ALDH2 Deficiency Promotes Mitochondrial Oxidative Stress and Adverse Limb Outcomes via VDAC–HSP60 Signaling

Figures

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  FIGURE 1. ALDH2 deficiency impairs perfusion recovery and enhances oxidative stress following limb ischemia. (A) Representative laser Doppler perfusion images of wild‐type (WT) and ALDH2 knock‐in (KI) mice at baseline (Day 0) and after hindlimb ischemia (Days 1, 14, and 28). (B) Quantitative analysis of perfusion recovery, expressed as perfusion intensity index (n = 5 per group). (C) Representative immunofluorescence staining of ischemic gastrocnemius muscle showing CD31 (red), 4‐HNE (green), and DAPI (blue). Scale bars are 100 and 20 µm at 400× and 1000× magnification, respectively. (D) Quantification of CD31 fluorescence intensity demonstrating impaired angiogenesis in ALDH2 KI mice. (E) Quantification of 4‐HNE fluorescence intensity showing elevated oxidative stress in ALDH2 KI mice. (F‐H) Schematic representation and quantification of DNA damage (8OHdG) accumulation in ischemic tissue. Data are presented as mean ± SEM. Statistical significance was determined using two‐way ANOVA followed by post hoc testing; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

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  FIGURE 2. ALDH2 knockdown impairs endothelial migration, proliferation, and angiogenic capacity under hypoxic conditions, rescued by AD5591. (A) ALDH2 enzymatic activity assay showing dose‐dependent activation by AD5591 in HUVECs (n = 5 per group). (B) Endothelial proliferation assessed by BrdU incorporation. siALDH2 combined with OGD significantly reduced proliferation, which was restored by AD5591 (n = 12 per group). (C‐D) Representative images and quantification of migration assays at 0 and 5 h under the indicated conditions. Red lines indicate scratch edges (n = 4–6 per group). (E‐F) Representative tube formation images and quantification demonstrating reduced angiogenic network formation in siALDH2 and OGD groups, with AD5591 treatment improving tube formation. Representative images and quantification of (G‐H) DHE (Dihydroethidium), cellular reactive oxygen species (ROS), and (I‐J) 8OHdG, DNA damage, expression in the indicated groups. Data are presented as mean ± SEM. Statistical significance was determined using one‐way ANOVA with post hoc testing; * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

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  FIGURE 3. Integrated transcriptomic and experimental analyses identify HSP60 as a downstream effector of ALDH2 deficiency during ischemic stress. (A) Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) highlights processes related to apoptosis regulation, transcriptional control, and inflammatory response. (B) Heatmap of representative DEGs shows altered expression of oxidative stress–associated genes such as APOE, CAT, GSTP1, and HSPD1 (HSP60). (C) STRING protein–protein interaction network reveals functional connections between ALDH2 and oxidative stress–modulating proteins, notably GSTP1 and HSPD1 (HSP60). (D‐E) Representative immunofluorescence staining of CD31 (red) and HSP60 (green) in sham and ischemic muscle from WT and ALDH2 KI mice (n = 5 per group). Scale bars are 100 and 20 µm at 400× and 1000× magnification, respectively. Data are presented as mean ± SEM. Statistical significance was determined using one‐way ANOVA with post hoc testing; *** p < 0.005, **** p < 0.001.

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  FIGURE 4. ALDH2 deficiency enhances 4‐HNE‐mediated HSP60 modification and promotes mitochondrial redistribution of HSP60 under ischemic stress. (A) Representative immunofluorescence images of HUVECs stained for mitochondrial marker TOM20 (green), HSP60 (red), and 4‐HNE (gray) under control, si‐Aldh2, OGD, si‐Aldh2 + OGD, and si‐Aldh2 + OGD + AD5591 conditions. Quantitative analysis of cytosolic HSP60 intensity, total HSP60 expression, and intracellular 4‐HNE levels is shown on the right (n = 4–6/group). (B) Representative immunoblot analysis of mitochondrial and cytosolic fractions showing redistribution of ALDH2 and HSP60 following OGD and ALDH2 silencing. GAPDH was used as a cytosolic loading control. (C) Covalent docking model of 4‐HNE with human HSP60. Covalent docking of 4‐HNE to human HSP60 was performed using SeeSAR. The overall docking pose is shown on the left, with HSP60 represented as a gray cartoon and 4‐HNE shown as cyan sticks. The enlarged view highlights the predicted binding environment of 4‐HNE near K335. The putative covalent interaction site is indicated by the red dashed circle, and the hydrogen bond is shown as a green dashed line. (D) Co‐immunoprecipitation analysis…

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  FIGURE 5. AD5591 mitigated ALDH2 deficiency‐exacerbated mitochondrial dysfunction under ischemic stress. (A) Mitochondrial DNA (mtDNA) damage was evaluated by qPCR of the mtDNA/β‐globin ratio in HUVECs under various conditions: control, siALDH2, OGD, siALDH2 + OGD, and siALDH2 + OGD + AD5591 (ALDH2 activator) (n = 5–8 per group). (B) Flow cytometric assessment of mitochondrial membrane potential using JC‐1 staining demonstrated significant depolarization in siALDH2 + OGD cells, which was restored by AD5591 but reversed by HSP60 knockdown. (C) MitoSOX Red fluorescence indicated excessive mitochondrial ROS generation in siALDH2 + OGD‐treated cells, attenuated by AD5591 and aggravated by HSP60 silencing. (D) Representative confocal images showing mitochondrial morphology by MitoTracker staining. Quantitative analysis of (E) mitochondrial morphology, including fusion/fission states, (F) mitochondrial area, (G) branch length, and (H) aspect ratio, is shown. Quantitative analysis using ImageJ revealed a shift from fused to fragmented mitochondrial networks in ALDH2‐deficient cells. Data are presented as mean ± SEM. Statistical significance was determined using one‐way ANOVA with post hoc…

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  FIGURE 6. VDAC inhibition attenuates ALDH2 deficiency‐induced mitochondrial dysfunction and preserves mitochondrial HSP60 under ischemic stress. (A) Representative confocal and 3D reconstructed images showing subcellular localization of HSP60 (green) and mitochondria labeled with MitoTracker Red in endothelial cells under control, si‐Aldh2 + OGD, and si‐Aldh2 + OGD + VDAC inhibitor (VDACi) conditions. Quantitative analysis demonstrates changes in total HSP60 intensity, mitochondrial localization, and cytosolic redistribution of HSP60 (n = 5–8/group). (B–C) Representative JC‐1 flow cytometry and quantitative analysis of mitochondrial membrane potential. VDAC inhibition partially restored mitochondrial polarization in ALDH2‐deficient cells exposed to OGD. (D) Representative mitochondrial morphology analysis using the MiNA toolbox. Quantitative analysis of (E) mitochondrial morphology, including fusion/fission states, (F) mitochondrial area, (G) branch length, and (H) aspect ratio, is shown. (I–K) Measurement of mitochondrial reactive oxygen species using MitoSOX, demonstrating increased mtROS accumulation under ALDH2 deficiency and ischemic stress, which was partially reduced by VDAC…

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  FIGURE 7. Genetic silencing of VDAC1 preserves mitochondrial integrity and prevents HSP60 redistribution under ALDH2 deficiency and ischemic stress. (A) Representative confocal and three‐dimensional reconstructed images showing the subcellular localization of HSP60 (green) and mitochondria labeled with MitoTracker Red in HUVECs under control, si‐Aldh2 + OGD, si‐Aldh2 + OGD + siVdac1, siVdac2, and siVdac3 conditions. Quantitative analysis of total HSP60 expression, mitochondrial localization, cytosolic redistribution, and mitochondrial fluorescence intensity is shown. (B) Representative MitoTracker images and MiNA (ImageJ) analysis of mitochondrial morphology. VDAC1 silencing attenuated mitochondrial fragmentation and preserved mitochondrial network architecture in ALDH2‐deficient cells exposed to OGD. Quantitative analysis of (C) mitochondrial morphology, including fusion/fission states, (D) mitochondrial area, (E) branch length, and (F) aspect ratio, is shown. Data are presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by post hoc multiple‐comparison testing. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001. (N = 4‐6 biologically independent…

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  FIGURE 8. K‐M plot showing that patients carrying ALDH2 rs671 variants (A/A or G/A polymorphism) exhibited higher rates of MALEs than wild‐type carriers in a 227‐patient PAD cohort.