Loss of DNA Polymerase β Delays Atherosclerosis in ApoE−/− Mice Due to Inhibition of Vascular Smooth Muscle Cell Migration

Figures

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  Figure 1. Pol β knockdown inhibits the formation of atherosclerotic plaques in ApoE−/− mice: (A) Oil Red O assay was used to detect the aortic plaque formation in ApoE−/− mice at different time points on a high-fat diet (n = 6 mice/treatment group). The mice were sampled after 6, 12, and 18 weeks and showed plaque progression induced by a high-fat diet; (B) Hematoxylin-eosin (HE) and Oil Red O staining experiments were used to observe the aortic roots of ApoE−/− mice. The arrow marks the observed area of arterial plaque, showing the location and size of the plaque. Scale bar, 1000 μm (n = 6 mice/treatment); (C) The expression of Pol β in atherosclerotic plaques of ApoE−/− mice was detected by immunofluorescence. The results showed that the expression of Pol β in AS plaques increased significantly; (D) Quantitative statistical analysis in (C) showed the change of Pol β expression in different treatment groups (n = 6 mice/treatment group); (E) Western blot analysis of Pol β expression in blood vessels and hearts of ApoE−/− mice after 18 weeks of high-fat diet. The results demonstrated the differential expression of Pol β in different tissues (n = 3–4 mice/treatment group); (F) Quantitative…

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  Figure 2. Pol β knockdown inhibits hCASMC cell migration and proliferative activity: (A) Cell scratch assay was used to measure the migration capacity of human coronary smooth muscle cells (hCASMC) in the control and shPol β groups. Scale bar, 100 μm (n = 3 independent experiments); (B) Representative images of hCASMC cell scratch tests in the control group and the shPol β group in (A), and a summary analysis of their migration data. Scale bar, 100 μm; (C) The migration of hCASMC cells was again examined by cell scratch assay to further verify the changes in cell migration ability in the shPol β group. Scale bar, 100 μm (n = 3 independent experiments); (D) Statistical analysis of the results in (C), showing a significant difference in hCASMC cell migration ability between the control and shPol β groups; (E,F) Western blot assay was used to detect the expression levels of migration-related proteins (such as Vimentin, N-cadherin, E-cadherin, etc.) in hCASMC cells, and the results were statistically analyzed. Experiments showed significant changes in the expression of migration-related proteins in the shPol β group (n = 3 independent experiments); (G) Transwell migration assay was used to…

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  Figure 3. Pol β knockdown inhibits hCASMC cell migration by inhibiting the expression of periostin: (A) RNAseq analysis was used to detect the expression levels of multiple cytokines in MEF cells, revealing the genes whose expression changed after Pol β knockout (n = 3 independent experiments); (B) qRT-PCR assay was used to detect mRNA expression levels of Pol β and POSTN (periostin) in MEF cell (n = 3 independent experiments); (C) qRT-PCR assay was used to detect the expression of collagen genes in MEF cell, including Col1α1, Col3α1, etc. (n = 3 independent experiments); (D) qRT-PCR assay was used to detect the mRNA expression levels of Pol β and POSTN in hCASMCs after Pol β knockdown, and to verify the expression changes of matrix protein periostin (n = 3 independent experiments); (E) Western blotting assay was used to detect the expression of Pol β and periostin in hCASMCs, and quantitative statistics were performed on the data (n = 3 independent experiments); (F) qRT-PCR analysis of collagen expression levels in hCASMC cells in control group, Pol β knockdown group, and Pol β recovery group revealed the changes of collagen under different conditions (n = 3 independent experiments); (G)…

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  Figure 4. Pol β knockdown inhibits periostin expression by inhibiting YY1-TGFβ1: (A) mRNA expression levels of Pol β, POSTN, TGFβ1, Twist1, and YY1 in the control group and Pol β-knockdown hCASMC cells were detected by qRT-PCR, revealing the changes in expression of these genes after Pol β knockdown (n = 3 independent experiments); (B) Western blot detection of YY1 protein expression in hCASMC cells after Pol β knockdown, and quantitative statistical analysis of the results showing the regulatory relationship between Pol β and YY1 (n = 3 independent experiments); (C–G) Western blotting assay was used to detect protein expression of other related proteins in hCASMC cells, including key proteins in TGFβ1 and its downstream signaling pathway. Experiments showed the effect of Pol β knockdown on the expression of these proteins, and the results were statistically analyzed (n = 3 independent experiments); (H,I) Immunofluorescence assay was used to detect the expression of TGF-β1 and YY1 at the heart base of ApoE+/+Pol β+/+ mice, ApoE−/−Pol β+/+ mice, and ApoE−/−Pol β+/− mice, and to reveal the effect of Pol β knockdown on these markers in atherosclerosis. Scale bar, 500 μm (n = 6 mice,…

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  Figure 5. Schematic diagram showing our proposed mechanisms of Pol β deficiency inhibiting vascular smooth muscle cell migration through activating the YY1/TGF-β1/POSTN pathway.