Endothelial Dysfunction in Murine Model of Systemic Sclerosis: Tight-skin Mice 1

We conducted this study to analyze endothelial cell function within intact thoracic aorta of the systemic sclerosis murine model, the heterozygous tight-skin mice 1: (i) assessing the distribution and activation intensity of endothelial cells, responsive to endothelium-dependent vasodilators (acetylcholine, adenosine triphosphate, bradykinin, and substance P) and Iloprost, using laser line confocal microscopy in combination with two Ca2+ fluorescent dyes; (ii) evaluating en-dothelium-dependent vasodilator- and Iloprostinduced relaxation, using isometric tension measurement; and (iii) investigating the role of nitric oxide in mediating relaxation to acetylcholine and adenosine triphosphate. The number of activated endothelial cells was significantly lower in heterozygous tight-skin mice 1, compared with controls, for adenosine triphosphate and Iloprost. Maximal increase of Ca2+ fluorescence intensity ratio in activated endothelial cells was decreased for adenosine triphosphate, bradykinin, and Iloprost, in heterozygous tight-skin mice 1. Adenosine triphosphate- and Iloprost-mediated aortic relaxation was further impaired in heterozygous tight-skin mice 1. Finally, aortic relaxation to acetylcholine and adenosine triphosphate was markedly decreased by nitric oxide synthase inhibitor in heterozygous tight-skin mice 1. This study suggests that endothelial cell receptors for endothelium-dependent vasodilators and Iloprost may not be homogeneously distributed or continuously expressed in thoracic aorta of heterozygous tight-skin mice 1, resulting in endothelium-dependent vasodilatation dysfunction. Moreover, because endothelium-dependent relaxation was highly dependent on nitric oxide release in heterozygous tight-skin mice 1, endothelium-dependent relaxation may differ from that of controls by increased production of nitric oxide. In turn, in heterozygous tight-skin mice 1, the resulting elevated nitric oxide levels may contribute to nitric oxide-mediated free radical endothelial cytotoxicity, although endothelium impairment may be related to other factors, particularly: Fbn-1 gene mutation and transforming growth factor-β

Connexins and M3 muscarinic receptors contribute to heterogeneous Ca(2+) signaling in mouse aortic endothelium.

Smooth muscle tone is controlled by Ca(2+) signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40) and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca(2+) signaling of the mouse aorta. Ca(2+) imaging was performed on intact aortic endothelium from both wild type (Cx40+/+) and Connexin40-deficient (Cx40 -/-) mice. Acetylcholine (ACh) induced early fast and high amplitude Ca(2+) transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca(2+) transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca(2+) waves, indicating that Cx40 contributes to the spreading of Ca(2+) signals. The propagation of those Ca(2+) responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca(2+) waves. Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca(2+) signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors

Geochemical Study of Natural CO2 Emissions in the French Massif Central: How to Predict Origin, Processes and Evolution of CO2 Leakage

International audienceThis study presents an overview of some results obtained within the French ANR (National Agency of Research) supported Géocarbone-Monitoring research program. The measurements were performed in Sainte-Marguerite, located in the French Massif Central. This site represents a natural laboratory for CO2/fluid/rock interactions studies, as well as CO2 migration mechanisms towards the surface. The CO2 leaking character of the studied area also allows to test and validate measurements methods and verifications for the future CO2 geological storage sites. During these surveys, we analyzed soil CO2 fluxes and concentrations. We sampled and analyzed soil gases, and gas from carbo-gaseous bubbling springs. A one-month continuous monitoring was also tested, to record the concentration of CO2 both in atmosphere and in the soil at a single point. We also developed a new methodology to collect soil gas samples for noble gas abundances and isotopic analyses, as well as carbon isotopic ratios. Our geochemical results, combined with structural geology, show that the leaking CO2 has a very deep origin, partially mantle derived. The gas rises rapidly along normal and strike-slip active faults. CO2 soil concentrations (also showing a mantle derived component) and CO2 fluxes are spatially variable, and reach high values. The recorded atmospheric CO2 is not very high, despite the important CO2 degassing throughout the whole area

Perinatal Hypoxia Enhances Cyclic Adenosine Monophosphate-mediated BKCa Channel Activation in Adult Murine Pulmonary Artery.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca-activated K (BKCa) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BKCa channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BKCa activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BKCa channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BKCa channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia

Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells.

Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K(+) channels play an important role in the regulation of vascular tone. In the fetus, Ca(2+)-activated K(+) channels (K(Ca)) are predominant, whereas from birth voltage-gated K(+) channels (K(V)) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K(+) channels in adulthood. We evaluated the effects of perinatal hypoxia on K(V) and K(Ca) channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only K(V) channels contributed to this current, whereas in mice born in hypoxia both K(V) and K(Ca) channels were implicated. K(V) channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased K(Ca) and K(V) channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance K(Ca) alpha-subunit and K(V)1.5 alpha-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,l-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by K(Ca) channels and only slightly by K(V) channels, whereas following perinatal hypoxia both K(Ca) and K(V) channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K(+) channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity

Connexins 43 and 26 are differentially increased after rat bladder outlet obstruction.

To evaluate the regulation of connexin expression by fluid pressure, we have studied the effects of elevated transmural urine pressure on Connexin43 (Cx43) and Cx26. We chose to focus on these two proteins out of the five connexins (Cx26, 43, 40, 37, and 45) which we found by RT-PCR to be expressed in the rat bladder, since in situ hybridization and immunofluorescence showed that Cx43 is the predominant connexin expressed by smooth muscle cells (SMC), whereas Cx26 is abundantly expressed only in the latter cell type. To evaluate whether these connexins are affected by changes in transmural urine pressure, we used a rat model of bladder outlet obstruction, in which a ligature is placed around the urethra. Under conditions of increased fluid pressure due to urine retention, we observed that the expression of both Cx43 and Cx26 increased at both transcript and protein levels, reaching a maximum 7-9 h after the ligature. Further analysis revealed that these changes were accounted for by a fourfold increase in Cx43 mRNA of SMC but not urothelial cell and by a fivefold increase in Cx26 mRNA of urothelium. Scrape-loading of propidium iodide showed that the latter change was paralleled by a twofold increase in coupling between urothelial cells. The data show that Cx43 and Cx26 are differentially regulated during bladder outlet obstruction and contribute to the response of the bladder wall to increased voiding pressure, possibly to control its elasticity