latent complex.77,82 TGFb1 treatment can increase TSP1 expression suggesting a reciprocal relationship,83 and interestingly, in the TM, administration of both TGFb1 and TGFb2 results in upregulation of TSP1.84–86 Biologically, TSP1 and TSP2 play a role in normal cutaneous wound healing and continued tissue turnover87; subsequently, when TSP1 expression is increased, the outcome can be fibrosis, wound healing, and excessive ECM production. 72,88,89 Reduction or loss of TSP1 expression can help reduce pathologic tissue remodeling,90–92 as validated by the fact that cultures of TSP1-null cell isolates revealed one-eighth the amount of active TGFb1.93 TSP1 is present in the TM, in which it increases with age and is manufactured by TM cells in vitro,94–96 and has also been observed in the TM of human eyes,94 as well as the corneal and stromal fibroblasts.97 Importantly, it is more intense in one-third of patients with POAG85 in agreement with the elevated levels of TGFb2 observed in the aqueous of POAG patients.73,74 TSP1 transcripts have been identified in a cDNA library generated from fresh TM.98 In the TM, TSP1 is primarily located to the JCT region (anatomical

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location of outflow resistance) and TSP2 localized to the uveoscleral meshwork72,98; therefore, TSP1 may help to regulate IOP through interactions between TM cells and their matrix.96 Stretch of TM cells results in an upregulation of TSP1,99 indicating an important role in IOP regulation. Studies from our laboratory investigating the role of LC cells in the fibrotic response associated with glaucoma have also implicated TSP1 as being an important factor in ECM deposition/turnover. Gene expression analysis comparing differentially expressed ECM genes between human primary LC cells obtained from both normal and glaucomatous donors showed an increase in expression of TSP1 in glaucomatous cells.39 This differential expression was also observed following mechanical stretch (mimicking elevated IOP) of LC cells100 and in response to exposure of LC cells to exogenous TGFb1.101 These data indicate that expression and secretion of TGFb mediated by TSP1 in LC cells may lead to progressive ECM accumulation and eventual damage in the LC region of the ONH.

Connective tissue growth factor The CCN family of proteins is named after its 3 original members; cysteine-rich protein 61 (Cyr61, CCN1), CTGF (CCN2), and nephroblastoma overexpressed protein (Nov, CCN3). Three additional Wnt-inducible secreted proteins were later identified (CCN4, CCN5, CCN6). Initially, it was thought that these proteins acted as growth factors; however, data now supports the function of CCN proteins as being matricellular proteins, which through modification of signals from other molecules such as TGFb can bind to the matrix and modulate cellular functions.102–104 It is well established that CTGF is strongly associated with fibrosis,105 is a TGFb1-inducible gene, and is significantly upregulated in experimental models of fibrosis associated with aberrant ECM deposition.106–110 Co-operation of CTGF and TGFb2 has been described to promote fibrosis, and inhibition of CTGF alone can ameliorate the observed fibrotic phenotype (eg, reduce the increased hydroxyproline:proline ratios) in a unilateral ureteral obstruction renal fibrosis model and in an intratracheal bleomycin instillation model of pulmonary fibrosis.111 CTGF mediates some TGFb-dependent effects on ECM production, including collagen and fibronectin,112,113 and transfection of CTGF siRNA before TGFb2 treatment inhibited the TGFb2-induced upregulation of CTGF and fibronectin.114 Tomarev et al. described CTGF and SPARClike 1 as being part of the 50 most abundant cDNA clones in the human TM cDNA library.98 Furthermore, CTGF is a critical mediator of the effects of TGFb2 on ECM synthesis in human TM cells, as siRNA knockdown of CTGF inhibited TGFb2-induced upregulation of fibronectin.115 It appears that coordinate expression of TGFb1/2 and CTGF is a normal feature of wound healing. However, pathological fibrosis is often attributed to uncontrolled matrix deposition, perhaps mediated by a CTGF-enriched microenvironment. This makes CTGF a very attractive therapeutic target for combating pathology due to the fibrotic ECM associated with glaucoma pathogenesis and would provide a novel disease modifying therapy for the treatment of both the glaucomatous TM and ONH and would avoid the pleiotropic effects of TGFb inhibition. TGFb alters ECM production and turnover in both the LC and TM and has been shown in numerous studies to play a

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role in ocular wound healing116,117; its role in the pathogenesis of glaucoma is also well documented.118–120 Several studies have reported elevated aqueous humor levels of TGFb2 in POAG73,74 and TGFb1 in pseudoexfoliation glaucoma (PXFG) patients.121 Pseudoexfoliation syndrome is currently the single most important identifiable risk factor for openangle glaucoma122 and is characterized by the production and progressive accumulation of fibrillar material (such as fibronectin and fibrillin-1) in ocular tissues and in the connective tissue portions of the various visceral organs.121,123 Our group has demonstrated that TGFb1 has an effect on global gene expression profiles, especially profibrotic ECM genes in nerve head LC cells,101 and that the CTGF level in the aqueous humor of patients with PXFG was significantly higher than in both POAG and normal control subjects.124,125 Recently, we investigated the ongoing fibrotic ECM pathology in the TM and LC regions by using a therapeutic anti-CTGF antibody126 (FG-3019; FibroGen, Inc.), previously shown to have therapeutic benefits in models of fibroproliferative renal disease.111 Exposure of TM cells to aqueous humor from both PXFG and POAG patients induced a significant increase in the expression of fibronectin, fibrillin-1, CTGF, collagen 1, and a-smooth muscle actin, all of which were significantly reduced by pretreatment with FG3019. The ability of FG-3019 to reduce protein expression of collagen 1A1 and a-smooth muscle actin in TM cells was also demonstrated.126 Thus, CTGF offers a potential novel therapeutic disease modifying strategy for PXFG and POAG.

Secreted protein acidic and rich in cysteine SPARC (osteonectin/BM-40) was first described as the main noncollagenous constituent of bone127,128 and is responsible for mediation of ECM organization and turn over in many human tissues. Its functions include regulation of cell function and tissue remodeling by exerting counteradhesive actions, by modulating growth factor signaling, and by serving as a cell cycle inhibitor.129 Growth factormediated effects appear to be implicated in the regulation of SPARC in injured tissues such as the TGFb family’s capacity to induce SPARC in various cell populations such as corneal fibroblasts130 and TM cells. Increased SPARC expression is observed in chronic fibrotic conditions131 suggestive of a profibrotic role for this matricellular protein and SPARC is expressed in TGFb2-treated TM cells.132,133 Accordingly, SPARC-null mice have decreased deposition of laminin and type I and IV collagens in the kidney in a diabetic nephropathy model,134 yet again indicating that the primary function of this matricellular protein is regulation of ECM production. Similar to the matricellular proteins TSPs 1 and 2, SPARC is also upregulated in LC cells obtained from glaucomatous human donors compared to normal cells and it is also increased following cyclical stretch of LC cells.39,100 SPARC is found in the aqueous humor and is highly expressed in the TM,70,98 especially in the JCT region. This is of importance as it is here that aqueous humor outflow resistance is the highest and believed to be the anatomic location of outflow resistance.65 The cause of this compromised aqueous drainage through the TM is not fully understood; however, it is well documented that patients with POAG have a much higher level of TGFb2 in their aqueous humor compared to agematched controls.73,74,117,135 Moreover, SPARC expression is

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significantly increased when TM cells are stretched in vitro,99 stretch being an in vivo consequence of elevated IOP. These findings in conjunction with our report that cyclical stretch of primary human LC cells induces SPARC100 are suggestive of a regulatory role in IOP regulation.

Periostin Similar to other matricellular proteins, periostin is upregulated in injured and remodeling tissues where it binds to ECM proteins. While it does not play a direct structural role, it does, however, modulate cell phenotype and function through integrin-mediated interactions.136 It is expressed in a wide variety of tissues137 and at extremely high levels in collagen-rich connective tissue subjected to mechanical stretch in vivo such as the cornea138 and LC.100 Comparable to other matricellular proteins, periostin is upregulated in TM cells in response to mechanical stretch99 and in the ONH of a rat model of elevated IOP.139 Studies from our group have shown that periostin is upregulated at the mRNA level39 in LC cells obtained from glaucomatous human donors compared to normal controls. Increased periostin expression associated with tissue injury, repair, and remodeling may be due to local activation of TGFb1 and bone morphogenetic protein signaling as both are potent inducers of periostin in cells, including fibroblasts,140 smooth muscle cells,141 and osteoblasts.142 In some cases, periostin appears to play an important role by enhancing profibrotic TGFb signaling.143 Periostin can bind directly to matrix proteins, such as collagens type I and V, fibronectin, and tenascin-C,144 regulate the assembly of collagen fibrils and, by doing so, modulate the biomechanical properties of connective tissues.138 These collagen fibrils are the ECM component allowing connective tissues to withstand tensile forces. As previously described, matricellular proteins are particularly important in collagen assembly145,146 with TSP2-null mice showing disruption of collagen fibrillogenesis145 and SPARC-null mice having significantly smaller collagen fibrils,147 all of which may affect aqueous humor outflow facility and subsequently IOP.

The effect of matricellular proteins on IOP CTGF is upregulated in both LC and TM cells under conditions of cell stretching, which are similar to conditions under elevated IOP.99,100 The role of TGFb2 and CTGF in aqueous humor homeostasis associated with raised IOP and glaucoma has been investigated.148,149 Through transgenic mouse models it was shown that CTGF expressed in the aqueous humor elevates IOP, which is associated with TM actin cytoskeleton modification.150 While some data are available regarding the role of CTGF in IOP, most studies to date have focused on the effects of TSP- and SPARC-null mice on IOP. Regarding the role of TSP1 in regulation of IOP, IOP of TSP1- and TSP2null mice has been measured in comparison to wild-type (WT) mice. Haddadin et al. demonstrated that the average IOP of TSP1- and TSP2-null mice was 10% and 7% (P < 0.05) less than that of the corresponding WT mice.151 The average IOP of TSP1-null mice and WT mice was 14.2 – 2.0 and 15.8 – 1.5 mmHg, respectively, and the average IOP of TSP2-null mice and WT mice was 16.8 – 2.0 and 18.1 – 1.6 mmHg, respectively. These lower IOP readings were thought to be owing to the TSP1- and TSP2-null mice demonstrating enhanced aqueous drainage. TSP levels in the TM were analyzed by immunofluorescence experiments and suggested that there

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may be a possible synergistic effect between TSP1 and TSP2 as TSP1-null mice had reduced expression of TSP2 and, similarly, TSP2-null mice had reduced levels of TSP1. TSP1 can also suppress activation of promatrix MMP9, while TSP1null mice exhibit higher levels of activated MMP9,152 and TSP2-null mice exhibit significantly greater MMP2 levels.153 The collagen fibril diameter in the JCT was also assessed and altered in the TSP1- and TSP2-null mice; it is proposed that this may be either a direct or indirect sign of altered matrix turnover and therefore affect outflow facility. These findings are suggestive of TSPs playing a role in aqueous humor outflow resistance through alteration of ECM in the JCT region. Local increased levels of TSP in the TM could also cause abnormally high levels of active TGFb in the TM leading to pathological increases in ECM deposition as studies have shown that implantation of subcutaneous TSP1-soaked sponges increased levels of active TGFb1.154 It remains to be determined if future therapies targeting the reduction of TSP1 or TSP2 to lower IOP may prove to be an attractive option for the treatment of glaucoma. Oh et al. described how overexpression of SPARC in human TM increases IOP in perfused cadaveric human anterior segments and SPARC alters ECM specifically at the JCT, with a selective decrease of MMP-9 activity thought to be the likely mechanism.155 Abnormal accumulations of ECM within the JCT have been identified in eyes with POAG compared with age-matched controls.156 Interestingly, SPARC also suppresses apoptosis in fibroblasts from patients with idiopathic pulmonary fibrosis157 and defective clearance of these cells may be important in disease progression. SPARC has also been shown to be significantly increased in the iris of primary angle closure glaucoma patients, suggesting that it could play a role in the development of primary angle closure glaucoma by influencing the biomechanical properties of the iris through a change in ECM organization.158 Studies on IOP levels in SPARC-null mice compared to the corresponding WT and heterozygous mice159 revealed that SPARC-null mice were found to have lower IOP than their corresponding WT mice with equal central corneal thickness. Heterozygous SPARC mice had intermediate IOP indicating that the transgenic deletion of SPARC significantly affects the aqueous humor outflow pattern. The collagen fibril diameter was significantly decreased in the JCT of SPARC-null mice reflecting the importance of SPARC in ECM processing and a potential mechanism by which IOP is reduced in SPARC-null mice. Significant changes in the collagen fibril diameter in other tissues of SPARC-null mice have been reported in other studies147,160,161; however, no significant morphological difference was detected between the TM of SPARC-null and WT tissues. Recent work by Rhee and colleagues has investigated the relationship between TGF-b2-mediated ocular hypertension and SPARC.162 Results showed that SPARC is essential for the regulation of TGF-b2-mediated ocular hypertension as deletion of SPARC significantly attenuates the effects by restricting collagen IV and fibronectin expression and maybe therefore has a significant role in IOP regulation and glaucoma pathogenesis.

Conclusion There is a fibrotic pathology associated with glaucoma, specifically in the TM tissue at the anterior of the eye and at

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the LC region of the ONH at the posterior. Changes in ECM deposition and turnover in the TM are speculated to play a vital role in outflow resistance in POAG and in the structural changes observed at the ONH. In POAG, resistance of aqueous humor outflow at the TM increases due to alterations in ECM homeostasis, which is responsible for elevation of IOP, deformation at the ONH, and subsequent nerve axon loss associated with progressive visual field loss. Matricellular proteins can govern such changes in ECM and there is growing interest in the role of this family of proteins in glaucoma pathophysiology. Matricellular proteins can also affect IOP readings. This knowledge provides a unique opportunity for the management/ treatment of glaucoma. Both TSP1- and SPARC-null mice exhibited a lower IOP reading compared to their WT counterparts151,159 through enhanced aqueous drainage. Therefore, approaches at altering the aqueous humor outflow pattern through modulation of TSP1 and SPARC expression could be a potential mechanism for lowering IOP in the disease state. Targeting matricellular proteins such as CTGF is a far more attractive strategy than targeting TGFb, as it avoids the pleiotropic effects associated with the latter. We have previously shown that elevated levels of CTGF are partnered to PXFG,124,125 whereas Junglas et al. demonstrated that CTGF expressed in the aqueous humor can elevate IOP correlated with TM actin cytoskeleton.150 The use of antiCTGF antibody technology such as FG-3019 may offer a realistic option for future maintenance of IOP as well as addressing fibrotic pathology. The function of matricellular proteins in glaucoma is still under exploration; however, targeting matricellular proteins represents an attractive strategy for regulation of ECM deposition and turnover in the disease setting particularly observed in the LC and TM regions in glaucoma.

Author Disclosure Statement No competing financial interests exist.

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Received: December 17, 2014 Accepted: February 23, 2015 Address correspondence to: Dr. Deborah. M. Wallace School of Medicine and Medical Science University College Dublin UCD Clinical Research Centre Nelson Street Dublin 7 Ireland E-mail: [email protected]