The role of myocardin in hypoxia-induced transdifferentiation of pulmonary artery endothelial cells into smooth muscle-like cells

Abstract

To investigate the transdifferentiation of pulmonary artery endothelial cells (PAECs) into smooth muscle-like cells under hypoxia and the role of myocardin therein. Recombinant plasmid psimyocardin (pSi), capable of silencing the expression of myocardin gene, was constructed by RNAi to be used to transfect the PAECs. Endothelial cells of adult pig pulmonary small arteries were purified by immunomagnetic purification technique, and divided into 4 groups: normoxia group (to be cultured in normoxic cell culture box containing 21% O(2), 5% CO2, and 74% N(2)), normoxia + pSi group, hypoxia group (to be cultured in cell box containing 1% O(2), 5% CO2, and 74% N(2)), and hypoxia + pSi group to be cultured for 1, 4, and 7 days respectively. Indirect fluorescence technique and morphological examination were used to identify the smooth muscle (SM) -like cells and the alpha-SM-actin positive cell ratio. RT-PCR was used to detect the mRNA expression of myocardin. Alpha-SM-actin positive cell could not be seen in the normoxia group and hypoxia 1 d group. The alpha-SM-actin positive cell rate of the hypoxia 7 days group was 2.07% +/- 0.06%, significantly higher than that of the hypoxia 4 d group (0.96% +/- 0.08%, P < 0.01). mRNA expression of myocardin gene could not be seen in the normoxia, normoxia + pSi, hypoxia 1 days, and hypoxia + pSi 1 days groups. The mRNA expression of myocardin gene of the hypoxia 7 days group was 0.23 +/- 0.03, significantly higher than that of the hypoxia 4 days group (0.14 +/- 0.01, P < 0.01). The mRNA expression levels of myocardin gene of the hypoxia + pSi 4 days and 7 days groups 0.03 +/- 0.02 and 0.05 +/- 0.01, both significantly lower than those of the hypoxia 4 days and 7 days groups respectively (0.14 +/- 0.01 and 0.23 +/- 0.03, both P < 0.01). The SM-like cell transdifferentiation rates of the hypoxia + pSi 4 days and 7 days groups were 0.19% +/- 0.07% and 0.21% +/- 0.04% respectively, both significantly lower than those of the corresponding hypoxia groups (0.96% +/- 0.08 and 2.07% +/- 0.06% respectively, both P < 0.01). Some PAECs have the potential to transdifferentiate into SM-like cells and may be one of the resources of muscularization of peripheral small vessels. Hypoxia remarkably promotes this transdifferentiation and myocardin may play an important role in this process.