0166: Changes in resistance arteries expression of extracellular nucleotides signaling partners during arterial hypertension

Cardiovascular diseases are the leading cause of mortality in industrialized countries and their prevalence increases with aging populations. Small arteries constitute the main site of peripheral vascular resistance and play a key role in the regulation of blood pressure. Vascular tone is exacerbated in hypertension (HBP) and accompanied by a hypertrophy of the arterial wall. Although signaling by extracellular nucleotides is important in vascular homeostasis its contribution to vascular pathologies affecting small arteries remains poorly understood. We evaluated here the expression pattern of nucleotides signaling pathway in resistance arteries vs aorta in mice. Genes of interest including P2 receptors, ectonucleotidases (CD39, CD73) and hemi channels (connexins, pannexins) were investigated by quantitative RT-PCR. Their expression in resistance arteries was assessed in Angiotensin II-treated mice, spontaneously hypertensive rats (SHR) and, since HBP is related to age, in 24-month old mice. Our results showed that several genes are more expressed in aorta (P2ry2 and connexin 43) while others are expressed specifically (P2rx1, connexin 37) or preferentially (P2ry6) in small arteries. The latest might be involved in pathologies affecting the small arteries. With HBP, we observed a decreased connexin 37 and 40 expression level in Angiotensin II-treated mice and in SHR respectively and both decreased with aging. Interestingly, CD39 (tone regulator) decreased in the two models of HBP and with aging. Such decrease in nucleotidase activity may enhance P2 receptors activation and increase vascular contractility/tone. This is especially true considering P2Y6 (tone promoter) that increased with aging. Further studies may allow us to evaluate the contribution of these mediators in the development of small arteries defect in aging associated or not with HBP. Signaling by extracellular nucleotides may constitute new therapeutic targets in the treatment of HBP

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

0343 : Essential role of P2Y6 UDP receptor in Angiotensin-II dependent arterial hypertension

Extracellular nucleotides are responsible for pleiotropic effects in the vasculature. Uracyl nucleotides are vasoactive and trophic agents and promote inflammation. The participation of specific P2 receptors in these effects remains undefined and their potential contribution in arterial hypertension is unknown.ObjectiveTo evaluate the contribution of the UDP receptor P2Y6 in hypertension in mouse.MethodsArterial contraction was evaluated using a wire myograph. Blood pressure was measured following nucleotides iv infusion and experimental hypertension was induced either by Angiotensine-II (Ang-II 1mg/kg/j) or DOCA-salt (1%) in uni-nephrectomized mice. Histological approaches, immunofluorescence and RTqPCR were used to evaluate the nature of vascular remodeling.ResultsP2Y6 displayed the highest arterial expression level among other P2Y receptors. Contraction of conductance (thoracic aorta) and resistance (mesenteric) arteries was abrogated in P2ry6-/- mice in response to UDP and UTP while other vasoconstrictor induced normal responses. P2Y6 receptor triggered a moderated intracellular calcium increase while RhoA (calcium facilitating pathway) activation was abrogated in P2ry6-/- mice. Both genetic deletion and pharmacological blockade of P2Y6 receptor abolished Ang-II-induced blood pressure increase (40 mmHg in wild type mice). By contrast, hypertensive response in DOCA-salt was equivalent in both genotypes. Following Ang-II treatment, P2ry6-/- mice developed a reduced arterial hypertrophic remodeling and fibrosis but equivalent immune cell recruitment/infiltration compared to wild type. These changes were corroborated to reduced mRNA expressions of TGFβ and NADPH oxidase subunits.ConclusionsVascular P2Y6 receptor contributes to exaggerated vascular tone, hypertrophy and fibrosis in the context of Ang-II-dependent hypertension. Its absence or pharmacological blockade limits vascular damages and prevents blood pressure increase associated to hypertension

The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin – a phosphoinositide 3-kinase-dependent mechanism

AbstractHigh concentrations of adenosine-5′-diphosphate ADP are able to induce partial aggregation without shape change of P2Y1 receptor-deficient mouse platelets through activation of the P2Y12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse αIIbβ3 integrin, revealed a low level activation of αIIbβ3 in P2Y1 receptor-deficient platelets in response to 100 μM ADP or 1 μM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of 32P-labeled platelets showed that P2Y12-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P20) or pleckstrin (P47) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y12 receptor is able to trigger a P2Y1 receptor-independent inside-out signal leading to αIIbβ3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the αIIbβ3 integrin, and (iii) the transduction pathway triggered by the P2Y12 receptor is independent of PKC but dependent on phosphoinositide 3-kinase

Alteration of Extracellular Nucleotide Metabolism in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a rare genetic condition primarily caused by hepatic ABCC6 transporter dysfunction. Most clinical manifestations of PXE are due to premature calcification of elastic fibers. However, the vascular impact of PXE is pleiotropic and remains ill defined. ABCC6 expression has recently been associated with cellular nucleotide export. We studied the impact of ABCC6 deficiency on blood levels of adenosine triphosphate and related metabolites and on soluble nucleotidase activities in PXE patients and Abcc6 mice. In addition, we investigated the expression of genes encoding ectocellular purinergic signaling proteins in mouse liver and aorta. Plasma adenosine triphosphate and pyrophosphate levels were significantly reduced in PXE patients and in Abcc6 mice, whereas adenosine concentration was not modified. Moreover, 5\u27-nucleotidase/CD73 activity was increased in the serum of PXE patients and Abcc6 mice. Consistent with alterations of purinergic signaling, the expression of genes involved in purine and phosphate transport/metabolism was dramatically modified in Abcc6 mouse aorta, with much less impact on the liver. ABCC6 deficiency causes impaired vascular homeostasis and tissue perfusion. Our findings suggest that these alterations are linked to changes in extracellular nucleotide metabolism that are remote from the liver. This opens new perspectives for the understanding of PXE pathophysiology

Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αβMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials

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

Selective involvement of serum response factor in pressure-induced myogenic tone in resistance arteries

OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9+/-2.3%) compared with control (16.3+/-3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries

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 contribution of arterial calcification to peripheral arterial disease in pseudoxanthoma elasticum

BACKGROUND AND AIMS: The contribution of arterial calcification (AC) in peripheral arterial disease (PAD) and arterial wall compressibility is a matter of debate. Pseudoxanthoma elasticum (PXE), an inherited metabolic disease due to ABCC6 gene mutations, combines elastic fiber fragmentation and calcification in various soft tissues including the arterial wall. Since AC is associated with PAD, a frequent complication of PXE, we sought to determine the role of AC in PAD and arterial wall compressibility in this group of patients. METHODS AND RESULTS: Arterial compressibility and patency were determined by ankle-brachial pressure index (ABI) in a cohort of 71 PXE patients (mean age 48 +/- SD 14 yrs, 45 women) and compared to 30 controls without PAD. Lower limb arterial calcification (LLAC) was determined by non-contrast enhanced helicoidal CT-scan. A calcification score (Ca-score) was computed for the femoral, popliteal and sub-popliteal artery segments of both legs. Forty patients with PXE had an ABI<0.90 and none had an ABI>1.40. LLAC increased with age, significantly more in PXE subjects than controls. A negative association was found between LLAC and ABI (r = -0.363, p = 0.002). The LLAC was independently associated with PXE and age, and ABI was not linked to cardiovascular risk factors. CONCLUSIONS: The presence of AC was associated with PAD and PXE without affecting arterial compressibility. PAD in PXE patients is probably due to proximal obstructive lesions developing independently from cardiovascular risk factors