The Role Of Myocardin In Hypoxia-induced Transdifferentiation Of Pulmonary Artery Endothelial Cells Into Smooth Muscle-like Cells

Objectives1. To investigate the transdifferentiation of pulmonary artery endothelial cells (PAECs) into smooth muscle-like cells under hypoxia.2. The role of myocardin in hypoxia-induced transdifferentiation of PAECs into smooth muscle-like cells.3. The effect of sildenafil in the hypoxia-induced transdifferentiation of PAECs into smooth muscle-like cells and the possible mechanism of Sildenafil in this progress.MethodsPAECs from adult pig pulmonary small arteries were purified by immunomagnetic purification techniques (based on PECAM-1 positive expression for sorting marker); RNA interference (RNAi) was used to silence the expression of myocardin gene by the constructed expression vector psimyocardinRNA. Purified PAECs were divided into the following groups:Normoxia group (N): in the cell culture box containing 21%O2, 5%CO2, 74%N2;Hypoxia group (H): in the cell culture box containing 1%O2, 5%CO2, 94%N2;Normoxia + psimyocardinRNA group (N+pSi);Hypoxia + psimyocardinRNA group (H+pSi);(Four groups were cultured for 1 d, 4 d and 7 d respectively)Normoxia + Sildenafil group (N+S);Hypoxia + Sildenafil group (H+S). (The two groups were cultured for 1 d, and 7 d respectively)RT-PCR was used to detect the levels of myocardin mRNA expression; the transdifferentiation of smooth muscle-like cells was identified by detecting the expression ofα-SM-actin (SM specific markers) in immunofluorescence and morphology.Results1. Theα-SM-actin positive ratio in hypoxia 4d group(0.96‰±0.08)was significantly lower than that in hypoxia 7d group(2.07‰±0.06)(P<0.05), theα-SM-actin positive cells appeared the fusiform shape or polygon, the ratio of transdifferentiated smooth muscle-like cells gradually increased and was time-dependent.2. The levels of myocardin mRNA in hypoxia 4d group (0.14±0.01) was markedly lower than that in hypoxia 7d group (0.23±0.03) (P<0.05). Myocardin mRNA expression were not detected both in normoxia groups and hypoxia 1d group.3. The levels of myocardin mRNA expression in hypoxia 4d group (0.03±0.02 ) and hypoxia 7d group (0.05±0.01 ) were significantly decreased after the expression of myocardin gene silenced by RNAi (P<0.05), and the ratios of transdifferentiated smooth muscle-like cells were markedly decreased (0.19‰±0.07; 0.21‰±0.04, P<0.05).4. The levels of myocardin mRNA expression (0.09±0.02; P<0.05) and the ratios of transdifferentiated smooth muscle-like cells (1.02‰±0.05; P<0.05) in hypoxia 7 d group were markedly decreased by sildenafil treatment.ConclusionsTherefore, we think that PAECs contain those cells which have the potential to transdifferentiate into smooth muscle-like cells in vitro; hypoxia could promote the transdifferentiation and myocardin probably plays an important role in this process. In addition, sildenafil could attenuate the transdifferentiation,which may be throught the mechanism of inhibiting expression of myocardin gene...