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.

NIMA-related kinase 7 (NEK7) is a serine/threonine kinase involved in cell cycle progression via mitotic spindle formation and cytokinesis. It has been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer, and colorectal cancer. Moreover, NEK7 regulated the NLRP3 inflammasome to activate Caspase-1, resulting in cell pyroptosis. In the present study, we investigated whether NEK7 is involved in cell pyroptosis of hepatocellular carcinoma (HCC). Interestingly, we found that NEK7 was significantly related to expression of pyroptosis marker GSDMD in HCC. We found that NEK7 expression was significantly correlated with GSDMD expression in bioinformatics analysis, and NEK7 expression was significantly co-expressed with GSDMD in our HCC specimens. Cell viability, migration, and invasion capacity of HCC cell lines were inhibited, and the tumor growth in the xenograft mouse model was also suppressed following knockdown of NEK7 expression. Mechanistic studies revealed that knockdown of NEK7 in HCC cells significantly upregulated the expression of pyroptosis markers such as NLRP3, Caspase-1, and GSDMD. Coculture of HCC cells stimulated hepatic stellate cell activation by increasing p-ERK1/2 and α-SMA. Knockdown of NEK7 impaired the stimulation of HCC cells. Therefore, downregulation of NEK7 inhibited cancer–stromal interaction by triggering cancer cell pyroptosis. Taken together, this study highlights the functional role of NEK7-regulated pyroptosis in tumor progression and cancer–stromal interaction of HCC, suggesting NEK7 as a potential target for a new therapeutic strategy of HCC treatment.

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 characterize the transcriptional profiles and metabolic pathways of pro-angiogenic microglia in a mouse model of oxygen-induced proliferative retinopathy (OIR). Using transcriptional single-cell sorting, we comprehensively map all microglia populations in retinas of room air (RA) and OIR mice. We unveil several unique types of PR-associated microglia (PRAM) and identify 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 hyper-metabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and pro-angiogenic insulin-like growth factor 1. Immunohistochemical staining shows these PRAMs were spatially located within or around neovascular (NV) 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.

Sepsis, a pathology resulting from excessive inflammatory response that leads to multiple organ failure, is a major cause of mortality in intensive care units. Macrophages play an important role in the pathophysiology of sepsis. Accumulating evidence has suggested an upregulated rate of aerobic glycolysis as a key common feature of activated proinflammatory macrophages. Here, we identified a crucial role of myeloid 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (Pfkfb3), a glycolytic activator in lipopolysaccharide (LPS)-induced endotoxemia in mice. Pfkfb3 expression is substantially increased in bone marrow derived macrophages (BMDMs) treated with LPS in vitro and in lung macrophages of mice challenged with LPS in vivo. Myeloid-specific knockout of Pfkfb3 in mice protects against LPS-induced lung edema, cardiac dysfunction and hypotension, which were associated with decreased expression of interleukin 1 beta (Il1b), interleukin 6 (Il6) and nitric oxide synthase 2 (Nos2), as well as reduced infiltration of neutrophils and macrophages in lung tissue. Pfkfb3 ablation in cultured macrophages attenuated LPS-induced glycolytic flux, resulting in a decrease in proinflammatory gene expression. Mechanistically, Pfkfb3 ablation or inhibition with a Pfkfb3 inhibitor AZ26 suppresses LPS-induced proinflammatory gene expression via the NF-κB signaling pathway. In summary, our study reveals the critical role of Pfkfb3 in LPS-induced sepsis via reprogramming macrophage metabolism and regulating proinflammatory gene expression. Therefore, PFKFB3 is a potential target for the prevention and treatment of inflammatory diseases such as sepsis.