HYPERHOMOCYSTEINEMIA ACCELERATES THROMBOSIS THROUGH ICAM-1 DEPENDENT ENDOTHELIAL ACTIVATION AND DNA HYPOMETHYLATION

Abstract

Background: Hyperhomocysteinemia (HHcy) is an established risk factor for thrombotic diseases yet the underlying mechanism remain unclear. In this study we investigated the effect of HHcy on endothelial cell-platelet interaction and its role in thrombosis. Methods and Results: We used a novel mouse model of HHcy (plasma homocysteine, Hcy 80 micromolar) in which a Zn2+ inducible human cystathionine beta-synthase (CBS) transgene was introduced to circumvent the neonatal lethality of the Cbs gene deficiency (Tg-hCBS Cbs-/- mice). Hcy-lowering therapy was performed by giving ZnSO4 water to induce human CBS transgene expression in adult mice. Thrombus formation was examined by photo dye-induced cremaster microvasculature thrombosis using intravital microscopy, in which endothelium was preserved, and by FeCl3-induced carotid artery thrombosis, which denudated the endothelium. HHcy accelerated cremaster arteriolar thrombosis and decreased blood flow cessation time from 41.8 min in control mice to 30.5 min in TghCBS Cbs-/- mice. Venular blood flow cessation time was slightly decreased from 5.6 to 5.0 min. Hcy-lowering therapy reduced Hcy level from 80 micromolar to 6.8 micromolar after 2 weeks of ZnSO4 water and prolonged arteriolar blood cessation time from 30.5 to 37.8 min. Interestingly, FeCl3-induced carotid artery thrombosis did not change the occlusion time. Hcy did not potentiate the aggregation and secretion function in washed human platelets from healthy donor treated with Hcy (50, 100 micromolar) or from Tg-hCBS Cbs-/- mice. However, inter-cellular adhesion molecule 1 (ICAM-1) levels, but not vascular adhesion molecule 1 (VCAM-1), were increased in cremaster tissues from Tg-hCBS Cbs-/- mice by western blot. In cultured human umbilical vein ECs (HUVEC), Hcy (100 micromolar, 24h) promoted human platelet adhesion by 200% in static adhesion assay. Using western blot, FACS and RT-PCR, we found that Hcy increased protein and mRNA levels of ICAM-1, but not that of VCAM-1, in HUVEC. ICAM-1 blocking antibody partially reversed Hcy increased platelets adhesion to HUVEC. Hcy induced ICAM-1 expression and reduced DNA methylation on ICAM-1 promoter, which were mimicked by DNA methyltransferase inhibitor azacytidine, and histone deacetylase inhibitors sodium butyrate and trichostatin A. Hcy treatment also increased intracellular Hcy, Sadenosylhomocysteine (SAH) accumulation and decreased SAM/SAH ratio in HUVECs. Hcy decreased methyl CpG binding protein 2 (MeCP2) binding and increased acetylated histone H3 (AcH3) binding to ICAM-1 core promoter region using chromatin immunoprecipitation. Pyrosequencing of ICAM-1 core promoter and adjacent region shows a decreased DNA methylation by Hcy treatment. In high methionine diet-induce HHcy in WT and Icam-/- mice, Icam-/- mice fed with HM diet only show moderately accelerated venular and barely accelerated arteriolar occlusion time compared with WT mice with CT diet using photo dye-induced thrombosis model. Conclusion: HHcy accelerates arteriolar thrombosis and increases EC-platelet interaction via ICAM-1 induction partially through DNA hypomethylation.