TMEM16A regulates the cell cycle of pulmonary artery smooth muscle cells in high‑flow‑induced pulmonary arterial hypertension rat model

Figures

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  Figure 1. High pulmonary blood flow-induced pulmonary hypertension leads to elevated right ventricular pressure and RVHI. (A) SRVP and MRVP, and (B) RVHI was measured in rats from the control, sham and shunt groups. Data are presented as the mean ± standard deviation (n=10). *P<0.05 vs. control and #P<0.05 vs. sham. RVHI, right ventricular hypertrophy index; SRVP, systolic right ventricular pressure; MRVP, mean right ventricular pressure.

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  Figure 2. Pathological changes of pulmonary arterioles by H&E staining. Images of H&E staining of pulmonary arteriole tissues of rats from (A) control, (B) sham, and (C) shunt groups (magnification, x100).

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  Figure 3. Characterization of primary PASMCs isolated from pulmonary arteries of rats. (A) PASMCs isolated from the pulmonary artery mass and aggregated into clusters (5 days after start of culture). (B) PASMCs exhibiting long fusiform shapes and ‘peak-valley’ appearance (10 days after start of culture). (C) Immunocytochemistry staining confirmed α-smooth muscle actin expression based on the specific myofilament structure of PASMCs (15 days after start of culture). (D) Specific TMEM16A-siRNA lentiviral vectors, which also encode GFP, were transfected into PASMCs and observed under a light microscope. PASMCs, pulmonary artery smooth muscle cells; TMEM16A, transmembrane member 16A; siRNA, small interfering RNA.

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  Figure 4. TMEM16A protein expression in primary PASMCs isolated from rats of the control, sham and shunt groups, in addition to PASMCs from the shunt group transfected with TMEM16A-siRNA. (A) Western blotting of TMEM16A and β-actin in primary PASMC samples from rats of the various experimental groups. (B) Quantified densitometry results of (A). Data are presented as mean ± standard deviation (n=10). *P<0.05 vs. control; #P<0.05 vs. sham; $P<0.05 vs. shunt; and &P<0.05 vs. shunt + siRNA-TMEM16A. TMEM16A, transmembrane member 16A; PASMCs, pulmonary artery smooth muscle cells; siRNA, small interfering RNA.

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  Figure 5. PASMC cell cycle progression as regulated by TMEM16A in high blood flow induced by PAH. (A) Representative FACS dot plots presenting cell cycle distribution of primary rat PASMCs from the four indicated experimental groups. (B) Quantified results of (A) presenting the proportion of primary rat PASMCs in G0/G1, S and G2/M phases from the four indicated experimental groups. Data are expressed as the mean ± standard deviation (n=10). *P<0.05 vs. control; #P<0.05 vs. shunt; $P<0.05 vs. shunt + siRNA-TMEM16A. PASMCs, pulmonary artery smooth muscle cells; TMEM16A, transmembrane member 16A; siRNA, small interfering RNA.

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  Figure 6. TMEM16A contributed to the regulation of PASMC cycle proteins. (A) Representative western blots presenting the expression of Cyclin D1, Cyclin E, CDK1 and p27KIP in the four indicated experimental groups. (B) Quantified densitometry results of (A). Data are presented as the mean ± standard deviation (n=10). *P<0.05 vs. control; #P<0.05 vs. shunt; and $P<0.05 vs. shunt + siRNA-TMEM16A. TMEM16A, transmembrane member 16A; PASMCs, pulmonary artery smooth muscle cells; siRNA, small interfering RNA; CDK1, cyclin-dependent kinase 1; p27KIP, cyclin-dependent kinase inhibitor P27.