Role Of Endothelial Nitric-oxide Synthase And Its Epigenetic Changes In Metabolic Memory Of Hyperglycemia

Aim Metabolic memory has been widely reported in diabetic cardiovascular complications. Here, we investigate the role of reactive oxygen species (ROS) derived from endothelial nitric oxide synthase (eNOS) in this phenomenon,.Methods and Results In human aortic endothelial cells (HAECs) and aortas of diabetic rats, the elevation of pro-inflammatory genes MCP-1and VACM-1and potent vasoconstrictive gene ET-1as well as reduction of nitric oxide (NO) bioavailability induced by hyperglycemia, persisted even after normalization of glucose level. Inhibition of NOXs with DPI or apocynin, or eNOS with L-NAME in HAECs returning to normoglycemia could interrupt reactive ROS mediated upregulation of MCP-1, VACM-1and ET-1and partially restored NO bioavailability. Further, consistent NADPH oxidase activation and upregulation of Nox4and p22phox were associated with continuous increment of ROS production. The active phosphorylation of eNOS at Ser-1177was decreased by hyperglycemia, and this alternation persisted even after glycemia normalization, making eNOS a contributor to ROS instead of NO. In addition, the persistent elevation of dysfuntional eNOS was also detected.Conclusion In the current study, we provided evidences that vascular ROS generated by NADPH oxidase and eNOS were implicated in cellular metabolic memory induced by high glucose. Objective:Epigenetic changes are implicated in the formation of metabolic memory of hyperglycemia. Our prior study has shown that transient hyperglycemia induced persistent up-regulation of eNOS gene expression, even after glucose level normalized. Therefore, we hypothesize that transient hyperglycemia induced persistent epigenetic changes in eNOS gene resulting in sustained increase of eNOS gene expression. Here, we investigate the epigenetic changes in eNOS gene promoter of HAECs exposed to prior hyperglycemia.Methods:HAECs were incubated in5.5or in30mmol/1glucose for4days or subjected to3days of normal glucose after being exposed to high glucose for24hours. HAECs were also transfected with Set7ShRNA or Set7over-expression plasmid to in vitro knockdown Set7or over-express Set7in HAECs. CHIP assay was performed to investigate the epigenetic modification of eNOS gene promoter region in HAECs.Results:the sustained up-regulation of the eNOS gene as a result of prior hyperglycemia was associated with increased H3K4ml but not H3K4m2or H3K4m3. Furthermore, the increased H3K4ml modification on eNOS promoter was mediated by methyl-transferase Set7. Finally, glucose was also shown to have other epigenetic effects, including the suppression of H3K9m2and H3K9m3methylation on the eNOS promoter,Conclusions:This study indicate that prior hyperglycemia induced active transcriptional state of the eNOS gene is linked with persisting epigenetic marks such as enhanced H3K4and reduced H3K9methylation. Furthermore, Set7mediates the increased H3K4ml modification on eNOS promoter.