Aldehyde dehydrogenase 2 protects against oxidative stress associated with pulmonary arterial hypertension

Abstract

The cardioprotective benefits of aldehyde dehydrogenase 2 (ALDH2) are well established, although the regulatory role of ALDH2 in vascular remodeling in pulmonary arterial hypertension (PAH) is largely unknown. ALDH2 potently regulates the metabolism of aldehydes such as 4-hydroxynonenal (4-HNE), the endogenous product of lipid peroxidation. Thus, we hypothesized that ALDH2 ameliorates the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting 4-HNE accumulation and regulating downstream signaling pathways, thereby ameliorating pulmonary vascular remodeling. We found that low concentrations of 4-HNE (0.1 and 1μM) stimulated cell proliferation by enhancing cyclin D1 and c-Myc expression in primary HPASMCs. Low 4-HNE concentrations also enhanced cell migration by activating the nuclear factor kappa B (NF-κB) signaling pathway, thereby regulating matrix metalloprotein (MMP)-9 and MMP2 expression in vitro. In vivo, Alda-1, an ALDH2 agonist, significantly stimulated ALDH2 activity, reducing elevated 4-HNE and malondialdehyde levels and right ventricular systolic pressure in a monocrotaline-induced PAH animal model to the level of control animals. Our findings indicate that 4-HNE plays an important role in the abnormal proliferation and migration of HPASMCs, and that ALDH2 activation can attenuate 4-HNE-induced PASMC proliferation and migration, possibly by regulating NF-κB activation, in turn ameliorating vascular remodeling in PAH. This mechanism might reflect a new molecular target for treating PAH.

Keywords: 4-hydroxynonenal; Alda-1; Aldehyde dehydrogenase 2; NF-κB; Oxidative stress; Pulmonary arterial hypertension.

Graphical abstract

Fig. 1

MCT induced 4-HNE accumulation in rat lung tissues. (A) 4-HNE was detected in pulmonary arterial smooth muscle cells (PASMCs) in the lungs of rats with MCT-induced PAH. Representative immunofluorescence images of lung sections stained with anti-4-HNE antibodies (green) and anti-α-SMA antibodies (red), at 0, 2, and 4 weeks post-MCT stimulation. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Compared with 0 week rats, 4-HNE levels in the pulmonary arterial media increased in rats with MCT-induced PAH, in terms of the percent nuclear localization. (B) 4-HNE expression in the lung tissue lysates, as determined by western blotting. (C) Quantification of protein levels from (B), relative to the control. ^*P<0.05 (n=3). (D) 4-HNE levels in tissue lysates measured by ELISA. Data are presented as the mean±SD (n=6 in each group). ^*P<0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The effect of 4-HNE on HPASMC proliferation in vitro. (A) The effect of different concentrations of 4-HNE (0.01, 0.1, and 1 μM) on HPASMC proliferation, as determined by performing MTT assays. Cells were treated for 0, 24, 48, or 72 h. (B) The effect of a 48-h 4-HNE treatment (0.01, 0.1, and 1 μM) on HPASMC DNA synthesis, as determined in BrdU assays. (C) HPASMC cyclin D1 and c-Myc expression, evaluated by western blotting. Cyclin D1 (D) and c-Myc (E) protein levels were quantified relative to the indicated control. ^*P<0.05 (n=3). (F) HPASMC cyclin D1 and (G) c-myc mRNA levels, determined by real-time PCR. ^*P<0.05 (n=3). (H) The effect of a 48-h 4-HNE treatment (0.01, 0.1, and 1 μmol/L) on HPASMC proliferation, evaluated by assessing the distribution of cells in various phases of the cell cycle by flow cytometry. ^*P<0.05 vs. control. Data are presented as the mean±SD (n=6 in each group).

Fig. 3

Effect of 4-HNE on HPASMC migration in vitro. (A) Representative photographs showing invading cells after a 24-h treatment with different 4-HNE concentrations (0.01, 0.1, and 1 μM). Wounds were induced by scraping confluent cell layers with a 1-mL pipet tip. Confluent HPASMC monolayers were starved for 24 h in serum-free SMCM and incubated with 4-HNE for 24 h, when the migrated areas were counted. (B) Bar graph showing the migrated wound area of HPASMCs in response to 4-HNE. ^*P<0.05 (n=6). (C) Migration of HPASMCs in response to a 24-h 4-HNE treatment, as determined in Boyden chamber assays. (D) Bar graph showing the number of migrated HPASMCs from (C). ^*P<0.05 (n=6). (E) Western blot analysis of MMP-9 and MMP-2 protein levels in HPASMCs. (F) Statistical analysis of MMP-9 and (G) MMP-2 protein levels in HPASMCs. ^*P<0.05 (n=3). (H) Levels of HPASMC MMP-9 and (I) MMP-2 mRNA, measured by real-time PCR. Data are presented as the mean±SD. ^*P<0.05 (n=3 in each group).

Fig. 4

The effect of 4-HNE on the nuclear translocation of NF-κB p65 in HPASMCs. Confluent HPASMC monolayers were starved for 24 h in serum-free SMCM and stimulated with 4-HNE (0.1 μM) for the indicated times. (A) Western blot analysis of phospho-IκBα, IκBα, phospho-NF-κB p65, NF-κB p65, and GAPDH protein levels in HPASMCs. (B) Statistical analysis of the phospho-IκBα/IκBα and (C) phospho-NF-κB/NF-κB levels in HPASMCs. ^*P<0.05 (n=6). (D) Representative western blot showing NF-κB p65 in nuclear extracts from cultured HPASMCs treated for various times with 0.1 μM 4-HNE, with lamin B1 used as a loading control. (E) Statistical analysis of the NF-κB p65/lamin B1 levels. ^*P<0.05 (n=3). (F) Western blot showing the effect of siRNA knockdown of RelA (NF-κB p65 siRNA) on MMP-9 and MMP-2 expression in response to 0.1 μM 4-HNE. (G) Statistical analysis of MMP-9 and (H) MMP-2 expression in HPASMCs. ^*P<0.05 (n=3). (I) The effect of NF-κB p65 siRNA on the migration of HPASMCs in response to 0.1 μM 4-HNE treatment. (J) Bar graph showing the number of migrated HPASMCs from I. Data are presented as the mean±SD. ^*P<0.05 (n=3 in each group).

Fig. 5

The role of ALDH2 in HPASMC proliferation and migration, and its effect on the response of NF-κB signaling to 4-HNE in vitro. (A) ALDH2 is expressed in HPASMCs upon 4-HNE treatment. Representative immunofluorescence images showing ALDH2 (red) and cell nuclei (blue) in HPASMCs. (B) Western blot analysis of ALDH2 levels in HPASMCs. (C) Statistical analysis of ALDH2 expression in HPASMCs from (B). ^*P<0.05 (n=3). (D) Real-time PCR analysis of ALDH2 mRNA levels in HPASMCs. ^*P<0.05 (n=3). (E) Western blot showing the effect of ALDH2 overexpression or Alda-1 (an ALDH2 agonist; 20 μM) on cyclin D1 and c-Myc HPASMC levels in response to 0.1 μM 4-HNE. (F) Statistical analysis of cyclin D1 and (G) c-Myc expression in HPASMCs. ^*P<0.05 (n=3). (H) The effect of ALDH2 overexpression or Alda-1 (20 μM) on HPASMC proliferation in response to a 48-h 4-HNE treatment (0.1 μM). Alda-1 was administered 30 min before 4-HNE treatment. (I) The effect of ALDH2 overexpression or Alda-1 (20 μM) on HPASMC proliferation in response to a 24-h 0.1 μM 4-HNE, evaluated by flow cytometry, enabled determination of the cell-population distribution in various phases of the cell cycle. ^*P<0.05 vs. control; ^#P<0.05 vs. 4-HNE + vector; ^∆P<0.05 vs. 4-HNE + DSMO (n=6). (J) Western blot showing the effects of ALDH2 overexpression or Alda-1 (20 μM) on the nuclear translocation of NF-κB p65 in response to 0.1 μM 4-HNE. (K) Statistical analysis of the cytoplasmic phospho-IκBα/IκBα levels and (L) nuclear NF-κB p65/lamin B1 levels. ^*P<0.05 (n=3). (M) The effect of ALDH2 overexpression or Alda-1 (20 μM) on HPASMC migration in response to a 24-h 0.1 μM 4-HNE treatment. (N) Bar graph showing the number of migrated HPASMCs from (M). Data are presented as the mean±SD. ^*P<0.05 (n=3 in each group). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Alda-1 prevents the development of PAH and pulmonary vascular remodeling in vivo. (A) Experimental protocol for examining the effect of Alda-1 treatment on MCT-induced PAH. (B) ALDH2 activity, and (C) 4-HNE and (D) MDA levels in the rat lung tissue, as measured by ELISA. Statistical analysis of (E) RVSP and (F) RVHI in PAH rats treated with Alda-1 (10 mg kg⁻¹ d⁻¹), at 4 weeks post-MCT challenge. ^*P<0.05 (n=6). (G) Representative images of H&E-stained pulmonary arteries from each group of animals. (H) Pulmonary artery medial wall thickness, measured in PAH rats treated with Alda-1, at 4 weeks post-MCT challenge. The medial wall thickness of the pulmonary arterioles (25–150 mm external diameter) was measured in H&E-stained lung sections. The medial wall thickness is expressed as specified in the Materials and Methods section (n=6). (I) The effect of ALDH2 on the nuclear translocation of NF-κB in the lungs. (J) Statistical analysis of cytoplasmic phospho-IκBα/IκBα levels and (K) nuclear NF-κB p65/lamin B1 levels. The data are presented as the mean±SD. ^*P<0.05 (n=3).