HK2 promotes angiogenesis through H3K18 lactylation in peripheral arterial disease

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  Fig. 1. Bioinformatics analysis and experimental evidence demonstrated that HK2 was downregulated in PAD. a Bioinformatics analysis workflow diagram. b Venn diagram shows signature gene (HK2) between IC hub genes and CLI hub genes. c The expression of HK2 between the HA, IC and CLI group. d ROC showed the diagnostic performance of HK2. e Typical images of limb perfusion using laser Doppler before and on days 0 after HLI. n = 6. f Quantitative RT-PCR analysis of HK2 mRNA levels in gastrocnemius muscle 3 days post-HLI. g, h Western blot and densitometric quantification of HK2 protein expression in gastrocnemius muscle 3 days post-HLI. f, h Data represent mean ± SEM; n = 3 (qRT-PCR), n = 3 (Western blot) per group. Statistical significance was assessed by a two-tailed unpaired Student’s t-test. HLI hindlimb ischemia, HA healthy, IC intermittent claudication, CLI critical limb ischemia, ROC receiver operating characteristic.

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  Fig. 2. HK2 overexpression restores angiogenesis in HSS-treated HUVECs. a GSEA enrichment plot of sprouting angiogenesis pathways in PAD. b, c Immunofluorescence staining and quantification of HK2 in murine gastrocnemius ECs under normal versus HLI conditions. n = 3. Scale bars, 20 μm. d Western blot and densitometry analysis of HK2 expression in HUVECs treated with normoxia or HSS. n = 3. e Western blot and densitometry analysis of HK2 expression in HUVECs under normoxia, HSS, and HSS with HK2 overexpression (HSS + HK2OE). n = 3. f, g EdU proliferation assay with statistical analysis of HUVEC growth in normoxia, HSS, and HSS + HK2OE. n = 6. Scale bars, 100 μm. h, i Scratch wound healing assay and migration quantification of HUVECs under normoxia, HSS, and HSS + HK2OE at 0, 6, and 12 h. n = 6. Scale bars, 100 μm. j Tube formation assay and angiogenic capacity quantification in HUVECs under normoxia, HSS, and HSS + HK2OE. n = 6. Scale bars, 100 μm. k, l Western blot and densitometry analysis of angiogenesis-related proteins (VEGFA, VEGFR2, p-VEGFR2, FAK, p-FAK, PI3K, p-PI3K, ERK, and p-ERK) in normoxia, HSS, and HSS + HK2OE HUVECs. n = 3. c, d Two-tailed unpaired Student’s t-test. e, g, l…

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  Fig. 3. Knockdown of HK2 in HUVECs inhibited angiogenesis under normoxia conditions. a Western blot and densitometry analysis of HK2 expression in HUVECs transfected with CRISPR/Cas9 HK2-targeting sgRNA (HK2KD) versus NC. n = 3. b EdU proliferation assay with statistical comparison between HK2KD and NC HUVECs. n = 6. Scale bars, 100 μm. c, d Scratch wound healing assay and migration rate quantification of HK2KD versus NC HUVECs at 0, 6, and 12 h. n = 6. Scale bars, 100 μm. e Tube formation assay and quantitative evaluation of angiogenic capacity in HK2KD versus NC HUVECs. n = 6. Scale bars, 100 μm. f, g Western blot and densitometry of angiogenesis-related proteins (VEGFA, VEGFR2, p-VEGFR2, FAK, p-FAK, PI3K, p-PI3K, ERK, and p-ERK) in HK2KD and NC HUVECs. n = 3. c Two-way ANOVA followed by Bonferroni’s multiple comparisons test. a, b, e, g Two-tailed unpaired Student’s t-test. HK2KD HK2-knockdown.

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  Fig. 4. HK2 modulates lactate metabolism and histone lactylation in HUVECs. a Intracellular lactate levels in NC and HK2KD HUVECs. n = 6. b Western blot and quantification of pan-histone lactylation and acetylation in NC and HK2KD HUVECs. n = 3. c, d WB analysis and quantification of histone lactylation sites (H3K9la, H3K14la, H3K18la, H3K27la, H4K5la, H4K8la, H4K12la, and H4K16la) in NC and HK2KD HUVECs. n = 3. e Intracellular lactate levels in normoxia, HSS, and HSS + HK2OE HUVECs. n = 6. f Western blot and densitometry analysis of pan-histone lactylation and acetylation in normoxia, HSS, and HSS + HK2OE HUVECs. n = 3. g, h Western blot and quantification of histone lactylation sites (H3K9la, H3K14la, H3K18la, H3K27la, H4K5la, H4K8la, H4K12la, and H4K16la) in normoxia, HSS, and HSS + HK2OE HUVECs. n = 3. a Two-tailed unpaired Student’s t-test. b, d, f, h Two-way ANOVA with Bonferroni’s multiple comparisons test. e One-way ANOVA followed by Tukey’s multiple comparisons test. Nor normoxia, HSS hypoxia/serum starvation, HK2KD HK2-knockdown, HK2OE HK2 overexpression.

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  Fig. 5. CUT& Tag analysis of H3K18la reveals HK2 regulates angiogenic genes. a Heatmap of altered H3K18la peaks associated with HK2KO. b Distribution of the H3K18la peaks across chromosomes. c, d The length and signals of H3K18la peaks. e Heatmaps and intensity profiles of H3K18la around ±2 kb of TSS throughout the genome. f Distribution of the altered H3K18la peaks in the genomic elements. g GO enrichment analysis of the elevated H3K18la binding peaks at candidate target genes. h Genome browser tracks of CUT& Tag signal at the VEGFA loci.

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  Fig. 6. Lactate rescued angiogenic dysfunction in HK2KD HUVECs. a EdU proliferation assay with statistical analysis of HK2KD HUVECs exposed to 5 mM lactate or control for 24 h. n = 6. Scale bars, 100 μm. b, c Scratch wound healing assay and migration rate quantification in HK2KD HUVECs treated with or without 5 mM lactate at 0, 6, and 12 h. n = 6. Scale bars, 100 μm. d Tube formation assay and angiogenic capacity quantification in HK2KD HUVECs with or without 5 mM lactate. n = 6. Scale bars, 100 μm. e, f Western blot and densitometry of angiogenesis-related proteins (VEGFA, VEGFR2, p-VEGFR2, FAK, p-FAK, PI3K, p-PI3K, ERK, and p-ERK) in HK2KD HUVECs treated with or without 5 mM lactate. n = 3. g Western blot analysis of histone pan-lactylation, pan-acetylation, and H3K18 lactylation (H3K18la) levels in HK2KD HUVECs with or without 5 mM lactate. n = 3. h HK2KD HUVECs treated with or without 5 mM lactate for 24 h. DNA fragments were immunoprecipitated with the H3K18la antibody and analyzed by qPCR. n = 3. Data were normalized to 1% input DNA. b Two-way ANOVA with Bonferroni’s multiple comparisons test. a, d, f, h Two-tailed unpaired Student’s t-test. Lac lactate, HK2KD HK2-knockdown.

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  Fig. 7. SIRT2 inhibition promoted angiogenesis and alleviated PAD. a Western blot analysis of H3K18la and SIRT2 protein levels in HK2KD HUVECs transfected with SIRT2-targeting (siSIRT2) or control siRNA (siNC). n = 3. b, c EdU proliferation assay and quantification of HK2KD HUVECs treated with siSIRT2 or siNC. n = 6. Scale bars, 100 μm. d, e Scratch wound healing assay and quantification of migration in HK2KD HUVECs with or without siSIRT2 at 0, 6, and 12 h. n = 6. Scale bars, 100 μm. f, g Tube formation assay and quantification of angiogenic capacity in HK2KD HUVECs with or without siSIRT2. n = 6. Scale bars, 100 μm. h, i Laser Doppler perfusion imaging and quantification in HLI mice treated with vehicle or AGK2 (SIRT2 inhibitor, 1 mg/kg/day) at 0, 3, 7, 14, and 21 days post-HLI. (n = 6 for each group). j, k Immunofluorescence imaging (CD31⁺ endothelial cells) and quantification of endothelial cell number in the gastrocnemius muscle of HLI mice treated with vehicle or AGK2. n = 3. Scale bars, 20 μm. l, m Western blot and densitometry analysis of SIRT2 and H3K18la levels in gastrocnemius muscle of HLI mice treated with vehicle or AGK2. n = 3. e, m Two-way ANOVA followed by Bonferroni’s…