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The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

The nucleoside adenosine is a potent regulator of vascular homeostasis, but it remains unclear how expression or function of the adenosine-metabolizing enzyme adenosine kinase (ADK) and the intracellular adenosine levels influence angiogenesis. We show here that hypoxia lowered the expression of ADK and increased the levels of intracellular adenosine in human endothelial cells. Knockdown (KD) of ADK elevated intracellular adenosine, promoted proliferation, migration, and angiogenic sprouting in human endothelial cells. Additionally, mice deficient in endothelial ADK displayed increased angiogenesis as evidenced by the rapid development of the retinal and hindbrain vasculature, increased healing of skin wounds, and prompt recovery of arterial blood flow in the ischemic hindlimb. Mechanistically, hypomethylation of the promoters of a series of pro-angiogenic genes, especially for VEGFR2 in ADK KD cells, was demonstrated by the Infinium methylation assay. Methylation-specific PCR, bisulfite sequencing, and methylated DNA immunoprecipitation further confirmed hypomethylation in the promoter region of VEGFR2 in ADK-deficient endothelial cells. Accordingly, loss or inactivation of ADK increased VEGFR2 expression and signaling in endothelial cells. Based on these findings, we propose that ADK downregulation-induced elevation of intracellular adenosine levels in endothelial cells in the setting of hypoxia is one of the crucial intrinsic mechanisms that promote angiogenesis.

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. [ABSTRACT FROM AUTHOR]

Background and Purpose: Macrophage infiltration into the lungs is a characteristic of pulmonary hypertension (PH). Glycolysis is the main metabolic pathway for macrophage activation. However, the effect of macrophage glycolysis on the development of PH remains unknown. We investigated the effect of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKBF3), a critical enzyme of macrophage glycolysis, on PH development.Experimental Approach: Lung tissues from PH patients were examined by immunostaining with macrophage markers. PH was induced in Wistar rats with SU5416/hypoxia and in mice with hypoxia. Lungs and macrophages were isolated for analysis by RT-PCR, western blot, flow cytometry, and immunostaining.Key Results: Expression of glycolytic molecules was increased in circulating peripheral blood mononuclear cells (PBMCs) and lung macrophages of PH patients. These results were also found in lung macrophages of SU5416/hypoxia (Su/Hx)-induced PH rats and hypoxia-induced PH mice. PH was ameliorated in myeloid-specific Pfkfb3-deficient mice (Pfkfb3ΔMϕ ) or mice treated with the PFKFB3 inhibitor 3PO, compared with their controls. Alveolar macrophages of PH Pfkfb3ΔMϕ mice produced lower levels of growth factors and pro-inflammatory cytokines than those of control mice. Circulating myeloid cells and lung myeloid cells were much fewer in PH Pfkfb3ΔMϕ mice than controls. Mechanistically, overexpression of Hif1a or Hif2a in bone marrow-derived macrophages (BMDMs) cultured with bone marrow of Pfkfb3ΔMϕ mice restored the decreased expression of pro-inflammatory cytokines and growth factors.Conclusions and Implications: Myeloid Pfkfb3 deficiency protects mice from PH, thereby suggesting that myeloid PFKFB3 is one of the important targets in the therapeutic effect of PFKFB3 inhibition in PH treatment. [ABSTRACT FROM AUTHOR]

Background: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis after angioplasty or stent placement. VSMCs reprogram their metabolism to meet the increased requirements of lipids, proteins, and nucleotides for their proliferation. De novo purine synthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined.Methods: De novo purine synthesis in proliferative VSMCs was evaluated by liquid chromatography-tandem mass spectrometry. The expression of ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last 2 steps of the de novo purine synthesis pathway, was assessed in VSMCs of proliferative arterial neointima. Global and VSMC-specific knockout of Atic mice were generated and used for examining the role of ATIC-associated purine metabolism in the formation of arterial neointima and atherosclerotic lesions.Results: In this study, we found that de novo purine synthesis was increased in proliferative VSMCs. Upregulated purine synthesis genes, including ATIC, were observed in the neointima of the injured vessels and atherosclerotic lesions both in mice and humans. Global or specific knockout of Atic in VSMCs inhibited cell proliferation, attenuating the arterial neointima in models of mouse atherosclerosis and arterial restenosis.Conclusions: These results reveal that de novo purine synthesis plays an important role in VSMC proliferation in arterial disease. These findings suggest that targeting ATIC is a promising therapeutic approach to combat arterial diseases. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is one of the leading causes of death in sepsis. Endothelial inflammation and dysfunction play a prominent role in development of ALI. Glycolysis is the predominant bioenergetic pathway for endothelial cells (ECs). However, the role of EC glycolysis in ALI of sepsis remains unclear. Here we show that both the expression and activity of PFKFB3, a key glycolytic activator, were markedly increased in lipopolysaccharide (LPS)-treated human pulmonary arterial ECs (HPAECs) in vitro and in lung ECs of mice challenged with LPS in vivo. PFKFB3 knockdown significantly reduced LPS-enhanced glycolysis in HPAECs. Compared with LPS-challenged wild-type mice, endothelial-specific Pfkfb3 knockout (Pfkfb3 ΔVEC) mice exhibited reduced endothelium permeability, lower pulmonary edema, and higher survival rate. This was accompanied by decreased expression of intracellular adhesion molecule-1 (Icam-1) and vascular cell adhesion molecule 1 (Vcam-1), as well as decreased neutrophil and macrophage infiltration to the lung. Consistently, PFKFB3 silencing or PFKFB3 inhibition in HPAECs and human pulmonary microvascular ECs (HPMVECs) significantly downregulated LPS-induced expression of ICAM-1 and VCAM-1, and monocyte adhesion to human pulmonary ECs. In contrast, adenovirus-mediated PFKFB3 overexpression upregulated ICAM-1 and VCAM-1 expression in HPAECs. Mechanistically, PFKFB3 silencing suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB)-p65, and NF-κB inhibitors abrogated PFKFB3-induced expression of ICAM-1 and VCAM-1. Finally, administration of the PFKFB3 inhibitor 3PO also reduced the inflammatory response of vascular endothelium and protected mice from LPS-induced ALI. Overall, these findings suggest that targeting PFKFB3-mediated EC glycolysis is an efficient therapeutic strategy for ALI in sepsis. [ABSTRACT FROM AUTHOR]