Background and Purpose: Inactivation of the gene for adenosine A_2A receptors (ADORA2A for humans and Adora2a for rodents) protects against brain injury in experimental stroke. However, the cell‐specific pathogenic effects of A_2A receptors in thromboembolic stroke and the underlying mechanisms remain undefined. Here, we tested the hypothesis that inhibition of endothelial A_2A receptors after thromboembolic stroke improves post‐stroke outcomes via down‐regulation of inflammation.Experimental Approach: Thromboembolic stroke was induced by embolic middle cerebral artery occlusion in mice. Post‐stroke outcomes were determined with neurological deficit scoring, infarct volume, inflammatory marker expression, brain leukocyte infiltration, blood–brain barrier (BBB) leakage, and oedema assessment. Anti‐inflammatory effects of silencing the gene for A_2A receptors or pharmacological antagonism of these receptors were assessed in vitro.Key Results: Thromboembolic stroke induced Adora2a expression in the brain. Mice globally deficient in Adora2a (Adora2a^−/−) were resistant to stroke injury. Mice specifically deficient in endothelial Adora2a (Adora2a^ΔVEC) showed reduced leukocyte infiltration, BBB leakage, and oedema after stroke, along with attenuated downstream proinflammatory markers, both in vivo and in vitro. The A_2A receptor antagonist, KW 6002, also reduced brain injury and inflammation after stroke. Inactivation of ADORA2A inhibited endothelial inflammation via suppression of the NLRP3 inflammasome, down‐regulating cleaved caspase 1 and IL‐1β expression.Conclusions and Implications: Specific inactivation of endothelial A_2A receptors mitigated ischaemic brain injury and improved post‐stroke outcomes, at least partly, through anti‐inflammatory effects via blockade of NLRP3 inflammasome activity. Our findings may open new approaches to vascular protection after ischaemic stroke.

Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and to characterize the transcriptional profiles and metabolic pathways of proangiogenic microglia in a mouse model of oxygen-induced PR (OIR). Using transcriptional single-cell sorting, we comprehensively mapped all microglia populations in retinas of room air (RA) and OIR mice. We have unveiled several unique types of PR-associated microglia (PRAM) and identified markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hypermetabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and proangiogenic Igf1 . IHC staining shows that these PRAM were spatially located within or around neovascular tufts. These unique types of microglia have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis–related diseases.

Background and Purpose: Pathological angiogenesis is a major cause of irreversible blindness in individuals with neovascular age‐related macular degeneration (nAMD). Macrophages and microglia (MΦ) contribute to aberrant ocular angiogenesis. However, the role of glucose metabolism of MΦ in nAMD is still undefined. Here, we have investigated the involvement of glycolysis, driven by the kinase/phosphatase PFKFB3, in the development of choroidal neovascularization (CNV).Experimental Approach: CNV was induced in mice with laser photocoagulation. Choroid/retinal pigment epithelium (RPE) complexes and MΦ were isolated for analysis by qRT‐PCR, western blot, flow cytometry, immunostaining, metabolic measurements and angiogenesis assays.Key Results: MΦ accumulated within the CNV of murine nAMD models and expressed high levels of glycolysis‐related enzymes and M1/M2 polarization markers. This phenotype of hyper‐glycolytic and activated MΦ was replicated in bone marrow‐derived macrophages stimulated by necrotic RPE in vitro. Myeloid cell‐specific knockout of PFKFB3, a key glycolytic activator, attenuated pathological neovascularization in laser‐induced CNV, which was associated with decreased expression of MΦ polarization markers and pro‐angiogenic factors, along with decreased sprouting of vessels in choroid/RPE complexes. Mechanistically, necrotic RPE increased PFKFB3‐driven glycolysis in macrophages, leading to activation of HIF‐1α/HIF‐2α and NF‐κB, and subsequent induction of M1/M2 markers and pro‐angiogenic cytokines, finally promoting macrophage reprogramming towards an angiogenic phenotype to facilitate development of CNV. The PFKFB3 inhibitor AZ67 also inhibited activation of HIF‐1α/HIF‐2α and NF‐κB signalling and almost completely prevented laser‐induced CNV in mice.Conclusions and Implications: Modulation of PFKFB3‐mediated macrophage glycolysis and activation is a promising strategy for the treatment of nAMD.

Aims: Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action.Methods and results: We found that ADK expression was higher in the neointima of injured vessels and in platelet-derived growth factor-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance.Conclusion: Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby down-regulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury.

Anti–vascular endothelial growth factor therapy has had a substantial impact on the treatment of choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (nAMD), the leading cause of vision loss in older adults. Despite treatment, many patients with nAMD still develop severe and irreversible visual impairment because of the development of subretinal fibrosis. We recently reported the anti-inflammatory and antiangiogenic effects of inhibiting the gene encoding adenosine receptor 2A (Adora2a), which has been implicated in cardiovascular disease. Here, using two mouse models of subretinal fibrosis (mice with laser injury–induced CNV or mice with a deficiency in the very low–density lipoprotein receptor), we found that deletion ofAdora2aeither globally or specifically in endothelial cells reduced subretinal fibrosis independently of angiogenesis. We showed thatAdora2a-dependent endothelial-to-mesenchymal transition contributed to the development of subretinal fibrosis in mice with laser injury–induced CNV. Deficiency ofAdora2ain cultured mouse and human choroidal endothelial cells suppressed induction of the endothelial-to-mesenchymal transition. A metabolomics analysis of cultured human choroidal endothelial cells showed thatADORA2Aknockdown with an siRNA reversed the increase in succinate because of decreased succinate dehydrogenase B expression under fibrotic conditions. Pharmacological inhibition of ADORA2A with a small-molecule KW6002 in both mouse models recapitulated the reduction in subretinal fibrosis observed in mice with genetic deletion ofAdora2a. ADORA2A inhibition may be a therapeutic approach to treat subretinal fibrosis associated with nAMD.