NiONPs-induced alteration in calcium signaling and mitochondrial function in pulmonary artery endothelial cells involves oxidative stress and TRPV4 channels disruption.

In New Caledonia, anthropic activities, such as mining, increase the natural erosion of soils in nickel mines, which in turn, releases nickel oxide nanoparticles (NiONPs) into the atmosphere. Pulmonary vascular endothelial cells represent one of the primary targets for inhaled nanoparticles. The objective of this in vitro study was to assess the cytotoxic effects of NiONPs on human pulmonary artery endothelial cells (HPAEC). Special attention will be given to the level of oxidative stress and calcium signaling, which are involved in the physiopathology of cardiovascular diseases. HPAEC were exposed to NiONPs (0.5-150 µg/cm2) for 4 or 24 h. The following different endpoints were studied: (i) ROS production using CM-H2DCF-DA probe, electron spin resonance, and MitoSOX probe; the SOD activity was also measured (ii) calcium signaling with Fluo4-AM, Rhod-2, and Fluo4-FF probes; (iii) inflammation by IL-6 production and secretion and, (iv) mitochondrial dysfunction and apoptosis with TMRM and MitoTracker probes, and AnnexinV/PI. Our results have evidenced that NiONPs induced oxidative stress in HPAEC. This was demonstrated by an increase in ROS production and a decrease in SOD activity, the two mechanisms seem to trigger a pro-inflammatory response with IL-6 secretion. In addition, NiONPs exposure altered calcium homeostasis inducing an increased cytosolic calcium concentration ([Ca2+]i) that was significantly reduced by the extracellular calcium chelator EGTA and the TRPV4 inhibitor HC-067047. Interestingly, exposure to NiONPs also altered TRPV4 activity. Finally, HPAEC exposure to NiONPs increased intracellular levels of both ROS and calcium ([Ca2+]m) in mitochondria, leading to mitochondrial dysfunction and HPAEC apoptosis.

Altered vasoreactivity in neonatal rats with pulmonary hypertension associated with bronchopulmonary dysplasia: Implication of both eNOS phosphorylation and calcium signaling.

Bronchopulmonary dysplasia (BPD) consists of an arrest of pulmonary vascular and alveolar growth, with persistent hypoplasia of the pulmonary microvasculature and alveolar simplification. In 25 to 40% of the cases, BPD is complicated by pulmonary hypertension (BPD-PH) that significantly increases the risk of morbidity. In vivo studies suggest that increased pulmonary vascular tone could contribute to late PH in BPD. Nevertheless, an alteration in vasoreactivity as well as the mechanisms involved remain to be confirmed. The purpose of this study was thus to assess changes in pulmonary vascular reactivity in a murine model of BPD-PH. Newborn Wistar rats were exposed to either room air (normoxia) or 90% O2 (hyperoxia) for 14 days. Exposure to hyperoxia induced the well-known features of BPD-PH such as elevated right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and decreased pulmonary vascular density. Intrapulmonary arteries from hyperoxic pups showed decreased endothelium-dependent relaxation to acetylcholine without any alteration of relaxation to the NO-donor sodium nitroprusside. This functional alteration was associated with a decrease of lung eNOS phosphorylation at the Ser1177 activating site. In pups exposed to hyperoxia, serotonin and phenylephrine induced exacerbated contractile responses of intrapulmonary arteries as well as intracellular calcium response in pulmonary arterial smooth muscle cells (PASMC). Moreover, the amplitude of the store-operated Ca2+ entry (SOCE), induced by store depletion using a SERCA inhibitor, was significantly greater in PASMC from hyperoxic pups. Altogether, hyperoxia-induced BPD-PH alters the pulmonary arterial reactivity, with effects on both endothelial and smooth muscle functions. Reduced activating eNOS phosphorylation and enhanced Ca2+ signaling likely account for alterations of pulmonary arterial reactivity.

Expression and role of connexin-based gap junctions in pulmonary inflammatory diseases.

Connexins are transmembrane proteins that can generate intercellular communication channels known as gap junctions. They contribute to the direct movement of ions and larger cytoplasmic solutes between various cell types. In the lung, connexins participate in a variety of physiological functions, such as tissue homeostasis and host defence. In addition, emerging evidence supports a role for connexins in various pulmonary inflammatory diseases, such as asthma, pulmonary hypertension, acute lung injury, lung fibrosis or cystic fibrosis. In these diseases, the altered expression of connexins leads to disruption of normal intercellular communication pathways, thus contributing to various pathophysiological aspects, such as inflammation or tissue altered reactivity and remodeling. The present review describes connexin structure and organization in gap junctions. It focuses on connexins in the lung, including pulmonary bronchial and arterial beds, by looking at their expression, regulation and physiological functions. This work also addresses the issue of connexin expression alteration in various pulmonary inflammatory diseases and describes how targeting connexin-based gap junctions with pharmacological tools, synthetic blocking peptides or genetic approaches, may open new therapeutic perspectives in the treatment of these diseases.

Mitochondria: roles in pulmonary hypertension.

Mitochondria are essential cell organelles responsible for ATP production in the presence of oxygen. In the pulmonary vasculature, mitochondria contribute to physiological intracellular signalling pathways through production of reactive oxygen species and play the role of oxygen sensors that coordinate hypoxic pulmonary vasoconstriction. Mitochondria also play a pathophysiological role in pulmonary hypertension (PH). This disease is characterized by increased pulmonary arterial pressure and remodelling of pulmonary arteries, leading to increased pulmonary vascular resistance, hypertrophy of the right ventricle, right heart failure and ultimately death. Mitochondrial alterations have been evidenced in PH in pulmonary arteries and in the right ventricle, in particular a chronic shift in energy production from mitochondrial oxidative phosphorylation to glycolysis. This shift, initially described in cancer cells, may play a central role in PH pathogenesis. Further studies of these metabolic mitochondrial alterations in PH may therefore open new therapeutic perspectives in this disease.

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.

Impairment of NO-dependent relaxation in intralobar pulmonary arteries: comparison of urban particulate matter and manufactured nanoparticles.

BACKGROUND AND OBJECTIVES: Because pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO-cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries. METHODS: We used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release. RESULTS: Endothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-alpha, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2. CONCLUSION: In contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress-independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM-induced cardiovascular dysfunction.

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 (> 300 microg/mL) exerted relaxation that was not inhibited by the NOS inhibitor N(omega)-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 >10 microg/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 10 microg/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.

Targeted and persistent effects of NO mediated by S-nitrosation of tissue thiols in arteries with endothelial dysfunction.

We have previously demonstrated that in endothelium-denuded arteries, S-nitrosation of cysteine residues is a mechanism of formation of releasable nitric oxide (NO) stores, accounting for the long-lasting relaxation induced by S-nitrosating agents like S-nitrosoglutathione (GSNO). Here, we have investigated whether such effects could also be obtained in arteries exhibiting oxidative stress-associated endothelial dysfunction. Rats were implanted or not with a minipump delivering saline or angiotensin II for 14 days. As expected, aorta from angiotensin II-infused rats exhibited increased level of superoxide anions (as evaluated with dihydroethidine as fluorescent probe) and a reduced relaxation to acetylcholine in comparison to saline group. Unlike aortic rings with endothelium from controls, those from angiotensin II-infused rats exhibited persistent hyporesponsiveness to phenylephrine after pre-exposure to GSNO, as well as relaxation upon addition of N-acetylcysteine (NAC, which can displace NO from cysteine-NO residues) or HgCl(2) (which cleaves S-NO bonds). In aorta from angiotensin II-infused rats, GSNO also induced a persistent increase in cysteine-NO residues (as determined using anti-cysteine-NO antiserum), which was blunted by NAC and HgCl(2). These data indicate that (i) the vasorelaxant influence of releasable NO stores is unmasked by endothelial dysfunction (ii) S-nitrosation of cysteine residues remains an effective mechanism of formation of releasable NO stores in arteries exhibited oxidative stress-associated endothelial dysfunction. Thus, formation of releasable NO stores by S-nitrosating agents allows targeted vasculoprotective effects of NO at sites of endothelial dysfunction.

Red wine polyphenols prevent angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase.

OBJECTIVE: Chronic administration of moderate amounts of red wine has been associated with a protective effect on the cardiovascular system. This study examined whether red wine polyphenols prevent the angiotensin II (Ang II)-induced hypertension and endothelial dysfunction in rats, and, if so, to elucidate the underlying mechanism. METHODS: Hypertensive rats were obtained by a 14-day infusion of Ang II. Red wine polyphenols were administered in the drinking water one week before and during the Ang II infusion. Arterial pressure was measured in conscious rats. Ex vivo vascular relaxation was assessed in organ chambers, vascular superoxide anion production by dihydroethidine and vascular NADPH oxidase expression by immunohistochemistry. RESULTS: Ang II-induced hypertension was associated with decreased relaxation to acetylcholine but not to red wine polyphenols. The Ang II treatment also increased vascular superoxide anion production and expression of nox1 and p22phox NADPH oxidase subunits. Intake of red wine polyphenols prevented the Ang II-induced hypertension and endothelial dysfunction and normalized vascular superoxide anion production and NADPH oxidase subunit expression. Red wine polyphenol treatment alone did not affect blood pressure. CONCLUSION: Intake of red wine polyphenols prevents Ang II-induced hypertension and endothelial dysfunction. Prevention of vascular NADPH oxidase induction and preservation of arterial nitric oxide availability during Ang II administration likely contribute to this effect.

Role of reactive oxygen species and gp91phox in endothelial dysfunction of pulmonary arteries induced by chronic hypoxia.

1. This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia-induced pulmonary hypertension. 2. In pulmonary arteries from control mice, the NO-synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) potentiated contraction to prostaglandin F2alpha (PGF2alpha) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine-induced relaxation was slightly inhibited by polyethyleneglycol-superoxide dismutase (PEG-SOD) or catalase. 3. In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L-NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2-induced contraction. PEG-SOD or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L-NAME-insensitive) relaxations in extra- and intrapulmonary arteries from hypoxic mice. 4. Exposure of gp91phox(-/-) mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox(-/-) mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5. Chronic hypoxia increases ROS levels and impairs endothelial NO-dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia-induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium-dependent relaxation appears to be mediated by ROS, in a gp91phox-independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO-dependent relaxation

Formation of releasable NO stores by S-nitrosoglutathione in arteries exhibiting tolerance to glyceryl-trinitrate.

S-Nitrosating nitric oxide (NO) donors like S-nitrosoglutathione (GSNO) induce a persistent inhibition of vascular tone, through the formation of releasable NO stores. In this study, we investigate whether GSNO also induces NO stores-related effects in vessels exhibiting tolerance to glyceryl-trinitrate. Rat aortic rings treated with glyceryl-trinitrate (100 microM for 1 h) exhibited increased level of superoxide and a decrease in NO elevation and relaxation induced by subsequent addition of glyceryl-trinitrate. In glyceryl-trinitrate-treated rings as in controls, pre-exposure to GSNO (1 microM for 30 min) induced a persistent hyporesponsiveness to noradrenaline and a relaxant response to N-acetylcysteine (a low molecular weight thiol which can displace NO from NO stores), both of which being inhibited by guanylyl-cyclase or cyclic GMP-dependent protein kinase inhibitors. These data indicate that GSNO can promote the formation of releasable NO stores in arteries exhibiting increased superoxide level and tolerance to glyceryl-trinitrate. Formation of releasable NO stores is of potential interest to restore the protective effect of NO in organic nitrate-tolerant blood vessels.

Inhibition of arterial contraction by dinitrosyl-iron complexes: critical role of the thiol ligand in determining rate of nitric oxide (NO) release and formation of releasable NO stores by S-nitrosation.

The inhibition of arterial tone produced by two nitric oxide (NO) derivatives of biological relevance, dinitrosyl-iron complexes with cysteine (DNIC-CYS) or with glutathione (DNIC-GSH), was compared. Both compounds induced vasorelaxation within the same concentration range (3-300 nM) in endothelium-denuded rat aortic rings. Consistent with a faster rate of NO release from DNIC-CYS than from DNIC-GSH, the relaxant effect of DNIC-CYS was rapid in onset and tended to recover with time, whereas the one of DNIC-GSH developed slowly and was sustained. In addition, DNIC-GSH (0.3 and 1 microM) but not DNIC-CYS (1 microM) induced, even after washout of the drug, a persistent hyporesponsiveness to vasoconstrictors and a relaxant effect of low molecular weight thiols like N-acetylcysteine (NAC, which can displace NO from preformed NO stores). Both effects of DNIC-GSH were associated with elevation of cyclic GMP content and were attenuated by NO scavengers or a cyclic GMP-dependent protein kinases inhibitor. In rings previously exposed to DNIC-GSH, addition of mercuric chloride (which can cleave the cysteine-NO bond of S-nitrosothiols) elicited relaxation, completely blunted the one of NAC and also abolished the persistent elevation of NO content. In conclusion, this study shows that whereas both DNIC-CYS and DNIC-GSH elicited a NO release-associated relaxant effect in isolated arteries, only DNIC-GSH induced an inhibition of contraction which persisted after drug removal. The persistent effect of DNIC-GSH was attributed to the formation of releasable NO stores in arterial tissue, most probably as S-nitrosothiols. Thus, the nature of the thiol ligand plays a critical role in determining the mechanisms and duration of the effect of LMW-DNIC in arteries.

S-nitrosating nitric oxide donors induce long-lasting inhibition of contraction in isolated arteries.

The ability of various nitric oxide (NO) donors to induce long-lasting inhibition of contraction in isolated arteries was compared. All the studied compounds elicited a relaxant effect in rat aortic rings precontracted with norepinephrine (NE). Almost maximal relaxation was obtained with 1 microM of each compound. The S-nitrosating agents S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, S-nitroso-N-acetylcysteine, and sodium nitroprusside (1 microM) produced a decrease of the maximal effect of NE that persisted after removal of the drug. This hyporesponsiveness to NE was associated with a relaxant effect of N-acetylcysteine, a low-molecular weight thiol that can displace NO from cysteine-NO bonds. Such modifications of contraction were not observed in aortic rings previously exposed to 1 microM S-nitrosocysteine, glyceryl trinitrate, 3-morpholinosydnonimine, or 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). The same differential effects of GSNO and DEA-NO on contraction were also observed in porcine coronary arteries. Rat aortic rings previously exposed to 100 microM GSNO, but not to 100 microM DEA-NO, displayed a persistent increase in NO content (determined by NO spin trapping) and cysteine-NO residues (determined by immunostaining with an anti-cysteine-NO antiserum). The GSNO-induced increase in cysteine-NO residues in aortic tissue was prevented by the thiolmodifying agent p-hydroxymercuribenzoic acid. This study shows that in isolated arteries, the effects of S-nitrosating agents differed from those of other NO-donating agents. S- Nitrosating agents induced a persistent inhibition of contraction, which was attributed to the formation of releasable NO stores by S-nitrosation of tissue thiols. These differential effects of NO donors may be important for orientating their therapeutic indications.

Role of S-nitrosation of cysteine residues in long-lasting inhibitory effect of nitric oxide on arterial tone.

S-Nitrosation of cysteine residues plays an important role in nitric oxide (NO) signaling and transport. The aim of the present study was to investigate the role of S-nitrosothiols as a storage form of NO, which may account for the long-lasting effects in the vasculature. Rat aorta exposed to S-nitrosoglutathione (GSNO) displayed, even after washout of the drug, a persistent increase in cysteine-NO residues (detected by immunostaining using an antiserum that selectively recognized S-nitrosoproteins) and in NO content (detected by NO spin-trapping), a persistent attenuation of the effect of vasoconstrictors, and a relaxant response upon addition of low molecular weight (LMW) thiols. Rat mesenteric and porcine coronary artery exposed in vitro to GSNO, as well as aorta and mesenteric arteries removed from rats treated in vivo with GSNO, displayed similar modifications of contraction. In isolated aorta exposed to GSNO, the decrease of the contractile response and the relaxant effect of LMW thiols were both blunted by NO scavengers (oxyhemoglobin or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) or by a cyclic GMP-dependent protein kinase inhibitor (Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate). In these arteries, mercuric chloride (which cleaves the cysteine-NO bond) exerted a transient relaxation, completely abolished the one of LMW thiols, and blunted the increase in cysteine-NO residues and NO content. Together, these data support the idea that S-nitrosation of cysteine residues is involved in long-lasting effects of NO on arterial tone. They suggest that S-nitrosation of tissue thiols is a mechanism of formation of local NO stores from which biologically active NO can subsequently be released.

Evidence that intrinsic iron but not intrinsic copper determines S-nitrosocysteine decomposition in buffer solution.

The present experiments were designed to analyze the influence of copper and iron ions on the process of decomposition of S-nitrosocysteine (cysNO), the most labile species among S-nitrosothiols (RSNO). CysNO fate in buffer solution was evaluated by optical and electron paramagnetic resonance (EPR) spectroscopy, and the consequences on its vasorelaxant effect were studied on noradrenaline-precontracted rat aortic rings. The main results are the following: (i) copper or iron ions, especially in the presence of the reducing agent ascorbate, accelerated the decomposition of cysNO and markedly attenuated the amplitude and duration of the relaxant effect of cysNO; (ii) by contrast, the iron and copper chelators bathophenantroline disulfonic acid (BPDS) and bathocuproine disulfonic acid (BCS) exerted a stabilizing effect on cysNO, prolonged its vasorelaxant effect, and abolished the influence of ascorbate; (iii) in the presence of ascorbate, BPDS displayed a selective inhibitory effect toward the influence of iron ions (but not toward copper ions) on cysNO decomposition and vasorelaxant effect, while BCS prevented the effects of both copper and iron ions; (iv) L-cysteine enhanced stability and prolonged the relaxant effect of cysNO; (v) the process of iron-induced decomposition of cysNO was associated with the formation of EPR-detectable dinitrosyl-iron complexes (DNIC) either with non-thiol- or thiol-containing ligands (depending on the presence of L-cysteine), both of which exhibiting vasorelaxant properties. From these data, it is concluded that the amount of intrinsic copper was probably too low to produce a destabilizing effect even on the most labile RSNO, cysNO, and that only intrinsic iron, through the formation of DNIC, was responsible for the process of cysNO decomposition and thus influenced its vasorelaxant properties.