Int6/eIF3e silencing promotes functional blood vessel outgrowth and enhances wound healing by upregulating hypoxia-induced factor 2alpha expression.
BACKGROUND
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.


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 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.


CONCLUSIONS
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.
Chen
Li
L
Department of Molecular Medical Research, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.
Endler
Alexander
A
Uchida
Kazuyo
K
Horiguchi
Shin-ichiro
S
Morizane
Yoshihito
Y
Iijima
Osamu
O
Toi
Masakazu
M
Shibasaki
Futoshi
F
D000199:Actins / Q000378:metabolism; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms; D039621:Eukaryotic Initiation Factor-3 / Q000235:genetics* / Q000378:metabolism; D025461:Feedback, Physiological / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000502:physiology / Q000637:transplantation; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D018919:Neovascularization, Physiologic / Q000502:physiology*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000502:physiology; D040521:Subcutaneous Tissue / Q000098:blood supply / Q000502:physiology; D014945:Wound Healing / Q000502:physiology*; D000099162:Endothelial PAS Domain-Containing Protein 1
D000199:Actins; D051792:Basic Helix-Loop-Helix Proteins; D039621:Eukaryotic Initiation Factor-3; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D034741:RNA, Small Interfering; D000099162:Endothelial PAS Domain-Containing Protein 1; C497443:Hif1a protein, mouse