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R Hera, M Keramidas, M Peoc'h, M Mouillon, J-PP Romanet, JJ Feige; Quantitative RT-PCR Analysis of Angiogenesis Markers (VEGF/VEGF Receptors; Angiopoietins/Tie2) in Subfoveal Membranes From Patients With Age-related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2531.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ARMD is characterized by active choroidal neovascularization(CNV). VEGF/VEGFR and angiopoietins/tie2 are key molecular regulators of angiogenesis. In this study, we used quantitative RT-PCR to determine the level of expression of these molecules in subfoveal membranes from patients with ARMD. Methods: Membranes were surgically excised from 24 patients (age 51-91years) that were diagnosed for classic CNV of ARMD since less than 6 months. 13 patients underwent recurrent CNV within 6 months after surgical excision. Two 8 µm sections were prepared from each membrane for IHC determination of vascular density (CD31 immunostaining) and the remaining tissue was used for total RNA preparation and reverse transcription into cDNA. The levels of VEGF-A, VEGF-R1, VEGF-R2, neuropilin-1, angiopoietin-1, angiopoietin-2, tie-2 and HPRT(taken as a control housekeeping gene) expression were determined by real-time PCR using a Light Cycler® apparatus. This allowed us to calculate, for each gene of interest, the amount of cDNA present in a volume of reverse transcription reaction that contained 10 -15 g of HPRT cDNA. Results: 23/24 samples express VEGF (mean=11fg[0,2-77fg]). Angiopoietin-2 expression was found in 13/24 samples ([0,01-7,5x10-3fg]). Neuropiline-1 expression was find in 13/24 samples ([0,002-0,47 fg]). Angiopoietin-1 expression was very low: 16/24 samples ([0,0001-3,4 x10-9fg]). Tie-2, VEGF-R1 and R2 were undetectable in most samples. Conclusion: The results indicate that, at the stage of surgical excision, subfoveal membranes strongly express VEGF and angiopoietin-2 but poorly or do not express the receptors for these pro-angiogenic factors suggesting that the phase of active angiogenesis is over. This suggest that anti-angiogenic therapy is more likely to be effective in early stages of ARMD.
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