Endocrine Abstracts

Objectives: Endothelial cells (ECs) have major role in post-ischemic angiogenesis. EZH2 (enhancer of zest homology) carries out trimethylation of lysine 27 on histone3 (H3K27me³) – repressive mark, thus modulating gene expression. We tested if the EZH2 inhibitor 3-deazaneplanocin (DZNep) regulates angiogenesis under hypoxia and limb ischemia (LI) in vivo in ECs and mouse limb muscles respectively.

Methods: Human umbilical vein ECs (HUVECs) were cultured in hypoxia (1.2% O₂, 6–48 h), to mimic ischemia, and treated with DZNep (ctr: 1% DMSO) or transfected with siEZH2 (ctr: scramble oligos). Migration was assessed by scratch assay, and network formation by Matrigel. Levels of eNOS and BDNF were measured by qPCR and western blot and ELISA respectively. Chromatin-immunoprecipitation of EZH2 and H3K27me³ at eNOS and BDNF promoters was coupled with qPCR. Further, limb ischemia was used as a mouse model of in vivo angiogenesis. CD1 male mice (12 weeks) received DZNep (1.5 mg/kg per 2 days) or vehicle 1 day pre-LI. Post-ischemic blood flow (BF) recovery was assessed by colour laser Doppler. Muscle tissue was collected for immunohistochemistry and for sorting endothelial like CD146⁺ cells to measure mRNA expression. We also tested whether DZNep influences mobilisation of bone marrow progenitor cells (based on target protein expression). Blood and bone marrow samples were collected for FACS analyses.

Results and conclusions: Hypoxia and ischemia increased levels of EZH2 and H3K27me³ and their enrichment at eNOS and BDNF promoter, while reducing eNOS expression. DZNep/siEZH2 treatment has reversed these effects by increasing the levels of eNOS and BDNF. In addition, inhibition of EZH2 improved HUVEC migration and network formation. Post-ischemic BF recovery (weeks 1–3) has significantly increased, due to DZNep, and increased the capillary density in the LI muscles. DZNep promotes angiogenesis by enhancing eNOS and BDNF expression in endothelial-like cells CD146⁺ of ischemic muscles. This is mediated through activation of pro-angiogenic hematopoietic cell in the bone marrow like Sca-1⁺CD11b⁺ and Lin- c-kit⁺.