Objective To examine the effects of hypoxia on endothelial-to-mesenchymal transition of porcine pulmonary arterial endothelial cells ( PAECs) .Methods The porcine PAECs were divided into a normoxia group and a hypoxia group. The cells in two groups were exposed to normoxic or hypoxic condition for 1,4, and 7 days respectively. The immunofluorescence,Western blot and RT-PCR were used to detect the protein and mRNA expressions of VE-cadherin and α-SMA. Results The porcine primary PAECs formed typical monolayer of cobblestone appearance on normoxia condition, and had a spindle-shaped appearance on hypoxia condition. Immunofluorescence results showed that these PAECs expressed mesenchymal cells specific marker of α-SMA. With the hypoxic time prolongation, the ratio of transdifferentiated smooth musclelike cells from PAECs was gradually increased ( P lt; 0. 01) . Western blot assay demonstrated that the expression level of VE-cadherin protein and mRNA was reduced gradually, but the expression level of α-SMA protein and mRNA was increased. Conclusion Hypoxia can induce endothelial-to-mesenchymal transition, which may be involved in the development of a variety of diseases.
Objective To investigate the molecular signal mechanism of transform growth factor (TGF)-β induced arterial endothelial-mesenchymal transition. Methods Rat arterial endothelial cells were primarily cultured by ex-transplant method. The endothelial cells were incubated by combinant TGF-β (10 ng/mL) for 48 hours and then were detected by immunofluorescence staining and western blotting to observe the cell surface marker expression profile and Akt/mTOR signal activation. On the other hand, the endothelial cells were preincubated by Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the Akt/mTOR signal, and then the cells were further treated with TGF-β (10 ng/mL) for 48 hours to observe the cell surface marker expression profile without Akt/mTOR signal activation. Results Rat artery endothelial cells by TGF-β after incubation, the expressions of smooth muscle cell markers α-smooth muscle actin (α-SMA) and smooth muscle-22α (SM-22α) were up-regulated, and the endothelial cell markers CD31 and vW factor were significantly down-regulated, at the same time, the expressions of phosphorylated Akt and mTOR were also up-regulated. However, after preincubation of Ly294002 (20 μmol/L) and rapamycin (10 nmol/L) to inhibit the phosphorylation of Akt and mTOR signal, above TGF-β-induced expressions of α-SMA and SM-22α in arterial endothelial cells were significantly suppressed and the expressions of CD31 and vWF were preserved. Conclusion TGF-β-induced arterial endothelial-mesenchymal transition is dependent on activation of Akt/mTOR signal, suggesting that Akt/mTOR-dependent arterial endothelial-mesenchymal transition would be one of the mechanisms for intima hyperplasia in transplant arteriosclerosis.