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J. M. Barnett, M. N. Young, D. P. Bingaman, J. S. Penn; Inhibition of Retinal Neovascularization by Anecortave Acetate Through PAI-1 and Matrix Metalloproteinases. Invest. Ophthalmol. Vis. Sci. 2007;48(13):84.
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© ARVO (1962-2015); The Authors (2016-present)
The angiostatic cortisene, anecortave acetate, has proven efficacy in the rat model of retinopathy of prematurity (ROP) (Penn et al. IOVS 2001;42:283). The present experiments were conducted to further elucidate the mechanisms by which anecortave acetate inhibits retinal neovascularization.
Human retinal microvascular endothelial cells (HRMECs) were grown in serum-free medium with 25 ng/ml VEGF, and treatment groups where given 100 uM anecortave desacetate (the active metabolite of anecortave acetate); controls received vehicle (PEG, EtOH, saline). Cell lysates and medium samples were collected at intervals from 0 to 36 hours. PAI-1 message and protein levels were analyzed by quantitative PCR and western blot analysis. Newborn (P0) Sprauge-Dawley rats were placed in environments alternating every 24 hours between 50% and 10% oxygen for 14 days. On day 14, the animals were removed to room air and their eyes were assigned to one of three treatments: no injection, 5 ul vehicle or 5 ul containing a 10% suspension of anecortave acetate in vehicle (Alcon Research, Ltd.). Rats were sacrificed 1 and 6 days following the injection to determine matrix metalloproteinase (MMP) activities by gel zymography.
Quantitative PCR showed a 1.6-fold peak increase in PAI-1 expression in the anecortave desacetate-treated HRMECs at 2 hours post-treatment when compared to controls. Media samples from HRMECs treated with anecortave desacetate demonstrated a significant increase in PAI-1 protein levels that peaked at 5.7-fold 36 hours after treatment when compared to untreated controls. Analysis of lysates indicated no change in cell-associated PAI-1. One day post-injection of the rat pups, a 33% reduction in pro-MMP-9 (p<0.05) and a 55% reduction in active MMP-9 enzyme levels were observed (p<0.02), in comparison to vehicle injected controls. At 6 days following injection, a 55% reduction in pro-MMP-9 (p<0.01) was observed. Additionally, a 39% and 42% reduction in pro-MMP-2 (p<0.05) and active MMP-2 levels (p<0.02), respectively, were found at 1 day post-injection. Moreover, a 50% reduction in pro-MMP-2 (p<0.05) and a 30% reduction in active MMP-2 (p<0.05) was observed at day 6 post-injection in comparison to controls.
Inhibition of the proteolytic aspect of the angiogenic cascade by up-regulation of endogenous PAI-1 and down-regulation of MMP levels and/or activities is of potential therapeutic value in sight-threatening conditions such as ROP, proliferative diabetic retinopathy and exudative macular degeneration.
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