Inhibition of proliferation, migration and tube formation of choroidal microvascular endothelial cells by targeting HIF-1α with short hairpin RNA-expressing plasmid DNA in human RPE cells in a coculture system

• DOI: 10.1007/s00417-008-0858-8
• Publication Date: 2008-06-03T09:14:53Z
• Authors: Wei Zhao; Yu-Sheng Wang; Yan-Nian Hui; Jie Zhu; Peng Zhang; Xia Li; Guo-Rui Dou
• Keywords: Vascular Endothelial Growth Factor A; Choroid; Reverse Transcriptase Polymerase Chain Reaction; Blotting, Western; Cell Culture Techniques; Enzyme-Linked Immunosorbent Assay; Retinal Pigment Epithelium; Hypoxia-Inducible Factor 1, alpha Subunit; Transfection; Choroidal Neovascularization; Coculture Techniques; 3. Good health; 03 medical and health sciences; 0302 clinical medicine; Cell Movement; Gene Targeting; Humans; RNA Interference; Endothelium, Vascular; Gene Silencing; RNA, Messenger; Cell Proliferation; Plasmids

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.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.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.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.