Activation of ALDH2 alleviates hypoxic pulmonary hypertension in mice by upregulating the SIRT1/PGC-1α signaling pathway

doi:10.12122/j.issn.1673-4254.2024.10.14

Abstract

Abstract:

Objective To investigate whether activation of mitochondrial acetal dehydrogenase 2 (ALDH2) alleviates hypoxic pulmonary hypertension by regulating the SIRT1/PGC-1α signaling pathway. Methods Thirty 8-week-old C57 BL/6 mice were randomized into control, hypoxia, and hypoxia +Alda-1 (an ALDH2 activator) group (n=10), and the mice in the latter two groups, along with 10 ALDH2 knockout (ALDH2^-/-) mice, were exposed to hypoxia (10% O₂, 90% N₂) with or without daily intraperitoneal injection of Alda-1 for 4 weeks. The changes in right ventricular function and pressure (RVSP) of the mice were evaluated by echocardiography and right ventricular catheter test, and pulmonary artery pressure was estimated based on RVSP. Pulmonary vascular remodeling, right ventricular injury, myocardial α-SMA expression, distal pulmonary arteriole muscle normalization, right ventricular cross-sectional area, myocardial cell hypertrophy, and right cardiac hypertrophy index were assessed with HE staining, immunofluorescence staining and WGA staining, and the expressions of ALDH2, SIRT1, PGC-1α, P16INK4A and P21^CIP1 were detected. In pulmonary artery smooth muscle cells with hypoxic exposure, the effect of Alda-1 and EX527 on cell senescence and protein expressions was evaluated using β-galactose staining and Western blotting. Results The wild-type mice with hypoxic exposure showed significantly increased RVSP, right ventricular free wall thickness and myocardial expressions of P16^INK4A and P21^CIP1, which were effectively lowered by treatment with Alda-1 but further increased in ALDH2^-/- mice. In cultured pulmonary artery smooth muscle cells, hypoxic exposure significantly increased senescent cell percentage and cellular expressions of P16^INK4A and P21^CIP1, which were all lowered by treatment with Alda-1, but its effect was obviously attenuated by EX527 treatment. Conclusion ALDH2 alleviates hypoxia-induced senescence of pulmonary artery smooth muscle cells by upregulating the SIRT1/PGC-1α signaling pathway to alleviate pulmonary hypertension in mice.

Key words: ALDH2, SIRT1, PGC-1α, smooth muscle cell senescence, pulmonary hypertension

Lei WANG, Fenlan BIAN, Feiyang MA, Shu FANG, Zihan LING, Mengran LIU, Hongyan SUN, Chengwen FU, Shiyao NI, Xiaoyang ZHAO, Xinru FENG, Zhengyu SUN, Guoqing LU, Pinfang KANG, Shili WU. Activation of ALDH2 alleviates hypoxic pulmonary hypertension in mice by upregulating the SIRT1/PGC-1α signaling pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1955-1964.

Figures/Tables 6

Fig.1 Comparison of right ventricular pressure (RVSP) and cardiac function of the mice among the groups. A: RVSP of the mice in each group. B: Heart color ultrasound in each group. C: Statistical diagram of RVSP in each group. D: Statistical diagram of maximum rise rate of RVSP in each group. E: Statistical map of right ventricular free wall thickness in each group. F: Statistical chart of pulmonary artery acceleration to ejection time ratio in each group. Data are presented as Mean±SD (n=6). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs hypoxia group.

Fig.2 Pulmonary artery remodeling in each group. A: HE staining of pulmonary arteries in each group (scale bar=10 μm). B: Pulmonary artery smooth muscle to total vascular area ratio in each group. C: Percentage of pulmonary artery wall thickness to vessel diameter in each group. D: Immunofluorescence staining of pulmonary artery smooth muscle in each group (scale bar=6 μm). E: Relative fluorescence intensity of α-SMA in each group. Data are presented as Mean±SD (n=6). **P<0.01 vs control group; #P<0.05, ##P<0.01 vs hypoxia group.

Fig.3 Right ventricular remodeling in each group. A: HE staining of the right ventricle in each group. B: Right ventricular cardiac hypertrophy index of each group. C: WGA staining of the right ventricle in each group. D: Cross-sectional area of mouse cardiomyocytes in each group. Data are presented as Mean±SD (n=6). **P<0.01 vs control group; #P<0.05, ##P<0.01 vs hypoxia group.

Fig.4 Wetsern blotting for detecting protein expressions of ALDH2, SIRT1, PGC-1α, P16INK4A and P21CIP1 in the lung tissues in each group. A, B: Western blots of ALDH2 protein and its relative expression levels. C-E: Western blots of SIRT1 and PGC-1α proteins and their relative expression levels. F-H: Western blots of P16INK4A and P21CIP1 and their relative protein expression levels. Data are presented as Mean±SD (n=6). **P<0.01 vs control group; #P<0.05, ##P<0.01 vs hypoxia group.

Fig.5 Protective effect of ALDH2 against pulmonary artery smooth muscle cell senescence mediated by the SIRT1 pathway. A: Immunofluorescence staining for α-SMA for identification of mouse pulmonary artery smooth muscle cells. B: β-galactose staining of pulmonary artery smooth muscle cells in each group. C: Percentage of senescent cells in each group. Data are presented as Mean±SD (n=3). **P<0.01 vs control group; ##P<0.01 vs hypoxia group; &&P<0.01 vs Hypoxia+Alda-1 group.

Fig.6 Protein expressions of ALDH2, SIRT1, PGC-1α, P16INK4A and P21CIP1 in mouse pulmonary artery smooth muscle cells in each group. A, B: Western blots of ALDH2 protien and its relative expression levels. C-E: Western blots of SIRT1 and PGC-1α proteins and their relative expression levels. F-H: Western blots of P16INK4A and P21CIP1 proteins and their relative expression levels. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01 vs control group; #P<0.05, ##P<0.01 vs Hypoxia group; &&P<0.01 vs Hypoxia+Alda-1 group.

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