Alda-1 attenuates hyperoxia-induced mitochondrial dysfunction in lung vascular endothelial cells

Abstract

Acute lung injury (ALI) is a major cause of morbidity and mortality worldwide, especially in aged populations. Mitochondrial damage is one of the key features of ALI. Hyperoxia-induced lung injury model in mice has been widely used for ALI study because it features many ALI phenotypes including, but not limited to, mitochondrial and vascular endothelial cell damage. Recently, accumulating evidence has shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) has a protective effect against oxidative stress mediated cell damage in epithelial cells. However, it is not known whether ALDH2 protects against oxidative stress in vascular endothelial cells. In this current study, we attempted to find the capacity of Alda-1 [(N-(1,3benzodioxol-5-ylmethyl)-2,6- dichloro-benzamide), an ALDH2 activator] to protect against oxidative stress in human microvascular endothelial cells (HMVEC). HMVEC pretreated with Alda-1 prior to hyperoxic exposure vs non-treated controls showed i) lower 4-hydroxynonenal (4-HNE) levels, ii) significantly decreased expressions of Bax and Cytochrome C, iii) partially restored activity and expression of ALDH2 and iv) significantly improved mitochondrial membrane potential. These results suggest that ALDH2 protein in lung vascular endothelial cells is a promising therapeutic target for the treatment of ALI and that Alda-1 is a potential treatment option.

Keywords: ALDH2; Alda-1; HMVEC; acute lung injury; hyperoxia.

Figure 1

Activation of ALDH2 diminishes hyperoxia-induced 4-HNE accumulation in lung vascular endothelial cells. HMVEC were cultured in three different conditions: NO (normoxia), HO (48 hrs of hyperoxia) or HO+Alda-1 (Alda-1 pretreatment followed by 48 hrs of hyperoxia). Whole cell lysates (50 µg protein equivalent) were subjected to ELISA to evaluate the amount of 4-HNE content. The results are shown in mean ± SEM (n=3). Data presented are representative of three independent experiments.

Figure 2

Activation of ALDH2 attenuates hyperoxia-induced increase in cytochrome c expression in lung vascular endothelial cells. (a) Whole cell lysates extracted from HMVEC cultured under different conditions (normoxia, 48 hrs of hyperoxia, or Alda-1 pretreatment followed by 48 hrs of hyperoxia) were evaluated for cytochrome C levels by Western blotting. Equal amounts of protein (20µg) were loaded per each lane. (b) Expression of Cytochrome C was normalized to β-actin and presented in arbitrary units. The results are shown in mean ± SEM (n=3). Data presented are representative of two independent experiments.

Figure 3

ALDH2 activation via Alda-1 attenuates hyperoxia-induced increase in Bax expression in lung vascular endothelial cells. (a) Whole cell lysates extracted from HMVEC cultured under different conditions (normoxia, 48 hrs of hyperoxia, or Alda-1 pretreatment followed by 48 hrs of hyperoxia) were evaluated for Bax levels by Western blotting. Equal amounts of protein (20µg) were loaded per each lane. (b) Expression of Bax was normalized to β-actin and presented in arbitrary units. The results are shown in mean ± SEM (n=3). Data presented are representative of two independent experiments.

Figure 4

ALDH2 activation through Alda-1 pretreatment enhances ALDH2 expression in lung vascular endothelial cells under hyperoxia. (a) Whole cell lysates extracted from HMVEC cultured under different conditions (normoxia, 48 hrs of hyperoxia, or Alda-1 pretreatment followed by 48 hrs of hyperoxia) were evaluated for ALDH2 levels by Western blotting. Equal amounts of protein (20µg) were loaded per each lane. (b) Expression of ALDH2 was normalized to β-actin and presented in arbitrary units. The results are shown in mean ± SEM (n=3). Data presented are representative of three independent experiments.

Figure 5

ALDH2 activation through Alda-1 pretreatment attenuates hyperoxia-induced decrease in ALDH2 activity in lung vascular endothelial cells. Mitochondrial lysates extracted from HMVEC cultured under different conditions (normoxia, 48 hrs of hyperoxia, or Alda-1 pretreatment followed by 48 hrs of hyperoxia) were subjected to an enzymatic assay to evaluate ALDH2 activity. The results are shown in mean ± SEM (n=3). Data presented are representative of three independent experiments

Figure 6

ALDH2 activation protects lung vascular endothelial cells against hyperoxia-induced mitochondrial membrane damage. (a) HMVEC cultured under different conditions (normoxia, 48 hrs of hyperoxia, or Alda-1 pretreatment followed by 48 hrs of hyperoxia) were subjected to JC-1 staining (magnification=200x). (b) Fluorescence intensities of green and red signals were quantified for each image using ImageJ software. The calculated ratios of red to green signals were expressed in arbitrary units. The results are shown in mean ± SEM (n=3). Data presented are representative of three independent experiments.