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Goldis Malek, Mayur Choudhary, David Forest, Lincoln V Johnson, Stephen H. Safe; The effect of pharmacologic activation of the aryl hydrocarbon receptor (AhR) on pathologies associated with age-related macular degeneration (AMD).. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4201.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we have shown that the activity of AhR, a transcription factor responsible for toxin metabolism, declines in human retinal pigment epithelial (RPE) cells with age. We also found that aged AhR knockout mice develop ocular phenotypic features of dry AMD and laser induced choroidal neovascular (CNV) lesions larger in size and volume as compared to wild type mice. Finally, we have shown that AhR regulates several AMD-related pathogenic pathways including extracellular matrix (ECM) remodeling, microglial activation, and angiogenesis. Given the lack of effective treatments for AMD, which is the leading cause of vision loss in the elderly, we tested the therapeutic potential of AhR activation using leflunomide, an FDA approved immunosuppressive drug and AhR agonist, which is emerging as a potential treatment for rheumatoid arthritis, breast cancer and melanoma.
Luciferase reporter assays and qPCR were used to assess AhR activity and target gene expression in human RPE (ARPE19), human fetal RPE (fRPE) and rhesus macaque choroidal endothelial (RF6A) cell lines treated with AhR ligands. ECM expression and secretion was evaluated by Western blot analysis. Sub-RPE ‘drusenoid’ deposit formation in a fRPE cell culture model was evaluated by light microscopy. Cell migration and endothelial tube formation were measured using a monolayer wound healing assay and GeltrexTM basement membrane matrix, respectively. Finally, the effect of AhR activation on severity of experimentally induced CNV lesion formation was evaluated in aged C57BL/6J mice.
Leflunomide treatment of ARPE19 and RF6A cells resulted in AhR activation (3 and 10 fold, respectively) and significant upregulation of the AhR target genes CYP1A1 and CYP1A2. Agonist treatment of ARPE19 cells inhibited secretion of the ECM proteins collagen IV (76%) and fibronectin (71%), and downregulated expression of monocyte chemo-attractant protein-1 (41%). A 71% reduction in sub-RPE deposit formation was seen in fRPE cell cultures treated with the agonist. Leflunomide also inhibited AhR knockdown-induced migration and tube formation of choroidal endothelial cells.
Our results support the hypothesis that pharmacologic modulation of AhR activity may be a viable path in ameliorating pathologies associated with both early dry- and wet-AMD.
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