Abstract
IntroductionSPARC 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.MethodsIn 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's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays.ResultsSPARC 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-β 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.ConclusionsSpecific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.
Highlights
secreted protein (SPARC) is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases
Gene and protein expression of Col1a2, connective growth factor (Ctgf) and SPARC in the fibroblasts from TBR1CA; Cre-ER mice with and without transfection of small interfering RNA (siRNA) of SPARC or Ctgf As measured by quantitative real-time RT-PCR, the transcripts of Col1a2, Ctgf and SPARC showed increased expression in the fibroblasts from TBR1CA; Cre-ER mice injected with 4-OHT, in which Tgfbr1 was constitutively active, compared with those in the cells from TBR1CA; Cre-ER mice injected with oil (Figure 1)
To study whether inhibition of SPARC induced a reduction of collagen in the fibroblasts from constitutively active Tgfbr1 mice, we transfected SPARC siRNA into cultured fibroblasts obtained from TBR1CA; Cre-ER mice injected with 4-OHT
Summary
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. Fibrosis is a general pathological process in which excessive deposition of extracellular matrix (ECM) occurs in the tissues. SPARC (secreted protein, acidic and rich in cysteine), a matricellular component of the ECM, has been reported as a bio-marker for fibrosis in multiple fibrotic diseases, such as interstitial pulmonary fibrosis, renal interstitial fibrosis, cirrhosis, atheroscle-. In animal studies, SPARC-null mice display a diminished amount of pulmonary fibrosis compared with control mice after
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