Department of Molecular Medical Research, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
Circulation. 2010 Aug 31;122(9):910-9. doi: 10.1161/CIRCULATIONAHA.109.931931. Epub 2010 Aug 16.
We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2alpha (HIF2alpha) activity. Small interfering RNA (siRNA)-Int6 adequately stabilized HIF2alpha, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo.
We demonstrated a 6- to 8-fold enhanced formation of normal arteries and veins in the subcutaneous regions of adult mice 5 days after a single siRNA-Int6 application. Subcutaneous fibroblasts were identified as the major source of secreted angiogenic factors that led to the formation of functional vessels during Int6 silencing. Fibroblasts transfected ex vivo with siRNA-Int6 induced potent neoangiogenesis when transplanted into a subcutaneous region of nude mice. Application of siRNA-Int6 promoted neoangiogenesis in the area surrounding the injury in wound healing models, including genetically diabetic mice, thereby accelerating the closure of the injury. HIF2alpha accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of the Int6 silencing-induced neoangiogenesis revealed that a negative feedback regulation of HIF2alpha stability was caused by HIF2alpha-induced transcription of Int6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2alpha protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis.
We suggest that the pathway involving INT6/HIF2alpha acts as a hypoxia-independent master switch of functional angiogenesis; therefore, siRNA-Int6 application might be of clinical value in treating ischemic diseases such as heart and brain ischemia, skin injury, and diseases involving obstructed vessels.
我们之前发现 INT6/eIF3e 是一种新型的低氧诱导因子 2α（HIF2α）活性调节剂。小干扰 RNA（siRNA）-Int6 在常氧条件下充分稳定 HIF2α，从而增强了几种血管生成因子的体外表达，这表明 siRNA-Int6 可能在体内诱导血管生成。
我们在单次 siRNA-Int6 应用后 5 天，在成年小鼠的皮下区域观察到正常动脉和静脉的形成增加了 6-8 倍。皮下成纤维细胞被鉴定为主要的分泌血管生成因子的来源，这些因子在 Int6 沉默期间导致功能性血管的形成。体外转染 siRNA-Int6 的成纤维细胞在裸鼠皮下区域移植时诱导强烈的新血管生成。siRNA-Int6 的应用促进了创伤愈合模型中损伤周围的新血管生成，包括遗传性糖尿病小鼠，从而加速了损伤的闭合。siRNA-Int6 引起的 HIF2α 积累被体内血管生成试验确认为血管生成的明确原因。对 Int6 沉默诱导的新血管生成的进一步分析表明，HIF2α 诱导的 Int6 转录通过其启动子中的缺氧反应元件对 HIF2α 稳定性的负反馈调节是造成这种情况的原因。因此，siRNA-Int6 暂时促进 HIF2α 蛋白的积累，导致缺氧非依赖性血管生成因子的转录和伴随的新血管生成。
我们认为，涉及 INT6/HIF2α 的途径作为功能性血管生成的缺氧非依赖性主开关起作用；因此，siRNA-Int6 的应用可能在治疗缺血性疾病（如心脏和大脑缺血、皮肤损伤和涉及阻塞血管的疾病）方面具有临床价值。
