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Vinson M. Wang, Ivo Francischetti, Yujuan Wang, De Fen Shen, Jingsheng Tuo, Jose Ribeiro, Chi-Chao Chan; Inhibition of Tissue Factor by Ixolaris in in vitro and in vivo Age-related Macular Degeneration models. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5561.
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Inflammation and angiogenesis play important roles in the development of Age-related macular degeneration (AMD), the leading cause of central, irreversible visual impairment in the elderly. Tissue factor (TF), a primary initiator in the blood coagulation cascade, has been found to be upregulated in human AMD eyes, stressed ARPE-19 cells, and in Ccl1/Cx3cr1 double knockout mice on rd8 background (DKO), an AMD model. Ixolaris, a recombinant protein with sequence homology to TF Pathway inhibitor, has recently been shown to inhibit TF activity and regulate angiogenesis. We evaluated the effect of Ixolaris on TF expression in RPE cells and in DKO mice.
Varying concentrations of Ixolaris were added to ARPE-19 cells along with H2O2 and coagulation factors FVIIa and FX, the scaffolds for the Ixolaris/TF binding complex. Quantitative RT-PCR was conducted to analyze the relative expression levels of TF and Vascular endothelial growth factor (VEGF). In an in vivo study, Ixolaris (70 ug) was injected intravitreally into the right eyes of 17 DKO mice and the contralateral left eyes were used as controls and injected with PBS. After 1 month, fundus images were taken and the eyes of 4 mice were measured for TF and VEGF by qRT-PCR and immunohistochemsitry. The remaining 13 mice were injected with another round of Ixolaris and PBS. At the 2-month time point, fundus examinations were taken and scored, and the eyes were subsequently studied for histopathology, qRT-PCR, immunohistochemistry, and A2E levels by HPLC.
In ARPE-19 cells stimulated with H2O2, the expression level of TF mRNA decreased in a dose-dependent manner while VEGF mRNA levels were not significantly affected when incubated with Ixolaris. At 1 month after received Ixolaris, fundus images of lesions were attenuated in 6 mice, stable in 4 mice, and worsened in 3 mice. After 2 months, 8 mice showed attenuation while 5 mice showed similar development following treatment. Histopathology confirmed the fundoscopic data with average lower scores in the treated right eyes compared to the control left eyes. The relative expression of TF in the treated eyes was 0.78 (n=4, p=0.07) and VEGF was 0.38 (n=4, p=0.14) as compared to control eyes (n=4). Immunoreactivities against TF and VEGF also decreased in the eyes treated with Ixolaris. The average A2E level in treated eyes was 19.8 pmol/eye while the average A2E level in untreated eyes was 22.3 pmol/eye (n=4, p=0.29).
These results suggest that Ixolaris suppresses TF expression in ARPE-19 cells under oxidative stress and may arrest retinal degeneration in DKO mice. Further studies of dose-response and longer follow-up in DKO and other AMD animal models are warranted.
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