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Acupuncture Promotes Angiogenesis after Myocardial Ischemia through H3K9 Acetylation Regulation at VEGF Gene |
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Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. |
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This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture. |
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RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. |
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Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. |
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Furthermore, increased H3K9 acetylation was also observed according with the VEGF. |
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ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter. |
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Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. |
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Pathway analysis indicated that these co-regulated genes were mainly involved in the calcium signaling pathway, cardiomyopathy related pathways, and cancer related pathways (Fig 4C and 4D), such as VEGF, an angiogenic factor, and VEGF-activated signaling component, including Ras, Akt and MAPK family members, decreased in the MI group, but significantly increased in the EA group (Table 1). |
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To verify that angiogenesis is a possible mechanism in the EA-induced myocardioprotective effect; angiogenic responses were examined by immunofluorescence staining of CD34, alpha-SMA and VEGF. |
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CD34-, alpha-SMA- and VEGF- positive cells were presented to the circulating endothelial progenitor cells (cEPCs), vascular smooth muscle cells, and endothelial cells, respectively. |
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Likewise, we also observed a significant increase in VEGF-positive staining after treatment with EA, whereas in the center and border regions of the MI heart VEGF protein level markedly decreased (P<0.01) (Fig.5A and 5B), suggesting that VEGF-induced angiogenesis is involved in the EA treatment. |
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Western blot results showed that VEGF protein expression was decreased obviously, accompanying with the decrease of Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt proteins in the MI group. |
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Surprisingly, in CEA group, the expressions of VEGF, phospho-p44/42 MAPK and phospho-p38 MAPK were also decreased, meanwhile phospho-SAPK/JNK and Akt expressions were significantly increased. |
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Elevation of VEGF are regulated directly by H3K9 acetylation |
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After we validated that VEGF was related to EA-induced angiogenesis, we further explored the mechanism underlying the EA-induced increase of VEGF expression. |
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Western blot results showed that acetylation level of H3K9, an epigenetic marker, was altered according to the expression level of VEGF. |
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The expression of H3K9ace in myocardium decreased in the MI group and the CEA group but increased significantly after EA treatment (P<0.01), which corresponded with the changes in VEGF (Fig.6B, 6D). |
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We measured H3K9ace chromatin occupancy at the VEGF promoter region in the infarct myocardial zone (Fig.7). |
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Our results indicated that H3K9ace occupancy at -500 bp to +200 bp region was augmented prominently by EA treatment (P<0.01), suggesting a direct modification of H3K9 acetylation at the VEGF gene. |
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EA at PC6 promoted angiogenesis in ischemic myocardium through activation of VEGF signaling |
