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S. Parameswaran, R. Kannan, C. Spee, S. J. Ryan, D. R. Hinton; Differential Apoptotic and Angiogenic Factor Regulatory Response of Nonpolarized and Highly Polarized RPE to N-(4-Hydroxyphenyl) Retinamide (4-HPR) Treatment. Invest. Ophthalmol. Vis. Sci. 2008;49(13):232. doi: https://doi.org/.
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4-HPR, a retinoic acid derivative, is in Phase II clinical trial for treatment of geographic atrophy in patients with AMD. We previously showed that 4-HPR promotes laser-induced CNV but does not affect normal retina. We studied the effect of 4-HPR in vitro on induction of cell death and regulation of angiogenic factors in cultured nonpolarized and highly polarized human RPE cells.
Confluent nonpolarized human RPE and long-term culture of highly polarizedhuman RPE grown on Transwell filters were incubated with varying doses of 4-HPR (0-5 µM) for 24 h in either 0% or 5% FBS containing medium. Apoptosis was studied by TUNEL staining and induction of caspase 3 by confocal microscopy and Western blot analysis. Gene expression of VEGF-A and -C, Ang-1, Ang-2, PEDF and TSP-1 was determined by real time PCR. Cellular protein levels and secretion of VEGF-A and -C, thrombospondin-1 and PEDF were also measured.
Increase in TUNEL positive cells and increased expression of active caspase 3 were observed in nonpolarized RPE treated with 4-HPR as compared to controls only in the presence of serum. 4-HPR did not induce apoptosis or caspase-3 activation in highly polarized RPE (with mean transepithelial resistance of 399 ± 19.98 Ω.cm2 which was not affected by 4-HPR treatment) with 0% or 5% FBS. In nonpolarized RPE, protein secretion and gene expression of VEGF-A and -C were significantly increased (p<0.01) with 5 µM 4-HPR while that of PEDF and TSP-1 was significantly decreased. In highly differentiated, polarized RPE, expression of VEGF and PEDF and apical and basolateral secretion were not altered in the 4-HPR group compared to controls.
4-HPR treatment causes cell death and regulates angiogenic factors in nonpolarized RPE but not in highly differentiated RPE. This finding needs to be taken into consideration in exploring therapeutic applications of 4-HPR to treat AMD.
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