The role of yes activated protein (YAP) in melanoma metastasis.

Hippo was first identified in a genetic screen as a protein that suppressed proliferation and cell growth. Subsequently, it was shown that hippo acted in a so-called canonical cascade to suppress Yorkie, the Drosophila equivalent of Yes-activated protein (YAP), a mechanosensitive transcriptional cofactor that enhances the activity of the TEAD family of transcription factors. YAP promotes fibrosis, activation of cancer-associated fibroblasts, angiogenesis and cancer cell invasion. YAP activates the expression of the matricellular proteins CCN1 (cyr61) and CCN2 (ctgf), themselves mediators of fibrogenesis and oncogenesis, and coordination of matrix deposition and angiogenesis. This review discusses how therapeutically targeting YAP through YAP inhibitors verteporfin and celastrol and its downstream mediators CCN1 and CCN2 might be useful in treating melanoma.

Using the Bleomycin-Induced Model of Fibrosis to Study the Contribution of CCN Proteins to Scleroderma Fibrosis.

Approximately 45% of the deaths in the developed world result from conditions with a fibrotic component. Although no specific, focused anti-fibrotic therapies have been approved for clinical use, a long-standing concept is that targeting CCN proteins may be useful to treat fibrosis. Herein, we summarize current data supporting the concept that targeting CCN2 may be a viable anti-fibrotic approach to treat scleroderma. Testing this hypothesis has been made possible by using a mouse model of inflammation-driven skin and lung fibrosis.

CCN-Based Therapeutic Peptides Modify Pancreatic Ductal Adenocarcinoma Microenvironment and Decrease Tumor Growth in Combination with Chemotherapy.

The prominent desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a determinant factor in tumor progression and a major barrier to the access of chemotherapy. The PDAC microenvironment therefore appears to be a promising therapeutic target. CCN2/CTGF is a profibrotic matricellular protein, highly present in the PDAC microenvironment and associated with disease progression. Here we have investigated the therapeutic value of the CCN2-targeting BLR100 and BLR200, two modified synthetic peptides derived from active regions of CCN3, an endogenous inhibitor of CCN2. In a murine orthotopic PDAC model, the two peptides, administered as monotherapy at low doses (approximating physiological levels of CCN3), had tumor inhibitory activity that increased with the dose. The peptides affected the tumor microenvironment, inhibiting fibrosis and vessel formation and reducing necrosis. Both peptides were active in preventing ascites formation. An increased activity was obtained in combination regimens, administering BLR100 or BLR200 with the chemotherapeutic drug gemcitabine. Pharmacokinetic analysis indicated that the improved activity of the combination was not mainly determined by the substantial increase in gemcitabine delivery to tumors, suggesting other effects on the tumor microenvironment. The beneficial remodeling of the tumor stroma supports the potential value of these CCN3-derived peptides for targeting pathways regulated by CCN2 in PDAC.

Can Intestinal Phosphate Binding or Inhibition of Hydroxyapatite Growth in the Vascular Wall Halt the Progression of Established Aortic Calcification in Chronic Kidney Disease?

Vascular calcification significantly contributes to mortality in chronic kidney disease (CKD) patients. Sevelamer and pyrophosphate (PPi) have proven to be effective in preventing vascular calcification, the former by controlling intestinal phosphate absorption, the latter by directly interfering with the hydroxyapatite crystal formation. Since most patients present with established vascular calcification, it is important to evaluate whether these compounds may also halt or reverse the progression of preexisting vascular calcification. CKD and vascular calcification were induced in male Wistar rats by a 0.75 % adenine low protein diet for 4 weeks. Treatment with PPi (30 or 120 µmol/kg/day), sevelamer carbonate (1500 mg/kg/day) or vehicle was started at the time point at which vascular calcification was present and continued for 3 weeks. Hyperphosphatemia and vascular calcification developed prior to treatment. A significant progression of aortic calcification in vehicle-treated rats with CKD was observed over the final 3-week period. Sevelamer treatment significantly reduced further progression of aortic calcification as compared to the vehicle control. No such an effect was seen for either PPi dose. Sevelamer but not PPi treatment resulted in an increase in both osteoblast and osteoid perimeter. Our study shows that sevelamer was able to reduce the progression of moderate to severe preexisting aortic calcification in a CKD rat model. Higher doses of PPi may be required to induce a similar reduction of severe established arterial calcification in this CKD model.

Balanced regulation of the CCN family of matricellular proteins: a novel approach to the prevention and treatment of fibrosis and cancer.

The CCN family of matricellular signaling proteins is emerging as a unique common link across multiple diseases and organs related to injury and repair. They are now being shown to play a central role in regulating the pathways to the initiation and resolution of normal wound healing and fibrosis in response to multiple forms of injury. Similarly, it is also emerging that they play a key role in regulating the establishment, growth, metastases and tissue regeneration in many forms of cancer via the interaction of cancer cells with the tumor stroma. Evidence has been recently provided that these proteins do not act independently but are co-regulated working in a yin/yang manner to alter the outcome of both normal physiological processes as well as pathology. The purpose of this review is to twofold. First, it will summarize work to date supporting CCN2 as a therapeutic target in the formation and progression of renal, skin, and other organ fibrosis, as well as cancer stroma formation. Second, it will highlight recent evidence for CCN3 as a counter-regulator and a potential therapeutic agent in these diseases with an exciting, novel potential to both treat and then restore tissue homeostasis in those afflicted by these devastating disorders.

Peritoneal delivery of sodium pyrophosphate blocks the progression of pre-existing vascular calcification in uremic apolipoprotein-E knockout mice.

Chronic kidney disease (CKD) is generally associated with disturbances of mineral and bone metabolism. They contribute to the development of vascular calcification (VC), a strong, independent predictor of cardiovascular risk. Pyrophosphate (PPi), an endogenous inhibitor of hydroxyapatite formation, has been shown to slow the progression of VC in uremic animals. Since in patients with CKD treatment is usually initiated for already existing calcifications, we aimed to compare the efficacy of PPi therapy with that of the phosphate binder sevelamer, using a uremic apolipoprotein-E knockout mouse model with advanced VCs. After CKD creation or sham surgery, 12-week-old female mice were randomized to one sham group and four CKD groups (n = 18-19/group). Treatment was initiated 8 weeks after left nephrectomy allowing prior VC development. Uremic groups received either intraperitoneal PPi (high dose, 1.65 mg/kg or low dose, 0.33 mg/kg per day), oral sevelamer (3 % in diet), or placebo treatment for 8 weeks. Both intima and media calcifications worsened with time in placebo-treated CKD mice, based on both quantitative image analysis and biochemical measurements. Progression of calcification between 8 and 16 weeks was entirely halted by PPi treatment, as it was by sevelamer treatment. PPi did not induce consistent bone histomorphometry changes. Finally, the beneficial vascular action of PPi probably involved mechanisms different from that of sevelamer. Further studies are needed to gain more precise insight into underlying mechanisms and to see whether PPi administration may also be useful in patients with CKD and VC.

Patients with encapsulating peritoneal sclerosis have increased peritoneal expression of connective tissue growth factor (CCN2), transforming growth factor-ß1, and vascular endothelial growth factor.

INTRODUCTION: Encapsulating peritoneal sclerosis (EPS) is a devastating complication of peritoneal dialysis (PD). The pathogenesis is not exactly known and no preventive strategy or targeted medical therapy is available. CCN2 has both pro-fibrotic and pro-angiogenic actions and appears an attractive target. Therefore, we studied peritoneal expression of CCN2, as well as TGFß1 and VEGF, in different stages of peritoneal fibrosis. MATERIALS AND METHODS: Sixteen PD patients were investigated and compared to 12 hemodialysis patients and four pre-emptively transplanted patients. Furthermore, expression was investigated in 12 EPS patients in comparison with 13 PD and 12 non-PD patients without EPS. Peritoneal tissue was taken during kidney transplantation procedure or during EPS surgery. In a subset of patients, CCN2 protein levels in peritoneal effluent and plasma were determined. Samples were examined by qPCR, histology, immunohistochemistry, and ELISA. RESULTS: Peritoneal CCN2 expression was 5-fold higher in PD patients compared to pre-emptively transplanted patients (P < 0.05), but did not differ from hemodialysis patients. Peritoneal expression of TGFß1 and VEGF were not different between the three groups; neither was peritoneal thickness. Peritoneum of EPS patients exhibited increased expression of CCN2 (35-fold, P < 0.001), TGFß1 (24-fold, P < 0.05), and VEGF (77-fold, P < 0.001) compared to PD patients without EPS. In EPS patients, CCN2 protein was mainly localized in peritoneal endothelial cells and fibroblasts. CCN2 protein levels were significantly higher in peritoneal effluent of EPS patients compared to levels in dialysate of PD patients (12.0 ± 4.5 vs. 0.91 ± 0.92 ng/ml, P < 0.01), while plasma CCN2 levels were not increased. CONCLUSIONS: Peritoneal expression of CCN2, TGFß1, and VEGF are significantly increased in EPS patients. In early stages of peritoneal fibrosis, only CCN2 expression is slightly increased. Peritoneal CCN2 overexpression in EPS patients is a locally driven response. The potential of CCN2 as biomarker and target for CCN2-inhibiting agents to prevent or treat EPS warrants further study.

Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 µg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-ß1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

Effects of pyrophosphate delivery in a peritoneal dialysis solution on bone tissue of apolipoprotein-E knockout mice with chronic kidney disease.

Vascular calcification (VC) is a risk factor for cardiovascular mortality in the setting of chronic kidney disease (CKD). Pyrophosphate (PPi), an endogenous molecule that inhibits hydroxyapatite crystal formation, has been shown to prevent the development of VC in animal models of CKD. However, the possibility of harmful effects of exogenous administration of PPi on bone requires further investigation. To this end, we examined by histomorphometry the bone of CKD mice after intraperitoneal PPi administration. After CKD creation or sham surgery, 10-week-old female apolipoprotein-E knockout (apoE(-/-)) mice were randomized to one non-CKD group or 4 CKD groups (n = 10-35/group) treated with placebo or three distinct doses of PPi, and fed with standard diet. Eight weeks later, the animals were killed. Serum and femurs were sampled. Femurs were processed for bone histomorphometry. Placebo-treated CKD mice had significantly higher values of osteoid volume, osteoid surface and bone formation rate than sham-placebo mice with normal renal function. Slightly higher osteoid values were observed in CKD mice in response to very low PPi dose (OV/BV, O.Th and ObS/BS) and, for one parameter measured, to high PPi dose (O.Th), compared to placebo-treated CKD mice. Treatment with PPi did not modify any other structural parameters. Mineral apposition rates, and other parameters of bone formation and resorption were not significantly different among the treated animal groups or control CKD placebo group. In conclusion, PPi does not appear to be deleterious to bone tissue in apoE(-/-) mice with CKD, although a possible stimulatory PPi effect on osteoid formation may be worth further investigation.

Gadolinium-induced fibrosis is counter-regulated by CCN3 in human dermal fibroblasts: a model for potential treatment of nephrogenic systemic fibrosis.

We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-ß stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-ß as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases.

Daily peritoneal administration of sodium pyrophosphate in a dialysis solution prevents the development of vascular calcification in a mouse model of uraemia.

BACKGROUND: The high rate of cardiovascular mortality in patients with end-stage renal disease (ESRD) is a significant barrier to improved life expectancy. Unique in this population is the marked development and aggressive worsening of vascular calcification (VC). Pyrophosphate (PPi), an endogenous molecule, appears to naturally inhibit soft tissue calcification, but may be depressed in chronic kidney disease (CKD) and ESRD. Although once thought to be a promising therapeutic, PPi's very short half-life in circulation curtailed earlier studies. We tested the possibility that a slow, continuous entry of PPi into the circulation and prevention of VC might be achieved by daily peritoneal dialysis (PD). METHODS: Pharmacokinetic studies were first carried out in rats with renal impairment resulting from a 5/6 nephrectomy. Efficacy studies were then performed in the apolipoprotein E gene knockout mouse model overlaid with CKD. PPi was delivered by means of a permanent peritoneal catheter in a solution simulating PD, but without the timed removal of spent dialysate. von Kossa's staining followed by semiquantitative morphological image processing, with separation of inside (intimal) and outside (presumed medial) lesions, was used to determine aortic root calcification. RESULTS: In comparison to an intravenous bolus, delivery of PPi in a PD solution resulted in a slower, extended delivery over >4 h. Next, the efficacy studies showed that a 6-day/week PD-simulated administration of PPi resulted in a dose-dependent inhibition of aortic calcification in both intimal and medial lesions. A dose-response effect on total aortic calcification was also documented, with a full inhibition seen at the highest dose. A limited peritoneal catheter-related inflammation was observed, as expected, and included the placebo-treated control groups. This inflammatory response could have masked a lower level PPi-specific adverse effect, but none was observed. CONCLUSIONS: Our findings suggest potential for PPi, administered during PD, to prevent the development of VC and to potentially extend the life of ESRD patients.

Treatment with pyrophosphate inhibits uremic vascular calcification.

Pyrophosphate, which may be deficient in advanced renal failure, is a potent inhibitor of vascular calcification. To explore its use as a potential therapeutic, we injected exogenous pyrophosphate subcutaneously or intraperitoneally in normal rats and found that their plasma pyrophosphate concentrations peaked within 15 min. There was a single exponential decay with a half-life of 33 min. The kinetics were indistinguishable between the two routes of administration or in anephric rats. The effect of daily intraperitoneal pyrophosphate injections on uremic vascular calcification was then tested in rats fed a high-phosphate diet containing adenine for 28 days to induce uremia. Although the incidence of aortic calcification varied and was not altered by pyrophosphate, the calcium content of calcified aortas was significantly reduced by 70%. Studies were repeated in uremic rats given calcitriol to produce more consistent aortic calcification and treated with sodium pyrophosphate delivered intraperitoneally in a larger volume of glucose-containing solution to prolong plasma pyrophosphate levels. This maneuver significantly reduced both the incidence and amount of calcification. Quantitative histomorphometry of bone samples after double-labeling with calcein indicated that there was no effect of pyrophosphate on the rates of bone formation or mineralization. Thus, exogenous pyrophosphate can inhibit uremic vascular calcification without producing adverse effects on bone.

CCN2 (CTGF) gene polymorphism is a novel prognostic risk factor for cardiovascular outcomes in hemodialysis patients.

BACKGROUND: The very high cardiovascular (CV) mortality and morbidity rates in hemodialysis (HD) patients are greatly related to atherosclerosis. CCN2 (connective tissue growth factor/CTGF) is a profibrotic factor that is secreted by endothelial cells, involved in atherogenesis, promoting fibroblast proliferation and matrix production. CCN2 protein is significantly increased in complicated fibrous plaques and enhances monocyte migration into atherosclerotic lesions. The aim of this study was to investigate a possible association between CCN2 gene polymorphism and CV morbidity and mortality in HD patients. METHODS: 98 HD patients, followed for 24 months, were genotyped for the common polymorphism on the CCN2 gene (G-945C). HD patient characteristics were: age 64 ± 13 years, males 64%, diabetes 24%, hypertension 62%, smokers 38%, dyslipidemia 28%, all undergoing standard HD three times weekly. RESULTS: All-cause mortality was not associated with CCN2 polymorphism (G-945C). In contrast, however, the GG genotype was strongly associated with CV mortality: OR 13 (1.49-155), p = 0.0048. Interestingly, the GG genotype was also greatly associated with the serious CV events of stroke and myocardial infarction in surviving HD patients: OR 13.3 (2.5-87.08), p = 0.0001. CONCLUSIONS: We demonstrate for the first time that CCN2 gene polymorphism is a prognostic risk factor for CV morbidity and mortality in HD patients. These data may have important implications for better understanding the link between accelerated atherosclerosis and increased mortality in HD population.

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease.

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-beta1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-beta1. Conversely, TGF-beta1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-beta1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.

Urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers for progression of diabetic nephropathy.

Profibrotic growth factors and inflammatory chemokines have been implicated in the pathogenesis of diabetic nephropathy (DN). However, measurement of urinary monocyte chemoattractant protein-1 (MCP-1) and connective tissue growth factor (CCN2) as prognostic markers has not previously been reported, and neither have two such molecules in urine been examined in a single study of DN. In this prospective observational study, 43 adult diabetic patients were studied, 40 were followed up for 6years. Urinary MCP-1/creatinine ratios were found to be significantly higher in patients with macroalbuminuria (3.3- and 2.1-fold higher (p<0.01) than normoalbuminuric and microalbuminuric patients, respectively). CCN2 exhibited a pattern different from that of urinary MCP-1. Urinary CCN2/creatinine ratios were greatly elevated in both microalbuminuric and macroalbuminuric patients (125- and 74-fold higher than normoalbuminuric patients, respectively, p<0.01 and p<0.05, respectively). Further, urinary CCN2, but not MCP-1, correlated with progression of microalbuminuria (R=0.49, p<0.05). In contrast, MCP-1, but not CCN2, correlated with the rate of eGFR decline for all patients (R=0.61, p<0.0001), reflective of its predictive value in patients with macroalbuminuria, but not for patients with microalbuminuria or normoalbuminuria. In conclusion, increased urinary CCN2 is associated with the early progression of DN, whereas MCP-1 is associated with later stage disease.

CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.

Fibrosis is a major cause of end-stage renal disease, and although initiation factors have been elucidated, uncertainty concerning the downstream pathways has hampered the development of anti-fibrotic therapies. CCN2 (CTGF) functions downstream of transforming growth factor (TGF)-beta, driving increased extracellular matrix (ECM) accumulation and fibrosis. We examined the possibility that CCN3 (NOV), another CCN family member with reported biological activities that differ from CCN2, might act as an endogenous negative regulator of ECM and fibrosis. We show that cultured rat mesangial cells express CCN3 mRNA and protein, and that TGF-beta treatment reduced CCN3 expression levels while increasing CCN2 and collagen type I activities. Conversely, either the addition of CCN3 or CCN3 overexpression produced a marked down-regulation of CCN2 followed by virtual blockade of both collagen type I transcription and its accumulation. This finding occurred in both growth-arrested and CCN3-transfected cells under normal growth conditions after TGF-beta treatment. These effects were not attributable to altered cellular proliferation as determined by cell cycle analysis, nor were they attributable to interference of Smad signaling as shown by analysis of phosphorylated Smad3 levels. In conclusion, both CCN2 and CCN3 appear to act in a yin/yang manner to regulate ECM metabolism. CCN3, acting downstream of TGF-beta to block CCN2 and the up-regulation of ECM, may therefore serve to naturally limit fibrosis in vivo and provide opportunities for novel, endogenous-based therapeutic treatments.

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1.

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.

TNF-alpha, but not IFN-gamma, regulates CCN2 (CTGF), collagen type I, and proliferation in mesangial cells: possible roles in the progression of renal fibrosis.

Connective tissue growth factor (CCN2) is a profibrotic factor acting downstream and independently of TGF-beta to mediate renal fibrosis. Although inflammation is often involved in the initiation and/or progression of fibrosis, the role of inflammatory cytokines in regulation of glomerular CCN2 expression, cellular proliferation, and extracellular matrix accumulation is unknown. We studied two such cytokines, TNF-alpha and IFN-gamma, for their effects on cultured mesangial cells in the presence or absence of TGF-beta, as a model for progressive renal fibrosis. Short-term treatment with TNF-alpha, like TGF-beta, significantly increased secreted CCN2 per cell, but unlike TGF-beta inhibited cellular replication. TNF-alpha combined with TGF-beta further increased CCN2 secretion and mRNA levels and reduced proliferation. Surprisingly, however, TNF-alpha treatment decreased baseline collagen type I protein and mRNA levels and largely blocked their stimulation by TGF-beta. Long-term treatment with TGF-beta or TNF-alpha alone no longer increased CCN2 protein levels. However, the combination synergistically increased CCN2. IFN-gamma had no effect on either CCN2 or collagen activity and produced a mild inhibition of TGF-beta-induced collagen only at a high concentration (500 U/ml). In summary, we report a strong positive regulatory role for TNF-alpha, but not IFN-gamma, in CCN2 production and secretion, including that driven by TGF-beta. The stimulation of CCN2 release by TNF-alpha, unlike TGF-beta, is independent of cellular proliferation and not linked to increased collagen type I accumulation. This suggests that the paradigm of TGF-beta-driven CCN2 with subsequent collagen production may be overridden by an as yet undefined inhibitory mechanism acting either directly or indirectly on matrix metabolism.

Urinary CCN2 (CTGF) as a possible predictor of diabetic nephropathy: preliminary report.

BACKGROUND: It is currently impossible to reliably predict which diabetic patients will develop nephropathy and progress to kidney failure. Microalbuminuria, often regarded as a predictor of overt diabetic renal disease is, in fact, an indicator of established glomerular damage. We have shown that glomerular expression of the prosclerotic cytokine CCN2 (CTGF) is greatly up-regulated early in experimental and in human diabetes and mesangial cell exposure to CCN2 increases its production of extracellular matrix (ECM) molecules responsible for glomerulosclerosis. As an early marker, we therefore investigated the presence of CCN2 in urine and the relationship to diabetes and/or renal disease in an experimental model of diabetes and in a limited patient population. METHODS: Urine samples from (1) healthy rats, (2) rats made diabetic by streptozotocin (STZ), (3) healthy human volunteers, (4) diabetic patients with renal disease, and (5) diabetic patients without renal disease were examined by Western blotting and/or enzyme-linked immunosorbent assay (ELISA) for qualitative and quantitative analysis of the of CCN2. RESULTS: Low levels of urinary CCN2 were present in healthy, control rats, but were increased approximately sevenfold overall in STZ-diabetic animals. CCN2 levels were the highest at week 3 of diabetes, then decreased with time, but remained significantly elevated over controls even after 32 weeks. Consistently low levels of urinary CCN2 were also detected in healthy volunteers (mean value, 7.1 CCN2/mg creatinine). However, levels were elevated approximately sixfold in the majority of diabetic patients with nephropathy. A small number of the diabetic patients not yet exhibiting evidence of renal involvement demonstrated CCN2 urinary levels that were ninefold greater than controls. The remaining normoalbuminuric diabetic patients demonstrated CCN2 levels indistinguishable from those of healthy volunteers. Analysis by Western blotting confirmed the identity of the urinary CCN2. A molecular species equivalent to full-length CCN2 (37/39 kD doublet) was present in healthy controls. In contrast, the nephropathic group demonstrated multiple CCN2 bands. CONCLUSION: These findings support our hypothesis that CCN2 is up-regulated early in the evolution of glomerulosclerosis, including that of diabetes. We contend that urinary CCN2 may both stage nephropathy and predict those patients who are destined for progressive glomerulosclerosis and end-stage renal disease (ESRD). Cross-sectional and prospective studies of larger, well-defined diabetic patients groups will be required to prove this hypothesis, and are ongoing.

Can glomerular mRNAs in human type 1 diabetes be used to predict transition from normoalbuminuria to microalbuminuria?

BACKGROUND: mRNAs of pathogenetic importance in the development of diabetic nephropathy were measured in subjects with type 1 diabetes to determine whether these might be used to predict progression from normoalbuminuria to microalbuminuria. We proposed that conversion from normoalbuminuria to microalbuminuria would be most likely in subjects whose connective tissue growth factor (CTGF) and collagen mRNAs were above the 95% confidence interval (CI) for live renal donors and within the 95% CI for subjects with abnormal albuminuria. METHODS: Glomerular CTGF, collagen alpha2(IV), and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured in microdissected glomeruli from living renal donors (n = 10), and subjects with normoalbuminuria (n = 12), microalbuminuria (n = 5), and overt proteinuria (n = 6). RESULTS: After 44 +/- 2 months of follow-up, one subject converted from normoalbuminuria to microalbuminuria. Although the data are limited, progression from normoalbuminuria to microalbuminuria occurred in the only normoalbuminuric subject whose mRNA levels were above the live renal donors' 95% CI for CTGF and collagen alpha2(IV) and within the 95% CI of subjects with abnormal albuminuria. No clinical or histopathologic finding distinguished the progressor from the nonprogressors at the time of biopsy. CONCLUSION: This case report provides proof-of-principle that a panel of glomerular mRNA markers chosen because of their pathogenetic relevance may be useful adjuncts to albuminuria and histology in predicting clinical stability or clinical progression in diabetic nephropathy.