Objective—: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. Approach and Results—: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X ₁ and P2Y ₆ was dominant. P2Y ₆ fully sustained UDP/UTP-induced contraction (abrogated in P2ry6 ^−/− arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y ₆ receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6 ^−/− mice were protected against MT elevation in myocardial infarction–induced heart failure. Although both P2Y ₆ and P2Y ₂ receptors contributed to calcium mobilization, P2Y ₆ activation was mandatory for RhoA–GTP binding, myosin light chain, P42–P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43 ^+/− and P2rx7 ^−/− mesenteric resistance arteries. Conclusions—: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y ₆ receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Tumor-associated macrophages (TAM) are immunosuppressive cells that can massively accumulate in the tumor microenvironment. In patients with ovarian cancer, their density is correlated with poor prognosis. Targeting mediators that control the generation or the differentiation of immunoregulatory macrophages represents a therapeutic challenge to overcome tumor-associated immunosuppression. The ectonucleotidase CD39 hydrolyzes ATP into extracellular adenosine that exhibits potent immunosuppressive properties when signaling through the A2A adenosine receptor. We report here that CD14⁺ CD163⁺ TAM isolated from ovarian cancer patients and macrophages generated in vitro with M-CSF, express high levels of the membrane ectonucleotidase CD39 compared to classically activated macrophages. The CD39 inhibitor POM-1 and adenosine deaminase (ADA) diminished some of the immunosuppressive functions of CD14^high CD163^high CD39^high macrophages, such as IL-10 secretion. We identified the cytokine IL-27, secreted by tumor-infiltrating neutrophils, located close to infiltrating CD163⁺ macrophages, as a major rheostat of CD39 expression and consequently, on the acquisition of immunoregulatory properties by macrophages. Accordingly, the depletion of IL-27 downregulated CD39 and PD-L1 expression as well as IL-10 secretion by M-CSF-macrophages. Collectively, these data suggest that CD39, drived by IL-27 and CD115 ligands in ovarian cancer, maintains the immunosuppressive phenotype of TAM. This work brings new information on the acquisition of immunosuppressive properties by tumor-infiltrating macrophages.

Macrophages fight infection and ensure tissue repair, often operating at nutrient-poor wound sites. We investigated the ability of human macrophages to metabolize glycogen. We observed that the cytokines GM-CSF and M-CSF plus IL-4 induced glycogenesis and the accumulation of glycogen by monocyte-derived macrophages. Glyconeogenesis occurs in cells cultured in the presence of the inflammatory cytokines GM-CSF and IFNγ (M1 cells), via phosphoenolpyruvate carboxykinase 2 (PCK2) and fructose-1,6-bisphosphatase 1 (FBP1). Enzyme inhibition with drugs or gene silencing techniques and ¹³ C-tracing demonstrate that glutamine (metabolized by the TCA cycle), lactic acid, and glycerol were substrates of glyconeogenesis only in M1 cells. Tumor-associated macrophages (TAMs) also store glycogen and can perform glyconeogenesis. Finally, macrophage glycogenolysis and the pentose phosphate pathway (PPP) support cytokine secretion and phagocytosis regardless of the availability of extracellular glucose. Thus, glycogen metabolism supports the functions of human M1 and M2 cells, with inflammatory M1 cells displaying a possible dependence on glyconeogenesis.

Pseudoxanthoma Elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Although arterial hypertension (AHT) has been reported in PXE patients, its impact on pathological manifestations has as yet been unexplored. We investigated the consequences of experimental AHT on Abcc6−/− PXE mouse models. Experimental AHT was induced by deoxycorticosterone acetate (DOCA-salt) in uni-nephrectomised mice. Blood pressure (BP) and vascular reactivity were monitored using tail-cuff plethysmography and myography respectively. Calcium content and fibrosis were assessed using colorimetry, Von Kossa and Sirius red staining respectively. The gene expression implicated in vascular biology was measured using quantitative polymerase chain reaction. DOCA-salt induced a matching rise in BP in Abcc6−/− and WT mice. Aortic ring contraction and relaxation in vitro were comparable. Calcium accumulated in the hearts of hypertensive Abcc6−/− mice along with significant fibrosis in the myocardium and aorta by contrast with the WT mice. In hypertensive Abcc6−/− mouse aortas, these results were corroborated by gene expression patterns favouring calcification, fibrosis and extracellular matrix remodelling. Abcc6 loss-of-function is associated with greater cardiovascular calcification and fibrosis in mice subjected to DOCA-Salt hypertension. These results suggest likely cardiovascular deterioration in PXE patients with AHT, necessitating diligent BP monitoring.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using Entpd1^−/− male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in Entpd1^−/− mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from Entpd1^−/− mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from Entpd1^−/− mice compared to wild-types. Finally, changes in vascular reactivity in Entpd1^−/− mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.