Long-term treatment with TGFβ ₁ impairs mechanotransduction in bovine aortic endothelial cells

Background and purpose: Vascular endothelial cells play a role in the physiological response to mechanical stress. Transforming growth factor β ₁ (TGFβ 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 TGFβ ₁ on hypotonic stress (HTS)-induced responses in bovine aortic endothelial cells (BAECs). Experimental approach: Cultured BAECs were treated with 3 ng ml ^-1 TGFβ ₁ for 24 h (24h-TGFβ ₁) or 7 days (7d-TGFβ ₁). Cytosolic actin fibres were stained with rhodamine-phalloidin. Intracellular Ca ²⁺ 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 Ca ²⁺ transients via endogenous ATP release in both control and 24h-TGFβ ₁ BAECs but not in 7d-TGFβ 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-TGFβ ₁ than in 24h-TGFβ ₁ or control BAECs. HTS increased the membrane-bound RhoA to the same fractional level in 24h-TGFβ ₁ and control BAECs, but its net maximal amount was significantly lower in 7d-TGFβ ₁. HTS-induced downstream signals of RhoA activation, i.e. the tyrosine phosphorylation of FAK and paxillin, were markedly suppressed in 7d-TGFβ ₁ BAECs. Conclusions and Implications: These results indicate that long-term treatment with TGFβ ₁ does not impair mechanoreception in BAECs but impairs mechanotransduction by affecting RhoA membrane translocation.