Int6 /eIF3e Silencing Promotes Functional Blood Vessel Outgrowth and Enhances Wound Healing by Upregulating Hypoxia-Induced Factor 2α Expression

Background— We previously identified INT6/eIF3e as a novel regulator of hypoxia-inducible factor 2α (HIF2α) activity. Small interfering RNA (siRNA)–Int6 adequately stabilized HIF2α, even under normoxic conditions, and thereby enhanced the expression of several angiogenic factors in vitro, suggesting that siRNA-Int6 may induce angiogenesis in vivo.Methods and Results— 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 duringInt 6 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. HIF2α accumulation caused by siRNA-Int6 was confirmed as the unequivocal cause of the angiogenesis by an in vivo angiogenesis assay. Further analysis of theInt 6 silencing–induced neoangiogenesis revealed that a negative feedback regulation of HIF2α stability was caused by HIF2α-induced transcription ofInt6 via hypoxia-response elements in its promoter. Thus, siRNA-Int6 temporarily facilitates an accumulation of HIF2α protein, leading to hypoxia-independent transcription of angiogenic factors and concomitant neoangiogenesis.Conclusions— We suggest that the pathway involving INT6/HIF2α 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.