A Novel HIF-2α/ARNT Signaling Pathway Protects Against Microvascular Dysfunction and heart failure After Myocardial Infarction

Experiments were conducted with mice carrying an inducible EC-specific Hif2α-knockout (ecHif2α–/–) mutation, with mouse cardiac microvascular endothelial cells (CMVECs) isolated from the hearts of ecHif2α–/– mice after the mutation was induced, and with human CMVECs and umbilical-vein endothelial cells transfected with ecHif2α siRNA. After MI induction, echocardiographic assessments of cardiac function were significantly lower, while measures of cardiac microvascular leakage (Evans blue assay), plasma IL6 levels, and cardiac neutrophil accumulation and fibrosis (histology) were significantly greater, in ecHif2α–/– mice than in control mice, and RNA-sequencing analysis of heart tissues from both groups indicated that the expression of genes involved in vascular permeability and collagen synthesis was enriched in ecHif2α–/– hearts. In cultured ECs, ecHif2α deficiency was associated with declines in endothelial barrier function (electrical cell impedance assay) and the reduced abundance of tight-junction proteins, as well as an increase in the expression of inflammatory markers, all of which were largely reversed by the overexpression of ARNT. We also found that ARNT, but not Hif2α, binds directly to the IL6 promoter and suppresses IL6 expression. To assess the functional role of HIF2α in ischemic heart injury, we ligated the proximal left anterior descending coronary artery to induce MI in tamoxifen inducible adult ecHIF2α-/- and control (Hif2aflox/flox) mice. Total RNA from controls and Hif2α-/- heart tissues was isolated 28 days after MI using RNeasy Fibrous Tissue Mini Kit (74704, Qiagen, MD) according to the manufacturer’s protocol.

本研究使用携带诱导型内皮细胞（endothelial cell, EC）特异性Hif2α敲除（ecHif2α–/–）突变的小鼠，在突变诱导后从ecHif2α–/–小鼠心脏中分离得到小鼠心脏微血管内皮细胞（cardiac microvascular endothelial cells, CMVECs），同时采用转染了ecHif2α小干扰RNA（siRNA）的人CMVECs与脐静脉内皮细胞开展实验；心肌梗死（myocardial infarction, MI）造模后，与对照组小鼠相比，ecHif2α–/–小鼠的心脏功能超声心动图评估结果显著降低，而心脏微血管渗漏（伊文思蓝染色实验）、血浆IL6水平、心脏中性粒细胞浸润与纤维化（组织学检测）相关指标均显著升高，对两组小鼠心脏组织的RNA测序分析显示，ecHif2α–/–小鼠心脏中参与血管通透性与胶原合成的基因表达显著富集；在体外培养的内皮细胞中，ecHif2α缺失会导致内皮屏障功能（细胞电阻抗检测）下降、紧密连接蛋白丰度降低，并伴随炎症标志物表达上调，上述表型均可通过过表达ARNT（芳香烃受体核转位蛋白）得到显著逆转，本研究同时发现，ARNT而非Hif2α可直接结合IL6启动子并抑制IL6的表达；为评估HIF2α在缺血性心脏损伤中的功能作用，本研究对他莫昔芬诱导型成年ecHIF2α–/–小鼠与对照（Hif2aflox/flox）小鼠结扎近端左前降支冠状动脉以构建心肌梗死模型，于心肌梗死造模后28天，按照试剂盒制造商的操作说明书，使用RNeasy Fibrous Tissue Mini Kit（货号74704，美国马里兰州Qiagen公司）提取对照组与Hif2α–/–小鼠的心脏组织总RNA。