CTGF IN DIABETIC RETINOPATHY

In various organs other than the eye, CTGF (in combination with TGF-b) is considered to cause ECM accumulation and fibrosis as a consequence of diabetic pathology [30]. This is based on experimental diabetic models, where CTGF mRNA and protein were found to be upregulated in kidney, heart, and liver [115]. TGF-b1 is generally considered to be the main profibrotic factor in diabetic nephropathy [75], with CTGF as an important downstream mediator. Diabetes-induced thickening of glomerular BL in mouse kidney, analogous to BL thickening of retinal capillaries, was shown to be diminished in CTGF-deficient mice [115]. CTGF expression is not only induced by TGF-b but also by high glucose levels, AGEs, RAAS, TNF-a, mechanical stress, and CTGF itself [15–17, 49, 116–118]. There is increasing evidence confirming this role of CTGF in diabetic nephropathy. In diabetic patients, glomerular CTGF mRNA levels were upregulated, both in patients with microalbuminuria as well as in overt nephropathy [18]. Moreover, CTGF mRNA levels correlated with the degree of albuminuria [119]. In a baboon model of type I diabetes, expression levels of tubular CTGF protein after 5 years predicted albuminuria after 10 years [120]. Accordingly, in human diabetic patients, CTGF levels in urine [19] and plasma [20] correlated with progression of diabetic nephropathy.

The role of CTGF in the development of DR was less clear. However, recent evidence suggests that CTGF is involved in both the early stages and in the late proliferative stage of DR.

CTGF in BL Thickening in PCDR

In the light of its known role in matrix remodeling in other diabetic microvascular complications, CTGF is a candidate causal factor in diabetic BL thickening in the human retina. We studied CTGF expression in a series of 36 diabetic patients and 18 nondiabetic controls [121]. 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 the CTGF protein. In the normal human retina, CTGF staining was present in paravascular microglia. However, in the retina of diabetic subjects, microglial staining was significantly decreased whereas expression of CTGF in microvascular pericytes was significantly increased. Therefore, two main patterns of CTGF expression can be distinguished: either predominant staining of microglia or predominant staining of pericytes. The predominant pericyte staining correlated almost exclusively with the presence of diabetes. The constitutive expression in paravascular microglia in the normal retina suggests a role in retinal microvascular physiology. In the light of known functions of CTGF in other cells and tissues, it is tempting to speculate that microglia-derived CTGF is involved in retinal matrix or vascular BL homeostasis in normal conditions. However, Abu El-Asrar et al. [104] did not find immunostaining of CTGF in the nondiabetic retina, whereas the diabetic retina showed CTGF staining in ganglion cells, cells in the inner nuclear layer, and in cells identified as microglia, in agreement with the study by Kuiper et al. The difference in the two studies may be explained by the different antibodies used. It was also investigated whether altered CTGF expression in diabetes was associated with established DR [121].

Fig. 3. Model of CTGF expression patterns during the development of DR. Progressive degrees of nonproliferative DR are indicated by an increase in PAL-E-positive endothelial cells (red). (A) Control subject without PAL-E staining, showing CTGF-positive microglia only (yellow). (B–D) Diabetic subjects with or without PAL-E staining, showing decreased CTGFpositive microglia (yellow) and increased CTGF-positive pericytes (orange). (Reproduced from [121] with permission from BMJ Publishing Group Ltd.).

Staining with the use of the endothelium-specific monoclonal antibody PAL-E recognizing plasmalemma vesicle-associated protein (PLVAP), a marker associated with local vascular leakage [122, 123], revealed no correlation with CTGF staining patterns in pericytes or microglia. In fact, CTGF seemed to be evenly distributed in diabetes, irrespective of PAL-E staining (Fig. 3). Apparently, CTGF expression patterns in pericytes of the diabetic retina are not related to clinical DR, but rather are associated with preclinical changes in the retina in diabetes. Increased pericyte CTGF expression may be related to BL thickening and/or pericyte apoptosis, both important early events in PCDR.

AGEs and CTGF in BL Thickening in PCDR

One of the proposed mechanisms of BL thickening in PCDR is the formation of AGEs. Treatment of diabetic rats with the AGE-inhibitor aminoguanidine markedly reduced AGE formation in the retinal vasculature, but also protected against retinal capillary BL thickening [46]. AGEs can also induce synthesis of ECM in diabetic rat kidney [117]. A similar induction of ECM synthesis is mediated by CTGF, both in diabetic kidney [115] and retina [124]. In the diabetic rat kidney, AGEs induce expression of fibronectin and collagen type IV, possibly partly through CTGF [125, 126]. Furthermore, AGEs induced CTGF expression in cultured retinal vascular cells [125].

Therefore, it seems likely that AGE-induced BL thickening in the retina is mediated by CTGF. We recently investigated the levels of CTGF and ECM-related molecules in both the STZ-induced diabetic rat retina, treated with or without aminoguanidine, and in the retina of mice infused with AGEs [49]. In rats, STZ treatment resulted in a significant increase in carboxy-methyl-lysine (CML) plasma levels, a marker for AGE formation, at 6 and 12 weeks of diabetes. Aminoguanidine treatment had no effect on CML levels at 6 weeks, but decreased CML levels by 25% after 12 weeks. At this time point, retinal CTGF mRNA levels were elevated twofold in diabetic rats compared to nondiabetic controls, but treatment with aminoguanidine almost completely prevented this increase. Similarly, CTGF protein levels were increased in the retina of diabetic rats, and aminoguanidine prevented this effect. Other ECM components, such as collagen type IV and TIMP-1, also showed elevated mRNA levels after 6 or 12 weeks of diabetes,

which were significantly reduced by aminoguanidine treatment. TGF-b and fibronectin levels in the retina were unaffected at 6 and 12 weeks of diabetes in this model.

Retinal mRNA analysis in mice that received exogenous AGEs daily for 7 consecutive days revealed a twofold increase in CTGF levels as compared with control mice. Expression levels of Cyr61 (CCN1) were also elevated in the AGE-treated animals, but other CCN family members were not affected [49]. Taken together, these data present evidence that AGEs are both necessary and sufficient to cause increased levels of CTGF in the diabetic retina, concomitantly with ECM-related molecules [49]. Therefore, CTGF, and possibly Cyr61 as well, may have a role in thickening of the BL.

Another crucial feature of PCDR is loss of retinal capillary pericytes. Pericytes maintain capillary structure and integrity and regulate homeostasis of the endothelium. Cultured rat retinal pericytes exposed to AGEs expressed increased levels of CTGF [125]. In these cells, AGEs induced anoikis, a form of apoptosis caused by loss of cell–matrix interactions. Likewise, overexpression of CTGF promoted detachment and anoikis of retinal pericytes. The authors suggested that accumulation of CTGF in the retinal capillaries at the onset of diabetes may alter vascular structure and organization and have a role in pericyte apoptosis in PCDR.

Role of VEGF in BL Thickening

VEGF, a potent vascular permeability and angiogenic factor in PDR, is also increased early in PCDR [6, 127, 128]. Neutralizing VEGF with an antibody partly prevented diabetes-induced BL thickening in the retina of obese type 2 diabetic mice [129]. To test whether VEGF itself is capable to induce expression of genes that contribute to BL thickening in PCDR, we investigated the effect of VEGF injected in the vitreous of rat eyes on the retinal expression of CTGF, other CCN family members, TGF-b, and ECM-related molecules [26]. Adult Wistar rats were injected intravitreously with recombinant rat VEGF164 in one eye and with solvent only in the contralateral eye. Retinal gene expression and protein levels were examined at various time points afterwards. At 24 h after injection, CTGF mRNA expression showed a 2.3-fold increase. TGF-b1 mRNA, but not TGF-b2 mRNA, was also induced significantly at 24 h after injection. Of the ECM-related molecules examined, fibronectin and TIMP-1 were significantly upregulated at 24 h. TIMP-2, collagen type IV, and laminin B1 mRNA levels were unaffected by VEGF. At the protein level, CTGF and fibronectin were clearly increased at 48 h after injection in VEGF-injected eyes. TGF-b and fibronectin immunostaining in retinal sections was more intense in the microvasculature in VEGF-injected eyes as compared to PBS-injected and noninjected eyes. VEGF stimulation in bovine retinal endothelial cells (BRECs) resulted in an early increase of CTGF, TGF-b1, TGF-b2, and fibronectin expression. At 24 h, TIMP-1 mRNA was significantly increased. In bovine retinal pericytes (BRPCs), fibronectin, collagen type IV, and TIMP-1 mRNA levels were significantly upregulated at 24 h after VEGF stimulation. CTGF, TGF-b1, and TGF-b2 expression was not affected by VEGF in BRPCs.

Overall, VEGF was able to induce expression of genes related to ECM remodeling in the rat retina. The specificity of this response was demonstrated by the fact that induction of expression of ECM-related genes was selective and that the expression profile correlated to changes in protein levels. In vitro, comparable gene expression profiles

Fig. 4. Hypothetical model of diabetes-induced BL thickening. The model was developed on the basis of data obtained in both the VEGF-induced retinopathy model and the STZ-induced diabetes study, and what is know from the literature [42]. During diabetes, levels of AGEs and VEGF increase, and ECM molecules are induced at different time points after the onset of diabetes. Both AGEs and VEGF contribute to the induction of CTGF expression.

were found in retinal endothelial cells and pericytes, suggesting that the retinal vasculature plays an important role in the altered gene expression profile found in rat retina. Thus, early expression of VEGF in PCDR may contribute directly, and/or via CTGF, to BL thickening and further development of DR. Based on the VEGF-induced retinopathy model and the STZ-induced diabetes study, we developed a model of the expression of profibrotic genes involved in diabetes-induced BL thickening (Fig. 4).

TGF-b and CTGF in BL Thickening

TGF-b plays a causal role in BL thickening in mouse brain capillaries [130] and in the diabetic kidney [131, 132]. However, evidence for such a role in DR is scarce or indirect. Recently, it was shown that two drugs that are effective in the suppression of experimental DR had in common that upregulation of expression of members of the TGF-b pathway was suppressed, suggesting that TGF-b signaling plays a major role in the early pathogenesis of DR [133]. More specifically, retinal vessels in diabetic rats showed both increased TGF-b activity and increased CTGF mRNA expression [133].

To further identify the possible role of TGF-b in BL thickening in DR, its downstream effects were characterized in cultured retinal vascular cells [134]. BRECs and BRPCs were incubated with both low and high concentrations of TGF-b1, and expression levels of ECM-related molecules downstream of TGF-b were analyzed. In BRECs, only high concentrations of TGF-b induced mRNA expression of specific downstream TGF- b effector genes, including fibronectin, but not of CTGF. In BRPCs, both low and high concentrations of TGF-b induced expression of fibronectin and CTGF. Specific inhibition of the TGF-b receptor ALK5 significantly decreased expression levels of fibronec-