The LSKL peptide also showed reduced the basal contractile force generated by normal fibroblasts by approximately 14%. These results suggested the intriguing notion that activation of endogenous latent TGFb played a key role in ECM con traction by both healthy and fibrotic fibroblasts. Blocking TSP1 activation of find protocol TGFb with LSKL peptide impacted on the mitogen activated protein kinase signalling pathways and reduced matrix protein expressions in SSc fibroblasts Lesional dermal SSc fibroblasts are characterised by the markedly enhanced ability to adhere to and contract extracellular matrix. To further investigate the mechanism underlying TSP1 dependent contractile activity, fibroblasts in fully contracted FPCL gel samples were analysed by western blotting to evaluate whether in this context TSP1 blocking peptide reduced expression of matrix proteins and the activation of procontractile sig nalling pathways.

Western blot analysis revealed that the TSP1 blocking peptide reduced expression of profibrotic proteins such as a SMA, integrin a3, integrin b5, and the activation of p ERK and p p38 kinase in SSc fibro blasts. Moreover, TGFb induced matrix gene expression and ERK and p38 phosphorylation in both normal and SSc fibroblasts were also reduced. TGFb causes fibroblasts to differentiate into myofibro blasts, the a SMA containing cells that are involve in the contraction processes in wound contraction and fibrosis tissue in vivo. ERK activation contributes to the enhanced contraction by lesional dermal scleroderma fibroblasts by promoting the assembly of a SMA stress fibres.

To extend our data obtained by western blot analyses indicating that LSKL peptide reduced ERK acti vation and a SMA expression in SSc fibroblasts, we employed indirect immunofluorescence analysis to show that a 24 h treatment of SSc fibroblasts with LSKL pep tide reduced the appearance of a SMA stress fibres and the intense p ERK staining, both key features characteris ing SSc fibroblasts, Moreover, the LSKL peptide also blocked TGFb induced a SMA expression and p ERK activity in normal and SSc fibroblasts. TSP1 is a key mediator promoting SSc fibroblast contraction Based on the above findings, it needed to be elucidated whether TSP1 could directly mediate the enhanced con tractile activities of SSc fibroblasts. To perform this ana lysis, we reduced TSP1 protein expression in normal and SSc fibroblasts using siRNA recognising TSP1.

Wes tern blot Cilengitide analysis was used to assess the ability of siRNA recognising TSP1, compared to control siRNA, to reduce TSP1 protein expression levels. The contractile ability of TSP1 knockdown cells was analysed using the CFM system. We found that the contractile ability of SSc fibroblast was reduced by 16% at the 24th hourly time point after TSP1 expression knockdown. in addition, TGFb induced contractility of both overnight delivery normal and SSc fibroblasts were diminished by 18% and 29%, respectively, at the 24 h time point.