Shi Yefei, Li Bo, Huang Xinru, Kou Wenxin, Zhai Ming, Zeng Yanxi, You Shuangjie, Yu Qing, Zhao Yifan, Zhuang Jianhui, Peng Wenhui, Jian Weixia
Department of Cardiology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Middle Yanchang Road, Shanghai, 200072, China.
Department of Endocrinology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai, 200092, China.
Cell Biosci. 2023 Jan 19;13(1):12. doi: 10.1186/s13578-023-00960-5.
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.
背景：缺血性疾病是全球主要的医疗负担。血管生成对于缺血性疾病中血流恢复和受损组织修复至关重要。10-11易位蛋白2（TET2）是DNA去甲基化酶家族成员，参与多种病理过程。然而，TET2在血管生成中的作用仍未明确。 方法：从参与5-羟甲基胞嘧啶（5-hmC）调控的三种DNA去甲基化酶（包括TET1、TET2和TET3）中筛选出TET2。采用小干扰RNA敲低和腺病毒过表达来评估TET2对内皮细胞功能的作用。分析内皮细胞特异性TET2缺陷小鼠的血流恢复情况和毛细血管密度。利用RNA测序确定缺氧条件下TET2介导的机制。进一步进行免疫共沉淀（Co-IP）、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与信号转导和转录激活因子3（STAT3）的相互作用。 结果：缺氧条件下内皮细胞中TET2显著下调，并导致5-hmC水平整体下降。TET2敲低加剧了缺氧诱导的内皮细胞功能障碍，而TET2过表达则减轻了缺氧诱导的功能障碍。同时，内皮细胞中TET2缺陷损害了后肢缺血小鼠模型的血流恢复和毛细血管密度。机制上，RNA测序表明TET2敲低显著抑制了STAT3信号通路。此外，Co-IP、ChIP-qPCR和GluMS-qPCR进一步表明STAT3招募并与TET2直接相互作用以激活STAT3靶基因。正如预期的那样，在体外，STAT3沉默完全抑制了TET2过表达的作用。 结论：我们的研究表明，内皮细胞中TET2缺陷通过抑制STAT3信号通路损害血管生成。这些发现为TET2成为缺血性疾病的治疗选择提供了确凿证据。
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