Gilles Kauffenstein

NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse

AIMS 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. METHODS AND RESULTS 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 P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). 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. CONCLUSION 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.

Identification of a Unique Co-operative Phosphoinositide 3-Kinase Signaling Mechanism Regulating Integrin αIIbβ3 Adhesive Function in Platelets

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 Gi-dependent integrin αIIbβ3 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 Gi-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 αIIbβ3 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 αIIbβ3 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 αIIbβ3 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/Gi-dependent integrinαIIbβ3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.

Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration.

The chemokine interleukin 8 (IL-8) is a major chemoattractant for human neutrophils. Here, we demonstrate novel evidence that IL-8-induced neutrophil chemotaxis requires a concurrent activation of P2 receptors, most likely the P2Y(2) which is dominantly expressed in these cells. Indeed, the migration of human neutrophils towards IL-8 was significantly inhibited by the P2Y receptor antagonists, suramin and reactive blue 2 (RB-2) and potentiated by a P2Y(2) ligand, ATP, but insensitive to specific antagonists of P2Y(1), P2Y(6) and P2Y(11) receptors. Adenosine had no effect on neutrophil migration towards IL-8 which contrasted with the stimulatory effect of this molecule on neutrophil chemotaxis caused by formyl-Met-Leu-Phe (fMLP or fMLF). Taken together, these data suggest that extracellular ATP is necessary for IL-8 to exert its chemotactic effect on neutrophils.

Emerging role of G protein-coupled receptors in microvascular myogenic tone

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.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke

AIMS 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. METHODS AND RESULTS 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. CONCLUSION 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.

The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation

Background and purpose:  Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto‐nucleotidase nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide‐dependent vasodilatation.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone

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 P2X1 and P2Y6 was dominant. P2Y6 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 P2Y6 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 P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 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 P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.

Quantification of the Calcification Phenotype of Abcc6-Deficient Mice with Microcomputed Tomography

Pseudoxanthoma elasticum in humans and dystrophic cardiac calcification in mice are heritable disorders characterized by dystrophic calcification of soft connective tissues related to the defective function of the ABCC6 (human)/Abcc6 (mouse) transporter. Of particular interest is the finding of calcified vibrissae in Abcc6(-/-) mice, which facilitates the study of dystrophic calcification by histological techniques. We aimed to determine whether mice prone to dystrophic cardiac calcification (C3H/HeOuJ and DBA/2J strains) presented similar vibrissae changes and to evaluate the value of microcomputed tomography to quantify the extent of mystacial vibrissae calcifications. These calcifications were absent in DBA/2J and C57BL/6J control mice. In both Abcc6(-/-) and C3H/HeOuJ mice, calcifications progressed in a caudal-rostral direction with aging. However, the calcification process was delayed in C3H/HeOuJ mice, indicating an incomplete expression of the calcification phenotype. We also found that the calcification process in the cephalic region was not limited to mystacial vibrissae but was also present in other periorbital sensorial vibrissae. The vibrissae calcification was circular and encompassed the medial region of the vibrissae capsule, adjacent to the ring and cavernous sinuses (the areas adjacent to blood and lymphatic vessels). Collectively, our findings confirm that Abcc6 acts as an inhibitor of spontaneous chronic mineralization and that microcomputed tomography is a valuable noninvasive tool for the assessment of the calcification phenotype in Abcc6-deficient mice.

Pseudoxanthoma elasticum: cardiac findings in patients and Abcc6-deficient mouse model.

Background Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, is a rare multiorgan disease characterized by the mineralization and fragmentation of elastic fibers in connective tissue. Cardiac complications reportedly associated with PXE are mainly based on case reports. Methods A cohort of 67 PXE patients was prospectively assessed. Patients underwent physical examination, electrocardiogram, transthoracic echocardiography, cardiac magnetic resonance imaging (CMR), treadmill testing, and perfusion myocardial scintigraphy (SPECT). Additionally, the hearts of a PXE mouse models (Abcc6−/−) and wild-type controls (WT) were analyzed. Results Three patients had a history of proven coronary artery disease. In total, 40 patients underwent exercise treadmill tests, and 28 SPECT. The treadmill tests were all negative. SPECT showed mild perfusion abnormalities in two patients. Mean left ventricular (LV) dimension and function values were within the normal range. LV hypertrophy was found in 7 (10.4%) patients, though the hypertrophy etiology was unknown for 3 of those patients. Echocardiography revealed frequent but insignificant mitral and tricuspid valvulopathies. Mitral valve prolapse was present in 3 patients (4.5%). Two patients exhibited significant aortic stenosis (3.0%). While none of the functional and histological parameters diverged significantly between the Abcc6−/− and WT mice groups at age of 6 and 12 months, the 24-month-old Abcc6−/− mice developed cardiac hypertrophy without contractile dysfunction. Conclusions Despite sporadic cases, PXE does not appear to be associated with frequent cardiac complications. However, the development of cardiac hypertrophy in the 24-month-old Abcc6−/− mice suggests that old PXE patients might be prone to developing late cardiopathy.

Disseminated Arterial Calcification and Enhanced Myogenic Response Are Associated With Abcc6 Deficiency in a Mouse Model of Pseudoxanthoma Elasticum

Objective—Pseudoxanthoma elasticum is an inherited metabolic disorder resulting from ABCC6 gene mutations. It is characterized by progressive calcification and fragmentation of elastic fibers in the skin, retina, and the arterial wall. Despite calcium accumulation in the arteries of patients with pseudoxanthoma elasticum, functional consequences remain unknown. In the present study, we investigated arterial structure and function in Abcc6−/− mice, a model of the human disease. Approach and Results—Arterial calcium accumulation was evaluated using alizarin red stain and atomic absorption spectrometry. Expression of genes involved in osteochondrogenic differentiation was measured by polymerase chain reaction. Elastic arterial properties were evaluated by carotid echotracking. Vascular reactivity was evaluated using wire and pressure myography and remodeling using histomorphometry. Arterial calcium accumulation was 1.5- to 2-fold higher in Abcc6−/− than in wild-type mice. Calcium accumulated locally leading to punctuate pattern. Old Abcc6−/− arteries expressed markers of both osteogenic (Runx2, osteopontin) and chondrogenic lineage (Sox9, type II collagen). Abcc6−/− arteries displayed slight increase in arterial stiffness and vasoconstrictor tone in vitro tended to be higher in response to phenylephrine and thromboxane A2. Pressure-induced (myogenic) tone was significantly higher in Abcc6−/− arteries than in wild type. Arterial blood pressure was not significantly changed in Abcc6−/−, despite higher variability. Conclusions—Scattered arterial calcium depositions are probably a result of osteochondrogenic transdifferentiation of vascular cells. Lower elasticity and increased myogenic tone without major changes in agonist-dependent contraction evidenced in aged Abcc6−/− mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis in the long term.