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EZH2 modulates angiogenesis in vitro and in a mouse model of limb ischemia.

ABSTRACT: Epigenetic mechanisms may regulate the expression of pro-angiogenic genes, thus affecting reparative angiogenesis in ischemic limbs. The enhancer of zest homolog-2 (EZH2) induces thtrimethylation of lysine 27 on histone H3 (H3K27me3), which represses gene transcription. We explored (i) if EZH2 expression is regulated by hypoxia and ischemia; (ii) the impact of EZH2 on the expression of two pro-angiogenic genes: eNOS and BDNF; (iii) the functional effect of EZH2 inhibition on cultured endothelial cells (ECs); (iv) the therapeutic potential of EZH2 inhibition in a mouse model of limb ischemia (LI). EZH2 expression was increased in cultured ECs exposed to hypoxia (control: normoxia) and in ECs extracted from mouse ischemic limb muscles (control: absence of ischemia). EZH2 increased the H3K27me3 abundance onto regulatory regions of eNOS and BDNF promoters. In vitro RNA silencing or pharmacological inhibition by 3-deazaneplanocin (DZNep) of EZH2 increased eNOS and BDNF mRNA and protein levels and enhanced functional capacities (migration, angiogenesis) of ECs under either normoxia or hypoxia. In mice with experimentally induced LI, DZNep increased angiogenesis in ischaemic muscles, the circulating levels of pro-angiogenic hematopoietic cells and blood flow recovery. Targeting EZH2 for inhibition may open new therapeutic avenues for patients with limb ischemia.

SUBMITTER: Mitic T

PROVIDER: S-EPMC4426795 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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EZH2 modulates angiogenesis in vitro and in a mouse model of limb ischemia.

Mitić Tijana T Caporali Andrea A Floris Ilaria I Meloni Marco M Marchetti Micol M Urrutia Raul R Angelini Gianni D GD Emanueli Costanza C

Molecular therapy : the journal of the American Society of Gene Therapy 20140905 1

Epigenetic mechanisms may regulate the expression of pro-angiogenic genes, thus affecting reparative angiogenesis in ischemic limbs. The enhancer of zest homolog-2 (EZH2) induces thtrimethylation of lysine 27 on histone H3 (H3K27me3), which represses gene transcription. We explored (i) if EZH2 expression is regulated by hypoxia and ischemia; (ii) the impact of EZH2 on the expression of two pro-angiogenic genes: eNOS and BDNF; (iii) the functional effect of EZH2 inhibition on cultured endothelial ...[more]

PMID: 25189741

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Project description:BackgroundPrevious work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model.MethodsWild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry.ResultsBy week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1? and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 ? compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO.ConclusionSimilar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.

Project description:Older patients are thought to tolerate acute ischemia more poorly than younger patients. Since aging may depress both angiogenesis and arteriogenesis, we determined the effects of age on both angiogenesis and arteriogenesis in a model of severe acute limb ischemia.Young adult (3-months-old) and aged (18-months-old) C57BL/6 mice underwent right common iliac artery and vein ligation and transection. Data were collected on days 0, 7, and 14. Perfusion was measured with a laser Doppler scan and compared to the contralateral limb. Functional deficits were evaluated with the Tarlov scale. Capillary density and endothelial progenitor cell (EPC) number were determined by direct counting lectin-positive/alpha-actin-negative cells and VEGFR2/CXCR4 dually-positive cells, respectively; angiography was performed to directly assess arteriogenesis.Young adult and aged mice had a similar degree of decreased perfusion after iliac ligation (young, n = 15: 20.4 +/- 1.9%, vs aged, n = 20: 19.6 +/- 1.3%; P = .72, analysis of variance [ANOVA]); however, young mice recovered faster and to a greater degree than aged mice (day 7, 35 +/- 6% vs 17 +/- 4%, P = .046; day 14, 60 +/- 5% vs 27 +/- 7%, P = .0014). Aged mice had worse functional recovery by day 14 compared to young mice (2.3 +/- 0.3 vs 4.3 +/- 0.4; P = .0021). Aged mice had increased capillary density (day 7, 12.9 +/- 4.4 vs 2.8 +/- 0.3 capillaries/hpf; P = .02) and increased number of EPC incorporated into the ischemic muscle (day 7, 8.1 +/- 0.9 vs 2.5 +/- 1.9 cells; P = .007) compared to young mice, but diminished numbers of collateral vessels to the ischemic limb (1 vs 9; P = .01), as seen on angiography.After severe hind limb ischemia, aged animals become ischemic to a similar degree as young animals, but aged animals have significantly impaired arteriogenesis and functional recovery compared to younger animals. These results suggest that strategies to stimulate arteriogenesis may complement those that increase angiogenesis, and may result in improved relief of ischemia.

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EZH2 modulates angiogenesis in vitro and in a mouse model of limb ischemia.

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EZH2 modulates angiogenesis in vitro and in a mouse model of limb ischemia.

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