Arnaud Courtois

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-α, 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.

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.

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.

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.

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.

In Vitro Glucuronidation and Sulfation of ε-Viniferin, a Resveratrol Dimer, in Humans and Rats

ε-Viniferin is a resveratrol dimer that possesses antioxidant or anti-inflammatory activities. However little is known about the metabolism of this oligostilbene. This study was thus undertaken as a first approach to identify and characterize the metabolites of ε-viniferin and to describe the kinetic profile of their appearance in humans and rats. The glucuronides and sulfates of ε-viniferin were first obtained by chemical hemi-synthesis and were fully characterized by UPLC-MS and NMR spectroscopy. Then, ε-viniferin was incubated with human or rat S9 liver fractions that led to the formation of four glucuronoconjugates and four sulfoconjugates. In both species, ε-viniferin was subjected to an intense metabolism as 70 to 80% of the molecule was converted to glucuronides and sulfates. In humans, the hepatic clearance of ε-viniferin (Vmax/Km) for glucuronidation and sulfation were 4.98 and 6.35 µL/min/mg protein, respectively, whereas, in rats, the hepatic clearance for glucuronidation was 20.08 vs. 2.59 µL/min/mg protein for sulfation. In humans, three major metabolites were observed: two glucuronides and one sulfate. By contrast, only one major glucuronide was observed in rats. This strong hepatic clearance of ε-viniferin in human and rat could explain its poor bioavailability and could help to characterize its active metabolites.

A review of dietary stilbenes: sources and bioavailability

Stilbenes are a class of phenolic metabolites found in various edible plants, such as grapevine, berries and peanuts. Their bioactivitiy and their potential benefits for human health have been the subject of several studies. Among all identified stilbenes, resveratrol has been particularly studied and results from literature showed that it presents several biological activities, including antioxidant, anti-inflammatory and antiproliferative effects. Likewise, some researches focused on other stilbenes and highlighted similar biological activity for those compounds. However, stilbenes present a high diversity in their phenolic structures (various chemical substituents and polymerization) which is a determining factor of their absorption and metabolism rates. Consequently, this could affect the effectiveness of stilbenes in vivo. In this context, an evaluation of the bioavailability and metabolism of stilbenes is necessary to move forward with pharmacological and clinical studies. Hence, this review aims to present recently obtained data and results concerning stilbenes sources and bioavailability, as a contribution to the valorization of the role of dietary stilbenes in the human diet.