Long-term treatment with TGFβ1 impairs mechanotransduction in bovine aortic endothelial

Background and purpose: Vascular endothelial cells play a role in the physiological response to mechanical stress. Transforming growth factor beta(1) (TGF beta(1)) induces morphological changes in endothelial cells, and this may alter their mechanosensitive responses. The aim of this study was to examine the effects of TGF beta(1) on hypotonic stress (HTS)-induced responses in bovine aortic endothelial cells (BAECs). Experimental approach: Cultured BAECs were treated with 3 ng ml(-1) TGF beta(1) for 24 h (24h-TGF beta(1)) or 7 days (7d-TGF beta(1)). Cytosolic actin fibres were stained with rhodamine-phalloidin. Intracellular Ca2+ concentration was measured using fura2. Tyrosine phosphorylation and RhoA expression were assessed by Western blotting. Expression of RhoA mRNA was assessed by real-time PCR. Key results: BAECs developed pseudopod-like processes within 24 h and showed a fibroblast-like appearance after 7 days. HTS induced Ca2+ transients via endogenous ATP release in both control and 24h-TGF beta(1) BAECs but not in 7d-TGF beta(1) BAECs. We have previously shown that HTS-induced ATP release is mediated by sequential activation of RhoA and tyrosine kinases. The basal amount of membrane-bound RhoA was significantly lower in 7d-TGF beta(1) than in 24h-TGF beta(1) or control BAECs. HTS increased the membrane-bound RhoA to the same fractional level in 24h-TGF beta(1) and control BAECs, but its net maximal amount was significantly lower in 7d-TGF beta(1). HTS-induced downstream signals of RhoA activation, i.e. the tyrosine phosphorylation of FAK and paxillin, were markedly suppressed in 7d-TGF beta(1) BAECs. Conclusions and Implications: These results indicate that long-term treatment with TGF beta(1) does not impair mechanoreception in BAECs but impairs mechanotransduction by affecting RhoA membrane translocation.