Endothelial cells (ECs) may display distorted epigenetic features in diabetes mellitus (DM) but this has not been investigated under combined conditions of diabetes and limb ischemia. Initial screening for histone H3 and H4 trimethylation (me3) and panacetylation of lysine residues (causing transcriptional repression and activation) were conducted using two models of hyperglycemia and ischemia. Human umbilical vein ECs (HUVECs) were cultured in EGM2 or EBM2 media (low growth factors) supplemented with high D-glucose (30 mM, 2%FBS, 24 h) to mimic hyperglycemia and ischemia, respectively. Low glucose (5.5 mM)-treated controls were included. CD1 mice underwent streptozotocin-mediated induction of DM for 3months. Left femoral artery ligation was performed (under anaesthesia) and unilateral limb ischemia (LI) was induced for 3 days. Age-matched normoglycemic mice with LI were included. Ischemic and contra-lateral adductors were collected at cull, for protein and RNA analyses. In a separate cohort of mice, adductors were formalin fixed. Results are compared with controls using 1-way ANOVA or t-test. Culture in HG and/or low growth factors increased nuclear intensity of H3K27me3 but not H3K9me3, H3K4me3 or H4K20me3 in HUVECs, and decreased H3 panacetylation (P<0.01). In adductors, association of ischemia and/or DM increased both H3K27me3 and H3K9me3 (P<0.05), repressive modifications that accumulate at the promoter of endothelial eNOS in DM, and are likely to silence eNOS promoter under LI too. Ischemia alone, increased the expression of murine Suv39 h1 and EzH2 enzymes, which bring about above trimethylations (P=0.05), but diabetes has decreased their expression slightly. Combined condition had no effect on expression levels of these enzymes, but increased their predicted activity. Thus, distinct epigenetic landscape may be present in the adductors and associated vascular cells during ischemia in DM. Further studies are needed to better understand action and regulation of individual methyl/acetyl-writing enzymes at the promoters of key endothelial regulatory genes (eg. eNOS) under here investigated chronic conditions.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: Declaration of Funding: British Heart Foundation (CE and MM), European Foundation for the Study of Diabetes (TM).