Department of Clinical Neuroscience, Section of Ophthalmology and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden.
Sci Rep. 2017 Feb 10;7:42546. doi: 10.1038/srep42546.
Cellular responses to hypoxia are mediated by the hypoxia-inducible factors (HIF). In normoxia, HIF-α proteins are regulated by a family of dioxygenases, through prolyl and asparagyl hydroxylation, culminating in proteasomal degradation and transcriptional inactivation. In hypoxia, the dioxygenases become inactive and allow formation of HIF transcription factor, responsible for upregulation of hypoxia genes. In ocular neoangiogenic diseases, such as neovascular age-related macular degeneration (nAMD), hypoxia seems pivotal. Here, we investigate the effects of HIF regulatory proteins on the hypoxia pathway in retinal pigment epithelium (RPE) cells, critically involved in nAMD pathogenesis. Our data indicates that, in ARPE-19 cells, prolyl hydroxylase domain (PHD)2 is the most potent negative-regulator of the HIF pathway. The negative effects of PHD2 on the hypoxia pathway were associated with decreased HIF-1α protein levels, and concomitant decrease in angiogenic factors. ARPE-19 cells stably expressing PHD2 impaired angiogenesis in vitro by wound healing, tubulogenesis, and sprouting assays, as well as in vivo by iris-induced angiogenesis. Gene transfer of PHD2 in vivo resulted in mitigation of HIF-mediated angiogenesis in a mouse model of nAMD. These results may have implications for the clinical treatment of nAMD patients, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis.
细胞对缺氧的反应是由缺氧诱导因子 (HIF) 介导的。在常氧条件下，HIF-α 蛋白通过一组双氧酶进行调节，通过脯氨酰和天冬酰胺羟化作用，最终导致蛋白酶体降解和转录失活。在缺氧条件下，双氧酶失活，允许 HIF 转录因子形成，负责上调缺氧基因。在眼部新生血管疾病中，如新生血管性年龄相关性黄斑变性 (nAMD)，缺氧似乎是关键因素。在这里，我们研究了 HIF 调节蛋白对视网膜色素上皮 (RPE) 细胞中缺氧途径的影响，RPE 细胞在 nAMD 发病机制中起着关键作用。我们的数据表明，在 ARPE-19 细胞中，脯氨酰羟化酶结构域 (PHD)2 是 HIF 途径的最强负调节剂。PHD2 对缺氧途径的负作用与 HIF-1α 蛋白水平降低以及血管生成因子的相应减少有关。稳定表达 PHD2 的 ARPE-19 细胞通过划痕愈合、小管形成和发芽测定在体外以及通过虹膜诱导的血管生成在体内损害血管生成。体内的 PHD2 基因转移导致 nAMD 小鼠模型中 HIF 介导的血管生成减轻。这些结果可能对 nAMD 患者的临床治疗具有重要意义，特别是在使用基因治疗来负调控新生血管生成方面。
