RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization

Abstract

Inflammation plays an important role in pathologic angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation, however its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization (CNV), Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis was mediated by caspase activation through a kinase-independent function of RIP1 and RIP3, and simultaneous caspase inhibition with RIP1 kinase inhibition abrogated the effects of RIP1 kinase inhibition on angiogenesis in vivo. Mechanistically, infiltrating macrophages are the key target for RIP1 kinase inhibition to attenuate pathological angiogenesis, and we observed that the inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of pro-angiogenic M2-like markers while M1 marker expressions were sustained. Similarly, in vitro, catalytic inhibition of RIP1 down-regulated M2 marker expressions in IL-4-activated bone marrow-derived macrophages, which was blocked by simultaneous caspase inhibition. Taken together, these results suggest a novel, non-necrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may represent a therapeutic target for the control of angiogenesis-related diseases.\n\nSignificancePathological angiogenesis has been implicated in diverse pathologies. Infiltrating macrophages, especially those activated to M2-like phenotype are critically important to support angiogenesis. Whereas the role of RIP1 kinase in the regulation of apoptosis, necroptosis, and inflammation have been well established, its role in angiogenesis remains elusive despite being abundantly expressed in angiogenesis-related infiltrating macrophages. This study demonstrated for the first time that RIP1 kinase inhibition attenuates angiogenesis in multiple mouse models of pathological angiogenesis in vivo. Mechanistically, the inhibitory effect on angiogenesis depends on RIP kinase inhibition-mediated caspase activation in infiltrating macrophages that suppresses M2-like polarization, thereby attenuating pathological angiogenesis.