Published in:
01-10-2008 | Basic Science
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
Published in: Graefe's Archive for Clinical and Experimental Ophthalmology | Issue 10/2008
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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 μM CoCl2, and RNA interference (RNAi) technique was used to knock down HIF-1α gene in RPE cells. mRNA and protein expression of HIF-1α 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-1α) on the proliferation, migration and tube formation of CECs respectively.
Results
Transfection of shRNA-expressing pDNA targeting HIF-1α to RPE cells resulted in the knock down of HIF-1α gene and reduction of the corresponding mRNA and protein of HIF-1α 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.
Conclusion
RNAi of HIF-1α in RPE cells can inhibit angiogenesis in vitro and provide a possible strategy for treatment of choroidal neovascularization diseases by targeting HIF-1α.