Nathalie Labranche

Vascular oxidative stress induced by diesel exhaust microparticles: synergism with hypertension.

Background: Epidemiological and clinical studies have shown that traffic-related air pollution and, particularly, diesel exhaust particles (DEP) are strongly linked to cardiovascular mortality. Methods: Vascular toxicity was studied by assessing vasomotor responses of aortas isolated from normotensive Wistar rats exposed in vitro to DEP (DEP suspension and aqueous DEP extract). In vivo experiments were performed on Wistar rats and spontaneously hypertensive rats (SHRs) exposed for 4 weeks via intratracheal instillation to either DEP or saline vehicle. After killing, vascular responses to acetylcholine (ACh) or sodium nitroprusside were assessed in vitro and the expression of p22phox, a major nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, was studied by real-time quantitative polymerase chain reaction. Results: In aortas from Wistar rats, in vitro DEP incubation (both preparations) markedly inhibited the relaxations to ACh and slightly to sodium nitroprusside; this effect was reversed in the presence of superoxide dismutase. In contrast, in aortas from in vivo–exposed animals, ACh-induced relaxations were only significantly impaired in the SHR group, accompanied with a significant upregulation of p22phox and no change in systolic blood pressure. Conclusions: Although in vitro exposure to DEP produces a vascular oxidative stress, repeated in vivo exposures to DEP only impair vascular function in SHR, via an upregulation of p22phox. This suggests a synergistic effect on endothelial dysfunction between particulate air pollution and hypertension.

Leptin-Induced Endothelium-Independent Vasoconstriction in Thoracic Aorta and Pulmonary Artery of Spontaneously Hypertensive Rats: Role of Calcium Channels and Stores

Decreased leptin-induced endothelium-dependent vasodilation has been reported in spontaneously hypertensive rats (SHR). Here, we report leptin-induced vasoconstriction in endothelium-denuded pulmonary artery and thoracic aorta from SHR and sought to characterize calcium handling underlying these mechanisms. Vasoreactivity to leptin was evaluated on pulmonary artery and thoracic aorta rings from 18 weeks old male SHR with or without calcium free medium, caffeine + thapsigargin + carbonyl cyanide-4-trifluoromethoxyphenylhydrazone emptying intracellular calcium stores, nifedipine a voltage-gated calcium channel inhibitor, SKF-96365 a transient receptor potential cation channels (TRPC) inhibitor, wortmaninn, a phosphatidylinositide 3-kinases (PI3K) inhibitor, or PD98059 a mitogen-activated protein kinase kinase (MAPKK) inhibitor. Calcium imaging was performed on cultured vascular smooth muscle cells incubated with leptin in presence or not of wortmaninn or PD98059. Leptin induced vasoconstriction in denuded pulmonary artery and thoracic aorta from SHR. Response was abolished when intra- or extracellular calcium stores were emptied, after blocking TRPC or voltage-dependent calcium channels or when using MAPKK or PI3K inhibitors. In vascular smooth muscle cells, leptin increased intracellular calcium. This rise was higher in SHR and abolished by MAPKK or PI3K inhibitors. TRPC6 gene expression was upregulated in arteries from SHR. Leptin-induced vasoconstriction in denuded arteries of SHR requires intracellular stores and is TRPC- and voltage-gated calcium channels dependent. Intracellular calcium increase is more pronounced in spontaneously hypertensive rats.

Effects of diesel exhaust particles on macrophage polarization

Background: Exposure to diesel exhaust particles (DEP) has long been associated with increased cardiovascular morbidity and mortality. The development of DEP toxicity seems to be linked to inflammation in which macrophages play a critical role. Macrophages can be polarized into proinflammatory M1 or anti-inflammatory M2 macrophages. The aim of this study was to identify the role of inflammation in DEP-induced toxicity by assessing the effects of DEP on macrophage polarization. Methods: Monocyte-derived macrophages (Mϕ) were stimulated with interferon γ and lipopolysaccharide or interleukin (IL)-4 to obtain M1 and M2 subtypes, respectively. To test the polarization capacity of DEP, Mϕ cells were exposed to DEP and compared to Mϕ, M1, and M2. We also studied the effects of DEP on already-polarized M1 or M2. The M1 markers assessed were tumor necrosis factor α (TNF-α) and IL-1β, while the M2 markers were the mannose receptor C type 1 (MRC-1) and transglutaminase 2 (TGM2). Results: Western blots revealed a 31 kDa band corresponding to pro-IL-1β, but only in M1-polarized macrophages. In M1, we also observed an upregulation of TNF-α messenger RNA (mRNA) expression. MRC-1 and TGM2 mRNA expression were only significantly enhanced in M2. DEP had no effect on any of the M1/M2 markers assessed. Moreover, DEP were not able to modify the phenotype of already-polarized M1 or M2. Conclusion: Mϕ incubation with DEP did not have any effect on macrophage polarization, at least on the markers assessed in this study, namely, TNF-α/IL-1β for M1, and MRC-1/TGM2 for M2. Hence, these data argue against an important role of inflammation in DEP-induced vascular toxicity.

Rosuvastatin and vascular oxidative stress induced by diesel exhaust particles

Objective: Exposure to diesel exhaust particles (DEP) is strongly linked to the development and exacerbation of cardiovascular diseases. Statins are effective drugs in the prevention and treatment of cardiovascular disorders. The aim of this study was to investigate the potential protective effect of rosuvastatin on DEP-induced endothelial dysfunction.Methods and results: Spontaneously hypertensive rats (SHR) were treated for 5 weeks with rosuvastatin and exposed, intratracheally, for the last 4 weeks, to either DEP suspensions or saline vehicle. Rings of thoracic aortas were used to assess superoxide anion production through the lucigenin-enhanced chemiluminescence technique. Real-time quantitative polymerase chain reaction analysis was performed on aortic segments to assess eNOS, iNOS, p22phox, gp91phox, Rac-1 and TNF-α mRNA expression. Human umbilical vein endothelial cells (HUVECs) were also used for the measurement of oxidative stress after DEP and/or rosuvastatin incubation. In thoracic aortic rings isolated from SHR, superoxide anion formation was increased after DEP exposure. This oxidative stress was markedly decreased in the rosuvastatin-treated group. DEP exposure also induced a downregulation of eNOS mRNA expression and a slight increase in gp91phox mRNA expression, which were reversed in the rosuvastatin group. In HUVECs, similar results were observed: DEP generated an accumulation of superoxide anion, which was significantly attenuated by rosuvastatin.Conclusions: Our results suggest that rosuvastatin interacts with the eNOS and NADPH oxidase pathways in hypertensive rats and therefore might counteract the oxidative stress induced by DEP. This effect was also observed in vitro in human endothelial cells (HUVECs).

A STUDY OF THE FEASIBILITY OF L NAME IONTOPHORESIS: EFFECTS OF L NAME CONCENTRATION AND SMOKING: PP.6.224

Introduction: Early detection of endothelial dysfunction, characterized by a reduced bioavailability of NO, is essential as it is a key phenomenon in the genesis of cardiovascular diseases. Heat-induced vasodilatation is mediated mainly by NO, as shown by L Name (L) microdialysis. L Name is a competitive inhibitor of cNOS, produced by the vascular endothelium, and is polarized negatively. The application of a microcurrent on the skin allows polarized molecules, such as sodium nitroprusside and acetylcholine, to penetrate into the dermis. Whether L Name iontophoresis is feasible, and will reduce skin blood flow measured by a laser Doppler imager (LDI) in response to heating, is not known. We also assessed whether this response is dose-dependent. Last, we compared this response between habitual smokers and non smokers. Methods: In a population of 17 healthy male smokers and 17 matched healthy male non-smokers (aged 28 years ± 5 yrs, BMI 22 ± 3 kg/m2), we conducted an iontophoresis of L Name and placebo on the two arms on Day 0. This was repeated with the same dose, and a double dose of L, on Day 4, on both arms. After iontophoresis, the sites were heated to 44°C to induce vasodilation, and scanned to measure blood flow in the microcirculation. We also determined pulse wave velocity in both groups. Smoking was forbidden 12 hours before the experiments. Results: L Name decreased equally blood flow during heating in the non smokers and smokers (p < 0.01, table 1). Both concentrations of 20 and 40 mM of L Name were equally effective. Pulse wave velocity was normal at 9 m/s, and did not differ between groups. Figure 1. No caption available. Conclusion: L Name iontophoresis is feasible and is able to inhibit vascular endothelial NOS. Higher concentrations of L Name than 20 mM do not provide further vasodilatation. NO-mediated skin blood flow vasodilatation is not altered in healthy young sportive smokers investigated after 12 hours of smoking cessation.