Effects And Mechanisms Of JMJD2A On Neointimal Hyperglycemia In Diabetic Rat Artery

Objectives:1. To explore the potential effect and underlying mechanisms of demethylases JMJD2A inhibition on vascular smooth muscle cells (VSMCs) proliferation and migration induced by high glucose (HG).2. To examine the proliferative, migratory, tube forming and apoptotic behavior as well as the underlying signaling molecules of human umbilical vein endothelial cell (HUVECs) in response to high glucose after JMJD2A inhibition.3. To evaluate the role of JMJD2A inhibition in neointimal hyperplasia and reendothelialization in diabetic rat carotid artery after balloon injury.Methods:1. The primary VSMCs were cultured under normal glucose condition (NG). After stimulation by HG (25mM) for 0-24h, JMJD2A and H3K9me3 were detected with Western blot. JMJD2A inhibition was achieved by the chemical inhibitor 2,4-pyridinedicarboxylic acid (2,4-PDCA) and small interfering RNA (siRNA). We investigate the proliferation, migration and the expression of inflammatory genes (MCP-1 and IL-6) of VSMCs in response to JMJD2A inhibition plus HG stimulation. Chromatin Immunoprecipitation (ChIP) was conducted to detect modification of histone H3 lysine 9 trimethylation (H3K9me3) at inflammatory genes promoters.2. The expression changes of JMJD2A and H3K9me3 in HUVECs stimulated with HG for 0-48h were detected firstly. After JMJD2A inhibition by 2,4-PDCA and siRNA, we examine the proliferative, migratory and apoptotic capacities of HUVECs in response to HG, as well as the expression of TNF-? and apoptosis-associated genes. ChIP assay was conducted to examine the modification of H3K9me3 at related genes' promoters.3. Diabetic rats induced using high-fat diet plus intraperitoneal injection of low-dose streptozotocin (35mg/kg) endure carotid artery balloon injury. All experimental animals were randomly received the injection of 2,4-PDCA (7.5mg/kg/d,15 mg/kg/d, 30 mg/kg/d) or saline, or transfection of siRNAJMJD2A?siRNASCR.The expression of JMJD2A, H3K9me3, MCP-1, IL-6 and TNF-? were detected. Morphometric analysis was performed using hematein eosin staining and immumohistochemical staining for the examination of neointimal hyperplasia and reendothelialization.Results:1. The global JMJD2A was increased in HG-stimulated VSMCs, accompanied by decreased H3K9me3. Compared with NG, HG stimulated the proliferation and migration of VSMCs significantly, and promoted the expression of MCP-1 and IL-6. Such the induced proliferation and migration were repressed by the JMJD2A inhibition. Moreover JMJD2A inhibition promoted the restoration of H3K9me3 levels at the promoters of MCP-1 and IL-6, and then inhibited the MCP-1 and IL-6 expression.2. HG encouraged the protein expression of JMJD2A in HUVECs, whereas discouraged H3K9me3. The restraint of proliferation and migration in HUVECs cultured under HG were increased by JMJD2A inhibition, whereas the raised apoptosis was prevented markedly. In addition, JMJD2A inhibition suppressed the expression of TNF-a via restoring H3K9me3 in the proximal promoter region. Corresponding decreased changes in expression of apoptosis-associated genes were observed after JMJD2A inhibition.3. Compared with normal rats, JMJD2A was elevated in carotid artery of diabetic rats? After balloon injury, higher level of JMJD2A was detected in carotid artery of diabetic rats, accompanied by decreased H3K9me3.7 days after injury, JMJD2A inhibition suppress MCP-1, IL-6 and TNF-a both in mRNA and protein. Compared to the control group, arteries displayed reduced neointimal formation and reduced percentage of PCNA-positive cells in the neointima at 28 days after injury in groups of JMJD2A inhibition. H3K9me3 level was regulated by JMJD2A inbition in an opposite direction. Moreover JMJD2A inhibition groups showed more extensive levels of reendothelialization.Conclusions:1. The JMJD2A inhibition significantly suppress the HG-induced proliferation and migration of VSMCs via the down-regulation of MCP-1 and IL-6 by restoring H3K9me3 at their promoters.2. The JMJD2A inhibition promote HUVECs proliferation and migration which were suffered under HG condition. The molecular mechanism may be partly ascribed to downregulation of TNF-a via restoring H3K9me3 in the proximal promoter region and the suppressed apoptosis.3. The inhibition of MJD2A attenuates neointimal hyperplasia and promotes reendothelialization after balloon injury in diabetic rat carotid artery. It introduces positive effects in vascular remodeling due to the repressed VSMCs proliferation and ECs apoptosis.