Our previous data had found that dermal fibroblasts from SSc lesions are char acterised by enhanced contractile ability of SSc fibro blasts and expression of a cohort sellckchem of overexpress profibrotic genes, including a SMA and integrins. TGFb1 is a key factor in mediating both in fibroblasts participation in wound repair and in a promoting patho logical fibrosis, including SSc. Treatment of fibroblasts with TGFb results in their differentiation into myofibro blasts and also stimulates their production of extracellu lar matrix, and adhesive proteins such as integrins. In monolayer culture, TGFb is partially responsi ble for the phenotype of lesional SSc fibroblasts. However, it remains unclear whether activation of TGFb signalling plays a role in ECM contraction in three dimensional models of contraction.

The data presented in this investigation shown that TSP1 is tightly linked with the enhanced contractility of SSc fibroblasts in the context of a three dimensional culture system, as knock down of the TSP1 gene or a blocking anti TSP1 peptide, which prevents activation of latent TGFb, reduced the cell contractility of fibrotic SSc fibroblasts. In parallel, antagonising TSP1 impaired expression of a SMA, integrin a3, and integrin b5. Blocking TSP1 expression and activity also reduced the basal contractility of nor mal fibroblasts. We have found that endogenous TGFb signalling contributes to the basal contractility of normal and SSc fibroblasts in three dimensional FPCL.

The results from our current report indicate that increased activation of latent TGFb by TSP1 contributes to the overall activity of exogenous TGFb during the process of ECM contraction in a three dimensional culture. After mechanical loading of fibroblasts within the FPCL system, TGFb activity and TSP1 expression were increased. All these results indicate that TSP1 contri butes to the contractile ability of fibroblasts by promot ing myofibroblast differentiation by TGFb. Our data are also consistent with the notion that TSP1 is a key med iator Dacomitinib contributing to the enhanced contractile ability dis played by lesional SSc dermal fibroblasts. In summary, blocking TSP1 may be a viable antifibrotic strategy. The ability of TGFb1 to induce TSP1 in fibroblasts is ERK dependent. TSP1 can also induce ERK phos phorylation via b1 integrin.

Prior data from our laboratory have shown heparan sulfate dependent ERK activation contributes to the enhanced contractile ability demonstrated by lesional dermal scleroderma fibroblasts. Erlotinib Consistent with these results, in the current study we have shown that anti TSP1 strategies not only reduced fibroblast contractility but also decreased ERK activation in fibroblasts subjected to ECM contraction and mechan ical loading. We have also shown that TGFb and PDGF induced contractility in normal and SSc fibroblasts corresponded with elevated expression of TSP1 and ERK activation.