Bernard Muller

Hypoxia-induced hyperreactivity of pulmonary arteries: role of cyclooxygenase-2, isoprostanes, and thromboxane receptors

AIMSnThis study investigates the role of the cyclooxygenase (COX)/prostanoid pathway in chronic hypoxia-induced hyperreactivity of pulmonary arteries.nnnMETHODS AND RESULTSnPulmonary arteries were removed from normoxic or hypoxic (0.5 atm for 21 days) mice and studied for protein expression/localization of COX-1, COX-2, and thromboxane A2 (TXA2)-synthase, release of TXA2, prostacyclin (PGI2) and the isoprostane 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), and vasomotor responses. COX-2 expression was increased in all layers of pulmonary arteries from hypoxic mice. In contrast, COX-1 expression was not significantly modified following chronic hypoxia, whereas TXA2-synthase was decreased. Chronic hypoxia differentially affected prostanoid release from pulmonary arteries: TXA2 secretion was not significantly modified; PGI2 secretion was decreased, whereas 8-iso-PGF2alpha secretion was increased. A selective COX-2 inhibitor decreased 8-iso-PGF2alpha release. Arachidonic acid elicited an endothelium- and COX-1-dependent relaxation in pulmonary arteries from normoxic mice. In contrast, arachidonic acid induced an endothelium-independent contraction in pulmonary arteries from hypoxic mice that was partially reduced by catalase, COX-1, COX-2, or TXA2-synthase inhibitors and was totally abolished by blockade of the thromboxane (TP) receptor. Hyperresponsiveness to phenylephrine (PE) of pulmonary arteries from hypoxic mice was also decreased by COX-2 inhibitors, TP receptor antagonists or catalase, but not by TXA2-synthase inhibitors. Finally, 8-iso-PGF2alpha induced a TP receptor-dependent contraction in pulmonary arteries and markedly potentiated the contractile response to PE.nnnCONCLUSIONnChronic hypoxia up-regulates COX-2 expression, increases 8-iso-PGF2alpha release, and shifts arachidonic acid-induced, endothelium-dependent relaxation to an endothelium-independent and TP receptor-dependent contraction in pulmonary arteries. COX-2-dependent production of 8-iso-PGF2alpha, by activating TP receptors, participates in hypoxia-induced hyperreactivity of pulmonary arteries.

Improved energy supply regulation in chronic hypoxic mouse counteracts hypoxia-induced altered cardiac energetics.

Background Hypoxic states of the cardiovacular system are undoubtedly associated with the most frequent diseases of modern time. Therefore, understanding hypoxic resistance encountered after physiological adaptation such as chronic hypoxia, is crucial to better deal with hypoxic insult. In this study, we examine the role of energetic modifications induced by chronic hypoxia (CH) in the higher tolerance to oxygen deprivation. Methodology/Principal Findings Swiss mice were exposed to a simulated altitude of 5500 m in a barochamber for 21 days. Isolated perfused hearts were used to study the effects of a decreased oxygen concentration in the perfusate on contractile performance (RPP) and phosphocreatine (PCr) concentration (assessed by 31P-NMR), and to describe the integrated changes in cardiac energetics regulation by using Modular Control Analysis (MoCA). Oxygen reduction induced a concomitant decrease in RPP (−46%) and in [PCr] (−23%) in Control hearts while CH hearts energetics was unchanged. MoCA demonstrated that this adaptation to hypoxia is the direct consequence of the higher responsiveness (elasticity) of ATP production of CH hearts compared with Controls (−1.88±0.38 vs −0.89±0.41, p<0.01) measured under low oxygen perfusion. This higher elasticity induces an improved response of energy supply to cellular energy demand. The result is the conservation of a healthy control pattern of contraction in CH hearts, whereas Control hearts are severely controlled by energy supply. Conclusions/Significance As suggested by the present study, the mechanisms responsible for this increase in elasticity and the consequent improved ability of CH heart metabolism to respond to oxygen deprivation could participate to limit the damages induced by hypoxia.

Relaxation induced by red wine polyphenolic compounds in rat pulmonary arteries: lack of inhibition by NO‐synthase inhibitor

Some red wine polyphenols exert nitric oxide (NO)‐dependent relaxation in systemic arteries, following activation of endothelial NO synthase (eNOS). In this study, the effect of red wine polyphenols was determined in rat intrapulmonary arteries, and the effect of some of these compounds was compared with the responses obtained in rat aorta. In pulmonary arteries, red wine polyphenolic extract (>u2003300u2003μg/mL) exerted relaxation that was not inhibited by the NOS inhibitor Nω‐nitro‐l‐arginine methylester (l‐NAME) or endothelium removal. Among the several fractions obtained from the extract, the one enriched with anthocyanins was less active than fractions containing non‐anthocyanins. Among the latter, the most active for relaxing pulmonary arteries was the one enriched in the stilbene derivative trans‐resveratrol (relaxation for concentration >10u2003μg/mL). Trans‐piceid, the glucoside derivative of trans‐resveratrol, was almost inactive. Trans‐resveratrol‐induced relaxation, as well as relaxation to the anthocyanin delphinidin, was l‐NAME‐insensitive in pulmonary arteries. In aorta, trans‐resveratrol and trans‐piceid exerted similar effects to those in pulmonary arteries that were also not inhibited by l‐NAME. However, red wine polyphenolic extract and delphinidin induced relaxation of aorta at much lower concentrations (about 10u2003μg/mL) than in pulmonary arteries, and their effects were inhibited by l‐NAME. These data show differences between small intrapulmonary arteries and systemic conductance arteries in their responses to red wine polyphenols, the major difference being that the relaxant effect of these compounds is not blunted by NOS inhibitor in pulmonary arteries. They suggest that red wine polyphenols act directly on smooth muscle to promote pulmonary artery relaxation.

Effect of engineered nanoparticles on vasomotor responses in rat intrapulmonary artery.

Pulmonary circulation could be one of the primary vascular targets of finest particles that can deeply penetrate into the lungs after inhalation. We investigated the effects of engineered nanoparticles on vasomotor responses of small intrapulmonary arteries using isometric tension measurements. Acute in vitro exposure to carbon nanoparticles (CNP) decreased, and in some case abolished, the vasomotor responses induced by several vasoactive agents, whereas acute exposure to titanium dioxide nanoparticles (TiO(2)NP) did not. This could be attributed to a decrease in the activity of those vasoactive agents (including PGF(2)(alpha), serotonin, endothelin-1 and acetylcholine), as suggested when they were exposed to CNP before being applied to arteries. Also, CNP decreased the contraction induced by 30 mM KCl, without decreasing its activity. After endoplasmic reticulum calcium stores depletion (by caffeine and thapsigargin), CaCl(2) addition induced a contraction, dependent on Store-Operated Calcium Channels that was not modified by acute CNP exposure. Further addition of 30 mM KCl elicited a contraction, originating from activation of Voltage-Operated Calcium Channels that was diminished by CNP. Contractile responses to PGF(2)(alpha) or KCl, and relaxation to acetylcholine were modified neither in pulmonary arteries exposed in vitro for prolonged time to CNP or TiO(2)NP, nor in those removed from rats intratracheally instilled with CNP or TiO(2)NP. In conclusion, prolonged in vitro or in vivo exposure to CNP or TiO(2)NP does not affect vasomotor responses of pulmonary arteries. However, acute exposure to CNP decreases contraction mediated by activation of Voltage-Operated, but not Store-Operated, Calcium Channels. Moreover, interaction of some vasoactive agents with CNP decreases their biological activity that might lead to misinterpretation of experimental data.

Modular control analysis of effects of chronic hypoxia on mouse heart

Modular control analysis (MoCA; Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007) was applied here on perfused hearts to describe the modifications of the regulation of heart energetics induced in mice exposed to 3-wk chronic hypoxia. MoCA combines 31P-NMR spectroscopy and modular (top down) control analysis to describe the integrative regulation of energy metabolism in the intact beating heart, on the basis of two modules [ATP/phosphocreatine (PCr) production and ATP/PCr consumption] connected by the energetic intermediates. In contrast with previous results in rat heart, in which all control of contraction was on ATP demand, mouse heart energetics presented a shared control of contraction between ATP/PCr-producing and -consuming modules. In chronic hypoxic mice, the decrease in heart contractile activity and PCr-to-ATP ratio was surprisingly associated with an important and significant higher response of ATP/PCr production (elasticity) to PCr changes compared with control hearts (-10.4 vs. -2.46). By contrast, no changes were observed in ATP/PCr consumption since comparable elasticities were observed. Since elasticities determine the regulation of energetics of heart contraction, the present results show that this new parameter may be used to uncover the origin of the observed dysfunctions under chronic hypoxia conditions. Considering the decrease in mitochondrial content reported after exposure to chronic hypoxia, it appears that the improvement of ATP/PCr production response to ATP demand may be viewed as a positive adaptative mechanism. It now appears crucial to understand the very processes responsible for ATP/PCr producer elasticity toward the energetic intermediates, as well as their regulation.

Calcium signalling induced by in vitro exposure to silicium dioxide nanoparticles in rat pulmonary artery smooth muscle cells

The development and use of nanomaterials, especially engineered nanoparticles (NP), is expected to provide many benefits. But at the same time the development of such materials is also feared because of their potential human health risks. Indeed, NP display some characteristics similar to ultrafine environmental particles which are known to exert deleterious cardiovascular effects including pro-hypertensive ones. In this context, the effect of NP on calcium signalling, whose deregulation is often involved in hypertensive diseases, remain poorly described. We thus assessed the effect of SiO2 NP on calcium signalling by fluorescence imaging and on the proliferation response in rat pulmonary artery smooth muscle cells (PASMC). In PASMC, acute exposure to SiO2 NP, from 1 to 500μg/mL, produced an increase of the [Ca2+]i. In addition, when PASMC were exposed to NP at 200μg/mL, a proliferative response was observed. This calcium increase was even greater in PASMC isolated from rats suffering from pulmonary hypertension. The absence of extracellular calcium, addition of diltiazem or nicardipine (L-type voltage-operated calcium channel inhibitors both used at 10μM), and addition of capsazepine or HC067047 (TRPV1 and TRPV4 inhibitors used at 10μM and 5μM, respectively) significantly reduced this response. Moreover, this response was also inhibited by thapsigargin (SERCA inhibitor, 1μM), ryanodine (100μM) and dantrolene (ryanodine receptor antagonists, 10μM) but not by xestospongin C (IP3 receptor antagonist, 10μM). Thus, NP induce an intracellular calcium rise in rat PASMC originating from both extracellular and intracellular calcium sources. This study also provides evidence for the implication of TRPV channels in NP induced calcium rise that may highlight the role of these channels in the deleterious cardiovascular effects of NP.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries.

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries. Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively. SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC. In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation.

Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2-) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2-) and a 4h-exposure to PM2.5 (5-15μg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2- levels. The basal intracellular calcium ion level [Ca2+]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress.

J012 Cyclooxygenase-2-dependent isoprostane production in hypoxia-induced pulmonary hypertension

This study investigates the contribution of contractile prostanoids in hyper-reactivity of pulmonary arteries to vasoconstrictors, in a mice model of hypoxic pulmonary arterial hypertension. Male C57BL/6 mice were exposed or not to hypobaric hypoxia (0.5xa0atm) for 21 days. Extrapulmonary arteries were removed and used for evaluation of vasomotor responses (using wire myograph), for expression and localisation of cyclooxygenases and thromboxane A2 (TXA2)-synthase (by western blotting and immunofluoresnce) and for release of vasoactive prostanoids (by ELISA). In pulmonary arteries from hypoxic mice (but not in those from normoxic mice), arachidonic acid (30xa0μM) induced a contractile effect, that was converted into relaxation in the presence of SQ29548 (0.5xa0μM), a thromboxane receptor (TP) antagonist. In these arteries, contraction to phenylephrine (3xa0μM) was enhanced about 1.8 fold increase compared to controls, in both endothelium-intact and denuded preparations. This hyper-reactivity to phenylephrine was diminished by SQ29548, by the selective COX-2 inhibitor NS398 (1xa0μM), but not by the phospholipase A2 inhibitor AACOCF3 (30xa0μM), the COX-1 inhibitor SC560 (0.1xa0μM) or the TXA2-synthase inhibitor furegrelate (100xa0μM). None of these agents affected contraction to phenylephrine in pulmonary arteries from normoxic mice. Expression of COX-1, which was found in all layers of pulmonary arteries, was decreased by chronic hypoxia as well as expression of TXA2-synthase. COX-2 expression was restricted to the medial layer of pulmonary arteries. Hypoxia decreased the release of TXA2 and the release of PGI2 from pulmonary arteries, while it increased the release of 8-iso-PGF2α (an isoprostane derivative that is a marker of oxidative stress). NS398 abolished hypoxia-induced elevation of 8-iso-PGF2α release from pulmonary arteries. Finally, 8-iso-PGF2α induced a contractile effect in pulmonary arteries, which was blunted by SQ29548. Moreover, 8-iso-PGF2α markedly potentiated contraction to phenylephrine. These data show that following chronic hypoxia, pulmonary arteries exhibited alterations in arachidonic acid pathway, and hyper-responsiveness to phenylephrine. The latter is likely mediated by COX-2-dependent production of 8-iso-PGF2α, which in turn activates TP receptor. Such mechanisms probably contribute to elevation in pulmonary arterial resistance in hypoxia-induced pulmonary arterial hypertension.

C006 Nadph-oxidases and uncoupled endothelial NO-synthase in pulmonary arterial hypertension induced by chronic hypoxia

Nitric oxide (NO) production by endothelial NO-synthase (eNOS) is critically dependent on the cofactor, tetrahydrobiopterin (BH4). Depletion in BH4 consecutive to an increase of reactive oxygen species (ROS) production by NADPH-oxidases and/or eNOS over-expression, favour eNOS uncoupling. This study investigates the potential role of NADPH-oxidases and uncoupled eNOS in pulmonary arterial hypertension induced by chronic hypoxia. Male C57BL/6 and eNOS knockout (eNOS-/-) mice were exposed or not to hypobaric hypoxia (0.5xa0atm) for 21 days. Fulton index (right ventricular / left ventricular + septum weight ratio) was determined. Lungs were used for measurement of BH4 (by HPLC), for expression of eNOS (by western-blotting) and of the NADPH-oxidases subunits Nox1, Nox2 and Nox4 (by RT-PCR). Pulmonary arteries were also mounted in a wire myograph for evaluation of vasomotor responses. Chronic hypoxia induced a marked up-regulation of Nox1, Nox2 and Nox4 mRNAs in lungs, and an increase of ROS levels in pulmonary arteries. BH4 levels, as well as eNOS expression, were enhanced in lungs from hypoxic WT mice (1.25 and 4 fold increase compared to normoxic WT mice, respectively). In pulmonary arteries from hypoxic WT mice, the contractile response to phenylephrine was about 1.8 greater than in those from normoxic WT mice. The use of ROS scavengers (PEG-SOD or catalase) and NOS inhibitor (L-NAME) revealed the involvement of ROS in hypoxia-induced hyper-reactivity to phenylephrine, and a loss of NO-dependent relaxation. Chronic treatment of hypoxic WT mice with the BH4 precursor sepiapterin preserved the vasorelaxant effect of NO. This treatment and the deletion of eNOS gene abolished the inhibitory effect of catalase on phenylephrine-induced contraction, and also attenuated hypoxia-induced right ventricular hypertrophy. These data show that chronic hypoxia induced an up-regulation of Nox isoforms and eNOS in lungs. They suggest that uncoupled eNOS participates to right ventricular hypertrophy and to alterations of vasomotor responses in pulmonary arteries in hypoxia-induced pulmonary hypertension. The weak increase in BH4 and the large over-expression of eNOS suggest the existence of compensatory mechanisms on BH4 synthesis, which may moderate eNOS dysfunction. Grants: Fondation de France, ANR, GRRC (E.D PhD grant)