RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization

Abstract

word limit; 250)Inflammation plays an important role in pathologic angiogenesis. Receptorinteracting 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 RIP1 K45A/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 kinaseindependent 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 proangiogenic 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. Significance (word limit; 120)Pathological 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. \body

A Nano “Immune‐Guide” Recruiting Lymphocytes and Modulating the Ratio of Macrophages from Different Origins to Enhance Cancer Immunotherapy

“…[ 37 ] Therefore, we first evaluated the stimulatory effect of HA‐PDA@Fe 3 O 4 on macrophages in vitro. Bone‐marrow‐derived macrophages obtained from Balb/c mice were induced to an M2‐like phenotype by interleukin‐4, [ 38 ] and then treated with HA‐PDA@Fe 3 O 4 , PDA@Fe 3 O 4 , or free HA for 24 h. Macrophage activation was evaluated using multicolor FCM and immunofluorescence staining. The macrophages exposed to HA‐PDA@Fe 3 O 4 , PDA@Fe 3 O 4 , or free HA upregulated M1‐related CD80 marker compared to that in the untreated control group (Figure S8a,b, Supporting Information), but simultaneously and significantly downregulated M2‐related CD206 marker, with HA‐PDA@Fe 3 O 4 being more efficient.…”

“…For in vitro macrophage polarization study, bone marrow‐derived macrophages were first pretreated with interleukin‐4 (Sigma, Merck KGaA, Darmstadt, Germany) at a concentration of 50 ng mL −1 to induce the M2 phenotype, [ 38,58 ] and then incubated with free HA (14 µg mL −1 ), PDA@Fe 3 O 4 (100 µg Fe mL −1 ), or HA‐PDA@Fe 3 O 4 (100 µg Fe mL −1 ) for 24 h. After incubation, the macrophages were stained with the M1‐related marker anti‐CD80‐PE/Cy7 (Biolegend, cat. no.…”

Neuroprotection for Age-Related Macular Degeneration

“…[RIPK]-regulated necrosis) that may be involved in photoreceptor degeneration. Mechanistic studies have revealed that blocking an individual cellular death pathway may activate another and vice versa 27,28,[80][81][82][83] . Furthermore, we have seen that different cellular death pathways may predominate in different cell types [28][29][30]81,82,84 .…”

“…For example, when ROS are the predominant cell death stimulus, ferroptosis (iron-dependent death) is the predominant pathway [87][88][89][90][91][92] ; whereas when tumor necrosis factor alpha (TNF-alpha) or FASL are the predominant stimuli, apoptosis and/or RIPK-dependent necrosis are the predominant pathways 24,25,31,32,86,[93][94][95][96] . To make things more complex, infiltrating immune cells also contribute to neurodegeneration via the inflammasome and cross-talk with several other pathways 80,85,[97][98][99][100][101][102][103][104][105][106] .…”

Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas

“…After corneal injury, neutrophils and macrophages infiltrate the cornea. Compared with other inflammatory cells, macrophages are presumed to be an important source of pro-angiogenic molecules, and they can support vascular endothelial sprouting (9,12,13). Furthermore, recent studies have revealed that the development of CNV can be suppressed by inhibiting macrophage infiltration but not neutrophil infiltration (14,15).…”