背景:缺血性疾病是全球卫生保健的主要负担。血管生成在缺血性疾病中的血流恢复和损伤组织的修复中至关重要。十-十一易位蛋白2(TET2),DNA去甲基酶的成员,参与了许多病理过程。然而,TET2在血管生成中的作用仍未被揭示.
方法:从参与5-羟甲基胞嘧啶(5-hmC)调节的三种DNA去甲基酶中筛选出TET2,包括TET1、TET2和TET3。通过小干扰RNA的敲除和腺病毒的过表达来评估TET2对内皮细胞功能的作用。在内皮细胞特异性TET2缺陷小鼠中分析血流恢复和毛细血管密度。RNA测序用于鉴定缺氧下TET2介导的机制。免疫共沉淀(Co-IP),进一步进行染色质免疫沉淀qPCR(ChIP-qPCR)和糖基化羟甲基敏感qPCR(GluMS-qPCR)以揭示TET2和STAT3的相互作用。
结果:TET2在缺氧条件下在内皮细胞中显著下调,并导致5-hmC水平整体降低。TET2敲低加重缺氧诱导的内皮细胞功能障碍,而TET2过表达减轻了缺氧诱导的功能障碍。同时,在后肢缺血小鼠模型中,内皮细胞中TET2的缺乏会损害血流恢复和毛细血管密度。机械上,RNA测序表明STAT3信号通路被TET2敲低显著抑制。此外,共同IP,ChIP-qPCR和GluMS-qPCR进一步说明STAT3募集并与TET2物理相互作用以激活STAT3靶基因。不出所料,体外STAT3沉默完全抑制了TET2过表达的影响。
结论:我们的研究表明,内皮细胞中TET2的缺乏通过抑制STAT3信号通路损害血管生成。这些发现为TET2是缺血性疾病的治疗替代品提供了坚实的证据。
BACKGROUND: Ischemic diseases represent a major global health care burden. Angiogenesis is critical in recovery of blood flow and repair of injured tissue in ischemic diseases. Ten-eleven translocation protein 2 (TET2), a member of DNA demethylases, is involved in many pathological processes. However, the role of TET2 in angiogenesis is still unrevealed.
METHODS: TET2 was screened out from three DNA demethylases involved in 5-hydroxylmethylcytosine (5-hmC) regulation, including TET1, TET2 and TET3. Knockdown by small interfering RNAs and overexpression by adenovirus were used to evaluate the role of TET2 on the function of endothelial cells. The blood flow recovery and density of capillary were analyzed in the endothelial cells-specific TET2-deficient mice. RNA sequencing was used to identify the TET2-mediated mechanisms under hypoxia. Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation-qPCR (ChIP-qPCR) and glucosylated hydroxymethyl-sensitive-qPCR (GluMS-qPCR) were further performed to reveal the interaction of TET2 and STAT3.
RESULTS: TET2 was significantly downregulated in endothelial cells under hypoxia and led to a global decrease of 5-hmC level. TET2 knockdown aggravated the hypoxia-induced dysfunction of endothelial cells, while TET2 overexpression alleviated the hypoxia-induced dysfunction. Meanwhile, the deficiency of TET2 in endothelial cells impaired blood flow recovery and the density of capillary in the mouse model of hindlimb ischemia. Mechanistically, RNA sequencing indicated that the STAT3 signaling pathway was significantly inhibited by TET2 knockdown. Additionally, Co-IP, ChIP-qPCR and GluMS-qPCR further illustrated that STAT3 recruited and physically interacted with TET2 to activate STAT3 target genes. As expected, the effects of TET2 overexpression were completely suppressed by STAT3 silencing in vitro.
CONCLUSIONS: Our study suggests that the deficiency of TET2 in endothelial cells impairs angiogenesis via suppression of the STAT3 signaling pathway. These findings give solid evidence for TET2 to be a therapeutic alternative for ischemic diseases.