The role of CTGF in diabetic retinopathy.

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)-ß. In PDR, the switch from neovascularization to a fibrotic phase - the angio-fibrotic switch - 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.

Connective tissue growth factor is involved in structural retinal vascular changes in long-term experimental diabetes.

Early retinal vascular changes in the development of diabetic retinopathy (DR) include capillary basal lamina (BL) thickening, pericyte loss and the development of acellular capillaries. Expression of the CCN (connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed) family member CCN2 or connective tissue growth factor (CTGF), a potent inducer of the expression of BL components, is upregulated early in diabetes. Diabetic mice lacking one functional CTGF allele (CTGF⁺/⁻) do not show this BL thickening. As early events in DR may be interrelated, we hypothesized that CTGF plays a role in the pathological changes of retinal capillaries other than BL thickening. We studied the effects of long-term (6-8 months) streptozotocin-induced diabetes on retinal capillary BL thickness, numbers of pericytes and the development of acellular capillaries in wild type and CTGF⁺/⁻ mice. Our results show that an absence of BL thickening of retinal capillaries in long-term diabetic CTGF⁺/⁻ mice is associated with reduced pericyte dropout and reduced formation of acellular capillaries. We conclude that CTGF is involved in structural retinal vascular changes in diabetic rodents. Inhibition of CTGF in the eye may therefore be protective against the development of DR.

Vitreous TIMP-1 levels associate with neovascularization and TGF-ß2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy.

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-ß2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-ß2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-ß2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-ß2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.

Hemizygous deletion of CTGF/CCN2 does not suffice to prevent fibrosis of the severely injured kidney.

BACKGROUND: Connective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis. METHODS: Wild-type (WT, CCN2(+/+)) and hemizygous CCN2(+/-) C57Bl/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio. RESULTS: CCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2(+/-) mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2(+/-) mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2(+/-) mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2(+/-) mice (all differences between WT and CCN2(+/-) mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2(+/-) mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6), and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models. CONCLUSION: Unlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney.

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.

INTRODUCTION: In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) may cause blindness by neovascularisation followed by fibrosis of the retina. It has previously been shown that a shift in the balance between levels of CTGF and VEGF in the eye is associated with this angiofibrotic switch. This study investigated whether anti-VEGF agents induce accelerated fibrosis in patients with PDR, as predicted by this model. METHODS: CTGF and VEGF levels were measured by 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. RESULTS: CTGF correlated positively, and VEGF correlated negatively with the degree of fibrosis. The CTGF/VEGF ratio was the strongest predictor of fibrosis. Clinically, increased fibrosis was observed after intravitreal bevacizumab. CONCLUSIONS: These results confirm that the CTGF/VEGF ratio is a strong predictor of vitreoretinal fibrosis in PDR, and show that intravitreal anti-VEGF treatment causes increased fibrosis in PDR patients. These findings provide strong support for the model that the balance of CTGF and VEGF determines the angiofibrotic switch, and identify CTGF as a possible therapeutic target in the clinical management of PDR.

Differential TGF-{beta} signaling in retinal vascular cells: a role in diabetic retinopathy?

Purpose. An early hallmark of preclinical diabetic retinopathy is thickening of the capillary basal lamina (BL). TGF-beta, a multipotent cytokine acting through its receptors ALK5 and -1, has been postulated to be involved in this phenomenon. In light of this possible role, TGF-beta signaling and its downstream molecular effects were characterized in cultured vascular endothelial cells and pericytes of the retina. Methods. Bovine retinal endothelial cells and pericytes were stimulated with TGF-beta1 in the presence or absence of SD-208, a specific inhibitor of the TGF-beta type I receptor ALK5, or ALK5 small interfering (si)RNA. TGF-beta-signaling pathways were characterized by analysis of phosphorylated Smad2 or -1/5/8 proteins and TGF-beta target genes (PAI-1, fibronectin, CTGF, Smad7, and Id1) and protein (fibronectin). Results. ALK5 was expressed in both cell types, whereas ALK1 was exclusively expressed in endothelial cells. In endothelial cells, TGF-beta induced Smad2 phosphorylation at high concentrations, which was efficiently blocked by ALK5 inhibition. In contrast, in pericytes, Smad2 phosphorylation was rapidly induced at low concentrations of TGF-beta. The ALK1-Smad1/5/8 pathway was activated by TGF-beta in endothelial cells only. TGF-beta caused ALK5-mediated upregulation of PAI-1, Smad7, and fibronectin and in pericytes at lower TGF-beta concentrations than in endothelial cells. CTGF mRNA expression was induced only in pericytes. Fibronectin protein was confirmed to be regulated by TGF-beta in both cell types. Conclusions. TGF-beta signaling in retinal endothelial cells and pericytes show that these cells, and in particular the pericytes, have the essential characteristics to allow for a role of TGF-beta in BL thickening in preclinical diabetic retinopathy.

Sodium profiling, but not cool dialysate, increases the absolute plasma refill rate during hemodialysis.

Intradialytic hypotension is often caused by a discrepancy between ultrafiltration and plasma refilling. Increasing the plasma refill rate could therefore reduce intradialytic hypotension. We used a recently developed method to measure the effect of cool dialysate and sodium (Na) profiling on refill during hemodialysis (HD). Using a Gambro AK200 with blood volume (BV) sensor plus computer-guided external pump, a high ultrafiltration rate quickly induced a preset BV reduction. A software feedback mechanism subsequently adjusted the ultrafiltration rate continuously to maintain BV between very narrow preset boundaries. The continuously changing, software-generated ultrafiltration rate then quantitatively equalled refill. Absolute plasma refill rate was measured in six stable patients without intradialytic hypotension, undergoing HD without intervention, with cool dialysate (1 degrees C below core temperature), and with Na profiling (gradually declining from 150 to 140 mmol/l). Baseline refill rate was 20.1 + or - 4.0 ml/min (mean + or - SD). Although cool dialysate did not affect refill (22.2 + or - 4.1 ml/min, p = 0.27 vs. baseline), Na profiling induced a significant improvement (26.8 + or - 3.7 ml/min, p = 0.006 vs. baseline). Using our method to measure absolute plasma refill rate during HD, we demonstrated that Na profiling indeed improves the plasma refill rate. A potential effect of cool dialysate could not be established.

Methylprednisolone pulse therapy for patients with moderately severe Graves' orbitopathy: a prospective, randomized, placebo-controlled study.

OBJECTIVE: To assess whether methylprednisolone (MP) pulse therapy is efficacious in the treatment of moderately severe Graves' orbitopathy (GO). DESIGN: Prospective, placebo (PL)-controlled, double-blind, randomized study. METHODS: Fifteen previously untreated patients with active, moderately severe GO participated in the study; 6 patients received MP and 9 patients a PL. Moderately severe disease was defined using the NOSPECS classification of clinical signs of GO . Activity was measured with the clinical activity score (CAS). A dose of 500 mg MP or only solvent was administered intravenously, over three consecutive days, in four cycles at 4 weekly intervals (6 g of MP in total). Qualitatively, a successful treatment outcome was defined as an improvement in one major and/or two minor criteria in the worst eye at week 48. The major criteria were: improvement in diplopia grade; improvement in eye movement; a decrease in CAS of three points. The minor criteria were: decrease of eyelid retraction; decrease of proptosis; improvement in grade of soft tissue swelling; a decrease in CAS of two points. RESULTS: The qualitative treatment outcome was successful at the end of the trial in five out of six (83%) patients receiving MP and in one out of nine (11%) patients given the PL (relative risk=7.5; (95% confidence interval 1.1-49.3), P=0.005). The treatment was well tolerated. CONCLUSIONS: In spite of the small number of patients, a significant difference in outcome was observed between MP- and PL-treated patients. We conclude that MP pulse therapy appears to be an effective treatment for active, moderately severe GO.

Effect of VEGF-A on expression of profibrotic growth factor and extracellular matrix genes in the retina.

PURPOSE: Vascular endothelial growth factor-A (VEGF) causes increased vascular permeability and leukocyte adhesion in preclinical diabetic retinopathy (PCDR). Another hallmark of PCDR is thickening of the capillary basement membrane (BM). Recently, VEGF has been shown to induce expression of profibrotic genes such as transforming growth factor (TGF)-beta1 and connective tissue growth factor (CTGF or CCN2) in cultured endothelial cells. Moreover, neutralization of VEGF prevented BM thickening in diabetic mice in vivo. The authors hypothesize that VEGF directly contributes to BM thickening in the diabetic retina by inducing expression of profibrotic growth factors and extracellular matrix (ECM) components. METHODS: Transcription and protein levels of ECM-related genes were evaluated in the rat retina after intravitreal VEGF injection by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. In addition, expression profiles of the same genes in response to VEGF stimulation were investigated in bovine retinal vascular cells in vitro. RESULTS: Intravitreal VEGF injection induced retinal transcription of CYR61 (CCN1), CTGF, TGF-beta1, tissue inhibitor of metalloproteases (TIMP)-1 and fibronectin, and protein expression of CYR61, CTGF, TGF-beta1 and fibronectin. In bovine retinal endothelial cells and pericytes stimulated by VEGF in vitro, gene expression profiles were similar to those in the intact retina in vivo. CONCLUSIONS: VEGF induces profibrotic growth factors and extracellular matrix genes in the retina in vivo, as well as in cultured retinal vascular cells in vitro. The current findings have relevance for understanding the pathogenesis of preclinical DR, where early upregulation of VEGF may cause BM thickening by induction of ECM-related genes.