Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Klotho May Ameliorate Proteinuria by Targeting TRPC6 Channels in Podocytes.

Klotho is a type-1 membrane protein predominantly produced in the kidney, the extracellular domain of which is secreted into the systemic circulation. Membranous and secreted Klotho protect organs, including the kidney, but whether and how Klotho directly protects the glomerular filter is unknown. Here, we report that secreted Klotho suppressed transient receptor potential channel 6 (TRPC6)-mediated Ca2+ influx in cultured mouse podocytes by inhibiting phosphoinositide 3-kinase-dependent exocytosis of the channel. Furthermore, soluble Klotho reduced ATP-stimulated actin cytoskeletal remodeling and transepithelial albumin leakage in these cells. Overexpression of TRPC6 by gene delivery in mice induced albuminuria, and exogenous administration of Klotho ameliorated the albuminuria. Notably, immunofluorescence and in situ hybridization revealed Klotho expression in podocytes of mouse and human kidney. Heterozygous Klotho-deficient CKD mice had aggravated albuminuria compared with that in wild-type CKD mice with a similar degree of hypertension and reduced clearance function. Finally, disrupting the integrity of glomerular filter by saline infusion-mediated extracellular fluid volume expansion increased urinary Klotho excretion. These results reveal a potential novel function of Klotho in protecting the glomerular filter, and may offer a new therapeutic strategy for treatment of proteinuria.

Inhibition of TRPC6 channels ameliorates renal fibrosis and contributes to renal protection by soluble klotho.

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.

Connective tissue growth factor antagonizes transforming growth factor-ß1/Smad signalling in renal mesangial cells.

The critical involvement of TGF-ß1 (transforming growth factor-ß1) in DN (diabetic nephropathy) is well established. However, the role of CTGF (connective tissue growth factor) in regulating the complex interplay of TGF-ß1 signalling networks is poorly understood. The purpose of the present study was to investigate co-operative signalling between CTGF and TGF-ß1 and its physiological significance. CTGF was determined to bind directly to the TßRIII (TGF-ß type III receptor) and antagonize TGF-ß1-induced Smad phosphorylation and transcriptional responses via its N-terminal half. Furthermore, TGF-ß1 binding to its receptor was inhibited by CTGF. A consequent shift towards non-canonical TGF-ß1 signalling and expression of a unique profile of differentially regulated genes was observed in CTGF/TGF-ß1-treated mesangial cells. Decreased levels of Smad2/3 phosphorylation were evident in STZ (streptozotocin)-induced diabetic mice, concomitant with increased levels of CTGF. Knockdown of TßRIII restored TGF-ß1-mediated Smad signalling and cell contractility, suggesting that TßRIII is key for CTGF-mediated regulation of TGF-ß1. Comparison of gene expression profiles from CTGF/TGF-ß1-treated mesangial cells and human renal biopsy material with histological diagnosis of DN revealed significant correlation among gene clusters. In summary, mesangial cell responses to TGF-ß1 are regulated by cross-talk with CTGF, emphasizing the potential utility of targeting CTGF in DN.

Connective tissue growth factor (CTGF/CCN2) is increased in peritoneal dialysis patients with high peritoneal solute transport rate.

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis (PD) therapy that often occurs in association with peritoneal high transport rate and ultrafiltration failure (UFF). To study the possible pathogenic role of connective tissue growth factor (CTGF) in the relationship of PF and UFF, dialysate CTGF contents (n = 178) and tissue CTGF expression (n = 61) were investigated by ELISA, real-time PCR, immunohistochemistry, and in situ hybridization. CTGF production with and without TGF-beta1 stimulation in human peritoneal mesothelial cells (HPMC) from the spent patients' peritoneal dialysate (n = 32) was studied in vitro. The dialysate-to-plasma ratio for creatinine (D/P Cr) was positively correlated to dialysate CTGF concentration and estimated local peritoneal production of CTGF. CTGF mRNA expression was 11.4-fold higher in peritoneal membranes with UFF than in pre-PD renal failure peritoneum and was correlated with thickness of the peritoneum. CTGF protein and mRNA were detected in mesothelium and in fibroblast-like cells. In cultured HPMC, TGF-beta(1)-induced expression of CTGF mRNA was increased at 12 and 24 h and was correlated with D/P Cr. In contrast, bone morphogenic protein-4 mRNA expression was inversely correlated with D/P Cr. Our results suggest that high peritoneal transport state is associated with fibrosis and increased peritoneal CTGF expression and production by mesothelial cells, which can be stimulated by TGF-beta1. Dialysate CTGF concentration could be a biomarker for both peritoneal fibrosis and membrane function. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state.

Renal proximal tubular dysfunction is a major determinant of urinary connective tissue growth factor excretion.

Connective tissue growth factor (CTGF) plays a key role in renal fibrosis. Urinary CTGF is elevated in various renal diseases and may have biomarker potential. However, it is unknown which processes contribute to elevated urinary CTGF levels. Thus far, urinary CTGF was considered to reflect renal expression. We investigated how tubular dysfunction affects urinary CTGF levels. To study this, we administered recombinant CTGF intravenously to rodents. We used both full-length CTGF and the NH(2)-terminal fragment, since the NH(2)-fragment is the predominant form detected in urine. Renal CTGF extraction, determined by simultaneous arterial and renal vein sampling, was 18 +/- 3% for full-length CTGF and 21 +/- 1% for the NH(2)-fragment. Fractional excretion was very low for both CTGFs (0.02 +/- 0.006% and 0.10 +/- 0.02%, respectively), indicating that >99% of the extracted CTGF was metabolized by the kidney. Immunohistochemistry revealed extensive proximal tubular uptake of CTGF in apical endocytic vesicles and colocalization with megalin. Urinary CTGF was elevated in megalin- and cubilin-deficient mice but not in cubilin-deficient mice. Inhibition of tubular reabsorption by Gelofusine reduced renal uptake of CTGF and increased urinary CTGF. In healthy volunteers, Gelofusine also induced an increase of urinary CTGF excretion, comparable to the increase of beta(2)-microglobulin excretion (r = 0.99). Furthermore, urinary CTGF correlated with beta(2)-microglobulin (r = 0.85) in renal disease patients (n = 108), and only beta(2)-microglobulin emerged as an independent determinant of urinary CTGF. Thus filtered CTGF is normally reabsorbed almost completely in proximal tubules via megalin, and elevated urinary CTGF may largely reflect proximal tubular dysfunction.

CTGF inhibits BMP-7 signaling in diabetic nephropathy.

In diabetic nephropathy, connective tissue growth factor (CTGF) is upregulated and bone morphogenetic protein 7 (BMP-7) is downregulated. CTGF is known to inhibit BMP-4, but similar cross-talk between BMP-7 and CTGF has not been studied. In this study, it was hypothesized that CTGF acts as an inhibitor of BMP-7 signaling activity in diabetic nephropathy. Compared with diabetic wild-type CTGF(+/+) mice, diabetic CTGF(+/-) mice had approximately 50% lower CTGF mRNA and protein, less severe albuminuria, no thickening of the glomerular basement membrane, and preserved matrix metalloproteinase (MMP) activity. Although the amount of BMP-7 mRNA was similar in the kidneys of diabetic CTGF(+/+) and CTGF(+/-) mice, phosphorylation of the BMP signal transduction protein Smad1/5 and expression of the BMP target gene Id1 were lower in diabetic CTGF(+/+) mice. Moreover, renal Id1 mRNA expression correlated with albuminuria (R = -0.86) and MMP activity (R = 0.76). In normoglycemic mice, intraperitoneal injection of CTGF led to a decrease of pSmad1/5 in the renal cortex. In cultured renal glomerular and tubulointerstitial cells, CTGF diminished BMP-7 signaling activity, evidenced by lower levels of pSmad1/5, Id1 mRNA, and BMP-responsive element-luciferase activity. Co-immunoprecipitation, solid-phase binding assay, and surface plasmon resonance analysis showed that CTGF binds BMP-7 with high affinity (Kd approximately 14 nM). In conclusion, upregulation of CTGF inhibits BMP-7 signal transduction in the diabetic kidney and contributes to altered gene transcription, reduced MMP activity, glomerular basement membrane thickening, and albuminuria, all of which are hallmarks of diabetic nephropathy.

Association of connective tissue growth factor with fibrosis in vitreoretinal disorders in the human eye.

OBJECTIVE: To investigate the expression of the profibrotic connective tissue growth factor (CTGF) in relation to severity of intraocular fibrosis and neovascularization in human vitreoretinal disorders for the identification of potential therapeutic targets to prevent fibrosis. METHODS: Concentrations of CTGF were measured by enzyme-linked immunosorbent assay in 119 vitreous samples from patients with proliferative diabetic retinopathy, proliferative vitreoretinopathy, epiretinal membrane, and macular hole. Clinical data, including degree of intraocular fibrosis and neovascularization, were collected using standardized forms. RESULTS: Multifactorial analysis revealed that only CTGF levels correlated highly significantly with degree of fibrosis in the various vitreoretinal disorders studied (P<.001; R2= 47.7%). Likewise, variation in degree of fibrosis was best predicted by CTGF levels (P<.001). CONCLUSION: The strong correlation between CTGF levels and degree of fibrosis in vitreoretinal disorders suggests that CTGF is an important factor in ocular fibrosis, similar to its role in pathologic fibrosis in other organs. CLINICAL RELEVANCE: Connective tissue growth factor may be a therapeutic target for prevention of sight-threatening vitreoretinal scarring in the eye.

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Accumulation of NH2-terminal fragment of connective tissue growth factor in the vitreous of patients with proliferative diabetic retinopathy.

OBJECTIVE: To evaluate the expression of connective tissue growth factor (CTGF) and its fragments in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to localize CTGF expression in associated preretinal membranes. RESEARCH DESIGN AND METHODS: Vitreous was obtained from 24 patients with active PDR, 4 patients with quiescent PDR, and 23 patients with other retinal diseases and no diabetes, including 5 patients with vitreous hemorrhage. Enzyme-linked immunosorbent assay was used to determine levels of whole CTGF and its NH2- and COOH-terminal fragments. Preretinal membranes from three patients with active PDR were stained immunohistochemically for the presence of CTGF and cell type-specific markers. RESULTS: A significant increase in NH2-terminal CTGF fragment content was found in vitreous samples from patients with active PDR when compared with samples from nondiabetic patients (P<0.0001) or patients with quiescent PDR (P=0.02). Levels of NH2-terminal CTGF were also greater in vitreous samples from diabetic patients with vitreous hemorrhage compared with samples from nondiabetic patients with vitreous hemorrhage (P=0.02). Vitreous levels of whole CTGF were similar in all groups. COOH-terminal fragments of CTGF were not detected. CTGF immunoreactivity was predominantly localized to smooth muscle actin-positive myofibroblasts within active PDR membranes. CONCLUSIONS: -NH2-terminal CTGF fragment content is increased in the vitreous of patients with active PDR, suggesting that it plays a pathogenic role or represents a surrogate marker of CTGF activity in the disorder. The localization of CTGF in myofibroblasts suggests a local paracrine mechanism for induction of fibrosis and neovascularization.

Connective tissue growth factor is increased in plasma of type 1 diabetic patients with nephropathy.

OBJECTIVE: Connective tissue growth factor (CTGF) is strongly upregulated in fibrotic disorders and has been hypothesized to play a role in the development and progression of diabetes complications. The aim of the present study was to investigate the possible association of plasma CTGF levels in type 1 diabetic patients with markers relevant to development of diabetes complications. RESEARCH DESIGN AND METHODS: Plasma CTGF levels (full-length and NH2-terminal fragments) were determined in 62 well-characterized patients with type 1 diabetes and in 21 healthy control subjects. Correlations of these plasma CTGF levels with markers of glycemic control, platelet activation, endothelial activation, nephropathy, and retinopathy were investigated. RESULTS: -Elevated plasma NH2-terminal fragment of CTGF (CTGF-N) levels were detected in a subpopulation of type 1 diabetic patients and were associated with diabetic nephropathy. Stepwise regression analysis revealed contribution of albuminuria, creatinine clearance, and duration of diabetes as predictors of plasma CTGF-N level. Elevation of plasma CTGF-N levels in patients with retinopathy was probably due to renal comorbidity. CONCLUSIONS: Plasma CTGF-N levels are elevated in type 1 diabetic patients with nephropathy and appear to be correlated with proteinuria and creatinine clearance. Further studies will be needed to determine the relevance of plasma CTGF as a clinical marker and/or pathogenic factor in diabetic nephropathy.

Regulation of fibronectin-EDA through CTGF domain-specific interactions with TGFß2 and its receptor TGFßRII.

PURPOSE: To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)-ß and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells. METHODS: Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGFß and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGFß2 or with its type II receptor TGFßRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays. RESULTS: FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGFß2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGFß2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGFß2 or TGFßRII. Interaction of TGFß2 or TGFßRII with CTGF was dose dependent and specific. CTGF directly bound TGFß2 and TGFßRII at its N- and C-terminal domains, respectively. CONCLUSIONS: These findings suggest that CTGF promotes the profibrotic activities of TGFß acting as a cofactor through direct protein interactions and complex regulatory mechanisms.

Connective tissue growth factor is increased in pseudoexfoliation glaucoma.

PURPOSE: Pseudoexfoliation (PXF) syndrome is a generalized disorder of the extracellular matrix (ECM) involving the trabecular meshwork (TM), associated with raised intraocular pressure, glaucoma, and cataract. The purposes of this study were to quantify aqueous humor connective tissue growth factor (CTGF) in PXF glaucoma, to determine the effect of CTGF on ECM production in TM cells, and to identify intracellular CTGF signaling pathways. METHODS: Aqueous humor samples were obtained from patients undergoing routine cataract surgery or trabeculectomy. CTGF levels were quantified by ELISA. The effect of CTGF on fibrillin-1 expression in TM cells was investigated by real-time PCR. Western immunoblot analysis was used to investigate CTGF signaling. c-Jun/AP-1 activation was measured in CHO cells by ELISA after stimulation with CTGF. RESULTS: PXF with glaucoma had the highest aqueous humor level of CTGF (n = 18; 5.15 ± 0.79 ng/mL [SEM]; P < 0.01) compared with PXF without glaucoma (n = 15; 2.76 ± 0.64 ng/mL), primary open-angle glaucoma (POAG; n = 20; 3.05 ± 0.40 ng/mL), and the control (n = 21; 2.60 ± 0.29 ng/mL). In vitro exposure of TM cells to CTGF resulted in a 50% upregulation of fibrillin-1, which was partially blocked with the MEK (mitogen-activated protein extracellular kinase) inhibitor PD098059. Western blot analysis demonstrated increased phosphorylation of p42/44 MAPK, p38 MAPK, and the JNK pathways in response to CTGF. c-Jun/AP-1 activity was significantly increased in response to CTGF treatment. CONCLUSIONS: Increased levels of CTGF in the aqueous humor of PXF patients likely has pathologic significance through increased production of fibrillin-1 by TM cells through activation of p42/44 MAPK, p38 MAPK, and JNK pathways.

Cooperative interaction of CTGF and TGF-ß in animal models of fibrotic disease.

BACKGROUND: Connective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-ß; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-ß2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis. RESULTS: Intraperitoneal coadministration of CTGF and TGF-ß2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-ß and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models. CONCLUSION: Cooperative interactions between CTGF and TGF-ß signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.

Could aging human skin use a connective tissue growth factor boost to increase collagen content?

The roles of connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-beta), both well-known collagen production stimulators, were examined in skin aging. Aged skin and fibroblasts exhibited a coordinate decrease in CTGF, TGF-beta, and type I procollagen expression and content. CTGF knockdown and TGF-beta blockade in normal dermal fibroblasts reduced procollagen expression, whereas overexpressing CTGF increased procollagen by a TGF-beta/Smad signaling-dependent mechanism without involving Smad2/3.

TGF-ß-stimulated CTGF production enhanced by collagen and associated with biogenesis of a novel 31-kDa CTGF form in human corneal fibroblasts.

PURPOSE: Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-ß) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-ß. METHODS: Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-ß1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs. RESULTS: TGF-ß-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-ß-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)ß(3) and α(5)ß(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes. CONCLUSIONS: Enhanced CTGF secretion induced by TGF-ß in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.

Connective tissue growth factor as a mediator of intraocular fibrosis.

PURPOSE: To investigate the role of connective tissue growth factor (CTGF) in the pathogenesis of proliferative vitreoretinopathy (PVR). METHODS: Expression of CTGF was evaluated immunohistochemically in human PVR membranes, and the accumulation of CTGF in the vitreous was evaluated by ELISA. The effects of CTGF on type I collagen mRNA and protein expression in RPE were assayed by real-time PCR and ELISA, and migration was assayed with a Boyden chamber assay. Experimental PVR was induced in rabbits with vitreous injection of RPE cells plus rhCTGF; injection of RPE cells plus platelet derived-growth factor, with or without rhCTGF, or by injection of RPE cells infected with an adenoviral vector that expressed CTGF. RESULTS: CTGF was highly expressed in human PVR membranes and partially colocalized with cytokeratin-positive RPE cells. Treatment of RPE with rhCTGF stimulated migration with a peak response at 50 ng/mL (P < 0.05) and increased expression of type I collagen (P < 0.05). There was a prominent accumulation of the N-terminal half of CTGF in the vitreous of patients with PVR. Intravitreous injection of rhCTGF alone did not produce PVR, whereas such injections into rabbits with mild, nonfibrotic PVR promoted the development of dense, fibrotic epiretinal membranes. Similarly, intravitreous injection of RPE cells infected with adenoviral vectors that overexpress CTGF induced fibrotic PVR. Experimental PVR was associated with increased CTGF mRNA in PVR membranes and accumulation of CTGF half fragments in the vitreous. CONCLUSIONS: The results identify CTGF as a major mediator of retinal fibrosis and potentially an effective therapeutic target for PVR.

Plasma connective tissue growth factor is an independent predictor of end-stage renal disease and mortality in type 1 diabetic nephropathy.

OBJECTIVE: We evaluated the predictive value of baseline plasma connective tissue growth factor (CTGF) in a prospective study of patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Subjects were 198 type 1 diabetic patients with established diabetic nephropathy and 188 type 1 diabetic patients with persistent normoalbuminuria. Follow-up time was 12.8 years. Prediction of end-stage renal disease (ESRD) and mortality by plasma CTGF was analyzed in conjunction with conventional risk factors. RESULTS: Plasma CTGF was higher in patients with nephropathy than in patients with normoalbuminuria (median 381 [interquartile range 270-630] vs. 235 [168-353] pmol/l). In patients with nephropathy, elevated plasma CTGF was an independent predictor of ESRD (covariate-adjusted hazard ratio [HR] 1.6 [95% CI 1.1-2.5]) and correlated with the rate of decline in glomerular filtration rate (GFR) (cumulative R = 0.46). Area under the receiver operating characteristic curve for prediction of ESRD was 0.72. Plasma CTGF above a cutoff level of 413 pmol/l predicted ESRD with a sensitivity of 73% and a specificity of 63% and was associated with a higher rate of decline in GFR (mean +/- SD 5.4 +/- 4.9 vs. 3.3 +/- 3.5 ml/min per 1.73 m(2) per year). Moreover, in patients with nephrotic range albuminuria (>3 g/day), plasma CTGF was the only predictor of ESRD (covariate-adjusted HR 4.5 [2.0-10.4]). Plasma CTGF was an independent predictor also of overall mortality (covariate-adjusted HR 1.4 [1.1-1.7]). In contrast, in normoalbuminuric patients, plasma CTGF did not correlate with clinical parameters and did not predict outcome. CONCLUSIONS: Plasma CTGF contributes significantly to prediction of ESRD and mortality in patients with type 1 diabetic nephropathy.

The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy.

BACKGROUND: In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring. METHODS/PRINCIPAL FINDINGS: VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment. CONCLUSIONS/SIGNIFICANCE: CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.

Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice.

Connective tissue growth factor (CTGF) is overexpressed in diabetic nephropathy (DN) and has therefore been implicated in its pathogenesis. The objective of the present study was to determine the tissue distribution of increased CTGF expression and the relationship of plasma, urinary, and renal CTGF levels to the development and severity of DN. We studied the relationship between CTGF and renal pathology in streptozotocin (STZ)-induced diabetes in C57BL/6J mice. Diabetic and age-matched control mice were killed after 1, 2, 4, and 9 wk of diabetes. In addition, key parameters of diabetes and DN were analyzed in 10-mo-old diabetic ob/ob mice and their ob/+ littermates. STZ-induced diabetic mice showed a significantly increased urinary albumin excretion after 1 wk and increased mesangial matrix score after 2 wk. Increased renal fibronectin, fibronectin ED-A, and collagen IValpha1 expression, as well as elevated plasma creatinine levels, were observed after 9 wk. After 2 wk, CTGF mRNA was upregulated threefold in the renal cortex. By 9 wk, CTGF mRNA was also increased in the heart and liver. In contrast, transforming growth factor-beta1 mRNA content was significantly increased only in the kidney by 9 wk. Renal CTGF expression was mainly localized in podocytes and parietal glomerular epithelial cells, and less prominent in mesangial cells. In addition, plasma CTGF levels and urinary CTGF excretion were increased in diabetic mice. Moreover, albuminuria strongly correlated with urinary CTGF excretion (R = 0.83, P < 0.0001). Increased CTGF expression was also demonstrated in type 2 diabetic ob/ob mice, which points to a causal relationship between diabetes and CTGF and thus argues against a role of STZ in this process. The observed relationship of podocyte and urinary CTGF to markers of DN suggests a pathogenic role of CTGF in the development of DN.