Abstract 12890: Epigenetic Modulation of Endothelial Cell Responses to Hypoxia and Post-ischemic Angiogenesis by the Component of Polycomb Repressive Complex 2 (PRC2), Enhancer of Zest Homology (EZH2)
The enhancer of zest homology (EZH2) is the key enzymatic component of polycomb complex 2 (PRC2) along with the suppressor of zest 12 homolog (SUZ12). EZH2 induces the trimethylation of lysine 27 of histone3 (H3K27me3), which epigenetically represses gene expression. Here, we investigated whether EZH2 regulates endothelial cell (EC) responses to in vitro hypoxia and post-ischemic angiogenesis in a mouse model of unilateral limb ischemia (LI). Human umbilical vein ECs (HUVECs) were cultured under normoxia or hypoxia (1.2%O2, 6-48h). The RNA and protein expression of PCR2 components were measured by q-RT-PCR and western blots. EZH2 expression was inhibited by transfecting HUVECs with silencing RNA (control: scramble oligos). To inhibit EZH2 enzymatic activity, 3-deazaneplanocin A (DZNep, 5μM; control: 0.01% DMSO) was used and HUVEC function was studied by scratch and Matrigel assays. The expression levels of target proangiogenic and endothelial genes: eNOS, brain-derived neurotrophic factor (BDNF), VEGF and VEGF-receptor 2 (KDR) were measured by qRT-PCR. Chromatin-immunoprecipitation (ChIP) for PRC2 and H3K27me3 was coupled with qPCR to amplify 50-200bp promoter regions of the aforementioned genes. Further, DZNep (1.5mg/kg, i.p. every 2days since the day before surgery) or vehicle was delivered to CD1 mice with experimentally induced LI. Blood flow (BF) recovery was assessed by sequential colour laser Doppler analyzes. Mice were culled at 7 and 21 day post-LI; tissues were snap-frozen or formalin-fixed for molecular biology and histology analyses. Hypoxia increased EZH2 and H3K27me3 but not SUZ12 levels in HUVECs. Both EZH2 knock-down and DZNep reduced PRC2 expression and improved HUVEC functions. The same treatments decreased PRC2 and H3K27me3 levels at the promoters of eNOS and BDNF, but not of VEGF or KDR. LI increased EZH2 and H3K27me3 levels in the limb muscles, which was prevented by DZNep. DZnep improved the post-LI BF recovery and increased the capillary density in ischemic muscles. In summary, EZH2 inhibition enhances the expression of pro-angiogenic genes and improves the angiogenic capacity of ECs under hypoxia and LI. EZH2 is a novel therapeutic target in the setting of ischemic diseases.
- © 2013 by American Heart Association, Inc.