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The key players of the hypoxic response are the hypoxia-inducible factors (Hif), whose α-subunits are tightly regulated by Prolyl-4-hydroxylases (PHD), predominantly by PHD2. Monocytes/Macrophages are involved in atherosclerosis but also restenosis and were found at hypoxic and sites of the lesion. Little is known about the role of the myeloid PHD2 in atherosclerosis and neointima formation. The study aimed to investigate the consequences of a myeloid deficiency of PHD2 in the process of neointima formation using an arterial denudation model. LysM-cre mice were crossed with PHD2 fl/fl , PHD2 fl/fl /Hif1α fl/fl and PHD2 fl/fl /Hif2α fl/fl to get myeloid specific knockout of PHD2 and the Hif-α subunits. Denudation of the femoral artery was performed and animals were fed a western type diet afterwards with analysis of neointima formation 5 and 35 days after denudation. Increased neointima formation in myeloid PHD2 knockouts was observed, which was blunted by double-knockout of PHD2 and Hif1α whereas double knockout of PHD2 and Hif-2α showed comparable lesions to the PHD2 knockouts. Macrophage infiltration was comparable to the neointima formation, suggesting a more inflammatory reaction, and was accompanied by increased intimal VEGF-A expression. Collagen-content inversely correlated to the extent of neointima formation suggesting a destabilization of the plaque. This effect might be triggered by macrophage polarization. Therefore, in vitro results showed a distinct expression pattern in differentially polarized macrophages with high expression of Hif-1α, VEGF and MMP-1 in proinflammatory M1 macrophages. In conclusion, the results show that myeloid Hif-1α is involved in neointima hyperplasia. Our in vivo and in vitro data reveal a central role for this transcription factor in driving plaque-vascularization accompanied by matrix-degradation leading to plaque destabilization., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Resolution of inflammation is essential for tissue homeostasis and represents a promising approach to inflammatory disorders. Here we found that developmental endothelial locus-1 (DEL-1), a secreted protein that inhibits leukocyte-endothelial adhesion and inflammation initiation, also functions as a non-redundant downstream effector in inflammation clearance. In human and mouse periodontitis, waning of inflammation was correlated with DEL-1 upregulation, whereas resolution of experimental periodontitis failed in DEL-1 deficiency. This concept was mechanistically substantiated in acute monosodium-urate-crystal-induced inflammation, where the pro-resolution function of DEL-1 was attributed to effective apoptotic neutrophil clearance (efferocytosis). DEL-1-mediated efferocytosis induced liver X receptor-dependent macrophage reprogramming to a pro-resolving phenotype and was required for optimal production of at least certain specific pro-resolving mediators. Experiments in transgenic mice with cell-specific overexpression of DEL-1 linked its anti-leukocyte-recruitment action to endothelial cell-derived DEL-1 and its efferocytic/pro-resolving action to macrophage-derived DEL-1. Thus, the compartmentalized expression of DEL-1 facilitates distinct homeostatic functions in an appropriate context that can be harnessed therapeutically.

Angiogenesis and inflammation go hand in hand in various (patho-)physiological conditions. Several studies have highlighted the interconnection between endothelial cells (ECs) and macrophages in these conditions at the level of growth factor and cytokine signaling, yet the importance of metabolism and metabolic signaling has been largely overlooked. Modulating macrophage and/or endothelial functions by interfering with metabolic pathways offers new perspectives for therapeutic strategies. In this review, we highlight the complexity of the interrelationship between the inflammatory response and angiogenesis. More in particular, the interaction between macrophages and ECs will be discussed with a special focus on how their metabolism can contribute to (patho-)physiological conditions.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1 f/ f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2 f/+ Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.f/ f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior., (Copyright © 2016 American Society for Microbiology.)

Brucellosis is a common zoonotic disease caused by intracellular pathogens of the genus Brucella. Brucella infects macrophages and evades clearance mechanisms, thus resulting in chronic parasitism. Herein, we studied the molecular changes that take place in human brucellosis both in vitro and ex vivo. RNA sequencing was performed in primary human macrophages (Mj) and polymorphonuclear neutrophils (PMNs) infected with a clinical strain of Brucella spp. We observed a downregulation in the expression of genes involved in host response, such as TNF signaling, IL-1b production, and phagosome formation in Mj, and phosphatidylinositol signaling and TNF signaling in PMNs, being in line with the ability of the pathogen to survive within phagocytes. Further transcriptomic analysis of isolated peripheral blood mononuclear cells (PBMCs) and PMNs from patients with acute brucellosis before treatment initiation and after successful treatment revealed a positive correlation of the molecular signature of active disease with pathways associated with response to interferons (IFN). We identified 24 common genes that were significantly altered in both PMNs and PBMCs, including genes involved in IFN signaling that were downregulated after treatment in both cell populations, and IL1R1 that was upregulated. The concentration of several inflammatory mediators was measured in the serum of these patients, and levels of IFN-g, IL-1b and IL-6 were found significantly increased before the treatment of acute brucellosis. An independent cohort of patients with chronic brucellosis also revealed increased levels of IFN-g during relapse compared to remissions. Taken together, this study provides for the first time an in-depth analysis of the transcriptomic alterations that take place in human phagocytes upon infection, and in peripheral blood immune populations during active disease. [ABSTRACT FROM AUTHOR]

Angiogenesis and inflammation go hand in hand in various (patho-)physiological conditions. Several studies have highlighted the interconnection between endothelial cells (ECs) and macrophages in these conditions at the level of growth factor and cytokine signaling, yet the importance of metabolism and metabolic signaling has been largely overlooked. Modulating macrophage and/or endothelial functions by interfering with metabolic pathways offers new perspectives for therapeutic strategies. In this review, we highlight the complexity of the interrelationship between the inflammatory response and angiogenesis. More in particular, the interaction between macrophages and ECs will be discussed with a special focus on how their metabolism can contribute to (patho-)physiological conditions. [ABSTRACT FROM AUTHOR]

The prolyl-4-hydroxylase domain (PHD) enzymes are regarded as the molecular oxygen sensors. There is an interplay between oxygen availability and cellular metabolism, which in turn has significant effects on the functionality of innate immune cells, such as macrophages. However, if and how PHD enzymes affect macrophage metabolism are enigmatic. We hypothesized that macrophage metabolism and function can be controlled via manipulation of PHD2. We characterized the metabolic phenotypes of PHD2-deficient RAW cells and primary PHD2 knockout bone marrow-derived macrophages (BMDM). Both showed typical features of anaerobic glycolysis, which were paralleled by increased pyruvate dehydrogenase kinase 1 (PDK1) protein levels and a decreased pyruvate dehydrogenase enzyme activity. Metabolic alterations were associated with an impaired cellular functionality. Inhibition of PDK1 or knockout of hypoxia-inducible factor 1α (HIF-1α) reversed the metabolic phenotype and impaired the functionality of the PHD2-deficient RAW cells and BMDM. Taking these results together, we identified a critical role of PHD2 for a reversible glycolytic reprogramming in macrophages with a direct impact on their function. We suggest that PHD2 serves as an adjustable switch to control macrophage behavior. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) primarily regulates red blood cell formation, and EPO serum levels are increased on hypoxic stress (e.g., anemia and altitude). In addition to anemia, recent discoveries suggest new therapeutic indications for EPO, unrelated to erythropoiesis. We investigated the skeletal role of EPO using several models of overexpression (Tg6 mice) and EPO administration (intermittent/continuous, high/low doses) in adult C57Bl6 female mice. Using microcomputed tomography, histology, and serum markers, we found that EPO induced a 32%-61% trabecular bone loss caused by increased bone resorption (+60%-88% osteoclast number) and reduced bone formation rate (-19 to -74%; P < 0.05 throughout). EPO targeted the monocytic lineage by increasing the number of bonemonocytes/macrophages, preosteoclasts, and mature osteoclasts. In contrast to the attenuated bone formation in vivo, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, suggesting an indirect effect of EPO on osteoblasts. However, EPO had a direct effect on preosteoclasts by stimulating osteoclastogenesis in isolated cultures (+60%) via the Jak2 and PI3K pathways. In summary, our findings demonstrate that EPO negatively regulates bone mass and thus bears significant clinical implications for the potential management of patients with endogenously or therapeutically elevated EPO levels. [ABSTRACT FROM AUTHOR]