Suppression of retinal neovascularization by shRNA targeting HIF-1alpha.
OBJECTIVE
To investigate whether vector-based HIF-1alpha -targeted shRNA expression system (pSUPER(siHIF-1alpha)) can inhibit HIF-1alpha and VEGF expression in vitro and suppress retinal neovascularization in the murine model of oxygen-induced retinopathy.


METHODS
pSUPER(siHIF-1alpha) from which siRNA targeting HIF-1alpha could be generated was constructed and transfected to human umbilical vein endothelial cell lines (HUVECs). Then the expression levels of HIF-1alpha and VEGF in the cultured cells were measured by RT-PCR, immunoblot, and ELISA assays. Subsequently, pSUPER(siHIF-1alpha)was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated by angiography using fluorescein-labeled dextran and quantitated histologically. Moreover, RT-PCR and immunoblot analysis were used to determine whether local administration of pSUPER(siHIF-1alpha)could affect the expression levels of HIF-1alpha and VEGF in murine retinas.


RESULTS
HIF-1alpha and hypoxia-induced vascular endothelial growth factor (VEGF) increase in cultured cells were greatly abolished by pSUPER(siHIF-1alpha). Meanwhile, retinal neovascularization in the eye with pSUPER(siHIF-1alpha)injection was significantly reduced compared with that of the contralateral control eye. Histological analysis indicates that neovascular nuclei protruding into the vitreous cavity were decreased by nearly 65%. Furthermore, HIF-1alpha and VEGF expression levels were down-regulated in the murine retinas treated with pSUPER(siHIF-1alpha).


CONCLUSIONS
RNAi targeting HIF-1alpha could inhibit the retinal neovascularization by approximately 65% through down-regulating the expression of HIF-1alpha and VEGF in the murine retinas, which may provide a powerful and novel therapeutic tool for ischemic-induced retinal diseases.
Xia
Xiao-bo
XB
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, P. R. China.
Xiong
Si-qi
SQ
Xu
Hui-zhuo
HZ
Jiang
Jian
J
Li
Yan
Y
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005451:Fluorescein Angiography; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D007231:Infant, Newborn; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen / Q000633:toxicity; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015861:Retinal Neovascularization / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D012178:Retinopathy of Prematurity / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D010100:Oxygen