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Mayur Choudhary, Amanda Bednar, Peter Saloupis, Peng Hu, Goldis Malek; Aryl-hydrocarbon receptor plays a role in choroidal neovascularization by regulating endothelial migration and tube formation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):324.
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Exudative age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV) and is the leading cause of irreversible visual impairment in the elderly in the United States. The aryl hydrocarbon receptor (AhR) has been shown to play a regulatory role in VEGF expression, HIF1α and TGFβ activity, pathways also important in the pathobiology of neovascular AMD. The purpose of the study is to understand the role of endogenous activation of the AhR in the pathology of neovascular AMD.
Experimental CNV was induced in 11-month old C57BL/6J and AhR-/- mice (n=18). A red diode laser (810 nm) was used to create four choroidal thermal burns in each eye. Mice were euthanized after 3 weeks and eyes were harvested for visualization of laser-induced CNV via flatmounts or cryopreserved for immunohistochemistry. Flatmounts were stained with propidium iodide to visualize area of the neovascular lesion. Cryosections were probed with antibodies to F4/80, collagen I and collagen IV. ARPE19 and RF/A6 cells were used for in vitro studies. AhR siRNA transfection was performed, followed by western blot analysis and qPCR to validate knockdown. Luciferase-based reporter assay was performed following transfection with the AhR-XRE domain and pCMV plasmids, in the presence of AhR ligands to measure AhR activity. Cell migration and endothelial tube formation was measured using monolayer wound healing assay and GeltrexTM basement membrane matrix, respectively. Total tube length was quantified per field using ImageJ.
The area of laser-induced neovascular lesions was 2-fold higher in Ahr-/- versus wild type mice. Ahr-/- mice displayed increased immunoreactivity to collagen IV in RPE, Bruch’s membrane, choroid and lesions compared to wild type mice. Increased AhR activity following treatment with AhR ligands confirmed the presence of the AhR signaling pathway in ARPE19 and RF/A6 cells. Endogenous AhR knockdown in RF/A6 cells resulted in increased collagen IV and TGFβ message levels. Collagen IV protein level and secretion was also higher after AhR knockdown. A 400% increase in endothelial migration and 50% increase in tube formation was observed as a result of AhR knockdown.
Our results show that endogenous AhR plays a role in CNV. Possible mechanisms involved include endothelial migration, tube formation and ECM regulation.
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