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Y. Fan, X. Zhang, X. Xu, F. Wang, Q. Huang; Effect of Antisense VEGF121 on the Expression of Vascular Endothelial Growth Factor in Human Retinal Pigment Epithelial Cell . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3581.
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Purpose: Vascular endothelial growth factor (VEGF) appears to be a major mediator of ischemia-induced ocular neovascularization. Inhibition of the secretion of VEGF may be a potential therapeutic target for the neovascularization. We proposed to determine if antisense VEGF121 suppresses the expression of VEGF in RPE cells under hypoxia condition. Methods: HVEGF121 cDNA was amplified by reverse-transcription PCR and was inserted into an expression vector pCR3.1 to construct the plasmid that encoded antisense VEGF121(pCR3.1/anti-VEGF121). RPE cells were transfected with this plasmid mediated by liposome. Select the stable cell lines. The transfected cells were exposed to hypoxia or normoxia conditions. Untreated cells were served as control. The protein expression of VEGF was measured by sandwich enzyme-linked immunosorbent assay (ELISA). The VEGF-specific proliferative potential of the medium was measured by means of human umbilical vein endothelial cell (HUVEC) growth assays. Results: pCR3.1/anti-VEGF121 contained the cDNA of hVEGF121 in an antisense orientation. The sequence was almost the same as reported except for a mutation of the 108bp from G to A and lost of base pairs from 430bp to 444bp. The transfected cells were represented just as their parental cells except for the difference in some organelles associated with the protein secretion function. Antisense VEGF decreased the level of secreted VEGF protein in the transtected RPE cells. The inhibitory rate was about 62.7%. Under hypoxia condition, the increased VEGF protein level in RPE cells was detected. Medium conditioned by RPE cells under hypoxia condition produced a 18.4% increase in HUVEC growth. But the protein level in the antisense VEGF transfected cells did not increase to the level of that of the parental cells under the same condition. And the HUVEC cells were not stimulated statistically when they were added by the medium of the transfected RPE cells under hypoxia condition. Conclusions: The construct encoding antisense VEGF121 that we've recombined could successfully inhibit the expression of VEGF in RPE cells under hypoxia condition. And it could also suppress the stimulatory effect of hypoxia RPE-conditioned medium on the growth of HUVEC cells through the down-regulation of the VEGF protein level in these cells. It provided effective vectors for gene therapy research of VEGF-induced neovascularization in vivo.
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