Attenuation of fibrosis in vitro and in vivo with SPARC siRNA

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson\u27s trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases

β-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis

Objectives: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). β-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of β-catenin in fibroblasts for the development of SSc dermal fibrosis. Methods: Nuclear accumulation of β-catenin in fibroblasts was assessed by triple staining for β-catenin, prolyl-4-hydroxylase-β and 4′,6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of β-catenin in fibrosis. Results: The auhors found significantly increased nuclear levels of β-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of β-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of β-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of β-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of β-catenin significantly reduced bleomycin-induced dermal fibrosis. Conclusions: The present study findings identify β-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of β-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of β-catenin/Wnt signaling have recently entered clinical trials

Rac Inhibition Reverses the Phenotype of Fibrotic Fibroblasts

Background: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.Methods and Findings: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.Conclusion: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc

Interstitial fibrosis is associated with increased COL1A2 transcription in AA-injured renal tubular epithelial cells in vivo

Accumulation of type I collagen is a key event in renal interstitial fibrosis. As there is no effective treatment, understanding the site where collagen is transcribed and the factors driving it in response to disease in vivo is critical for designing future therapies. The present research investigated the transcriptional activity of the COL1A2 gene in a mouse model of progressive fibrosis induced by aristolochic acid (aristolochic acid nephropathy, AAN). To achieve this we genetically modified mice to express a reporter gene (LacZ) and CCN2 (connective tissue growth factor) under the transcriptional control of the COL1A2 promoter /enhancer sequences. Using these mice we asked where is collagen actively transcribed and secondly, what is the role of CCN2 in AAN. Here, we report that de-novo transcription of the COL1A2 gene occurred predominantly in damaged tubular epithelial cells during progressive interstitial fibrosis in vivo. The activation of COL1A2 was studied by detection of the reporter gene LacZ and COL1A2 mRNA in interstitial, glomerular, vascular, and tubular epithelial tissue from laser capture microscopy. We also demonstrated that LacZ-positive cells co-express E-Cadherin a marker of epithelial origin which is consistent with an epithelial phenotype which is capable of collagen expression during injury. There was no evidence of detachment of these cells from tubules to become myofibroblasts. Moreover, we showed that the transgenic mice show a modest enhancement of CCN2 expression; however fibrosis induced by AA is the same in transgenics and controls suggesting that CCN2, at this level of expression, is not sufficient to enhance fibrogenesis. Overall our study provides a better understanding into the expression patterns and roles of two major extracellular matrix proteins: type I collagen and CCN2

Rac inhibition reduces the enhanced migration of lesional SSc fibroblasts.

<p>Fibroblasts were subjected to a scratch wound assay in the presence or absence of 50 µM NSC23766. Cells were cultured on fibronectin until reaching confluence. A uniform linear scrape wound was made across the cell layer. Three independent experiments were performed Gap size expressed as a percentage of the original wound is shown (average +/− standard deviation) (* = p<0.05 statistically different relative to untreated healthy controls).</p

Overexpression of rac in normal fibroblasts results in a profibrotic phenotype phenotype in a PI3kinase/Akt-dependent fashion: collagen gel contraction analysis.

<p>(A) Transfection of constitutively active rac (ca rac), compared to empty expression vector (empty), increases ECM contraction by normal fibroblasts. Eighteen hours post-transfection, cells were subjected to the floating collagen gel model of ECM contraction in the presence or absence of 100 nM wortmannin, 10 µM Ly294002, 10 µM U0126 or 10 µM SP600125. Average +/− standard deviation is shown (N = 3, * = p<0.05). (B) Rac overexpression rescues the reduced ECM contraction in rac knockout fibroblasts (K/K) versus control fibroblasts (C/C). Cells were transfected with empty expression vector or constitutively active rac (ca rac) in the presence or absence of wortmannin or Ly294002. (*, p<0.05, Student's t test).</p

Overexpression of rac in normal fibroblasts results in a profibrotic phenotype in a PI3kinase/Akt-dependent fashion: protein analysis.

<p>(A) Transfection of constitutively active rac (ca rac), compared to empty expression vector (empty), increases profibrotic protein expression. Twenty four hours post-transfection, protein was harvested and subjected to Western blot analysis was conducted with antibodies detecting the proteins indicated. Average +/− standard deviation is shown (N = 3, * = p<0.05). (B) The effect of rac overexpression is reduced by PI3 kinase inhibition. Cells were transfected with empty expression vector or constitutively active rac (ca rac) in the presence or absence of 100 nM wortmannin, 10 µM Ly294002, 10 µM U0126 and 10 µM SP600125. Cells were then processed for Western blot analysis as described in (A).</p

Rac activity is elevated in dermal fibroblasts cultured from scars of SSc patients.

<p>Cells were loaded with or without GTPγS (positive control, to assess maximal Rac activation) or GDP (as a negative control). Rac-GTP was immunoprecipitated from cell lysates. The precipitated Rac-GTP was detected by immunoblot analysis using anti-Rac. In parallel, whole cell lysates were subjected to SDS/PAGE and Western blot analysis with an anti-rac antibody. Quantitative densitometry data is indicated on the right. Lanes are: GTPγS, GDP, cells from three normal individuals (N1, N2, N3) and three individuals with SSc (SScF1, SScF2, SScF3). Experiments were performed on cells derived from 6 normal individuals and 6 SSc patients. Quantitative densitometry data is indicated on the right (* = p<0.05 relative to wild-type control).</p

Rac inhibition suppresses the pro-fibrotic phenotype of lesional SSc fibroblasts. Gel contraction analysis.

<p>Rac inhibitor 50 µM NSC23766 (24 hour treatment) reduced the ability of lesional SSc fibroblasts to contract a collagen gel matrix: (A) FPCL analysis. The effect of loss of rac inhibition on the ability of fibroblasts to exert contractiles force in a fixed, tethered floating collagen gel lattice was investigated using a Culture Force Monitor. Forces generated by fibroblasts were measured over 24 hours; a representative trace is shown (N = 3). (B) Floating gel analysis. The effect of rac inhibition on ECM contraction over 24 hours generated by fibroblasts embedded in a floating collagen gel matrix was evaluated. Contraction was assessed photographically and by measuring gel weight iameter of contracted gels (n = 6; Average +/− standard deviation is indicated; * = p<0.05, statistically different from control untreated normal fibroblasts, ANOVA). Note that NSC23766 suppressed the enhanced ECM contraction by lesional SSc fibroblasts.</p