Lactate Dehydrogenase A Crotonylation and Mono‐Ubiquitination Maintains Vascular Smooth Muscle Cell Growth and Migration and Promotes Neointima Hyperplasia

Figures

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  Figure 1. LDHA was screened that may regulate VSMC phenotypic switching by crotonylome and ubiquitylome analysis. A, A systematic workflow for quantitative profiling of global crotonylome and ubiquitylome. VSMCs pre‐ and post‐treatment with PDGF‐BB were extracted (step 1) and digested with trypsin enzyme (step 2). HPLC was used to separate the peptides (step 3), and we then analyzed the peptides with MS (step 5) after affinity enrichment using the pan anti‐crotonyl‐lysine monoclonal antibody and the pan anti‐ubiquitinate‐lysine monoclonal antibody (step 4). Finally, we used the data from MS for bioinformatics analysis (step 6). B, Differentially crotonylated and ubiquitinated proteins and modification sites in the PDGF‐BB group relative to the control group. C, Crotonylated proteins and ubiquitinated proteins in glycolysis/gluconeogenesis. D, Functions of GAPDH, PKM, and LDHA in glycolysis/gluconeogenesis. E, Comparison of GAPDH, PKM and LDHA crotonylation and ubiquitination level. HPLC indicates high‐performance liquid chromatography; LC/MS‐MS, liquid chromatography–mass spectrometry; LDHA, lactate dehydrogenase A; MS, mass spectrometry; PDGF‐BB, platelet‐derived growth factor‐BB; PKM,…

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  Figure 2. Lysine crotonylation and ubiquitination of LDHA increases in proliferative/synthetic VSMCs. A, Immunoblotting analysis of protein expression in VSMCs stimulated by PDGF‐BB for different durations (12, 24, and 48 h). The expression of LDHA, PCNA, and SM22α were analyzed (n=3). B, Immunofluorescence staining analysis of LDHA in VSMCs with (+) or without (−) PDGF‐BB teatment (n=16). The scale bar was 25 μm. C, Quantitative analysis of lactate levels in VSMCs stimulated by PDGF‐BB for different durations (12, 24 and 48 h) (n=6). D, Complexes immunoprecipitated with anti‐LDHA antibody were immunoblotted with anti‐crotonyl‐lysine antibodies in VSMCs with (+) PDGF‐BB treatment for different durations (12, 24, and 48 h) (n=3). E, Complexes immunoprecipitated with anti‐LDHA antibodies were immunoblotted with ubiquitin antibodies in VSMCs with (+) PDGF‐BB treatment for different durations (12, 24, and 48 h) (n=3). IP indicates immunoprecipitation; LDHA, lactate dehydrogenase A; PCNA, proliferating cell nuclear antigen; PDGF‐BB, platelet‐derived growth factor‐BB; SM22α, smooth muscle 22 alpha; and VSMCs, vascular smooth muscle cells.

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  Figure 3. LDHA is necessary for the growth and migration of VSMCs. A, Immunoblotting analysis of PCNA protein expression upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h). B, Cell growth of VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h) (n=3). C, Cell viability of VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h) (n=3). D, Wound‐healing assay in VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h) was done blinded. Quantitation of relative width of injury line was shown (n=3). E, Immunofluorescence staining analysis of LDHA and Actin filaments with phalloidine (pha) in VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h). The scale bar was 25 μm. F, Immunofluorescence staining analysis of VSMCs with phalloidine upon depletion of LDHA with (+) or without (−) PDGF‐BB stimulation (24 h). The scale bar was 25 μm. The arrow refers to podosomes. LDHA indicates lactate dehydrogenase A; ns, not significant; PCNA, proliferating cell nuclear antigen; PDGF‐BB,…

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  Figure 4. LDHA is crotonylated at lysine 5 in proliferative/synthetic VSMCs. A and B, Identification of crotonylated LDHA peptides by mass spectrometry analysis of VSMCs upon PDGF‐BB stimulation. VSMCs without PDGF‐BB treatment were examined as the control group. C, Sequence analysis of LDHA K5 from Xenopus tropicalis to Homo sapiens using the software of Bioedit. D, Analysis of LDHA crotonylation in VSMCs upon treatment with different concentrations of crotonate (n=3). E, Analysis of LDHA crotonylation in HEK293A cells transfected with HA‐LDHA constructs followed by different concentrations of crotonate treatment (n=3). F, Analysis of LDHA crotonylation in HEK293A cells transfected with HA‐LDHA or HA‐K5R constructs followed by crotonate treatment (n=3). G, Analysis of LDHA crotonylation in VSMCs transduced with the LDHA WT or K5R adenovirus followed by PDGF‐BB stimulation (n=3). H, Analysis of LDHA crotonylation in VSMCs transduced with the LDHA WT or K5R adenovirus followed by crotonate treatment (n=3). IP indicates immunoprecipitation; K, lysine; LDHA, lactate dehydrogenase A; MS‐MS, tandem mass spectrometry; PDGF‐BB, platelet‐derived growth factor‐BB; R, arginine; VSMCs, vascular…

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  Figure 5. LDHA K5 crotonylation promotes tetramer formation and VSMC growth. A, Docking analysis of LDHA using PyMol software. The sequence was obtained from PBD (DOI: 10.2210/pdb4AJJ/pdb). B, Analysis of LDHA tetramerization in cells using a glutaraldehyde crosslinked method. VSMCs with (+) or without (−) PDGF‐BB stimulation were crosslinked by 0.05% glutaraldehyde and analyzed by Western blotting using LDHA antibody (n=3). Tetrameric, dimeric, and monomeric LDHA were indicated. C, Analysis of LDHA tetramerization upon treatment with different concentrations of crotonate in VSMCs (n=3). D, Analysis of LDHA tetramerization in HEK293A cells transfected with HA‐LDHA or HA‐K5R constructs followed by crotonate treatment (n=3). E, Analysis of LDHA tetramerization in VSMCs transduced with the LDHA WT or K5R adenovirus followed by PDGF‐BB stimulation (n=3). F, Analysis of LDHA tetramerization in VSMCs transduced with the LDHA WT or K5R adenovirus followed by crotonate treatment (n=3). G, Quantitative analysis of lactate levels in VSMCs transduced with the LDHA WT or K5R adenovirus followed by PDGF‐BB stimulation (n=6). H, Cell viability of VSMCs transduced with the LDHA WT or K5R adenovirus…

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  Figure 6. LDHA is mono‐ubiquitinated at K76. A and B, Identification of ubiquitinated LDHA peptides by mass spectrometry analysis of VSMCs upon PDGF‐BB stimulation. VSMCs without PDGF‐BB treatment were examined as the control group. C, Sequence analysis of LDHA K76 from Xenopus tropicalis to Homo sapiens using the software of Bioedit. D, Analysis of LDHA ubiquitination in HEK293A cells transfected with His‐Ub along with HA‐LDHA‐WT, HA‐LDHA‐K5R, or HA‐LDHA‐K76R plasmids using Ni‐NTA agarose beads (n=3). E, Analysis of LDHA ubiquitination in VSMCs transduced with the LDHA WT, K5R, or K76R adenovirus followed by PDGF‐BB stimulation (n=3). His‐Ub indicates histone‐ubiquitin; IP, immunoprecipitation; K, lysine; LDHA lactate dehydrogenase A; Ni‐NTA, His(6)‐Ni‐nitrilotriaceate; NS, not significant; PDGF‐BB, platelet‐derived growth factor‐BB; R, arginine; VSMCs, vascular smooth muscle cells; and WT, wild type.

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  Figure 7. Mono‐ubiquitination of LDHA K76 promotes mitochondrial division and VSMC migration and growth. A, Immunofluorescence staining analysis of LDHA and mitochondria with Mito tracker in VSMCs with (+) or without (−) PDGF‐BB stimulation. The scale bar was 25 μm. B, LDHA expression level in the mitochondria fractions which were extracted from VSMCs with (+) or without (−) PDGF‐BB treatment (n=3). C, Immunofluorescence staining analysis of LDHA and mitochondria in VSMCs upon depletion of LDHA using siLDHA with (+) or without (−) PDGF‐BB stimulation. The scale bar was 25 μm. D, Quantitative analysis of mitochondria mean branch length in VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h). E, Immunoblotting analysis of DRP1 expression in VSMCs upon depletion of LDHA using sicon/siLDHA with (+) or without (−) PDGF‐BB stimulation (24 h) (n=3). F, Immunofluorescence staining analysis of LDHA and mitochondria in VSMCs transduced with the LDHA WT or K76R adenovirus followed by PDGF‐BB stimulation. The scale bar was 25 μm. G, Quantitative analysis of mitochondria mean branch length in VSMCs transduced with the LDHA WT or K76R adenovirus followed by…

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  Figure 8. LDHA crotonylation and ubiquitination exacerbates post‐injury neointima hyperplasia in vivo. A, Immunoblotting analysis of protein expression in injured carotid arteries at days 7, 14, and 21 after ligation injury/sham procedure. The expression of LDHA, PCNA, and SM22α were analyzed (n=3). B, Immunofluorescence staining analysis of LDHA in sham/injured carotid arteries. The scale bar was 25 μm (n=6). C, Immunofluorescence staining analysis of PCNA in sham/injured carotid arteries. The scale bar was 25 μm (n=6). D, Analysis of LDHA crotonylation in injured carotid arteries at days 7, 14, and 21 after ligation injury/sham procedure (n=3). E, Analysis of LDHA ubiquitination in injured carotid arteries at days 7, 14, and 21 after ligation injury/sham procedure (n=3). F, hematoxylin and eosin staining analysis of wire‐injured carotid arteries transduced with the LDHA control, WT, K5R, or K76R adenovirus at day 14. Quantitative analysis of (G) the intima area, (H) intima‐to‐media ratio, (I) media area, and (J) external elastic lamina circumference in wire‐injured carotid arteries transduced with the LDHA control, WT, K5R, or K76R adenovirus at day 14 (n=6). K and L, Immunofluorescence…

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  Figure 9. LDHA K5 crotonylation and K76 mono‐ubiquitination in regulating VSMC phenotypic switching. Crotonylation at K5 activates LDHA enzymatic activity and mono‐ubiquitination at K76 mediates the translocation of LDHA into mitochondria. Both glycolytic and nonglycolytic functions of LDHA maintain the growth and migration of VSMCs and promote neointima hyperplasia. K indicates lysine; LDHA, lactate dehydrogenase A; PDGF‐BB, platelet‐derived growth factor‐BB; PDGFR, platelet‐derived growth factor receptors; and VSMCs, vascular smooth muscle cells.