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Endocrine Abstracts (2013) 31 P180 | DOI: 10.1530/endoabs.31.P180

SFEBES2013 Poster Presentations Obesity, diabetes, metabolism and cardiovascular (67 abstracts)

A component of transcriptional PRC2 complex, enhancer of zest homology, modulates endothelial cell responses to hypoxia and post-ischemic angiogenesis in a mouse model of limb ischemia

Tijana Mitic , Orchi Anannya & Costanza Emanueli

Bristol Heart Institute, University of Bristol, Bristol, UK.

Endothelial cells (ECs) have major role in post-ischemic angiogenesis. Trimethylation of lysine 27 of histone3 (H3K27me3) is a repressive epigenetic mark carried by EZH2 enzyme, the catalytic part of Polycomb complex2 (PRC2, also comprising the suppressor of zeste 12 homolog (Suz12)). We investigated if in vitro hypoxia and in vivo limb ischemia affect the expression of PRC2 in ECs and mouse limb muscles, respectively. Then, modulation of angiogenic genes by EZH2 and angiogenic responses were assessed using EZH2 inhibitor, 3-deazaneplanocin (DZNep).

Human umbilical vein ECs (HUVECs) were cultured under normoxia or hypoxia (1.2 %O2, 6–48 h), to mimic ischemia, and treated with DZNep (control: 1% DMSO) or transfected with siRNA against EZH2 (control: scramble oligos). Migration was assessed by scratch assay. The expressions of PRC2 and proangiogenic genes, eNOS, VEGF and VEGF-receptor2, were measured by qPCR. Chromatin-immunoprecipitation coupled with qPCR was performed for EZH2, Suz12 or H3K27me3 at promoters of aforementioned genes. Further, limb ischemia (LI) was used as a mouse model of in vivo angiogenesis. CD1 male mice (aged 15 weeks) received DZNep (1.5 mg/kg, i.p. every 2 days) or vehicle 1 day pre-LI. Post-ischemic blood flow (BF) recovery was assessed by colour laser doppler at 30 min and weekly thereafter for 3 weeks. Mice were culled and tissue was snap-frozen or formalin-fixed for analyses.

Hypoxia amplified levels of EZH2 and H3K27me3 in HUVECs. DZNep and EZH2 knock-down reduced expression of EZH2 but not Suz12, and improved migration of HUVECs. Same treatments reversed enrichment of EZH2 or H3K27me3, thus unlocking the expression of above proangiogenic genes. LI has increased the expression of EZH2 and levels of H3K27me3; which were reversed by DZNep. Post-ischaemic BF recovery (weeks 1–3) was increased (P<0.01) due to DZNep in line with increased capillary density in the LI-muscles. Therefore, inhibition of EZH2 promotes angiogenesis in both ECs and LI-muscles by enhancing the expression of pro-angiogenic genes.

Declaration of funding: This work was supported by the Society for Endocrinology Early Career Grant 2012, the European Federation for the Study of Diabetes and the FP7 RESOLVE project grant.

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