The COX-2-Selective Antagonist (NS-398) Inhibits Choroidal Neovascularization and Subretinal Fibrosis

<div><p>Choroidal neovascularization (CNV) is an important pathologic component of neovascular age-related macular degeneration (AMD), and CNV lesions later develop into fibrous scars, which contribute to the loss of central vision. Nowadays, the precise molecular and cellular mechanisms underlying CNV and subretinal fibrosis have yet to be fully elucidated. Cyclooxygenase-2 (COX-2) has previously been implicated in angiogenesis and fibrosis. However, the role of COX-2 in the pathogenesis of CNV and subretinal fibrosis is poorly understood. The present study reveals several important findings concerning the relationship of COX-2 signaling with CNV and subretinal fibrosis. Experimental CNV lesions were attenuated by the administration of NS-398, a COX-2-selective antagonist. NS-398-induced CNV suppression was found to be mediated by the attenuation of macrophage infiltration and down-regulation of VEGF in the retinal pigment epithelium–choroid complex. Additionally, NS-398 attenuated subretinal fibrosis, in an experimental model of subretinal scarring observed in neovascular AMD, by down-regulation of TGF-β₂ in the retinal pigment epithelium–choroid complex. Moreover, we cultured mouse RPE cells and found that NS-398 decreased the secretion of VEGF and TGF-β₂ in mouse RPE cells. The results of the present study provide new findings regarding the molecular basis of CNV and subretinal fibrosis, and provide a proof-of-concept approach for the efficacy of COX-2 inhibition in treating subretinal fibrosis.</p></div

Suppression of VEGF and TGF-β2 expression with COX-2-selective Antagonist (NS-398) in RPE–choroid complexes and mouse RPE cells.

<p>Light microscope images of confluent RPE cultures on day 10 (A) (magnification = 100×). Immunofluorescence showed Cytokeratin 8-positive cells were the RPE cells (B) (magnification = 400×). In the RPE–choroid complexes, the mRNA and protein levels of VEGF and TGF-β₂ were suppressed significantly in the NS-398-treated mice (4C-4F). *<i>P</i><0.01 compared with vehicle-treated mice for all, after CNV and subretinal fibrosis model formation; vehicle n = 6 eyes for each time point, NS-398 n = 6 eyesfor each time point. In mouse RPE cells, the mRNA and protein levels of VEGF (G, I), TGF-β₂ (H, J) were significantly reduced respectively at 12h, 24h, 36h after added NS-398 (4G-4J). (^#<i>P</i><0.05, *<i>P</i><0.01). Error bars indicate mean ± SD; Experiments were conducted in triplicate with similar results.</p

Suppression of subretinal fibrosis with COX-2-selective Antagonist (NS-398) in a mouse model.

<p>Representative subretinal fibrosis of RPE–choroidal flat mounts of mice administered PBS vehicle (A), NS-398 intraperitoneally (B), on day 7 after PECs-injecting. Subretinal fibrosis lesions were assessed quantitatively(C), **<i>P</i><0.001 compared with vehicle-treated mice. Error bars represented ±SD. intraperitoneal vehicle, n = 16 burn spots, intraperitoneal NS-398, n = 18 burn spots, Scale bars in A, B are 500μm.</p

Suppression of CNV with COX-2-selective Antagonist (NS-398).

<p>Representative images of fluorescein dextran perfused RPE–choroidal flat mounts of mice administered PBS vehicle (A), NS-398 intraperitoneally (B), MF1+ DC101 (C) day 10 after photocoagulation. The CNV lesions were assessed quantitatively (D). **<i>P</i><0.001 compared with vehicle-treated mice. Error bars represented ±SD. intraperitoneal vehicle, n = 18 burn spots, intraperitoneal NS-398, n = 20 burn spots, intraperitoneal MF1+ DC101, n = 16 burn spots, Scale bars in A, B,C are 200μm.</p

Suppression of macrophage infiltration with COX-2-selective Antagonist (NS-398).

<p>Green fluorescence from isolectin B4 indicates CNV, red fluorescence indicates F4/80-positive macrophages. Representative macrophage infiltration lesions in the PBS vehicle-treated mice (A) and NS-398-treated mice (B). Areas of F4/80-positive cells were assessed quantitatively (C). **<i>P</i><0.001 compared with vehicle-treated mice. Error bars represented ±SD. intraperitoneal vehicle, n = 15 burn spots, intraperitoneal NS-398, n = 16 burn spots, Scale bars in A, B are 200μm.</p