| Purpose: To evaluate the ability of a lentivirus-vectored short hairpin (sh)RNA directed against HIF1á to inhibit retinal and choroidal neovascularization in vitro and in vivo in animal models.
Methods: We have designed 6 HIF1a.shRNA and use the lentiviral-delivered HIF1a.shRNA on human aorta vascular endothelial cells(HAEC), Human lung microvascular endothelial cells(HLMEC), HeLa cells and in a mouse model of oxygen induced retinopathy(OIR) and laser induced choroid neovascularization(CNV) model. To determine the transfection efficiency of lentivirus in vivo. Lenti.GFP was used to evaluate distribution of the lentivirus in the mouse CNV model and in the OIR model .
Results: We found that HIF1α.shRNA can specifically and efficiently silence HIF1α mRNA and protein expression in HeLa cells and HAEC and HLMEC(Real-time PCR, Western blot, ELISA, Immunocytochemistry). In mouse laser CNV model, after injection of Lenti.GFP
for 2 weeks , analyses of cryosections for GFP fluorescence showed high levels of GFP in all nuclear layers of the retina and in inner retinal vessels in laser-treated eye. we found that intravitreous and subretinal injection of lenti-H1.HIF1α.shRNA resulted in significantly reduction in the area of NV compared with lenti-negative control.shRNA(P=0.009). In mice with OIR model, after injection of lenti.GFP for 12 days, nearly all of the tissues in the eye had strong GFP levels. Immunohistochemistry for HIF1α on cryosections of OIR eyes harvested at P13 shows significant differences in HIF1α levels and distribution between the lenti.HIF1α.shRNA and lenti.negative.shRNA injected eyes. we found lenti.HIF1α.shRNA can reduce 31.3 % retinal NV compared with lenti-negative control.shRNA(P=0.027).
Conclusions: our data show that, lentivirus-delivered shRNA targeted to HIF1α can specifically knock down HIF1α mRNA and protein levels in vitro. Lentivirus can effectively target retinal cells, particularly in disease states such as models of oxygen-induced retinopathy and the laser photocoagulation model of CNV. After delivery of Lenti-HIF1α.shRNA in these models, there is significant reduction in the extent
of retinal and choroidal neovascularization, respectively. Vectored delivery of RNAi specific to HIF1α may thus ultimately have clinical application in the treatment of acute and chronic blinding ocular neovascular disease.
*Support: NIH RO1-EY10820, RO1-EY12156, P30
DK47757, EY01583, Foundation Fighting Blindness, the
Research to Prevent Blindness Lew R.Wasserman merit
award, the LIFE Foundation , the Paul and EM
Foundation, F.M.Kirby center foundation
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