Histidine Triad Nucleotide-Binding Protein 1 Improves Critical Limb Ischemia by Regulating Mitochondrial Homeostasis

Abstract

Critical limb ischemia (CLI) is a common complication of diabetes mellitus that typically occurs in the later stages of the disease. Vascularization is indeed an important physiological process involving the formation of new blood vessels from existing ones. It occurs in response to various normal and pathophysiological conditions, and one of its critical roles is to compensate for inadequate oxygen supply, which is often seen in situations like chronic limb ischemia (CLI). Histidine triad nucleotide-binding protein 1 (Hint1) is a member of the Hint family that has been shown to attenuate cardiac hypertrophy, but its role in vascularization still needs to be clarified. In this study, we investigated the role of Hint1 in CLI. We found that Hint1 is significantly reduced in the muscle tissue of STZ-induced diabetic mice and high-glucose (HG)-treated endothelial cells (ECs). Hint1 deletion impaired blood flow recovery and vascularization, whereas Hint1 overexpression promoted these processes. In addition, our in vitro study showed that Hint1 deficiency aggravated mitochondrial dysfunction in ECs, as evidenced by impaired mitochondrial respiration, decreased mitochondrial membrane potential, and increased reactive oxygen species. Our findings suggest that Hint1 deficiency impairs blood perfusion by damaging mitochondrial function and that Hint1 may represent a potential therapeutic target for treating CLI.

Keywords: critical limb ischemia; endothelial cells; histidine triad nucleotide-binding protein 1; mitochondrial dysfunction; neovascularization; reactive oxygen species.

Figure 1

Hint1 is reduced in STZ-induced mouse muscle and high-glucose (HG)-treated endothelial cells. (A) Blood glucose levels following STZ injection. (B) Immunoblot analysis of Hint1 level in the gastrocnemius muscle from control and STZ-treated mice. (C) quantification of Hint1 level. n = 5, unpaired 2-tailed t test, *** p < 0.001. (D) Immunoblot analysis of Hint1 level in the control and HG cultured HUVECs. (E) Quantification of Hint1 level. n = 5, unpaired 2-tailed t test, *** p < 0.001. (F) Representative gating plots for Ly6G high cells and percentages of Ly6G high cells within CD45+ cells. n = 6, unpaired 2-tailed t test, ns indicates no significant difference. (G) Representative gating plots for CD11b+; CD64+ cells and percentages of CD11b+; CD64+ cells within Ly6G+ cells. n = 6, unpaired 2-tailed t test, ns indicates no significant difference. (H) Representative gating plots for CD206+ macrophages and percentages of CD206+ macrophages within CD11b+; CD64+ cells. n = 6, unpaired 2-tailed t test, ns indicates no significant difference. (I) Representative gating plots for CD31+ cells and percentages of CD31+ cells within CD45- cells. n = 6, unpaired 2-tailed t test, **** p < 0.0001.

Figure 2

Hint1 deficiency impairs blood flow recovery. (A) Experimental protocol. (B) Representative immunoblot of Hint1 expression in gastrocnemius muscle from wild-type and Hint1 knockout mice. (C) Laser Doppler perfusion images (LDPI) of posterior limb perfusion at 1, 3, 7, 14, and 21 days after femoral artery ligation in each group. n = 5, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05. (D) Quantification of perfusion recovery. (E) Micrographs of representative hindlimb sections 14 days after surgery; CD31(+) staining is shown in red. Scar bar = 100 μm. (F) The ECs were treated with HG (25 mM) or vehicle after transfection with the siHint1 for 24 h, Micrograph of capillary-like tube formation in each group. (G) Quantification of tube length. n = 5, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01.

Figure 3

Endothelium-specific overexpression of Hint1 improves perfusion recovery. (A) Specific expression of EGFP-tagged Hint1 in endothelial cells in gastrocnemius muscles. Scar bar = 100 μm. (B) Laser Doppler perfusion images (LDPI) of posterior limb perfusion at 1, 3, 7, 14, and 21 days after femoral artery ligation in each group. (C) Quantification of perfusion recovery. n = 5, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05. (D) Micrographs of representative hindlimb sections 14 days after surgery; CD31(+) staining is shown in red. Scar bar = 100 μm. (E) Capillary density analysis. n = 5, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01. (F) Micrograph of capillary-like tube formation in each group. (G) Quantification of tube length. n = 5, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01.

Figure 4

Silencing of Hint1 damages mitochondrial function in EC. (A) An analysis of O₂ consumption in HUVECs using different inhibitors. The ECs were treated with HG (25 mM) or vehicle after transfection with the siHint1 for 24 h. Graphs present the basal OCR (B), maximal OCR (C), ATP production (D), and spare respiratory capacity OCR (E) in ECs; n = 3 per group, two-way ANOVA with Bonferroni multiple comparison test. ** p < 0.01. (F,G) MitoTracker staining was used to detect mitochondrial membrane potential (Δψm); Scar bar = 100 μm, n = 5 per group, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05, *** p < 0.001. (H,I) mitoSOX fluorescence staining was used to detect mitochondrial reactive oxygen species (mROS); Scar bar = 50 μm, n = 5 per group, two-way ANOVA with Bonferroni multiple comparison test. ** p < 0.01. (J) Representative Western blot analysis of SOD2, CATALASE, and CytoC in ECs are shown.

Figure 5

Hint1 overexpression ameliorates mitochondrial dysfunction in ECs. (A) An analysis of O₂ consumption in HUVECs using different inhibitors. The ECs were treated with HG (25 mM) or vehicle after being infected with AAV-Hint1 for 48 h. Graphs present the basal OCR (B), maximal OCR (C), ATP production (D), and spare respiratory capacity OCR (E) in ECs; n = 3 per group, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. (F,G) MitoTracker staining was used to detect mitochondrial membrane potential (Δψm); Scar bar = 100 μm, n = 5 per group, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05. (H,I) mitoSOX fluorescence staining was used to detect mitochondrial reactive oxygen species (mROS); Scar bar = 50 μm, n = 5 per group, two-way ANOVA with Bonferroni multiple comparison test. * p < 0.05. (J) Representative Western blot analysis of SOD2, CATALASE, and CytoC in ECs are shown.