Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1^−/− mice. Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT–PCR. Entpd1^−/− mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1^−/− mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y₆ receptor and was independent of other uracil nucleotide-responding receptors (P2Y₂ and P2Y₄). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1^−/− mice. In addition, pressurized mesenteric arteries from Entpd1^−/− mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110β and p110γ) have been implicated in G_i-dependent integrin α_IIbβ₃ regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110β is primarily responsible for G_i-dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin α_IIbβ₃ activation in p110γ^-/- platelets was not associated with alterations in the levels of PI(3,4)P₂ or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110γ principally regulated integrin α_IIbβ₃ activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110γ, led to a major reduction in integrin α_IIbβ₃ activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinase-independent signaling function for p110γ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110β and p110γ is critical for P2Y₁₂/G_i-dependent integrinα_IIbβ₃ regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.

Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2–VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.

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.

Blood flow autoregulation results from the ability of resistance arteries to reduce or increase their diameters in response to changes in intravascular pressure. The mechanism by which arteries maintain a constant blood flow to organs over a range of pressures relies on this myogenic response, which defines the intrinsic property of the smooth muscle to contract in response to stretch. The resistance to flow created by myogenic tone (MT) prevents tissue damage and allows the maintenance of a constant perfusion, despite fluctuations in arterial pressure. Interventions targeting MT may provide a more rational therapeutic approach in vascular disorders, such as hypertension, vasospasm, chronic heart failure, or diabetes. Despite its early description by Bayliss in 1902, the cellular and molecular mechanisms underlying MT remain poorly understood. We now appreciate that MT requires a complex mechanotransduction converting a physical stimulus (pressure) into a biological response (change in vessel diameter). Although smooth muscle cell depolarization and a rise in intracellular calcium concentration are recognized as cornerstones of the myogenic response, the role of wall strain-induced formation of vasoactive mediators is less well established. The vascular system expresses a large variety of Class 1 G protein-coupled receptors (GPCR) activated by an eclectic range of chemical entities, including peptides, lipids, nucleotides, and amines. These messengers can function in blood vessels as vasoconstrictors. This review focuses on locally generated GPCR agonists and their proposed contributions to MT. Their interplay with pivotal G_q-11 and G_12-13 protein signalling is also discussed.