Abstract WP551: Senescence-Induced Methylation Changes in Global DNA and Atherosclerosis-Related Genes in Human Vascular Endothelial Cells

Abstract

Background: Endothelial senescence has been known as a pathophysiological alteration related to atherosclerosis development. We investigated whether methylation changes in global DNA and atherosclerosis-related genes are developed in in-vitro senescence of human vascular endothelial cells. Methods: We induced in-vitro replicative senescence by subculturing 5 human umbilical vein endothelial cell (HUVEC) lines from three to fifteen passages. Using DNA extracted from the 5th, 10th, and 15th passages of the 5 HUVECs, we evaluated LINE1 methylation for global methylation status as well as promoter methylation status for 5 genes ( AIRE1, ALOX12, FANK1, NETO1 , and SERHL2 ) profiled from carotid endarterectomy plaques for gene-specific methylation evaluations. Differences in LINE1 methylations and promoter methylations of the 5 genes were evaluated using pyrosequencing and compared among the 5th, 10th, and 15th passages of the 5 HUVECs using analysis of variance (ANOVA) tests. Results: LINE1 global methylation subsequently decreased as passages were advanced from 5 to 15 (p<0.001). On gene-specific promoter methylation analysis, ALOX12 (p<0.001) and SERHL2 (p<0.001) showed significant passage-dependent increase of promoter methylation. AIRE1 and NETO1 also showed an increase of promoter methylation as passages increased, but without statistical significance. FANK1 exhibited an increase-decrease pattern of methylation changes showing an increase of methylation at passage 10 compared to passage 5, but showing a decrease in passage 15 (p=0.005). Conclusion: After replicative senescence for vascular endothelial cells, global methylation subsequently decreased. However, atherosclerosis-related genes showed either a increase or an increase-decrease of promoter methylations. In particular, the increase of ALOX12 methylation showed as a possible epigenetic marker related to vascular endothelial cell senescence.