Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling

Abstract

Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle alpha-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin.

Figure 1

Mean pulmonary artery pressure (mPAP) among the six groups of rats. n = 10 rats for all the groups. Results are mean ± SD. *P < 0.01 for normoxia vs. all the other groups but normoxia + sildenafil. **P < 0.01 for within hypoxia 4 weeks group (hypoxia vs. hypoxia + sildenafil) and for within hypoxia 8 weeks group (hypoxia vs. hypoxia + sildenafil), respectively.

Figure 2

Representative photomicrographs of rat lung after 4 and 8 weeks of normoxia, hyoxia, hyoxia + sildenafil treatment. SMA immunohistochemistry-stained sections (a, d and g) show a change in hypoxia-induced wall thickening and (j, k and l) in sildenafil treated ones; in situ hybridization analysis of myocardin mRNA expression (b, e and h) when exposed to hypoxia; myocardin mRNA expression is shown by sildenafil treatment (c, f and i). Bars represent a length of 50 μm.

Figure 3

In situ hybridization analysis of myocardin mRNA expression (OD value) among the six groups of rats as in Figure 1. *P < 0.01 for normoxia vs. all the other groups but normoxia + sildenafil. **P < 0.01 for within hypoxia 4 weeks group (hypoxia vs. hypoxia + sildenafil) and for within hypoxia 8 weeks group (hypoxia vs. hypoxia + sildenafil), respectively.

Figure 4

Immunohistochemistry (ICH) analysis of SMA protein expression (OD value) among the six groups of rats as in Figure 1. *P < 0.01 for normoxia vs. all the other groups but normoxia + sildenafil. **P < 0.01 for within hypoxia 4 weeks group (hypoxia vs. hypoxia + sildenafil) and for within hypoxia 8 weeks group (hypoxia vs. hypoxia + sildenafil), respectively.

Figure 5

Reverse transcription polymerase chain reaction (RT-PCR) analysis of myocardin among the six groups of rats. (a) RT-PCR analysis of myocardin and β-actin gene expression in representative rat lungs from six groups. (b) Quantitation of myocardin mRNA levels, results are means ± SD; n = 10 rats in each group. *P < 0.01 for normoxia vs. all the other groups but normoxia + sildenafil. **P < 0.01 for within hypoxia 4 weeks group (hypoxia vs. hypoxia + sildenafil) and for within hypoxia 8 weeks group (hypoxia vs. hypoxia + sildenafil) respectively.

Figure 6

RT-PCR analysis of SMA among the six groups of rats. (a) RT-PCR analysis of SMA and β-actin gene expression in representative rat lungs from six groups. (b) Quantitation of SMA mRNA levels, results are mean ± SD; n = 10 rats in each group. *P < 0.01 for normoxia vs. all the other groups but normoxia + sildenafil. **P < 0.01 for within hypoxia 4 weeks group (hypoxia vs. hypoxia + sildenafil) and for within hypoxia 8 weeks group (hypoxia vs. hypoxia + sildenafil), respectively.

Figure 7

The isolated endothelial cells exhibited typical ‘cobblestone’ morphology after purified culture.

Figure 8

Double-label immunofluorescence staining in isolated endothelial cells (in the same field) Hoechst fluorescence showing cell nuclei (left); VIII relative antigen expression is detected in cytoplasm (right).

Figure 9

Double-label immunofluorescence staining in cultured endothelial cells of hypoxic group (in the same field) Hoechst fluorescence showing cell nuclei (left), SMA expression is negative in the cytoplasm by day 1 (top, right); SMA is expressed in a cytoplasmic pattern by days 7, and the SMA-positive cells alter their morphological appearance toward an elongated, spindle-shaped form (bottom, right).

Figure 11

The results of RT-PCR in cultured endothelial cells of all groups there was no expression of myocardin mRNA in N group, H group by day 1, and H + pSi group, but myocardin mRNA expression was detected in H group by days 7, and the level of myocardin mRNA is much weaker in H + pSi group.

Figure 10

Double-label immunofluorescence staining in cultured endothelial cells of hypoxia + pSi group (in the same field) Hoechst fluorescence showing cell nuclei (left), SMA is not expressed in cytoplasm by day1 (top, right); SMA expression is detected in a cytoplasmic pattern by days 7 (bottom, right), but weaker than that in H group by days 7, and the SMA-positive cells are fewer than those in H group by days 7.