Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1alpha with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system.
BACKGROUND
Retinal pigment epithelial (RPE) cells and choroidal microvascular endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), and hypoxia plays an important role in CNV formation via hypoxia inducible factor 1 (HIF-1). Our aim was to evaluate the role of HIF-1 in human RPE cells with regard to proliferation, migration and tube formation of CECs under hypoxia.


METHODS
RPE cells were cultured under chemical hypoxia induced by 200 muM CoCl(2), and RNA interference (RNAi) technique was used to knock down HIF-1alpha gene in RPE cells. mRNA and protein expression of HIF-1alpha and VEGF in RPE cells were investigated by real-time RT-PCR and Western blot. Three kinds of coculture models were used to observe the effects of RPE cells transfected by short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) (pshHIF-1alpha) on the proliferation, migration and tube formation of CECs respectively.


RESULTS
Transfection of shRNA-expressing pDNA targeting HIF-1alpha to RPE cells resulted in the knock down of HIF-1alpha gene and reduction of the corresponding mRNA and protein of HIF-1alpha and VEGF under hypoxia. Consequently, the proliferation, migration and tube formation of CECs were significantly inhibited by the knocked-down RPE cells compared with the control in the coculture system. The proliferation rates of CECs decreased by 40.2%, 36.6% and 36.8% on days 3, 4 and 5 respectively. Migration reduced by 49.6% at 5 h, and tube formation decreased by 40.4% at 48 h.


CONCLUSIONS
RNAi of HIF-1alpha in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1alpha.
Department of Ophthalmology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi Province 710032, People's Republic of China.
Wei
W
Zhao
Yu-Sheng
YS
Wang
Yan-Nian
YN
Hui
Jie
J
Zhu
Peng
P
Zhang
Xia
X
Li
Guo-Rui
GR
Dou
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000502:physiology*; D049109:Cell Proliferation; D002829:Choroid / Q000098:blood supply; D020256:Choroidal Neovascularization / Q000517:prevention & control*; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing / Q000502:physiology; D018390:Gene Targeting; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology*; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A