The role of CTGF in diabetic retinopathy.

Experimental Eye Research 133 (2015) 37e48

Contents lists available at ScienceDirect

Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer

Review

The role of CTGF in diabetic retinopathy Ingeborg Klaassen a, b, *, Rob J. van Geest a, b, Esther J. Kuiper a, b, Cornelis J.F. van Noorden a, b, Reinier O. Schlingemann a, b, c a

Ocular Angiogenesis Group, Department of Ophthalmology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c Netherlands Institute for Neuroscience, Royal Academy of Sciences, Amsterdam, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 April 2014 Accepted in revised form 17 October 2014

Connective tissue growth factor (CTGF, CCN2) contributes to fibrotic responses in diabetic retinopathy, both before clinical manifestations occur in the pre-clinical stage of diabetic retinopathy (PCDR) and in proliferative diabetic retinopathy (PDR), the late clinical stage of the disease. CTGF is a secreted protein that modulates the actions of many growth factors and extracellular matrix (ECM) proteins, leading to tissue reorganization, such as ECM formation and remodeling, basal lamina (BL) thickening, pericyte apoptosis, angiogenesis, wound healing and fibrosis. In PCDR, CTGF contributes to thickening of the retinal capillary BL and is involved in loss of pericytes. In this stage, CTGF expression is induced by advanced glycation end products, and by growth factors such as vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-b. In PDR, the switch from neovascularization to a fibrotic phase e the angio-fibrotic switch e in PDR is driven by CTGF, in a critical balance with vascular endothelial growth factor (VEGF). We discuss here the roles of CTGF in the pathogenesis of DR in relation to ECM remodeling and wound healing mechanisms, and explore whether CTGF may be a potential novel therapeutic target in the clinical management of early as well as late stages of DR. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Connective tissue growth factor Pre-clinical diabetic retinopathy Proliferative diabetic retinopathy Extracellular matrix Basal lamina thickening Wound healing Angio-fibrotic switch

1. Introduction Diabetic retinopathy (DR) can cause blindness by macular ischemia, macular edema, intra-ocular angiogenesis and scarring (Frank, 2004). The pathogenesis of DR is only partly understood. Before clinical signs appear, in a stage called pre-clinical DR (PCDR), the retina is already changing. These changes eventually may lead to retinal vascular occlusion and ischemia, which cause the main clinical manifestations of the disease during proliferative DR (PDR): vision-threatening vascular leakage, macular edema, and preretinal neovascularization (Aiello and Wong, 2000; Schlingemann et al., 1997) that may then lead to a switch from neovascularization to fibrosis, similar to other wound healing-like responses. PCDR is characterized by capillary basal lamina (BL) thickening, pericyte, endothelial cell and neural cell apoptosis, and breakdown of the blood-retinal barrier (BRB) leading to diffuse vascular permeability (Hammes et al., 2011; Klaassen et al., 2013; Lorenzi and Gerhardinger, 2001). In experimental rodent models of DR,

* Corresponding author. E-mail address: [email protected] (I. Klaassen). http://dx.doi.org/10.1016/j.exer.2014.10.016 0014-4835/© 2014 Elsevier Ltd. All rights reserved.

retinal mRNA levels of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are increased in this stage, suggesting an early role of VEGF in DR, possibly as a result of high levels of glucose, advanced glycation end products (AGEs) and/or other factors altered by the diabetic milieu (Aiello and Wong, 2000; Schlingemann et al., 1997). Although the exact sequence of events and the relative importance of the various early changes are poorly understood, capillary BL thickening is a hallmark of PCDR, and may be causal in endothelial and pericyte dysfunction. BL thickening is the result of extracellular matrix (ECM) remodeling resulting in increased deposition of BL components such as collagen type IV, laminin and fibronectin (Kuiper et al., 2007a). PDR is potentially the most serious ocular complication of diabetes and can cause tractional retinal detachment and total blindness. PDR is a wound healing-like response in which neovascularization is accompanied by influx of inflammatory cells and myofibroblasts into the retina (Walshe et al., 1992) leading to fibrosis of the retina with concomitant fibrovascular contraction causing hemorrhages, retinal detachment and blindness. Retinal detachment often occurs late in the disease and is caused by neovascularization, development of neovascular epiretinal membranes and vitreous contraction. VEGF is a major angiogenic factor in this sequence of events (Aiello et al., 1997; Gariano and Gardner, 2005;

38

I. Klaassen et al. / Experimental Eye Research 133 (2015) 37e48

Witmer et al., 2003), but several other growth factors play a role in the course of PDR as well, such as transforming growth factor (TGF)-b, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and the pro-fibrotic connective tissue growth factor (CTGF, CCN2) (Cui et al., 2007; Hinton et al., 2002; Kuiper et al., 2006; Van Geest et al., 2010, 2013). CTGF acts as a mitogen for fibroblasts and induces ECM production (Abraham, 2008; Moussad and Brigstock, 2000). CTGF functions as a downstream mediator of TGF-b signaling in specific cell types and is involved in the pro-fibrotic actions of TGF-b, such as ECM production (Duncan et al., 1999). CTGF contributes to fibrotic responses in both PCDR and PDR. In long-standing diabetes, structural and functional ECM alterations, including BL thickening, lead to microvascular diabetic complications, such as DR, nephropathy, peripheral vascular disease, and cerebrovascular disorders (Khan and Chakrabarti, 2003; Zimmet et al., 2001). CTGF is involved in these diabetic microvascular complications (Murphy et al., 1999; Riser et al., 2000; Twigg et al., 2001). In diabetic nephropathy (DN), CTGF is strongly overexpressed in the kidney glomerulus (Riser et al., 2000; Umezono et al., 2006), and CTGF levels in urine and plasma correlate with progression of DN (Nguyen et al., 2006; Roestenberg et al., 2004). Plasma CTGF levels were also found to be elevated in patients with DR, but this seems to be caused by concomitant DN since plasma CTGF levels were low in DR patients without DN (Roestenberg et al., 2004). Plasma CTGF levels can thus not be used as a diagnostic marker for DR. Similar to its involvement in fibrosis in DN (Boor and Floege, 2011; Goldschmeding et al., 2000; Phanish et al., 2010), CTGF has a causal role in capillary BL thickening in PCDR and fibrosis in PDR. This review discusses these roles of CTGF in the pathogenesis of DR, in relation to ECM remodeling and wound healing responses in the retina, and explores whether CTGF may become a therapeutic target in the clinical management of PCDR and PDR.

2. CTGF structure and function CTGF is a secreted matricellular protein and a member of the CCN family of growth factors, which was named after the first three members identified, Cyr61 (CCN1), CTGF (CCN2) and Nov (CCN3), but which also includes CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3) (Brigstock et al., 2003; Lau and Lam, 1999; Perbal, 2004). The biological functions of CTGF are diverse and cell and context dependent. CTGF is most relevant during development. CTGF expression is mainly associated with pathological conditions in the adult. In normal adult tissue, CTGF is present at low or undetectable levels. It is highly upregulated during tissue remodeling such as wound healing (De Winter et al., 2008; Liu et al., 2007), fibrotic disorders (Leask, 2009), angiogenesis (Kubota and Takigawa, 2007) and in some types of cancer (Cicha and Goppelt-Struebe, 2009) and in transdifferentiation of cells, such as endothelial- or epithelial-tomesenchymal transition (EMT) (Lee et al., 2010). CTGF was first discovered in conditioned media of endothelial cells as a protein affecting the activity of fibroblasts (Bradham et al., 1991). The diverse actions of CTGF may be explained by its modular structure which CTGF shares with other CCN family members. It contains four modules, two modules in the N-terminal domain, a hinge region and two modules in the C-terminal domain (CT), that also contains a cysteine knot motif (Perbal, 2004). Each module has specific binding affinity for certain proteins, such as fibronectin and heparin sulfate proteoglycans, extracellular signaling molecules, and cell surface proteins, such as integrins as is indicated in Fig. 1 (Brigstock, 1999; Gao and Brigstock, 2004; Hoshijima et al., 2006; Lau and Lam, 1999; Nguyen and Goldschmeding, 2008; Oliver et al., 2010; Pi et al., 2008). CTGF also indirectly regulates signaling by modulating the activity of other growth factors. For example, binding of CTGF to VEGF suppresses VEGF-induced angiogenesis, whereas cleavage of CTGF by matrix metalloproteinases (MMPs) recovers the

Fig. 1. CTGF domain structure, activating factors, binding partners and cellular effects. CTGF consists of a secretory signal peptide (S), an insulin-like growth factor-binding protein domain (IGFBP), a von Willebrand type C domain (VWC), a thrombospondin-1 domain (TSP-1) and a cysteine knot (CT) domain. Domains are linked by hinge regions, susceptible to protease cleavage by matrix metalloproteinases (MMPs). CTGF is activated by multiple growth factors and environmental stimuli, has numerous binding partners and modulates a wide range of biological functions. AGEs, advanced glycation end products; BMP, bone morphogenetic protein; C, C-terminus; IGF, insulin-like growth factor; FN, fibronectin; FoxO, forkhead box O transcription factors; HIF-1, hypoxia-inducible factor-1; HSPG, heparan sulfate proteoglycan; JNK, c-Jun N-terminal kinase; LRP, lipoprotein receptor-related protein; MAPK, mitogen-activated protein kinase; N, N-terminus; RhoA, Ras homolog gene family, member A; TGF-b, transforming growth factor-b; VEGF, vascular endothelial growth factor.


angiogenic activity of VEGF (Inoki et al., 2002). CTGF binds to many other molecules, including TGF-b1 and 2, bone morphogenetic protein (BMP)-4 and -7, insulin-like growth factor (IGF)-1 and -2, lipoprotein receptor-related protein (LRP)-1, -5 and -6, von Willebrand factor (vWF), and PDGF-B (Abreu et al., 2002; Bradham et al., 1991; Inoki et al., 2002; Khankan et al., 2011; Kim et al., 1997; Lam et al., 2003; Pi et al., 2008; Yoshida et al., 2007). CTGF belongs to the cysteine knot superfamily, that also includes TGF-bs, BMPs, VEGFs, PDGFs, nerve growth factor (NGF), guidance cue slits, and the adhesion molecule vWF, and through this cysteine knot motif these proteins are able to dimerize with each other (Moussad and Brigstock, 2000; Murray-Rust et al., 1993; Pi et al., 2012). Because it binds to numerous proteins, CTGF is not considered to be a classical growth factor, but rather a modulator of activities of other growth factors (Cicha and Goppelt-Stuebe, 2009; Lipson et al., 2012). For example, CTGF acts as an essential downstream mediator for most of the pro-fibrotic activities of TGF-b, in particular in relation to ECM production (Leask and Abraham, 2003), and fibroblast proliferation (Grotendorst et al., 2004; Grotendorst and Duncan, 2005; Uchio et al., 2004). Furthermore, skin fibrosis in newborn mice was persistent only after co-injection of both TGF-b1 and CTGF, and not after injection of TGF-b1 or CTGF alone (Leask et al., 2001; Mori et al., 1999). CTGF enhances binding of TGF-b to its receptor complex, thereby increasing Smad2-dependent (Abreu et al., 2002) and Smad2/3-independent (Pannu et al., 2007; Quan et al., 2010) gene transcription. CTGF expression is induced by hypoxia in a context- and species-dependent manner. For example, hypoxia induces CTGF expression via hypoxia-inducible factor (HIF)-1a in mouse kidney tubular epithelial cells via a TGF-b1-independent pathway (Higgins et al., 2004), whereas hypoxia reduced CTGF levels via HIF-1a in human kidney tubular epithelial cells (Kroening et al., 2009). It is not exactly clear what causes this species difference but it may be due to the fact that the hypoxia-response element in the mouse promoter is not conserved in the human promoter (Higgins et al., 2004). In mouse endothelial cells, FoxO transcription factors are involved in HIF-1a-dependent hypoxic regulation of CTGF expression (Samarin et al., 2010). It is unknown whether this is the case in human endothelial cells. Furthermore, HIF-1a-mediated upregulation of CTGF expression may not only occur under hypoxic conditions, since HIF-1a can also be activated under non-hypoxic stimuli, by nitric oxide, TNF-a (Sandau et al., 2001), IL-1, angiotensin II, PDGF (Thornton et al., 2000), and HGF (Tacchini et al., 2001), all of which have been implicated to play a role in the development of DR. Proteolytic cleavage of CTGF results in the generation of fragments with different biological activities. CTGF has a proteolysissensitive hinge region (Hashimoto et al., 2002). Fragments of CTGF were discovered by Brigstock et al. (1997) in uterine flushings and have since then been identified in various cell types, tissues and body fluids. N-terminal and C-terminal fragments have distinct and sometimes opposing effects on cells, stimulating either proliferation (C-terminal) or differentiation (N-terminal) (Grotendorst and Duncan, 2005). MMP-1, 3, 7 and 13 (Hashimoto et al., 2002) and the secreted serine proteases kalikrein-related peptidases (GuillonMunos et al., 2011) cleave CTGF into smaller fragments. In the eye, large amounts of the 21-kDa and 25-kDa fragments are present in cornea, retina, iris, sclera, lens and vitreous (Robinson et al., 2012). Higher molecular weight forms have not been found in the eye, indicating that CTGF is either effectively processed into smaller fragments or the full length 38-kDa protein is tightly bound to ECM components. On the other hand, the 38-kDa protein and not the 21kDa fragment is associated with diabetic retinas in rat (Tikellis et al., 2004).

39

In conclusion, CTGF is not a growth factor, but rather a modulator of the effects of other growth factors. CTGF is composed of building blocks with different and sometimes opposing effects. 3. CTGF in PCDR 3.1. CTGF in BL thickening Constituents of the retinal capillary BL are collagen type IV, which is the predominant component, laminin and fibronectin (Lorenzi and Gerhardinger, 2001; Oshitari et al., 2006). Changes in the BL in diabetes are considered to be a result of disturbed balance between synthesis and degradation of the ECM components (Nishikawa et al., 2000; Roy et al., 1994; Spirin et al., 1999). The thickened BL contain ECM proteins that are normally found in the BL, but also proteins that are not present in the BL under physiological conditions (Kuiper et al., 2007a; Lorenzi and Gerhardinger, 2001; Ljubimov et al., 1996; Roy et al., 1990; Spirin et al., 1999). BL thickening is a hallmark in all organs that are affected by diabetes (Brownlee and Spiro, 1979), and is directly related to loss of function of these organs (Ban and Twigg, 2008). Early thickening of the retinal capillary BL in diabetes was already recognized over 60 years ago (Friedenwald and Day, 1950). Afterward, numerous electron microscopic (EM) studies have demonstrated increased thickness of the BL in diabetic humans and animals (Fig. 2) (Curtis et al., 2009; Mansour et al., 1990; Stitt et al., 1994). In a canine model of diabetes that displays all early features of diabetic retinal microvascular damage, the only clear structural retinal change after 1e3 years of diabetes was capillary BL thickening (Gardiner et al., 1994), whereas loss of pericytes was observed not sooner than 4 years after the onset of diabetes. In line with this sequence of events, BL thickening has been shown to contribute to progression of early DR. Inhibition of the expression of BL components in experimental models of diabetes not only prevented BL thickening, but also inhibited more advanced features of PCDR, such as apoptosis of pericytes and endothelial cells, development of acellular capillaries (Roy

Fig. 2. Electron micrograph of a retinal capillary analyzed for basal lamina (BL) thickness. The different layers are defined as outer BL, consisting of an endothelial BL domain (eBL; red) and a pericyte BL domain (pBL; blue), and as inner BL or joint BL (jBL; green) in between the endothelial cells and pericytes. Bar ¼ 2 mm.

40


et al., 2011, 2003) and vascular leakage (Oshitari et al., 2006). However, there are also reports that argue against a causal role of BL thickening in the progression of PCDR (Hammes et al., 2011; Pfister et al., 2013). Streptozotocin (STZ)-induced diabetes and ocular injection of exogenous VEGF in rodents both lead to a 2e3 fold increased expression of CTGF and other pro-fibrotic proteins in the retina (Berner et al., 2012; Hughes et al., 2007; Kuiper et al., 2007a; Tikellis et al., 2004), suggesting a role of CTGF in BL thickening. This role was shown as well in CTGFþ/ mice where reduced protein levels of CTGF prevented BL thickening of retinal capillaries in STZ-induced diabetes (Kuiper et al., 2008a; Van Geest et al., 2014) (Fig. 3AeC), which was in agreement with the outcome of an earlier study (Fischer and Gartner, 1983). After 4 months and 6e8 months of diabetes, the CTGFþ/ and CTGFþ/þ mice had similar blood glucose levels but approximately 50% lower CTGF protein expression levels in plasma and urine whereas BL thickening had not occurred in

CTGFþ/ diabetic mice. After long-term diabetes (Van Geest et al., 2014), prevention of BL thickening was accompanied by absence of pericyte loss and a reduced formation of acellular capillaries in CTGF haploinsufficient mice (Fig. 3). 3.2. AGEs and CTGF in BL thickening in PCDR Hyperglycaemia induces increased formation of glucosederived AGEs (Nishikawa et al., 2000; Goldin et al., 2006). In DR, AGEs have a causal role in changes in growth factor expression levels and ECM turnover (Roy et al., 1994; Brownlee et al., 1988). AGEs also induce increased synthesis of BL components, most likely via upregulation of TGF-b signaling and expression of its downstream effectors, including CTGF (Hughes et al., 2007). AGEs accumulate in the retinal vasculature and Müller cells during diabetes (Gardiner et al., 2003; Stitt et al., 1997; Curtis et al., 2011), and are associated with retinal disease progression (Beisswenger et al.,

Fig. 3. CTGF haplo-insufficiency is associated with reduced in diabetes-induced basal lamina (BL) thickening and formation of acellular capillaries. (A) Electron micrographs of retinal capillaries of diabetic (DM) and control (Con) wild type (WT) and CTGFþ/ (þ/) mice showing the basal lamina (BL) of pericytes (black arrow), endothelial cells (white arrow) and their joint BL (gray arrow). Bar ¼ 2 mm. (B, C) BL thickness measurements in nanometers of retinal capillaries in DM and Con WT and CTGFþ/ mice after 6e8 months of diabetes. (B) Thickness of the outer BL, as defined by the BL outside the endothelial cell and/or pericyte. (C) Endothelial cell BL (eBL) thickness. n ¼ 14e15 animals per group. Data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. (D) Light microscopical images of the retinal vasculature in digest preparations of diabetic (DM) and control (Con) wild type (WT) and CTGFþ/ mice. Arrows, acellular capillaries. Bar ¼ 4 mm. (E) Numbers of pericytes and (F) Acellular capillaries (ACs) in DM and Con WT and CTGFþ/ retinas after 8 months of diabetes expressed as numbers per unit area of retina (mm2). n ¼ 7e9 animals per group. Data are the mean ± SD. ***p < 0.001. Reprinted with permission from Van Geest et al. (2014).


2001; Fosmark et al., 2006; Yamaguchi et al., 1998). AGE inhibitors reduce the amount of retinal lesions (Bhatwadekar et al., 2008; Hammes et al., 1999, 2003) and protect against retinal capillary BL thickening (Gardiner et al., 2003). AGEs induce increased CTGF mRNA levels in vitro and vivo (Hughes et al., 2007; Liu et al., 2008). STZ-induced diabetes in rodents is associated with a 2e3 fold increase in CTGF gene expression in the retina (Berner et al., 2012; Hughes et al., 2007; Tikellis et al., 2004), which is attenuated by treatment with the ACE inhibitors perindopril and aminoguanidine, respectively (Hughes et al., 2007; Tikellis et al., 2004). Both inhibitors may share the same molecular mechanism in attenuating CTGF expression, since both inhibitors are able to reduce AGE accumulation (Forbes et al., 2002; Hughes et al., 2007). CTGF expression is also attenuated by overexpression of glyoxalase-1 (GLO1) (Berner et al., 2012), which is one of the two enzymes that metabolizes the AGE-precursor methylglyoxal (MG). MG-derived hydroimidazalone-1 (MG-H1) is one of the major AGEs found in the diabetic retina (Karachalias et al., 2003) around retinal blood vessels (Canning et al., 2007). GLO1 overexpression not only has beneficial effects on Müller cells, but also reduces the number of acellular capillaries after 6 months of diabetes, indicating that CTGF is upregulated by AGEs in the diabetic retina. Besides CTGF, ECM components such as collagen type IV and tissue inhibitor of MMPs (TIMP)-1 show elevated mRNA levels in the retina after 6 or 12 weeks of diabetes, which are significantly reduced by aminoguanidine treatment (Hughes et al., 2007). This suggests that AGEinduced elevation of the expression of ECM components and CTGF may contribute to BL thickening. Taken together, it can be concluded that AGE-induced BL thickening in the retina is likely meditated by CTGF. 3.3. VEGF in BL thickening Levels of VEGF, an important mediator of vascular permeability and angiogenesis in PDR, are increased early in PCDR (Boulton et al., 1998; Mathews et al., 1997; Witmer et al., 2003). Neutralization of VEGF with an antibody partly prevents diabetes-induced BL thickening in the retina of obese type 2 diabetic mice (Flyvbjerg et al., 2002). Furthermore, VEGF induces increased mRNA and protein levels of CTGF, fibronectin and TIMP-1 in the rat retina, and in cultured retinal endothelial cells and pericytes (Kuiper et al., 2007a). These findings indicate that VEGF can induce CTGF expression in PCDR contributing to BL thickening. 3.4. TGF-b in BL thickening TGF-b plays a causal role in BL thickening of kidney and brain capillaries (Fujimoto et al., 2003; Wolf et al., 2005; Wyss-Coray et al., 2000). However, evidence for such a role in DR is not yet established. Two drugs that are effective in the suppression of experimental DR, the aldose reductase inhibitor sorbinil and aspirin, have in common that upregulation of expression of members of the TGF-b pathway is suppressed, suggesting that TGF-b signaling plays a role in early pathogenesis of DR (Gerhardinger et al., 2009). Moreover, retinal vessels in diabetic rats show both increased TGF-b activity and increased CTGF mRNA expression. TGF-b-induced CTGF upregulation is cell-type specific, because it occurs in retinal pericytes and not in retinal endothelial cells, and can be blocked by an inhibitor of the TGF-b receptor ALK5 (Van Geest et al., 2010). This suggests that retinal pericytes in particular have the essential characteristics to allow for a role of TGF-b in BL thickening in PCDR via CTGF. Pericytes are of mesenchymal origin, like fibroblasts, which may explain their sensitivity for TGFb-dependent CTGF regulation. These results suggest that in retinal

41

endothelial cells, CTGF expression is regulated by other pathways and factors, such as VEGF, AGEs and/or high glucose levels acting independently of TGF-b whereas in retinal pericytes CTGF expression is regulated by TGF-b (Kuiper et al., 2007a; Van Geest et al., 2010). 3.5. CTGF and pericyte loss in PCDR Another crucial feature of PCDR is loss of pericytes in the retina. Pericytes maintain capillary structure and integrity of the BRB and regulate both homeostasis of the endothelium and local retinal blood flow (Klaassen et al., 2013; Pfister et al., 2013). Increased levels of angiotensin II and AGEs are associated with pericyte loss in PCDR (Wilkinson-Berka, 2006; Hammes et al., 1991), which has rez et al., 2003; been proposed to be mediated by CTGF (Rupe Hughes et al., 2007). Angiotensin causes functional uncoupling of pericytes from retinal microvessels (Kawamura et al., 2004) and induces their migration (Nadal et al., 1999). Cultured rat retinal pericytes exposed to AGEs express increased levels of CTGF (Liu et al., 2008) and go into anoikis, a form of apoptosis, caused by loss of cellematrix interactions. As experimental overexpression of CTGF also promotes detachment and anoikis of retinal pericytes, accumulation of CTGF in retinal capillaries at the onset of diabetes may alter vascular structure and organization, and have a role in pericyte apoptosis in PCDR. We studied CTGF protein expression in normal and diabetic human retinas in a series of 36 diabetic patients and 18 nondiabetic controls (Kuiper et al., 2004). Immunohistochemical staining with a highly-specific antibody against CTGF revealed a distinct and specific cellular cytoplasmic staining in the retina, suggesting local cellular expression of CTGF protein. In the normal human retina, CTGF staining was predominantly present in paravascular microglia, whereas in the retina of diabetic subjects, CTGF was predominantly present in pericytes. The constitutive expression of CTGF in paravascular microglia in the healthy retina suggests a role in retinal microvascular physiology where CTGF may be involved in retinal vascular BL homeostasis, whereas the predominant pericyte staining in diabetic subjects suggests a role for CTGF in these cells during pathology, possibly causing pericyte anoikis. Abu El-Asrar et al. (2007) did not find CTGF in the non-diabetic retina, whereas the diabetic retina showed CTGF staining in cells of the ganglion cell layer and the inner nuclear layer and in microglia, which is not in agreement with the data reported by Kuiper et al. (2004). By using transgenic mice expressing GFP under the control of the CTGF promoter (CTGFp-GFP), it was shown that in arteries and veins of the superficial retinal plexus CTGF was strongly expressed in smooth muscle-like perivascular cells in the arteries and in pericytes in the wall of venules (Pi et al., 2011). On the other hand, staining of CTGF in the adult mouse retina only showed faint staining in the ganglion cell layer and inner nuclear layer. It was suggested that this discrepancy was due to a high turnover of CTGF in the adult retina (Pi et al., 2011). Recently, we provided evidence for a role of CTGF in pericyte loss in diabetes in a study using the CTGFþ/ mouse. Reduced CTGF levels resulted in reduced pericyte loss after a period of 6e8 months of diabetes (Van Geest et al., 2014; Fig. 3DeE). Taken together, these studies suggest that increased CTGF expression in pericytes causes BL thickening and/or pericyte apoptosis as early events in the progression of PCDR. Whether BL thickening and pericyte loss are causal, sufficient and necessary for capillary loss, also called vasoregression, in DR remains controversial. There are other mechanisms that have been suggested to cause vasoregression (Hammes et al., 2011), including angiopoetin2 upregulation, inflammation, glial cell activation and an impaired repair function of circulating endothelial progenitor cells. In fact,

42


acellular capillaries have been found in the retina of humans with type 2 diabetes, whereas pericytes were still present (Pfister et al., 2013), suggesting independent loss of pericytes and vasoregression in the progression of PCDR. 4. CTGF in PDR In PDR, CTGF was found in fibrovascular membranes with a predominant localization in myofibroblasts (Abu El-Asrar et al., 2007; Hinton et al., 2004), and with a significant correlation between the number of a-smooth muscle actin (SMA)-positive myofibroblasts and the number of myofibroblasts expressing CTGF (Abu El-Asrar et al., 2007). Myofibroblasts are activated ECM-producing fibroblasts, associated with conditions of (persistent) fibrosis (Chen et al., 2005). Furthermore, CTGF was detected in endothelial cells in these membranes (Abu El-Asrar et al., 2007). In the vitreous of a small series of patients with active PDR, levels of the N-terminal CTGF fragment were increased as compared to non-diabetic patients and patients with quiescent PDR (Hinton et al., 2004). Vitreous levels of full-length CTGF were similar in all groups, whereas the C-terminal fragment was not detectable. N-terminal CTGF levels were also higher in diabetic patients with vitreous hemorrhage than in non-diabetic patients with vitreous hemorrhage, who had similar N-CTGF levels as non-diabetic controls. These findings suggests that local synthesis of CTGF plays a role in PDR. On the basis of the association between CTGF levels and PDR, Hinton et al. (2004) concluded that CTGF has a role in angiogenesis. However, the relation between CTGF and PDR may be more complicated. We investigated CTGF levels in a series of vitreous samples of 119 patients with varying degrees of intra-ocular fibrosis and angiogenesis, including PDR patients, and observed that CTGF levels were significantly associated with the degree of fibrosis, but not with angiogenic activity (Kuiper et al., 2006). The degree of fibrosis was best predicted by CTGF levels. In the next paragraphs, we describe the relation between CTGF and angiogenesis and fibrosis in PDR in more detail. 4.1. CTGF in ocular fibrosis Fibrosis is the deposition and cross-linking of collagen in the terminal phase of the normal wound healing response (Gabbiani et al., 2003; Van der Slot-Verhoeven et al., 2005), which has mainly been studied in the skin. Wound healing in the skin is initiated by tissue injury (Baum and Arpey, 2005; Blom et al., 2001; Franklin, 1997), which involves vascular damage, hemorrhages and activation of the clotting system. The subsequent response can be divided into three phases: an inflammatory phase, a proliferative phase and a maturation phase (Baum and Arpey, 2005). Most features of the wound healing response in human skin can also be recognized in pathological wound healing responses occurring in various disease states in other organs. These pathological conditions have in common that tissue-specific wound healing responses are initiated, but that the wound healing process is not properly terminated, leading to ‘pathological fibrosis’ (Gabbiani et al., 2003; Leask and Abraham, 2004). This is a situation in which normal scarring progresses to excessive production, limited degradation, altered deposition and/or contraction of the ECM, probably due to an imbalance between pro- and anti-fibrotic factors causing a profibrotic state. Several eye conditions lead to blindness by the involvement of wound healing-like responses culminating in scarring or excessive fibrosis (Kuiper et al., 2006). Although the initial wound healing response may have a functional meaning in restoring ocular integrity, it also results in loss of visual function, and is therefore

deemed to be pathological (Pastor et al., 2002; Schlingemann et al., 2004). In subretinal neovascularization in age-related macular degeneration, in pre-retinal neovascularization in PDR and other ischemic retinopathies, these responses are initially driven by angiogenesis as occurs in skin wound healing. In other conditions such as proliferative vitreo-retinopathy (PVR), these responses are mainly avascular. It has been suggested that CTGF functions in these ocular conditions primarily as a pro-fibrotic growth factor (He et al., 2008; Hinton et al., 2002; Kita et al., 2007a; Kuiper et al., 2006). In the human eye, CTGF has been identified in various diseases complicated by fibrosis, both in the anterior and posterior segments (Esson et al., 2004; Ho et al., 2005; Khaw et al., 1994; Kuiper et al., 2006; Nagai et al., 2009; Neumann et al., 2008; Razzigue et al., 2003; Van Setten et al., 2003; Yamanaka et al., 2007), and there is accumulating evidence that CTGF is an important pathogenic factor in these conditions. In the vitreous of patients with PVR, CTGF is present in higher levels as compared to non-proliferative retinal diseases (Kuiper et al., 2006; He et al., 2008), in correlation with TGF-b (Kita et al., 2007b). In human PVR membranes, CTGF has been identified as well (Abu El-Asrar et al., 2007; Cui et al., 2007; Hinton et al., 2002). 4.2. CTGF in ocular angiogenesis CTGF has been suggested to play a role in ocular angiogenesis but the evidence is ambiguous. Corneal micropocket implants in the rat eye containing murine CTGF induced neovascularization (Babic et al., 1999). Moreover, CTGF and VEGF co-localized in vascular cells in human choroidal neovascular membranes, and levels of CTGF were increased in the vitreous of patients with active PDR (Hinton et al., 2004). On the other hand, when CTGF is upregulated by VEGF (Suzuma et al., 2000), it can reduce the bioavailability of VEGF through direct binding. VEGF-induced angiogenesis was also inhibited by combined exogenous administration of CTGF and VEGF in the back of mice, as well as in a mouse model of hind limb ischemia, supposedly as a result of binding of VEGF by CTGF (Inoki et al., 2002; Jang et al., 2004). Therefore, a causative role of CTGF in angiogenesis in ocular diseases in general and in DR in particular is still a matter of debate (Kuiper et al., 2008a, 2007b, 2006). In human PDR, CTGF levels consistently correlated with the degree of fibrosis and not with angiogenesis (Kuiper et al., 2006, 2008b; Van Geest et al., 2012, 2013). Moreover, vascular outgrowth from metatarsals of 17-day-old CTGF/ embryos, cultured in the presence or absence of VEGF, did not differ significantly from wild-type or heterozygous CTGFþ/ metatarsals (Kuiper et al., 2007b). These data indicate that CTGF is not required for (VEGF-induced) angiogenesis in these models. The effect of CTGF gene deletion was also investigated in two ocular angiogenesis models. In the oxygen-induced retinopathy (OIR) model (Agostini et al., 2005), in which retinal hypoxia-induced VEGF overexpression causes pre-retinal angiogenesis, differences in the angiogenic response between CTGFþ/þ and CTGFþ/ mice were not observed. In another ocular angiogenesis model, choroidal neovascularization was induced in CTGFþ/þ and CTGFþ/ mice by laser burns (Lambert et al., 2003, 2001), but again differences in the degree of neovascularization between CTGFþ/þ and CTGFþ/ mice were not observed (Kuiper et al., 2007b). In other studies, attenuation by lentiviral inhibition of the normally increased CTGF levels in the mouse OIR model was associated with reduced neovascularization and prevention of MMP-2 upregulation in neovascular tufts (Chintala et al., 2012). Another study using the OIR model confirmed these findings, as ocular administration of dominant negative CTGF (Pi et al., 2012) resulted in


reduced vascular obliteration and retinal neovascularization. It was also shown that CTGF plays a role in postnatal retinal vascularization, where it mainly is expressed in endothelial cells and pericytes (Pi et al., 2011). Interestingly, CTGF was expressed in the filopodial extensions of tip cells in the superficial vascular plexus, and the appearance of these filopodia was blocked by a CTGF-specific antibody (Pi et al., 2011). In addition, HIF-1a, a transcription factor that induces the transcription of many pro-angiogenic genes (e.g. VEGF, erythropoietin) is also able to induce CTGF expression (Samarin et al., 2010; Smith et al., 2006; Higgins et al., 2004). Taken together, it can be concluded that when CTGF expression is partly reduced like in the heterozygous CTGFþ/ mice, the effect on angiogenesis is undetectable, whereas near to complete inhibition by lentiviral shRNA or dominant negative CTGF shows that CTGF is necessary for angiogenesis. This suggests that the role of CTGF in angiogenesis is subtle and probably works by facilitating the action of angiogenic factors such as VEGF in specific conditions (Chintala et al., 2012), whereas in other conditions CTGF can prevent VEGF-induced angiogenesis (Inoki et al., 2002). The multiple interactions of CTGF with angiogenesis-related factors (VEGF, HIF1a) and fibrosis-related factors (TGF-b, fibronectin) that induce its expression levels or bind CTGF directly suggest dual roles of CTGF in the regulation of angiogenesis and fibrosis in the eye. 4.3. Role of CTGF and VEGF in the angio-fibrotic switch in PDR In the course of PDR, neovascularization progresses to a fibrotic phase. VEGF is considered to be a major angiogenic factor in this process (Aiello and Wong, 2000; Witmer et al., 2003). In vitreoretinal disorders (including PDR), N-terminal CTGF levels in the vitreous are elevated (Hinton et al., 2004) and are strongly correlated with the degree of fibrosis (Kuiper et al., 2006). Therefore, it was proposed that CTGF is a causal factor of fibrosis and scarring in PDR. In vitreous of PDR and PVR patients, Kita et al. (2007b) did not find a correlation between levels of CTGF and VEGF, even though concentrations of CTGF and VEGF were both significantly higher as compared to those in vitreous of patients with non-proliferative

43

diseases. With respect to a possible role of CTGF in retinal neovascularization, it was concluded that CTGF may have no direct effect on retinal neovascularization, but possibly works indirectly by modulation of VEGF levels (Cicha and Goppelt-Struebe, 2009; Inoki et al., 2002). Our group was the first to investigate the correlation between VEGF and CTGF levels and the degree of neovascularization and fibrosis in the vitreous of a series of patients with PDR and other vitreoretinal disorders (macular hole and macular pucker) (Kuiper et al., 2008b). Neovascularization and fibrosis occurred in various degrees, almost exclusively in PDR patients, and vitreous CTGF levels were significantly associated with the degree of fibrosis and with VEGF levels, but not with neovascularization. On the other hand, VEGF levels were associated only with neovascularization, in agreement with the widely accepted role of VEGF as the major angiogenic factor in PDR. As the ratio of CTGF and VEGF levels was the strongest predictor of the degree of fibrosis, the results suggested that the balance of VEGF and CTGF levels in the vitreous determines the progression of fibrovascular proliferation in PDR. In the study of Hinton et al. (2004), increased amounts of the N-terminal fragment were found in the vitreous of patients with active PDR as compared with vitreous samples from non-diabetic patients or patients with quiescent PDR, whereas levels of full-length CTGF were similar in all groups and C-terminal fragments were undetectable. This indicates that the full-length protein is actively cleaved by proteases such as MMPs, which show increased levels in vitreous of patients with PDR as well (Abu El-Asrar et al., 2013; Salzmann et al., 2000). The absence of C-terminal fragments could mean a further degradation or binding to other proteins. Binding of the C-terminal fragment of CTGF to VEGF may be a good explanation, since it contains the VEGF-binding domain (Inoki et al., 2002). These findings led to the following concept of regulation of angiogenesis and fibrosis in ocular disease: angiogenesis in the vitreous is driven by VEGF, which upregulates production of the pro-fibrotic factor CTGF in various cell types in the newly formed neovascular membranes. The elevated CTGF levels do not significantly contribute to ocular angiogenesis. In contrast, increased levels of CTGF sequester VEGF, and when the balance between these two factors shifts to a certain threshold ratio, the angio-

Fig. 4. Example of an angio-fibrotic switch in a patient with proliferative diabetic retinopathy (PDR). Fundus photographs (A,D), red-free photographs (B,E) and fluorescein angiographs (C,F) of a patient with PDR show neovascularization growing from the disk along the vascular arcades (arrows), before (AeC) and 2 years after (DeF) an injection with bevacizumab followed by pan-retinal photocoagulation. Note the increase in fibrosis (arrows) and traction on the retina (arrowheads) after combined anti-VEGF and laser treatment (DeF).

44


fibrotic switch occurs: angiogenesis ceases and fibrosis driven by excess CTGF leads to scarring and blindness. This concept predicts that a decline in VEGF levels in a PDR patient with active neovascularization due to anti-VEGF treatment may inhibit angiogenesis and promote fibrosis because of the angio-fibrotic switch. This is supported by clinical observations in patients with active neovascularization treated with intravitreal inhibitors of VEGF, such as bevacizumab and ranibizumab (Fig. 4), and/or pan-retinal laser photocoagulation, which destroys large areas of the retina and reduces intra-ocular VEGF levels (Aiello et al., 1994). A regression of neovascularization and increased fibrosis as predicted by our concept was indeed found in a non-systematic survey of a small series of patients (Van Geest et al., 2012). CTGF and VEGF levels were measured using ELISA in 52 vitreous samples of PDR patients, of which 24 patients had received intravitreal bevacizumab 1 week to 3 months before vitrectomy, and were correlated with the degree of vitreoretinal fibrosis as determined clinically and intra-operatively. Like in our previous study (Kuiper et al., 2008b), CTGF correlated positively, and VEGF correlated negatively with the degree of fibrosis, and the CTGF/VEGF ratio was the strongest predictor of fibrosis. Two patients who had the highest CTGF levels and CTGF/VEGF ratios out of the 17 patients, developed tractional retinal detachment after intravitreal bevacizumab treatment even after the vitrectomy (Van Geest et al., 2012). This suggests that a high CTGF level in combination with low levels of VEGF after bevacizumab treatment is a risk factor for the development of postoperative fibrotic complications due to the angio-fibrotic switch. Others have also reported exacerbation of scarring and subsequent contraction of fibrous tissue leading to tractional retinal detachment in patients who received anti-VEGF treatment for active PDR (Arevalo et al., 2008; Moradian et al., 2008; Sohn et al., 2012). PDR patients treated with bevacizumab also showed a remarkable inhibition of angiogenesis, suggesting that the elevated CTGF levels, which remain in the vitreous after VEGF inhibition, are not able to maintain the angiogenic response, indicating that CTGF alone has no major pro-angiogenic role in PDR. To determine whether other pro-fibrotic factors play a role in the angio-fibrotic switch, we investigated next to VEGF and CTGF, the protein levels of TIMP-1, TGF-b2 and MMP-2 and -9 in vitreous of patients with PDR (Van Geest et al., 2013). Although TIMP-1 has also been associated with fibrosis, and its expression is induced by TGF-b in the eye (Connor et al., 1989; Guerin et al., 2001), no correlation of TIMP-1 or TGF-b2 with degree of fibrosis could be found. Again, we found that the CTGF/VEGF ratio is the strongest predictor of fibrosis in PDR (Van Geest et al., 2013). Recent experimental evidence underlines the importance of CTGF in the angio-fibrotic switch. In the laser-induced ischemic retinopathy model, 2 out of 5 mice that received both CTGF and VEGF developed fibrovascular membranes with abundant collagen fibers (Pi et al., 2011). Such fibrovascular membranes were not observed in control eyes of the same animals or in mice that received only VEGF. These results indicate that CTGF is necessary for fibrovascular proliferation caused by laser-induced ischemia.

retinal vein occlusion. In each of these conditions, anti-VEGF therapy has been shown to be successful in patients in inhibiting angiogenesis, but enhanced or persistent fibrotic responses are common and are regarded as one of the main factors preventing substantial visual improvement. Several studies indicate that the angio-fibrotic switch also plays a role in other neovascular eye diseases like neovascular age-related macular degeneration (Daniel et al., 2014; Hwang et al., 2011; Michalewski et al., 2014), since subretinal fibrosis developed after bevacizumab treatment (Hwang et al., 2011). In advanced retinopathy of prematurity, anti-VEGF treatment has been shown to cause fibrosis as well (Honda et al., 2008; Sun et al., 2012; Zepeda-Romero et al., 2010). We hypothesize that in these conditions, the balance between VEGF and CTGF is crucial in regulating the angio-fibrotic switch, and that anti-VEGF treatment can enhance the pro-fibrotic actions of CTGF. Recent experimental evidence suggests a role of CTGF in the angio-fibrotic switch in these conditions. In the laser-induced ischemic retinopathy model, 2 out of 5 mice that received both CTGF and VEGF developed fibrovascular membranes with abundant collagen fibers (Pi et al., 2011). Such fibrovascular membranes were not observed in control eyes of the same animals or in mice that received only VEGF. These results indicate that CTGF is necessary for fibrovascular proliferation during laser-induced ischemia. Future research should address the role of CTGF in these conditions, but extrapolation of the role of CTGF in PDR suggests that also in age-related macular degeneration and other conditions with choroidal neovascularization, targeting CTGF in addition to VEGF may prevent fibrosis-induced visual loss.

6. Concluding remarks We conclude on the basis of the available evidence that CTGF has a major role in two stages of DR. Early in the pathogenesis, before the clinical sight-threatening manifestations of DR occur, CTGF contributes to thickening of the retinal capillary BL and is involved in loss of pericytes. In this stage, CTGF is induced by AGEs, and by growth factors such as VEGF and TGF-b. Treatment strategies against TGF-b, a major inducer of fibrosis in many diabetic complications, are unattractive, as this growth factor also has beneficial immunosuppressive and anti-inflammatory activities. Targeting CTGF, a more specific downstream regulator of profibrotic TGF-b actions, may therefore be a more suitable therapeutic option for a preventive treatment aimed at the pre-clinical stage of the disease. Recently, this was tested in STZ-induced diabetic rats with 10 weeks of diabetes (Hu et al., 2014) in a therapy combining anti-VEGF (ranibuzimab) and anti-CTGF (by CTGF shRNA). This study showed improved microvessel ultrastructure after this dual-target intervention in PCDR as compared to singletarget intervention. More studies are needed to pursue this interesting therapeutic option. In the later clinical stages of DR, the switch from neovascularization to a fibrotic phase in PDR is also driven by CTGF, in a critical balance with VEGF. This indicates that CTGF-targeted therapy, in particular in combination with anti-VEGF agents, is a possible novel option to prevent sight-threatening fibrosis in PDR and other ocular diseases associated with neovascularization and fibrosis.

5. Implications for other ocular conditions and future research Acknowledgments Angiogenesis in the context of wound healing-like responses plays a major role in a wide array of ocular conditions other than DR, e.g. under the retina as in choroidal neovascularization in agerelated macular degeneration and high myopia, and in other ischemic retinopathies such as retinopathy of the prematurity and

We apologize to all colleagues whose work was not cited in this review because of space limitations. This study was financially supported by grant 2005.00.042 of the Dutch Diabetes Research Foundation.


References Abraham, D., 2008. Connective tissue growth factor: growth factor, matricellular organizer, fibrotic biomarker or molecular target for anti-fibrotic therapy in SSc? Rheumatology (Oxford) 47 (Suppl. 5), v8ev9. Abreu, J.G., Ketpura, N.I., Reversade, B., De Robertis, E.M., 2002. Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta. Nat. Cell Biol. 4, 599e604. Abu El-Asrar, A.M., Mohammad, G., Nawaz, M.I., Siddiquei, M.M., Van den Eynde, K., Mousa, A., De Hertogh, G., Opdenakker, G., 2013. Relationship between vitreous levels of matrix metalloproteinases and vascular endothelial growth factor in proliferative diabetic retinopathy. PLoS One 8, e85857. Abu El-Asrar, A.M., Van den Steen, P.E., Al-Amro, S.A., Missotten, L., Opdenakker, G., Geboes, K., 2007. Expression of angiogenic and fibrogenic factors in proliferative vitreoretinal disorders. Int. Ophthalmol. 27, 11e22. €ld, A., Martin, G., Hansen, L., Fiedler, U., Esser, N., Agostini, H., Boden, K., Unso , D., 2005. A single local injection of recombinant VEGF receptor 2 but Marme not of Tie2 inhibits retinal neovascularization in the mouse. Curr. Eye Res. 30, 249e257. Aiello, L.P., Avery, R.L., Arrigg, P.G., Keyt, B.A., Jampel, H.D., Shah, S.T., Pasquale, L.R., Thieme, H., Iwamoto, M.A., Park, J.E., et al., 1994. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331, 1480e1487. Aiello, L.P., Bursell, S.E., Clermont, A., Duh, E., Ishii, H., Takagi, C., Mori, F., Ciulla, T.A., Ways, K., Jirousek, M., Smith, L.E., King, G.L., 1997. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 46, 1473e1480. Aiello, L.P., Wong, J.S., 2000. Role of vascular endothelial growth factor in diabetic vascular complications. Kidney Int. Suppl. 77, S113eS119. Arevalo, J.F., Maia, M., Flynn Jr., H.W., Saravia, M., Avery, R.L., Wu, L., Eid Farah, M., Pieramici, D.J., Berrocal, M.H., Sanchez, J.G., 2008. Tractional retinal detachment following intravitreal bevacizumab (Avastin) in patients with severe proliferative diabetic retinopathy. Br. J. Ophthalmol. 92, 213e216. Babic, A.M., Chen, C.C., Lau, L.F., 1999. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol. Cell Biol. 19, 2958e2966. Ban, C.R., Twigg, S.M., 2008. Fibrosis in diabetes complications: pathogenic mechanisms and circulating and urinary markers. Vasc. Health Risk Manag. 4, 575e596. Baum, C.L., Arpey, C.J., 2005. Normal cutaneous wound healing: clinical correlation with cellular and molecular events. Dermatol. Surg. 31, 674e686. Beisswenger, P.J., Szwergold, B.S., Yeo, K.T., 2001. Glycated proteins in diabetes. Clin. Lab. Med. 21, 53e78 (vi). Berner, A.K., Brouwers, O., Pringle, R., Klaassen, I., Colhoun, L., McVicar, C., Brockbank, S., Curry, J.W., Miyata, T., Brownlee, M., Schlingemann, R.O., Schalkwijk, C., Stitt, A.W., 2012. Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology. Diabetologia 55, 845e854. Bhatwadekar, A., Glenn, J.V., Figarola, J.L., Scott, S., Gardiner, T.A., Rahbar, S., Stitt, A.W., 2008. A new advanced glycation inhibitor, LR-90, prevents experimental diabetic retinopathy in rats. Br. J. Ophthalmol. 92, 545e547. Blom, I.E., van Dijk, A.J., Wieten, L., Duran, K., Ito, Y., Kleij, L., deNichilo, M., Rabelink, T.J., Weening, J.J., Aten, J., Goldschmeding, R., 2001. In vitro evidence for differential involvement of CTGF, TGFbeta, and PDGF-BB in mesangial response to injury. Nephrol. Dial. Transpl. 16, 1139e1148. Boor, P., Floege, J., 2011. Chronic kidney disease growth factors in renal fibrosis. Clin. Exp. Pharmacol. Physiol. 38, 441e450. Boulton, M., Foreman, D., Williams, G., McLeod, D., 1998. VEGF localisation in diabetic retinopathy. Br. J. Ophthalmol. 82, 561e568. Bradham, D.M., Igarashi, A., Potter, R.L., Grotendorst, G.R., 1991. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J. Cell. Biol. 114, 1285e1294. Brigstock, D.R., Goldschmeding, R., Katsube, K.I., Lam, S.C., Lau, L.F., Lyons, K., Naus, C., Perbal, B., Riser, B., Takigawa, M., Yeger, H., 2003. Proposal for a unified CCN nomenclature. Mol. Pathol. 56, 127e128. Brigstock, D.R., Steffen, C.L., Kim, G.Y., Vegunta, R.K., Diehl, J.R., Harding, P.A., 1997. Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids. Identification as heparin-regulated Mr 10,000 forms of connective tissue growth factor. J. Biol. Chem. 272, 20275e20282. Brigstock, D.R., 1999. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr. Rev. 20, 189e206. Brownlee, M., Cerami, A., Vlassara, H., 1988. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N. Engl. J. Med. 318, 1315e1321. Brownlee, M., Spiro, R.G., 1979. Biochemistry of the basement membrane in diabetes mellitus. Adv. Exp. Med. Biol. 124, 141e156. Canning, P., Glenn, J.V., Hsu, D.K., Liu, F.T., Gardiner, T.A., Stitt, A.W., 2007. Inhibition of advanced glycation and absence of galectin-3 prevent blooderetinal barrier dysfunction during short-term diabetes. Exp. Diabetes Res. 51837, 1e10. Chen, Y., Shi-Wen, X., van Beek, J., Kennedy, L., McLeod, M., Renzoni, E.A., BouGharios, G., Wilcox-Adelman, S., Goetinck, P.F., Eastwood, M., Black, C.M.,

45

Abraham, D.J., Leask, A., 2005. Matrix contraction by dermal fibroblasts requires transforming growth factor-beta/activin-linked kinase 5, heparan sulfate-containing proteoglycans, and MEK/ERK: insights into pathological scarring in chronic fibrotic disease. Am. J. Pathol. 167, 1699e1711. Chintala, H., Liu, H., Parmar, R., Kamalska, M., Kim, Y.J., Lovett, D., Grant, M.B., Chaqour, B., 2012. Connective tissue growth factor regulates retinal neovascularization through p53 protein-dependent transactivation of the matrix metalloproteinase (MMP)-2 gene. J. Biol. Chem. 287, 40570e40585. Cicha, I., Goppelt-Struebe, M., 2009. Connective tissue growth factor: contextdependent functions and mechanisms of regulation. Biofactors 35, 200e208. Connor Jr., T.B., Roberts, A.B., Sporn, M.B., Danielpour, D., Dart, L.L., Michels, R.G., De Bustros, S., Enger, C., Kato, H., Lansing, M., Hayashi, H., Glaser, B.M., 1989. Correlation of fibrosis and transforming growth factor beta type 2 levels in the eye. J. Clin. Investig. 83, 1661e1666. Cui, J.Z., Chiu, A., Maberley, D., Ma, P., Samad, A., Matsubara, J.A., 2007. Stage specificity of novel growth factor expression during development of proliferative vitreoretinopathy. Eye 21, 200e208. Curtis, T.M., Gardiner, T.A., Stitt, A.W., 2009. Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis? Eye 23, 1496e1508. Curtis, T.M., Hamilton, R., Yong, P.H., McVicar, C.M., Berner, A., Pringle, R., Uchida, K., Nagai, R., Brockbank, S., Stitt, A.W., 2011. Muller glial dysfunction during diabetic retinopathy in rats is linked to accumulation of advanced glycation end-products and advanced lipoxidation end-products. Diabetologia 54, 690e698. Daniel, E., Toth, C.A., Grunwald, J.E., Jaffe, G.J., Martin, D.F., Fine, S.L., Huang, J., Ying, G.S., Hagstrom, S.A., Winter, K., Maguire, M.G., 2014. Comparison of Agerelated Macular Degeneration Treatments Trials Research Group. Risk of scar in the comparison of age-related macular degeneration treatments trials. Ophthalmology 121, 656e666. De Winter, P., Leoni, P., Abraham, D., 2008. Connective tissue growth factor: structure-function relationships of a mosaic, multifunctional protein. Growth Factors 26, 80e91. Duncan, M.R., Frazier, K.S., Abramson, S., Williams, S., Klapper, H., Huang, X., Grotendorst, G.R., 1999. Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: downregulation by cAMP. Faseb J. 13, 1774e1786. Esson, D.W., Neelakantan, A., Iyer, S.A., Blalock, T.D., Balasubramanian, L., Grotendorst, G.R., Schultz, G.S., Sherwood, M.B., 2004. Expression of connective tissue growth factor after glaucoma filtration surgery in a rabbit model. Investig. Ophthalmol. Vis. Sci. 45, 485e491. Fischer, F., Gartner, J., 1983. Morphometric analysis of basal laminae in rats with long-term streptozotocin diabetes L. II. Retinal capillaries. Exp. Eye Res. 37, 55e64. Flyvbjerg, A., Dagnaes-Hansen, F., De Vriese, A.S., Schrijvers, B.F., Tilton, R.G., Rasch, R., 2002. Amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. Diabetes 51, 3090e3094. Forbes, J.M., Cooper, M.E., Thallas, V., Burns, W.C., Thomas, M.C., Brammar, G.C., Lee, F., Grant, S.L., Burrell, L.M., Jerums, G., Osicka, T.M., 2002. Reduction of the accumulation of advanced glycation end products by ACE inhibition in experimental diabetic nephropathy. Diabetes 51, 3274e3282. Fosmark, D.S., Torjesen, P.A., Kilhovd, B.K., Berg, T.J., Sandvik, L., Hanssen, K.F., Agardh, C.D., Agardh, E., 2006. Increased serum levels of the specific advanced glycation end product methylglyoxal-derived hydroimidazolone are associated with retinopathy in patients with type 2 diabetes mellitus. Metabolism 55, 232e236. Frank, R.N., 2004. Diabetic retinopathy. N. Engl. J. Med. 350, 48e58. Franklin, T.J., 1997. Therapeutic approaches to organ fibrosis. Int. J. Biochem. Cell Biol. 29, 79e89. Friedenwald, J., Day, R., 1950. The vascular lesions of diabetic retinopathy. Bull. Johns Hopkins Hosp. 86, 253e254. Fujimoto, M., Maezawa, Y., Yokote, K., Joh, K., Kobayashi, K., Kawamura, H., Nishimura, M., Roberts, A.B., Saito, Y., Mori, S., 2003. Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy. Biochem. Biophys. Res. Commun. 305, 1002e1007. Gabbiani, G., 2003. The myofibroblast in wound healing and fibrocontractive diseases. J. Pathol. 200, 500e503. Gao, R., Brigstock, D.R., 2004. Connective tissue growth factor (CCN2) induces adhesion of rat activated hepatic stellate cells by binding of its C-terminal domain to integrin alpha(v)beta(3) and heparan sulfate proteoglycan. J. Biol. Chem. 279, 8848e8855. Gardiner, T.A., Anderson, H.R., Stitt, A.W., 2003. Inhibition of advanced glycation end-products protects against retinal capillary basement membrane expansion during long-term diabetes. J. Pathol. 201, 328e333. Gardiner, T.A., Stitt, A.W., Anderson, H.R., Archer, D.B., 1994. Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs. Br. J. Ophthalmol. 78, 54e60. Gariano, R.F., Gardner, T.W., 2005. Retinal angiogenesis in development and disease. Nature 438, 960e966. Gerhardinger, C., Dagher, Z., Sebastiani, P., Park, Y.S., Lorenzi, M., 2009. The transforming growth factor-beta pathway is a common target of drugs that prevent experimental diabetic retinopathy. Diabetes 58, 1659e1667. Goldin, A., Beckman, J.A., Schmidt, A.M., Creager, M.A., 2006. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation 114, 597e605.

46


Goldschmeding, R., Aten, J., Ito, Y., Blom, I., Rabelink, T., Weening, J.J., 2000. Connective tissue growth factor: just another factor in renal fibrosis? Nephrol. Dial. Transpl. 15, 296e299. Grotendorst, G.R., Duncan, M.R., 2005. Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. Faseb J. 19, 729e738. Grotendorst, G.R., Rahmanie, H., Duncan, M.R., 2004. Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation. Faseb J. 18, 469e479. Guerin, C.J., Hu, L., Scicli, G., Scicli, A.G., 2001. Transforming growth factor beta in experimentally detached retina and periretinal membranes. Exp. Eye Res. 73, 753e764. Guillon-Munos, A., Oikonomopoulou, K., Michel, N., Smith, C.R., Petit-Courty, A., -Vourc'h, N., Diamandis, E.P., Courty, Y., 2011. Canepa, S., Reverdiau, P., Heuze Kallikreinrelated peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors. J. Biol. Chem. 286, 25505e25518. Hammes, H.P., Martin, S., Federlin, K., Geisen, K., Brownlee, M., 1991. Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc. Natl. Acad. Sci. U. S. A. 88, 11555e11558. Hammes, H.P., Alt, A., Niwa, T., Clausen, J.T., Bretzel, R.G., Brownlee, M., Schleicher, E.D., 1999. Differential accumulation of advanced glycation end products in the course of diabetic retinopathy. Diabetologia 42, 728e736. Hammes, H.P., Du, X., Edelstein, D., Taguchi, T., Matsumura, T., Ju, Q., Lin, J., Bierhaus, A., Nawroth, P., Hannak, D., Neumaier, M., Bergfeld, R., Giardino, I., Brownlee, M., 2003. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat. Med. 9, 294e299. Hammes, H.P., Feng, Y., Pfister, F., Brownlee, M., 2011. Diabetic retinopathy: targeting vasoregression. Diabetes 60, 9e16. Hashimoto, G., Inoki, I., Fujii, Y., Aoki, T., Ikeda, E., Okada, Y., 2002. Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165. J. Biol. Chem. 277, 36288e36295. He, S., Chen, Y., Khankan, R., Barron, E., Burton, R., Zhu, D., Ryan, S.J., Oliver, N., Hinton, D.R., 2008. Connective tissue growth factor as a mediator of intraocular fibrosis. Investig. Ophthalmol. Vis. Sci. 49, 4078e4088. Higgins, D.F., Biju, M.P., Akai, Y., Wutz, A., Johnson, R.S., Haase, V.H., 2004. Hypoxic induction of Ctgf is directly mediated by Hif-1. Am. J. Physiol. Ren. Physiol. 287, F1223eF1232. Hinton, D.R., He, S., Jin, M.L., Barron, E., Ryan, S.J., 2002. Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy. Eye 16, 422e428. Hinton, D.R., Spee, C., He, S., Weitz, S., Usinger, W., LaBree, L., Oliver, N., Lim, J.I., 2004. Accumulation of NH2-terminal fragment of connective tissue growth factor in the vitreous of patients with proliferative diabetic retinopathy. Diabetes Care 27, 758e764. Ho, S.L., Dogar, G.F., Wang, J., Crean, J., Wu, Q.D., Oliver, N., Weitz, S., Murray, A., Cleary, P.E., O'Brien, C., 2005. Elevated aqueous humour tissue inhibitor of matrix metalloproteinase-1 and connective tissue growth factor in pseudoexfoliation syndrome. Br. J. Ophthalmol. 89, 169e173. Honda, S., Hirabayashi, H., Tsukahara, Y., Negi, A., 2008. Acute contraction of the proliferative membrane after an intravitreal injection of bevacizumab for advanced retinopathy of prematurity. Graefe’s Arch. Clin. Exp. Ophthalmol. 246, 1061e1063. Hoshijima, M., Hattori, T., Inoue, M., Araki, D., Hanagata, H., Miyauchi, A., Takigawa, M., 2006. CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1. Febs Lett. 580, 1376e1382. Hu, B., Zhang, Y., Zeng, Q., Han, Q., Zhang, L., Liu, M., Li, X., 2014. Intravitreal injection of ranibizumab and CTGF shRNA improves retinal gene expression and microvessel ultrastructure in a rodent model of diabetes. Int. J. Mol. Sci. 15, 1606e1624. Hughes, J.M., Kuiper, E.J., Klaassen, I., Canning, P., Stitt, A.W., Van Bezu, J., Schalkwijk, C.G., Van Noorden, C.J., Schlingemann, R.O., 2007. Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina. Diabetologia 50, 1089e1098. Hwang, J.C., Del Priore, L.V., Freund, K.B., Chang, S., Iranmanesh, R., 2011. Development of subretinal fibrosis after anti-VEGF treatment in neovascular agerelated macular degeneration. Ophthalmic Surg. Lasers Imaging 42, 6e11. Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K., Okada, Y., 2002. Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis. Faseb J. 16, 219e221. Jang, H.S., Kim, H.J., Kim, J.M., Lee, Y.S., Kim, K.L., Kim, J.A., Lee, J.Y., Suh, W., Choi, J.H., Jeon, E.S., Byun, J., Kim, D.K., 2004. A novel ex vivo angiogenesis assay based on electroporation-mediated delivery of naked plasmid DNA to skeletal muscle. Mol. Ther. 9, 464e474. Karachalias, N., Babaei-Jadidi, R., Ahmed, N., Thornalley, P.J., 2003. Accumulation of fructosyl-lysine and advanced glycation end products in the kidney, retina and peripheral nerve of streptozotocin-induced diabetic rats. Biochem. Soc. Trans. 31, 1423e1425. Kawamura, H., Kobayashi, M., Li, Q., Yamanishi, S., Katsumura, K., Minami, M., Wu, D.M., Puro, D.G., 2004. Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina. J. Physiol. 561, 671e683.

Khan, Z.A., Chakrabarti, S., 2003. Growth factors in proliferative diabetic retinopathy. Exp. Diabesity Res. 4, 287e301. Khankan, R., Oliver, N., He, S., Ryan, S.J., Hinton, D.R., 2011. Regulation of fibronectinEDA through CTGF domain-specific interactions with TGFb2 and its receptor TGFbRII. Investig. Ophthalmol. Vis. Sci. 52, 5068e5078. Khaw, P.T., Occleston, N.L., Schultz, G., Grierson, I., Sherwood, M.B., Larkin, G., 1994. Activation and suppression of fibroblast function. Eye 8, 188e195. Kim, H.S., Nagalla, S.R., Oh, Y., Wilson, E., Roberts Jr., C.T., Rosenfeld, R.G., 1997. Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): characterization of connective tissue growth factor as a member of the IGFBP superfamily. Proc. Natl. Acad. Sci. U. S. A. 94, 12981e12986. Kita, T., Hata, Y., Kano, K., Miura, M., Nakao, S., Noda, Y., Shimokawa, H., Ishibashi, T., 2007b. Transforming growth factor-beta2 and connective tissue growth factor in proliferative vitreoretinal diseases: possible involvement of hyalocytes and therapeutic potential of Rho kinase inhibitor. Diabetes 56, 231e238. Kita, T., Hata, Y., Miura, M., Kawahara, S., Nakao, S., Ishibashi, T., 2007a. Functional characteristics of connective tissue growth factor on vitreoretinal cells. Diabetes 56, 1421e1428. Klaassen, I., Van Noorden, C.J., Schlingemann, R.O., 2013. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog. Retin. Eye Res. 34, 19e48. Kroening, S., Neubauer, E., Wessel, J., Wiesener, M., Goppelt-Struebe, M., 2009. Hypoxia interferes with connective tissue growth factor (CTGF) gene expression in human proximal tubular cell lines. Nephrol. Dial. Transpl. 24, 3319e3325. Kubota, S., Takigawa, M., 2007. CCN family proteins and angiogenesis: from embryo to adulthood. Angiogenesis 10, 1e11. Kuiper, E.J., de Smet, M.D., van Meurs, J.C., Tan, H.S., Tanck, M.W., Oliver, N., van Nieuwenhoven, F.A., Goldschmeding, R., Schlingemann, R.O., 2006. Association of connective tissue growth factor with fibrosis in vitreoretinal disorders in the human eye. Arch. Ophthalmol. 124, 1457e1462. Kuiper, E.J., Hughes, J.M., Van Geest, R.J., Vogels, I.M., Goldschmeding, R., Van Noorden, C.J., Schlingemann, R.O., Klaassen, I., 2007a. Effect of VEGF-A on expression of profibrotic growth factor and extracellular matrix genes in the retina. Investig. Ophthalmol. Vis. Sci. 48, 4267e4276. Kuiper, E.J., Roestenberg, P., Ehlken, C., Lambert, V., van Treslong-de Groot, H.B., Lyons, K.M., Agostini, H.J., Rakic, J.M., Klaassen, I., Van Noorden, C.J., Goldschmeding, R., Schlingemann, R.O., 2007b. Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice. J. Histochem. Cytochem. 55, 1139e1147. Kuiper, E.J., Van Nieuwenhoven, F.A., de Smet, M.D., van Meurs, J.C., Tanck, M.W., Oliver, N., Klaassen, I., Van Noorden, C.J., Goldschmeding, R., Schlingemann, R.O., 2008b. The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy. PLoS One 3, e2675. Kuiper, E.J., van Zijderveld, R., Roestenberg, P., Lyons, K.M., Goldschmeding, R., Klaassen, I., Van Noorden, C.J., Schlingemann, R.O., 2008a. Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice. J. Histochem. Cytochem. 56, 785e792. Kuiper, E.J., Witmer, A.N., Klaassen, I., Oliver, N., Goldschmeding, R., Schlingemann, R.O., 2004. Differential expression of connective tissue growth factor in microglia and pericytes in the human diabetic retina. Br. J. Ophthalmol. 88, 1082e1087. Lam, S., van der Geest, R.N., Verhagen, N.A., van Nieuwenhoven, F.A., Blom, I.E., Aten, J., Goldschmeding, R., Daha, M.R., van Kooten, C., 2003. Connective tissue growth factor and igf-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose. Diabetes 52, 2975e2983. Lambert, V., Munaut, C., Carmeliet, P., Gerard, R.D., Declerck, P.J., Gils, A., Claes, C., €l, A., Rakic, J.M., 2003. Dose-dependent modulation of Foidart, J.M., Noe choroidal neovascularization by plasminogen activator inhibitor type I: implications for clinical trials. Investig. Ophthalmol. Vis. Sci. 44, 2791e2797. €l, A., Frankenne, F., Bajou, K., Gerard, R., Carmeliet, P., Lambert, V., Munaut, C., Noe Defresne, M.P., Foidart, J.M., Rakic, J.M., 2001. Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization. Faseb J. 15, 1021e1027. Lau, L.F., Lam, S.C., 1999. The CCN family of angiogenic regulators: the integrin connection. Exp. Cell Res. 248, 44e57. Leask, A., Abraham, D.J., 2004. TGF-beta signaling and the fibrotic response. Faseb J. 18, 816e827. Leask, A., Abraham, D.J., 2003. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. Biochem. Cell Biol. 81, 355e363. Leask, A., Sa, S., Holmes, A., Shiwen, X., Black, C.M., Abraham, D.J., 2001. The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts. Mol. Pathol. 54, 180e183. Leask, A., 2009. Trial by CCN2: a standardized test for fibroproliferative disease? J. Cell Commun. Signal. 3, 87e88. Lee, C.H., Shah, B., Moioli, E.K., Mao, J.J., 2010. CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J. Clin. Investig. 120, 3340e3349. Lipson, K.E., Wong, C., Teng, Y., Spong, S., 2012. CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenes. Tissue Repair 5 (Suppl. 1), S24. Liu, H., Yang, R., Tinner, B., Choudhry, A., Schutze, N., Chaqour, B., 2008. Cysteinerich protein 61 and connective tissue growth factor induce deadhesion and anoikis of retinal pericytes. Endocrinology 149, 1666e1677.

I. Klaassen et al. / Experimental Eye Research 133 (2015) 37e48 Liu, L.D., Shi, H.J., Jiang, L., Wang, L.C., Ma, S.H., Dong, C.H., Wang, J.J., Zhao, H.L., Liao, Y., Li, Q.H., 2007. The repairing effect of a recombinant human connectivetissue growth factor in a burn-wounded rhesus-monkey (Macaca mulatta) model. Biotechnol. Appl. Biochem. 47, 105e112. Ljubimov, A.V., Burgeson, R.E., Butkowski, R.J., Couchman, J.R., Zardi, L., Ninomiya, Y., Sado, Y., Huang, Z.S., Nesburn, A.B., Kenney, M.C., 1996. Basement membrane abnormalities in human eyes with diabetic retinopathy. J. Histochem. Cytochem. 44, 1469e1479. Lorenzi, M., Gerhardinger, C., 2001. Early cellular and molecular changes induced by diabetes in the retina. Diabetologia 44, 791e804. Mansour, S.Z., Hatchell, D.L., Chandler, D., Saloupis, P., Hatchell, M.C., 1990. Reduction of basement membrane thickening in diabetic cat retina by sulindac. Investig. Ophthalmol. Vis. Sci. 31, 457e463. Mathews, M.K., Merges, C., McLeod, D.S., Lutty, G.A., 1997. Vascular endothelial growth factor and vascular permeability changes in human diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 38, 2729e2741. Michalewski, J., Nawrocki, J., Izdebski, B., Michalewska, Z., 2014. Morphological changes in spectral domain optical coherence tomography guided bevacizumab injections in wet age-related macular degeneration, 12-months results. Indian J. Ophthalmol. 62, 554e560. Moradian, S., Ahmadieh, H., Malihi, M., Soheilian, M., Dehghan, M.H., Azarmina, M., 2008. Intravitreal bevacizumab in active progressive proliferative diabetic retinopathy. Graefe’s Arch. Clin. Exp. Ophthalmol. 246, 1699e1705. Mori, T., Kawara, S., Shinozaki, M., Hayashi, N., Kakinuma, T., Igarashi, A., Takigawa, M., Nakanishi, T., Takehara, K., 1999. Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: a mouse fibrosis model. J. Cell Physiol. 181, 153e159. Moussad, E.E., Brigstock, D.R., 2000. Connective tissue growth factor: what's in a name? Mol. Genet. Metab. 71, 276e292. Murphy, M., Godson, C., Cannon, S., Kato, S., Mackenzie, H.S., Martin, F., Brady, H.R., 1999. Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J. Biol. Chem. 274, 5830e5834. Murray-Rust, J., McDonald, N.Q., Blundell, T.L., Hosang, M., Oefner, C., Winkler, F., Bradshaw, R.A., 1993. Topological similarities in TGF-beta 2, PDGF-BB and NGF define a superfamily of polypeptide growth factors. Structure 1, 153e159. Nadal, J.A., Scicli, G.M., Carbini, L.A., Nussbaum, J.J., Scicli, A.G., 1999. Angiotensin II and retinal pericytes migration. Biochem. Biophys. Res. Commun. 266, 382e385. Nagai, N., Klimava, A., Lee, W.H., Izumi-Nagai, K., Handa, J.T., 2009. CTGF is increased in basal deposits and regulates matrix production through the ERK (p42/ p44mapk) MAPK and the p38 MAPK signaling pathways. Investig. Ophthalmol. Vis. Sci. 50, 1903e1910. Neumann, C., Yu, A., Welge-Lussen, U., Lutjen-Drecoll, E., Birke, M., 2008. The effect of TGF-beta2 on elastin, type VI collagen, and components of the proteolytic degradation system in human optic nerve astrocytes. Investig. Ophthalmol. Vis. Sci. 49, 1464e1472. Nguyen, T.Q., Goldschmeding, R., 2008. Bone morphogenetic protein-7 and connective tissue growth factor: novel targets for treatment of renal fibrosis? Pharm. Res. 25, 2416e2426. Nguyen, T.Q., Tarnow, L., Andersen, S., Hovind, P., Parving, H.H., Goldschmeding, R., van Nieuwenhoven, F.A., 2006. Urinary connective tissue growth factor excretion correlates with clinical markers of renal disease in a large population of type 1 diabetic patients with diabetic nephropathy. Diabetes Care 29, 83e88. Nishikawa, T., Edelstein, D., Du, X.L., Yamagishi, S., Matsumura, T., Kaneda, Y., Yorek, M.A., Beebe, D., Oates, P.J., Hammes, H.P., Giardino, I., Brownlee, M., 2000. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787e790. Oliver, N., Sternlicht, M., Gerritsen, K., Goldschmeding, R., 2010. Could aging human skin use a connective tissue growth factor boost to increase collagen content? J. Invest. Dermatol. 130, 338e341. Oshitari, T., Polewski, P., Chadda, M., Li, A., Sato, T., Roy, S., 2006. Effect of combined antisense oligonucleotides against high-glucose- and diabetes-induced overexpression of extracellular matrix components and increased vascular permeability. Diabetes 55, 86e92. Pannu, J., Nakerakanti, S., Smith, E., ten Dijke, P., Trojanowska, M., 2007. Transforming growth factor-beta receptor type I-dependent fibrogenic gene program is mediated via activation of Smad1 and ERK1/2 pathways. J. Biol. Chem. 282, 10405e10413. Pastor, J.C., de la Rua, E.R., Martin, F., 2002. Proliferative vitreoretinopathy: risk factors and pathobiology. Prog. Retin. Eye Res. 21, 127e144. Perbal, B., 2004. CCN proteins: multifunctional signalling regulators. Lancet 363, 62e64. Pfister, F., Przybyt, E., Harmsen, M.C., Hammes, H.P., 2013. Pericytes in the eye. Pflugers Arch. 465, 789e796. Phanish, M.K., Winn, S.K., Dockrell, M.E., 2010. Connective tissue growth factor(CTGF, CCN2)ea marker, mediator and therapeutic target for renal fibrosis. Nephron Exp. Nephrol. 114, e83ee92. Pi, L., Ding, X., Jorgensen, M., Pan, J.J., Oh, S.H., Pintilie, D., Brown, A., Song, W.Y., Petersen, B.E., 2008. Connective tissue growth factor with a novel fibronectin binding site promotes cell adhesion and migration during rat oval cell activation. Hepatology 47, 996e1004. Pi, L., Shenoy, A.K., Liu, J., Kim, S., Nelson, N., Xia, H., Hauswirth, W.W., Petersen, B.E., Schultz, G.S., Scott, E.W., 2012. CCN2/CTGF regulates neovessel formation via

47

targeting structurally conserved cystine knot motifs in multiple angiogenic regulators. Faseb J. 26, 3365e3379. Pi, L., Xia, H., Liu, J., Shenoy, A.K., Hauswirth, W.W., Scott, E.W., 2011. Role of connective tissue growth factor in the retinal vasculature during development and ischemia. Investig. Ophthalmol. Vis. Sci. 52, 8701e8710. Quan, T., Shao, Y., He, T., Voorhees, J.J., Fisher, G.J., 2010. Reduced expression of connective tissue growth factor (CTGF/CCN2) mediates collagen loss in chronologically aged human skin. J. Investig. Dermatol. 130, 415e424. Razzaque, M.S., Foster, C.S., Ahmed, A.R., 2003. Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid. Investig. Ophthalmol. Vis. Sci. 44, 1998e2003. Riser, B.L., Denichilo, M., Cortes, P., Baker, C., Grondin, J.M., Yee, J., Narins, R.G., 2000. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J. Am. Soc. Nephrol. 11, 25e38. Robinson, P.M., Smith, T.S., Patel, D., Dave, M., Lewin, A.S., Pi, L., Scott, E.W., Tuli, S.S., Schultz, G.S., 2012. Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing. Investig. Ophthalmol. Vis. Sci. 53, 8093e8103. Roestenberg, P., van Nieuwenhoven, F.A., Wieten, L., Boer, P., Diekman, T., Tiller, A.M., Wiersinga, W.M., Oliver, N., Usinger, W., Weitz, S., Schlingemann, R.O., Goldschmeding, R., 2004. Connective tissue growth factor is increased in plasma of type 1 diabetic patients with nephropathy. Diabetes Care 27, 1164e1170. Roy, S., Maiello, M., Lorenzi, M., 1994. Increased expression of basement-membrane collagen in human diabetic-retinopathy. J. Clin. Investig. 93, 438e442. Roy, S., Nasser, S., Yee, M., Graves, D.T., Roy, S., 2011. A long-term siRNA strategy regulates fibronectin overexpression and improves vascular lesions in retinas of diabetic rats. Mol. Vis. 17, 3166e3174. Roy, S., Sato, T., Paryani, G., Kao, R., 2003. Downregulation of fibronectin overexpression reduces basement membrane thickening and vascular lesions in retinas of galactose-fed rats. Diabetes 52, 1229e1234. Roy, S., Sala, R., Cagliero, E., Lorenzi, M., 1990. Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory. Proc. Natl. Acad. Sci. U. S. A. 87, 404e408. rez, M., Lorenzo, O., Blanco-Colio, L.M., Esteban, V., Egido, J., Ruiz-Ortega, M., Rupe 2003. Connective tissue growth factor is a mediator of angiotensin II-induced fibrosis. Circulation 108, 1499e1505. Salzmann, J., Limb, G.A., Khaw, P.T., Gregor, Z.J., Webster, L., Chignell, A.H., Charteris, D.G., 2000. Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy. Br. J. Ophthalmol. 84, 1091e1096. Samarin, J., Wessel, J., Cicha, I., Kroening, S., Warnecke, C., Goppelt-Struebe, M., 2010. FoxO proteins mediate hypoxic induction of connective tissue growth factor in endothelial cells. J. Biol. Chem. 285, 4328e4336. Sandau, K.B., Zhou, J., Kietzmann, T., Brune, B., 2001. Regulation of the hypoxiainducible factor 1alpha by the inflammatory mediators nitric oxide and tumor necrosis factor-alpha in contrast to desferroxamine and phenylarsine oxide. J. Biol. Chem. 276, 39805e39811. Schlingemann, R.O., van Hinsbergh, V.W., 1997. Role of vascular permeability factor/ vascular endothelial growth factor in eye disease. Br. J. Ophthalmol. 81, 501e512. Schlingemann, R.O., 2004. Role of growth factors and the wound healing response in age-related macular degeneration. Graefe’s Arch. Clin. Exp. Ophthalmol. 242, 91e101. Smith, E.K., Price, D.K., Figg, W.D., 2006. Piecing together the HIF-1 puzzle: the role of the CTGF as a molecular mechanism of HIF-1 regulation. Cancer Biol. Ther. 5, 1443e1444. Sohn, E.H., He, S., Kim, L.A., Salehi-Had, H., Javaheri, M., Spee, C., Dustin, L., Hinton, D.R., Eliott, D., 2012. Angiofibrotic response to vascular endothelial growth factor inhibition in diabetic retinal detachment: report no. 1. Arch. Ophthalmol. 130, 1127e1134. Spirin, K.S., Saghizadeh, M., Lewin, S.L., Zardi, L., Kenney, M.C., Ljubimov, A.V., 1999. Basement membrane and growth factor gene expression in normal and diabetic human retinas. Curr. Eye Res. 18, 490e499. Stitt, A.W., Anderson, H.R., Gardiner, T.A., Archer, D.B., 1994. Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments. Br. J. Ophthalmol. 78, 133e137. Stitt, A.W., Li, Y.M., Gardiner, T.A., Bucala, R., Archer, D.B., Vlassara, H., 1997. Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGEinfused rats. Am. J. Pathol. 150, 523e531. Sun, H.J., Choi, K.S., Lee, S.J., 2012. Adjunctive effect of intravitreal bevacizumab prior to lens-sparing vitrectomy in aggressive posterior retinopathy of prematurity: a case report. Jpn. J. Ophthalmol. 56, 476e480. Suzuma, K., Naruse, K., Suzuma, I., Takahara, N., Ueki, K., Aiello, L.P., King, G.L., 2000. Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells. J. Biol. Chem. 275, 40725e40731. Tacchini, L., Dansi, P., Matteucci, E., Desiderio, M.A., 2001. Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells. Carcinogenesis 22, 1363e1371. Thornton, R.D., Lane, P., Borghaei, R.C., Pease, E.A., Caro, J., Mochan, E., 2000. Interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts. Biochem. J. 350, 307e312.

48


Tikellis, C., Cooper, M.E., Twigg, S.M., Burns, W.C., Tolcos, M., 2004. Connective tissue growth factor is up-regulated in the diabetic retina: amelioration by angiotensin-converting enzyme inhibition. Endocrinology 145, 860e866. Twigg, S.M., Chen, M.M., Joly, A.H., Chakrapani, S.D., Tsubaki, J., Kim, H.S., Oh, Y., Rosenfeld, R.G., 2001. Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus. Endocrinology 142, 1760e1769. Uchio, K., Graham, M., Dean, N.M., Rosenbaum, J., Desmouliere, A., 2004. Downregulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis. Wound Repair Regen. 12, 60e66. Umezono, T., Toyoda, M., Kato, M., Miyauchi, M., Kimura, M., Maruyama, M., Honma, M., Yagame, M., Suzuki, D., 2006. Glomerular expression of CTGF, TGFbeta 1 and type IV collagen in diabetic nephropathy. J. Nephrol. 19, 751e757. Van der Slot-Verhoeven, A.J., van Dura, E.A., Attema, J., Blauw, B., Degroot, J., Huizinga, T.W., Zuurmond, A.M., Bank, R.A., 2005. The type of collagen crosslink determines the reversibility of experimental skin fibrosis. Biochim. Biophys. Acta 1740, 60e67. Van Geest, R.J., Klaassen, I., Lesnik-Oberstein, S.Y., Tan, H.S., Mura, M., Goldschmeding, R., Van Noorden, C.J., Schlingemann, R.O., 2013. Vitreous TIMP1 levels associate with neovascularization and TGF-b2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy. J. Cell Commun. Signal. 7, 1e9. Van Geest, R.J., Klaassen, I., Vogels, I.M., Van Noorden, C.J., Schlingemann, R.O., 2010. Differential TGF-b signaling in retinal vascular cells: a role in diabetic retinopathy? Investig. Ophthalmol. Vis. Sci. 51, 1857e1865. Van Geest, R.J., Leeuwis, J.W., Dendooven, A., Pfister, F., Bosch, K., Hoeben, K.A., Vogels, I.M., Van der Giezen, D.M., Dietrich, N., Hammes, H.P., Goldschmeding, R., Klaassen, I., Van Noorden, C.J., Schlingemann, R.O., 2014. Connective tissue growth factor is involved in structural retinal vascular changes in long-term experimental diabetes. J. Histochem. Cytochem. 62, 109e118. Van Geest, R.J., Lesnik-Oberstein, S.Y., Tan, H.S., Mura, M., Goldschmeding, R., Van Noorden, C.J., Klaassen, I., Schlingemann, R.O., 2012. A shift in the balance of vascular endothelial growth factor and connective tissue growth factor by

bevacizumab causes the angiofibrotic switch in proliferative diabetic retinopathy. Br. J. Ophthalmol. 96, 587e590. Van Setten, G., Aspiotis, M., Blalock, T.D., Grotendorst, G., Schultz, G., 2003. Connective tissue growth factor in pterygium: simultaneous presence with vascular endothelial growth factor e possible contributing factor to conjunctival scarring. Graefe’s Arch. Clin. Exp. Ophthalmol. 241, 135e139. Walshe, R., Esser, P., Wiedemann, P., Heimann, K., 1992. Proliferative retinal diseases: myofibroblasts cause chronic vitreoretinal traction. Br. J. Ophthalmol. 76, 550e552. Wilkinson-Berka, J.L., 2006. Angiotensin and diabetic retinopathy. Int. J. Biochem. Cell Biol. 38, 752e765. Witmer, A.N., Vrensen, G.F.J.M., Van Noorden, C.J.F., Schlingemann, R.O., 2003. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 22, 1e29. Wolf, G., Chen, S., Ziyadeh, F.N., 2005. From the periphery of the glomerular capillary wall toward the center of disease: podocyte injury comes of age in diabetic nephropathy. Diabetes 54, 1626e1634. Wyss-Coray, T., Lin, C., von Euw, D., Masliah, E., Mucke, L., Lacombe, P., 2000. Alzheimer's disease-like cerebrovascular pathology in transforming growth factorbeta 1 transgenic mice and functional metabolic correlates. Ann. N. Y. Acad. Sci. 903, 317e323. Yamaguchi, M., Nakamura, N., Nakano, K., Kitagawa, Y., Shigeta, H., Hasegawa, G., Ienaga, K., Nakamura, K., Nakazawa, Y., Fukui, I., Obayashi, H., Kondo, M., 1998. Immunochemical quantification of crossline as a fluorescent advanced glycation endproduct in erythrocyte membrane proteins from diabetic patients with or without retinopathy. Diabet. Med. 15, 458e462. Yamanaka, O., Saika, S., Ohnishi, Y., Kim-Mitsuyama, S., Kamaraju, A.K., Ikeda, K., 2007. Inhibition of p38MAP kinase suppresses fibrogenic reaction in conjunctiva in mice. Mol. Vis. 13, 1730e1739. Yoshida, K., Munakata, H., 2007. Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin. Biochim. Biophys. Acta 1770, 672e680. rrez-Padilla, J.A., ValtierraZepeda-Romero, L.C., Liera-Garcia, J.A., Gutie mez, C.D., 2010. Paradoxical vascular-fibrotic reaction Santiago, C.I., Avila-Go after intravitreal bevacizumab for retinopathy of prematurity. Eye 24, 931e933. Zimmet, P., Alberti, K.G.M.M., Shaw, J., 2001. Global and societal implications of the diabetes epidemic. Nature 414, 782e787.

The role of microglia in diabetic retinopathy.

Role of FAM18B in diabetic retinopathy.

Role of diabetologist in evaluating diabetic retinopathy.

Role of microRNAs in the modulation of diabetic retinopathy.

The Role of Systemic Risk Factors in Diabetic Retinopathy.

Diabetic retinopathy. The primary care physician's role in management.

Role of Angiopoietins and Tie-2 in Diabetic Retinopathy.

Potential role of polyunsaturated fatty acids in diabetic retinopathy.

Role of macular xanthophylls in prevention of common neovascular retinopathies: retinopathy of prematurity and diabetic retinopathy.

NRF2 plays a protective role in diabetic retinopathy in mice.

Diabetic retinopathy.

Imaging in diabetic retinopathy.

Diabetic retinopathy.

Biomarkers in Diabetic Retinopathy.

Advances in diabetic retinopathy.

Diabetic retinopathy in Swaziland.

Diabetic retinopathy in acromegaly.

Perspectives in the treatment of diabetic retinopathy.

CCN2 in Diabetic Nephropathy.

Genetics of diabetic retinopathy.

The role of abnormal hemorrheodynamics in the pathogenesis of diabetic retinopathy.

Pathophysiology of diabetic retinopathy.

Management of diabetic retinopathy.

The role of O-GlcNAc signaling in the pathogenesis of diabetic retinopathy.