Endothelial cell (EC) injury accelerates the progression of diabetic macrovascular complications. Hypoxia is an important cause of EC injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is an important hypoxia regulatory protein. Our previous studies showed that high-glucose and hypoxic conditions could upregulate HIF-1α expression and enhance EC inflammatory injury, independently of the nuclear factor kappa-B (NF-κB) pathway. However, it is not clear whether HIF-1α plays a role in vascular disease through epigenetic-related mechanisms. We conducted gene expression analysis and molecular mechanistic studies in human umbilical vein endothelial cells (HUVECs) induced by hyperglycemia and hypoxia using RNA sequencing (RNA-seq) and small interfering HIF-1α (si-HIF-1α). We determined HIF-1α and Jumonji domain-containing protein 1 A (JMJD1A) expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, analyzed inflammatory protein secretion in the cell supernatant by enzymelinked immunosorbent assay (ELISA), and assessed protein interaction between HIF-1α and JMJD1A by chromatin immunoprecipitation (Ch-IP). We used the Cell Counting Kit8 (CCK-8) assay to analyze cell viability, and assessed oxidative stress indicators by using a detection kit and flow cytometry. High glucose and hypoxia up-regulated HIF-1α expression, and down-regulated HIF-1α decreased the level of inflammation and oxidative stress in HUVECs. To determine the downstream pathways, we observed histone demethylases genes and related pathway by RNA-sEq. Among these, JMJD1A was the most upregulated gene in histone demethylases. Moreover, we observed that HIF-1α bound to the promoter of JMJD1A, and the ameliorative effects of si-HIF-1α on oxidative stress and inflammatory cytokines in high-glucose and hypoxia-induced HUVECs were reversed by JMJD1A overexpression. Furthermore, knockdown of JMJD1A decreased inflammatory and oxidative stress injury. To determine the JMJD1A-related factors, we conducted gene expression analysis on JMJD1A-knockdown HUVECs. We observed that downregulation of inflammation and the oxidative stress pathway were enriched and FOS and FOSB might be important protective transcription factors. These findings provide novel evidence that the HIF-1α/JMJD1A signaling pathway is involved in inflammation and oxidative stress in HUVECs induced by high glucose and hypoxia. Also, this pathway might act as a novel regulator of oxidative stress and inflammatory-related events in response to diabetic vascular injury and thus contribute to the pathological progression of diabetes and vascular disease.

中文翻译：

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HIF-1α/JMJD1A 信号调节高血糖和缺氧诱导的血管细胞损伤后的炎症和氧化应激

内皮细胞 (EC) 损伤加速了糖尿病大血管并发症的进展。缺氧是EC损伤的重要原因。缺氧诱导因子-1α（HIF-1α）是一种重要的缺氧调节蛋白。我们之前的研究表明，高葡萄糖和缺氧条件可以上调 HIF-1α 表达并增强 EC 炎症损伤，与核因子 kappa-B (NF-κB) 通路无关。然而，尚不清楚 HIF-1α 是否通过表观遗传相关机制在血管疾病中发挥作用。我们使用 RNA 测序 (RNA-seq) 和小干扰 HIF-1α (si-HIF-1α) 对高血糖和缺氧诱导的人脐静脉内皮细胞 (HUVEC) 进行基因表达分析和分子机制研究。我们通过定量逆转录聚合酶链反应 (qRT-PCR) 和蛋白质印迹测定 HIF-1α 和含有 Jumonji 结构域的蛋白 1 A (JMJD1A) 表达，通过酶联免疫吸附试验 (ELISA) 分析细胞上清液中的炎症蛋白分泌，并通过染色质免疫沉淀 (Ch-IP) 评估 HIF-1α 和 JMJD1A 之间的蛋白质相互作用。我们使用 Cell Counting Kit8 (CCK-8) 测定法分析细胞活力，并使用检测试剂盒和流式细胞仪评估氧化应激指标。高糖和缺氧上调 HIF-1α 表达，下调 HIF-1α 降低 HUVECs 的炎症和氧化应激水平。为了确定下游通路，我们通过 RNA-sEq 观察了组蛋白去甲基化酶基因和相关通路。在这些当中，JMJD1A 是组蛋白去甲基化酶中上调最多的基因。此外，我们观察到 HIF-1α 与 JMJD1A 的启动子结合，并且 si-HIF-1α 对高糖和缺氧诱导的 HUVEC 中氧化应激和炎性细胞因子的改善作用被 JMJD1A 过表达逆转。此外，JMJD1A 的敲低减少了炎症和氧化应激损伤。为了确定 JMJD1A 相关因素，我们对 JMJD1A 敲低 HUVEC 进行了基因表达分析。我们观察到炎症和氧化应激途径的下调被富集，FOS 和 FOSB 可能是重要的保护性转录因子。这些发现提供了新的证据，表明 HIF-1α/JMJD1A 信号通路参与了高糖和缺氧诱导的 HUVEC 中的炎症和氧化应激。还，