Vascular Smooth Muscle Cell-Specific BCAT2 Deficiency Attenuates Diabetic Atherosclerotic Calcification via Histone Propionylation

Figures

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  Fig. 1.. Altered BCAA catabolism in calcification of diabetic plaque. (A) Orthogonal partial least squares discriminant analysis (OPLS-DA) models based on positive and negative ion mode AFADESI-MSI data. Each dot represented one pixel from MSI. VC samples are displayed by green dots. NVC samples are represented by blue dots. Goodness of fit R2 (X) = 0.775, R2 (Y) = 0.234, goodness of prediction Q2 = 0.18. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of identified metabolites with variable importance in projection (VIP) value more than 1 between VC and NVC groups. The top 18 enriched pathways are shown. (C) Left panel: Adjacent von Kossa-stained tissue images. Scale bar, 200 μm. Right panel: Array of MS images of BCAA pathway metabolites from VC (above) and NVC (below) groups by airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI. (D) VC tissue section zoomed in to show Ca and NearCa group corresponding to the H&E staining and MS image. (E) OPLS-DA score plot showing distribution based on metabolite profiles of Ca and NearCa groups. Each dot represented one pixel from MSI. Ca groups are displayed by green dots. NearCa…

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  Fig. 2.. BCAT2 is markedly increased in VSMCs during diabetic atherosclerotic calcification. (A) t-Distributed stochastic neighbor embedding (t-SNE) showing the main cell types of anterior tibial arteries collected from individuals subjected to diabetic foot amputation. Population identities were determined based on marker gene expression. (B) Box plot showing the activity level of each gene in BCAA catabolism by Qusage analysis. The horizontal axis represents gene names, and the vertical axis represents gene activity. An activity level greater than zero indicates that the corresponding gene is up-regulated in the VC group. On the contrary, the expression is down-regulated. Blue dashed line represents the overall activity of the gene set, and gray box represents the confidence interval of the gene set. (C) Pie graphs showing the expression distribution of BCAT2 among the different subgroups in the cells of anterior tibial arteries collected from individuals subjected to diabetic foot amputation. (D) Representative images and quantification of immunofluorescence staining of BCAT2 and VSMC marker α-SMA in anterior tibial arteries from diabetic foot amputation. Red, BCAT2; green, α-SMA;…

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  Fig. 3.. VSMC-specific BCAT2 deficiency inhibits VSMC osteogenic differentiation and diabetic atherosclerotic calcification. (A) Western blot image and analysis showing SM22α, α-SMA, BMP2, and RUNX2 protein expression level in the aorta of ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice with NC or DM. n = 6. (B and C) Biochemical measurement of aortic calcium content and ALP activity for indicated mice. n = 6. (D) Representative images of alizarin red S staining in the whole aorta for indicated mice. (E) Micro-CT was used to detect aortic calcification for indicated mice. (F) Representative images and quantification of H&E and von Kossa staining in aortic arch and aortic root tissue sections. Scale bars, 200 μm. n = 6. (G) Western blot image and analysis showing SM22α, α-SMA, BMP2, RUNX2, and BCAT2 protein expression level in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under NM or OM treatment for 14 d. n = 6. (H) Representative images of alizarin red S staining in VSMCs isolated from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under NM or OM treatment for 14 d. Scale bars, 100 μm. n = 6. (I) Quantification of calcium deposits. n = 6. Data are presented as mean ± SD. *P <…

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  Fig. 4.. BCKA supplement promotes VSMC osteogenic differentiation and diabetic atherosclerotic calcification. (A) Schematic protocol: ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice were intraperitoneally injected with streptozotocin (STZ) (40 mg/kg) for 5 d and then supplemented with 1 mg/ml BCKA in drinking water together with the high-fat diet for 24 weeks. (B) Western blot image and analysis showing SM22α, α-SMA, BMP2, and RUNX2 protein expression level in the aorta of ApoE−/−/BCAT2fl/fl, ApoE−/−/BCAT2ΔSMC, and ApoE−/−/BCAT2ΔSMC supplemented with 1 mg/ml BCKA in drinking water. n = 6. (C and D) Biochemical measurement of aortic calcium content and ALP activity for indicated mice. n = 6. (E) Representative images of alizarin red S staining in the whole aorta for indicated mice. (F) Micro-CT was used to detect aortic calcification for indicated mice. (G) Representative images and quantification of H&E and von Kossa staining in aortic arch and aortic root tissue sections. Scale bars, 200 μm. n = 6. (H) Western blot image and analysis showing SM22α, α-SMA, BMP2, and RUNX2 protein expression level in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC supplemented with or without BCKA under…

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  Fig. 5.. KMV or KIV supplement, but not KIC, promotes VSMC osteogenic differentiation and diabetic atherosclerotic calcification. (A) Western blot image and analysis showing BCKDH, SM22α, α-SMA, BMP2, and RUNX2 protein expression level in VSMCs from ApoE−/−/BCAT2ΔSMC mice supplemented with vehicle, BCKA, KMV, KIV, or KIC for 72 h under OM treatment for 14 d. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle. (B) Representative images of alizarin red S staining in VSMCs from ApoE−/−/BCAT2ΔSMC mice supplemented with vehicle, 0.6 mM BCKA, 0.6 mM KMV, 0.6 mM KIV, or 0.6 mM KIC for 72 h under OM treatment for 14 d. Scale bars, 200 μm. n = 6. (C) Quantification of calcium deposits. n = 6. ****P < 0.0001 versus vehicle. (D) Schematic map for BCAA catabolism pathway. BCAA contains valine, leucine, and isoleucine. BCKA contains KMV, KIC, and KIV. (E) Western blot image and analysis showing BCKDH, SM22α, α-SMA, BMP2, and RUNX2 protein expression level in VSMCs from ApoE−/−/BCAT2ΔSMC mice supplemented with or without prop-CoA under OM treatment for 14 d. n = 6. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus ApoE−/−/BCAT2fl/fl or ApoE−/−/BCAT2ΔSMC. (F) Representative images of…

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  Fig. 6.. VSMC-specific BCAT2 deficiency inhibits histone propionylation. (A) Western blot images and analysis show protein propionylation levels in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under OM treatment for 14 d. n = 6. (B) Western blot image and analysis showing H3K23pr, H3K14pr, H3K18pr, and H3K56pr protein expression levels in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under OM treatment for 14 d. n = 6. (C) Representative images and quantification of immunofluorescence staining of Kpr and α-SMA in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under OM treatment for 14 d. Red, Kpr; green, α-SMA; blue, DAPI. Scale bar, 50 μm. n = 6. (D) Representative images and quantification of immunofluorescence staining of H3K23pr and α-SMA in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice under OM treatment for 14 d. Red, H3K23pr; green, α-SMA; blue, DAPI. Scale bar, 50 μm. n = 6. (E) Left panel: Adjacent von Kossa-stained tissue images. Medium panel: Representative images of immunofluorescence staining of Kpr and α-SMA in the aorta of ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice. Right panel: Representative images of immunofluorescence…

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  Fig. 7.. BCAT2–BCKA axis regulates RUNX2 expression via histone propionylation. (A) Heatmap showing the differentially expressed genes of RNA-seq from the aorta of ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC mice in terms of osteoblast differentiation. (B) qRT-PCR analysis of RUNX2 gene expression in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC supplemented with or without BCKA under OM treatment for 14 d. n = 6. (C) Western blot images and analysis show H3K23pr protein expression levels in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC supplemented with or without BCKA under OM treatment for 14 d. n = 6. (D) Genome browser tracks of the ChIP-seq signals from GSE229131 at representative H3K23pr peaks at the RUNX2 locus. Scale bar, 20 kb. (E) ChIP-qPCR assay of H3K23pr in the RUNX2 locus in VSMCs from ApoE−/−/BCAT2fl/fl and ApoE−/−/BCAT2ΔSMC supplemented with or without BCKA under OM treatment for 14 d. n = 6. (F) Western blot image and analysis showing SM22α, α-SMA, BMP2, and RUNX2 protein expression levels in VSMCs transfected with negative control siRNA (siNC) or RUNX2 siRNA (siRUNX2) for 48 h followed by supplementation with or without BCKA for 72 h under OM treatment for 14…

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  Fig. 8.. BCAT2 inhibitor impedes diabetic atherosclerotic calcification. (A) Docking model of BAY-069 (BCAT2 inhibitor) and BCAT2. (B) qRT-PCR analysis of BCAT2 gene expression in the aorta of NC or DM ApoE−/− mice supplemented with DMSO or BAY-069 (0.3 mg/kg). n = 6. (C) Western blot image and analysis showing α-SMA, SM22α, BMP2, RUNX2, and BCAT2 protein expression level in the aorta of NC or DM ApoE−/− mice supplemented with DMSO or BAY-069 (0.3 mg/kg). n = 6. (D) BCAT2 enzyme activity in the aorta of indicated mice. n = 6. (E and F) Biochemical measurement of aortic calcium content and ALP activity for indicated mice. n = 6. (G) Representative images of alizarin red S staining in the whole aorta for indicated mice. (H) Micro-CT was used to detect aortic calcification for indicated mice. (I) Representative images and quantification of von Kossa staining in aortic arch and aortic root tissue sections, and immunohistochemistry staining of RUNX2 in aortic root tissue sections. Scale bars, 200 μm for von Kossa staining and 50 μm for immunohistochemistry staining. n = 6. (J) Western blot image and analysis showing α-SMA, SM22α, BMP2, RUNX2, and BCAT2 protein expression level in Movas…

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  Fig. 9.. Schematic illustration of BCAT2-mediated BCAA catabolism contributing to the pathogenesis of diabetic atherosclerotic calcification. High glucose stimulates up-regulation of BCAT2 expression and enhances BCAA catabolism. The up-regulation of propionyl-CoA derived from BCKA promotes histone propionylation, thus activating the expression of RUNX2, thereby promoting VSMC osteogenic differentiation and diabetic atherosclerotic plaques.