The Omega-3 Fatty Acid Metabolite 4-HDHA Reduces VEGF-Induced Choroidal Neovascularization in a VEGFR2-independent manner

Abstract

Purpose: : Age-related macular degeneration (AMD) and its neovascular "wet" form is the leading cause of vision loss among adults above the age of 65. We have recently demonstrated that the ω-3 PUFA metabolite, 4-hydroxydocosahexaenoic acid (4-HDHA), has potent anti-angiogenic properties. Here we investigated whether 4-HDHA can directly inhibit VEGF-induced angiogenesis in models of choroidal neovascularization (CNV).

Methods: : The ability of 4-HDHA to inhibit pathological angiogenesis was assessed in a mouse model of laser-induced CNV. Adult C57BL/6 male mice received daily intravenous (iv) injections of 4-HDHA (5uM) or vehicle (EtOH 0,01%) following laser impact until sacrifice (14 days later). The extent of CNV was compared to mice receiving defined isocaloric diets enriched in either ω-3 or ω-6 PUFAs. The capacity of 4-HDHA to directly inhibit VEGF-induced angiogenesis was studied ex vivo in choroidal explants and in a series of in vitro assays using Human Retinal Microvascular Endothelial Cells (HRMECs) including, the scratch-wound migration assay and the CyQUANT proliferation assay. The ability of 4-HDHA to interfere with VEGF signalling was investigated by Western blot and immunoprecipitation analysis. Treatment concentrations where as follows: VEGF (40 ng/mL), vehicle (EtOH 0,01%), 4-HDHA (5 uM).

Results: : Daily (iv) treatment with 4-HDHA significantly reduced CNV by 43% when compared to vehicle injected controls. The magnitude of the effect was similar to the 40% reduction in CNV noted in mice receiving ω-3 rich diets when compared to ω-6 rich diets. Correspondingly, 4-HDHA efficiently attenuated VEGF-induced vascular sprouting in choroidal explants by 84% and completely abrogated VEGF-induced HRMEC migration in the scratch-wound assay and proliferation as determined by CyQUANT assay. Importantly, the classic effectors of the VEGFR2 signalling where not perturbed by treatment with 4-HDHA.

Conclusions: : Our study demonstrates that 4-HDHA acts as a potent and direct inhibitor of VEGF-induced choroidal neovascularization while operating in a VEGFR2-independent manner. 4-HDHA shows promise as an adjunct to anti-VEGF therapies for neovascular AMD.

Keywords: age-related macular degeneration • lipids • vascular endothelial growth factor