Circulating microparticles from pulmonary hypertensive rats are vectors of oxidative stress

Date du colloque : 04/2010National audienc

Circulating microparticles from a rat model of pulmonary arterial hypertension induce endothelial dysfunction

Pulmonary arterial hypertension (PAH) is a rare and severe disease characterized by an increase of pulmonary vascular resistance and right heart failure. Chronic hypoxia induces PAH, which is accompanied by functional (endothelial dysfunction, increased vasoconstriction) and structural (thickening of media) changes in pulmonary arteries. However, the mechanisms of these alterations remain unsolved. Among biological hallmark of this disease, level of circulating microparticles (MPs), small vesicles of plasma membrane released during cell activation and apoptosis, is increased in PAH patients. Although MPs can act as biological vectors of endothelial dysfunction, their role in PAH are not elucidated yet. We studied circulating MP effects on endothelial function during hypoxic PAH. Male Wistar rats were exposed or not to chronic hypoxia (3 weeks, 0.5 atmosphere) and normoxic or hypoxic MPs were isolated from peripheral blood and characterized by flow cytometry. Endothelial cells from rat normoxic aorta or pulmonary arteries were incubated for 24 h with MPs. We studied also effects of in vivo treatment of MPs on vasomotricity, for this, normoxic or hypoxic MPs or vehicle were i.v. injected into rats, and 24 h after, endothelial function were studied. Levels of circulating MPs from hypoxic rats was twice than MPs from normoxic rats (1568 ± 174 vs 852 ± 80 MPs/Âμl of plasma). In vitro treatment of endothelial cells with hypoxic MPs reduced NO production both in aortas and pulmonary arteries ; these effects were associated with enhanced phosphorylation of endothelial NO-synthase at their inhibitory site. By contrast, O2- production was increased only in endothelial cells from pulmonary arteries. In vivo injection of normoxic or hypoxic MPs into rats impaired to the same extent the endotheliumdependent relaxation induced by acetylcholine in aorta. Although pulmonary arteries from rat treated either with normoxic or hypoxic MPs displayed reduction of endothelium-dependent relaxation to carbachol compared to control, the deleterious effect of hypoxic MPs was greater than normoxic MPs. These data provide evidence that hypoxic circulating MPs induce in vitro and in vivo endothelial dysfunction by increasing oxydative stress and by decreasing NO production

Circulating microparticles from pulmonary hypertensive rats induce endothelial dysfunction

RATIONALE: Pulmonary arterial hypertension (PAH) is a severe disease characterized by an increase of pulmonary vascular resistance, which is accompanied by functional and structural changes in pulmonary arteries. Microparticles (MPs) have been described as biological vector of endothelial dysfunction in other pathologies.OBJECTIVES: The purpose of this work was to characterize circulating MPs during hypoxic PAH and to study their effects on endothelial function. METHODS: Male Wistar rats were exposed or not to chronic hypoxia, and normoxic or hypoxic MPs from blood were characterized by flow cytometry. Endothelial cells (ECs) from rat aorta or pulmonary arteries were incubated with MPs, and then expression and phosphorylation of enzymes involved in nitric oxide (NO) and reactive oxygen species productions were analyzed. Hypoxic MPs were injected into rats, and endothelium-dependent relaxation was assessed. MEASUREMENTS AND MAIN RESULTS: Circulating levels of MPs from hypoxic rats were twofold higher than those present in normoxic rats. In vitro treatment of ECs with hypoxic MPs reduced NO production in aortas and pulmonary arteries by enhancing phosphorylation of endothelial NO synthase at the inhibitory site. Hypoxic MPs increased oxidative stress only in pulmonary ECs via xanthine oxidase and mitochondrial implication. In vivo injection of hypoxic MPs into rat impaired endothelium-dependent relaxation both in aorta and pulmonary arteries. CONCLUSIONS: These data provide evidence that hypoxic circulating MPs induce endothelial dysfunction in rat aorta and pulmonary arteries by decreasing NO production. Moreover, MPs display tissue specificity with respect to increased oxidative stress, which occurs only in pulmonary ECs

Stretch-Activated Piezo1 Channel in Endothelial Cells Relaxes Mouse Intrapulmonary Arteries

In intrapulmonary artery (IPA), endothelial cells (EC) respond to mechanical stimuli by releasing vasoactive factors to set the vascular tone. Piezo1, a stretch-activated calcium permeable channel is a sensor of mechanical stress in EC. The present study was undertaken to investigate the implication of Piezo1 in the endothelium-dependent regulation of IPA tone and its potential involvement in pulmonary hypertension, the main disease of this circulation. IPA tone was quantified by means of a myograph in control Piezo1+/+ mouse and in mouse lacking endothelial Piezo1 (EC-Piezo1-/-). Endothelial intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) production were measured, in mouse or human EC, with fluo-4 and DAF-fm probes, respectively. Immunofluorescence labeling and patch-clamp experiments revealed the presence of Piezo1 channels in EC. Yoda1, a Piezo1 agonist, induced an endothelium-dependent relaxation that was significantly reduced in pulmonary arteries in EC-Piezo1-/- compared to Piezo1+/+ mouse. Yoda1 as well as mechanical stimulation (by osmotic stress) increased [Ca2+]i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca2+]i increases were reduced in EC from Piezo1-/- mouse or in the presence of Piezo1 inhibitors. Furthermore, deletion of Piezo1 increased alpha-adrenergic mediated contraction. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, Piezo1 still mediated arterial relaxation and deletion of this channel did not impair the development of the disease. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i and NO production and that this effect is still present in pulmonary hypertension

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

International audienceRecent 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 (O2radical dot−) 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, O2radical dot−) and a 4 h-exposure to PM2.5 (5–15 μg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2radical dot− levels. The basal intracellular calcium ion level [Ca2 +]i was also increased after 4 h-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