目的α-平滑肌-肌动蛋白(a-SM-actin)的表达是血管外膜成纤维细胞/肌成纤维细胞表型转化的分子标记。本研究观察阻断RhoA-ROKα信号转导通路对于转化生长因子β1(transfor- ming growth factorβ1,TGF-β1)诱导的血管外膜成纤维细胞α-SM-肌动蛋白(actin)表达的影响,以揭示RhoA-ROKα信号通路在血管外膜成纤维细胞表型转化为肌成纤维细胞过程中的作用。方法使用贴壁法体外培养大鼠胸主动脉外膜成纤维细胞；用Western blot技术测定RhoA和ROKα在血管外膜成纤维细胞表型转化为肌成纤维细胞过程中的表达。结果RhoA-ROKα在血管外膜成纤维细胞表达,TGF-β1可以诱导RhoA表达上调。用反义寡核苷酸技术抑制RhoA或ROKα的表达后能够抑制α-SM-actin的表达。结论RhoA-ROKα信号转导通路参与了TGF-β1诱导的血管外膜成纤维细胞表型转化为肌成纤维细胞的过程。
Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψ_m) reduction, indicative of mitochondrial permeability transition pore (PTP) opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψ_m reduction and apoptosis, suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψ_m reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B1 was downregulated only in cardiac fibroblasts, which may be associated with G_2/M accumulation in response to mechanical stretch.
