RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization

Significance

Pathological angiogenesis has been implicated in diverse pathologies. Infiltrating macrophages, especially those activated to M2-like phenotype, are critically important for angiogenesis. Although the role of RIP1 kinase in the regulation of apoptosis, necroptosis, and inflammation has been well-established, its role in angiogenesis remains elusive, despite being abundantly expressed in angiogenesis-related infiltrating macrophages. This study demonstrates that RIP1 kinase inhibition attenuates angiogenesis in multiple mouse models of pathological angiogenesis in vivo and suggests a therapeutic role of RIP1 kinase inhibition in pathological angiogenesis. Mechanistically, the inhibitory effect on angiogenesis depends on RIP kinase inhibition-mediated caspase activation in infiltrating macrophages through suppression of M2-like polarization, and subsequent attenuation of pathological angiogenesis.

Abstract

Inflammation plays an important role in pathological 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, a comprehensive description of 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 RIP1 ^K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4–activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.