The pro-cell death RIP1 kinase mediates angiogenesis by facilitating M2-like macrophages

Abstract

Purpose : Angiogenesis, including choroidal neovascularization (CNV), has been implicated in diverse pathologies. Whereas multiple roles for receptor-interacting protein 1 (RIP1) in the regulation of apoptosis, necroptosis, and inflammation have been reported, its role in angiogenesis remains elusive.

Methods : The animal model of laser-induced CNV in mice was used for this study. Necrostatin-1 (Nec-1), GSK’872, and Z-VAD-FMK (Z-VAD) was intravitreally injected after CNV induction to inhibit RIP1 kinase, RIP3 kinase, and caspase activity, respectively. Kinase-inactive RIP1^K45A/K45A knock-in mice and RIP3^-/- mice were also used to assess the involvement of RIP1/RIP3 pathway in the development of laser-induced CNV. CNV size was evaluated after 7 days of CNV induction. Tissue samples of 4–5 days after CNV inductions were used to evaluate the effect of RIP kinases and caspase on the ongoing angiogenesis with infiltrated macrophages. Mouse bone marrow-derived macrophages (BMDMs) were polarized to M2 phenotype by IL-4 activation, and the effect of RIP1 kinase and caspase inhibition on polarization was evaluated.

Results : RIP1 protein expression was significantly up-regulated in CNV lesions with ongoing angiogenesis. Genetic or pharmacological inhibitions of RIP1 kinase as well as pharmacological inhibition of RIP3 kinase, but not total deletion of RIP3, attenuate angiogenesis in laser-induced CNV, suggesting a role for kinase independent function of RIP3 in the attenuation of CNV development. The catalytic inhibition of RIP1 activates caspases in infiltrating macrophages, thereby dampening CNV development. Furthermore, caspase activation by the catalytic inhibition of RIP1 suppressed the pro-angiogenic M2-like polarization in macrophages, while M1-like marker expressions were up-regulated.

Conclusions : These results suggest a novel, non-necrotic function of RIP kinases and suggest that RIP1-mediated modulation of macrophage activation may represent a therapeutic target for the control of angiogenesis-related diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.