Circulating microparticles from septic shock patients exert differential tissue expression of enzymes related to inflammation and oxidative stress

Objective: Septic shock is characterized by hypotension and multiple organ failure after infection of microorganisms. Septic shock patients display high levels of circulating microparticles. These are small vesicles released from the plasma membrane of activated or apoptotic cells. Here, we have investigated the effects of in vivo injection of microparticles from nonseptic or septic subjects on protein expression in mouse tissues.Design: Prospective, controlled experiments. Setting: Animal basic science laboratory. Subjects: Male Swiss mice were randomly assigned to one of two groups: 11 animals injected with microparticles isolated from healthy subjects and 15 animals injected with microparticles isolated from septic patients. Interventions: Microparticles were extracted from whole blood of septic and nonseptic subjects and were intravenously injected in mice. After 24 hrs, mice were killed and heart, lungs, liver, and kidneys were isolated for Western blot assays. Organs were also used for direct measurements of nitric oxide and superoxide anion production by electron paramagnetic resonance. Measurements and Main Results: In heart and lungs, microparticles from septic shock patients increased the expression of endothelial and inducible nitric oxide synthases, cyclooxygenase-2, and nuclear factor-κB. However, extracellular superoxide dismutase was only increased in the heart. These effects were associated either with a greater oxidative or nitrative stress in heart and lungs, without affecting nitric oxide production. The liver exhibited an increase in oxidative stress linked to decreased endothelial nitric oxide synthase and manganese superoxide dismutase expression. However, cyclooxygenase-2 expression and IκBα phosphorylation were decreased. Septic microparticles did not change superoxide anion and nitric oxide productions in kidneys. Conclusions: Results suggest that microparticles from septic shock patients exert pleiotropic and differential effects depending on target tissues with regard to the expression of proinflammatory proteins related with nitrative and oxidative stresses. Thus, microparticles might participate in organ dysfunction observed in septic shock patients

Sonic hedgehog carried by microparticles prevents angiotensin II-induced hypertension and endothelial dysfunction in mice

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation. Among the candidate proteins harbored by MPs, we recently showed that morphogen Sonic hedgehog is present in MPs generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release (Agouni et al. FASEB J 2007). The present study further investigates whether MPs bearing Shh prevent angiotensin II (Ang II)-induced hypertension and endothelial dysfunction in mice. Male Swiss mice (6–8 week old) were subcutaneously implanted with osmotic minipumps delivering Ang II (0.5 mg/kg per day) or NaCl (0.9%, control group). Systolic blood pressure and heart rate were measured daily during 21 consecutive days using tail cuff plethysmography connected to a computerized system (LE 5002 # , BIOSEB). Mice were tained for 7 days. After 7 day of minipump implantation, mice received i.v. injections of MPs (10 μg/mL) or i.p. Sonic Hedgehog receptor antagonist cyclopamine (10 mg/kg per 2 days) during 1 week prior sacrifice. Thoracic aorta was removed, cleaned of connective tissue and cut in rings (3 mm length) and mounted in a myograph to study vascular reactivity. Ang II induced a significant rise in systolic blood pressure without affecting heart rate when compared to control mice. Interestingly, MPs alone did not modify both parameters but reversed Ang II-induced hypertension. Moreover, cyclopamine prevented the effects of MPs on Ang II-induced hypertension, suggesting the involvement of a Sonic Hedgehog-dependent mechanism. In the aorta, MPs alone slightly increased the sensitivity of endothelium-dependent relaxation to acetylcholine and completely reversed the impairment of acetylcholine-induced relaxation in aorta from Ang II-infused mice. The improvement of endothelial function induced by MPs was completely prevented by cyclopamine treatment. Moreover, measurement of NO production showed that MPs alone did not modify NO production in aorta, but significantly restored its decrease in Ang II-treated mice. Altogether, these results show that MPs bearing Sonic hedgehog prevent Ang II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Sonic hedgehog-induced NO production. These MPs may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production

Microparticles from apoptotic monocytes enhance nitrosative stress in human endothelial cells.

Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation or apoptosis. Microparticles from monocytes have been implicated in atherosclerosis and vascular inflammation, but their direct effects on endothelial cells are not completely elucidated. The present study was designed to dissect the signaling pathways of monocytic microparticles in endothelial cells with respect to both NO pathway and reactive oxygen species. Microparticles were produced by treatment of human monocytic cell line THP-1 with the apoptotic agent VP-16. Human endothelial cells were treated with monocytic microparticles and then, we studied their effects on nitrosative and oxidative stresses. Incubation of human endothelial cells with microparticles enhanced the production of NO without affecting superoxide anions generation. Microparticles did not affect endothelial NO synthase expression and its phosphorylation. Interestingly, microparticles decreased caveolin-1 expression and increased its phosphorylation. Inhibition of PI-3-kinase or MEK1/2 reversed the effects of microparticles on caveolin-1 expression but not its phosphorylation. Moreover, microparticles increased nitration of several proteins, reflecting peroxynitrite production, which was prevented by blockade of PI-3-kinase pathway. In summary, monocyte microparticles active multiple pathways related to nitrosative stress in endothelial cells including both PI-3-kinase and ERK1/2 in the regulation of caveolin-1 expression. These data underscore the pleiotropic effect of microparticles on endothelial cells and suggest that they probably play a critical role on vascular function

Influence of micropaticles harvested from patients affected by obstructive sleep apnea syndrome on endothelial function and vascular reactivity

Obstructive sleep apnea syndrome (OSAS) is a highly prevalent disease characterized by recurrent episodes of partial or complete obstruction of the upper airways during sleep, leading to oxygen desaturation, sleep fragmentation and clinical endothelial dysfunction. Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. Elevated levels of circulating MPs have been detected in pathologies associated with vascular alterations. We investigated the effects of MPs on endothelial function and vascular reactivity in OSAS. Blood samples were obtained either from 38 OSAS patients without any other cardiovascular comorbidities and 23 healthy subjects. A desaturation index above 10 per hour defined OSAS patients. MPs concentration and origin were assessed using flow cytometer. Male Swiss mice were injected i.v. with MPs from OSAS or healthy subjects, or with saline solution, and sacrified after 24hours. Endothelial function and vascular reactivity were studied on aortic rings and small mesenteric resistance (SMA) arteries by myography and arteriography, respectively. Patients with OSAS did not display increased circulating levels of MPs compared to healthy subjects including those from pro-coagulant, platelet, endothelial, leukocyte and erythrocyte origins. Interestingly, MPs from granulocytes and activated leukocytes were significantly enhanced in OSAS patients. Activated leukocyte MPs positively correlated with oxygen desaturation index. In aorta, MPs from OSAS patients but not those from healthy subjects significantly reduced endothelium-dependent relaxation to acetylcholine. MPs from OSAS increased sensitivity of the aorta in response to serotonin that was greater compared to the effect of MPs from healthy subjects. In SMA, MPs from OSAS but not those from healthy subjects impaired flow-induced dilation without any effect on myogenic tone. Although SMA from mice treated with healthy subjects MPs did not affect flow-induced dilation, these vessels showed a reduced prostacyclin-component that was completely compensated by the NO-component of the response. The endothelial dysfunction induced by MPs from OSAS was caused by the reduction of both NO- and prostacyclin- but not the endothelium-derived hyperpolarizing factor-components of the response in SMA. These data provide evidence that circulating MPs from OSAS patients influence both endothelial function and vascular reactivity

Sonic Hedgehog Carried by Microparticles Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice

Microparticles are small fragments of the plasma membrane generated after cell stimulation. We recently showed that Sonic hedgehog (Shh) is present in microparticles generated from activated/apoptotic human T lymphocytes and corrects endothelial injury through nitric oxide (NO) release. This study investigates whether microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in mice. Male Swiss mice were implanted with osmotic minipumps delivering angiotensin II (0.5 mg/kg/day) or NaCl (0.9%). Systolic blood pressure and heart rate were measured daily during 21 days. After 7 day of minipump implantation, mice received i.v. injections of microparticles (10 µg/ml) or i.p. Shh receptor antagonist cyclopamine (10 mg/kg/2 days) during one week. Angiotensin II induced a significant rise in systolic blood pressure without affecting heart rate. Microparticles reversed angiotensin II-induced hypertension, and cyclopamine prevented the effects of microparticles. Microparticles completely corrected the impairment of acetylcholine- and flow-induced relaxation in vessels from angiotensin II-infused mice. The improvement of endothelial function induced by microparticles was completely prevented by cyclopamine treatment. Moreover, microparticles alone did not modify NO and O2. - production in aorta, but significantly increased NO and reduced O2. - productions in aorta from angiotensin II-treated mice, and these effects were blocked by cyclopamine. Altogether, these results show that microparticles bearing Shh correct angiotensin II-induced hypertension and endothelial dysfunction in aorta through a mechanism associated with Shh-induced NO production and reduction of oxidative stress. These microparticles may represent a new therapeutic approach in cardiovascular diseases associated with decreased NO production

Phosphatidylinositol 3-Kinase and Xanthine Oxidase Regulate Nitric Oxide and Reactive Oxygen Species Productions by Apoptotic Lymphocyte Microparticles in Endothelial Cells

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. We have previously shown that MPs from apoptotic T cells induce endothelial dysfunction, but the mechanisms implicated are not completely elucidated. In this study, we dissect the pathways involved in endothelial cells with respect to both NO and reactive oxygen species (ROS). Incubation of endothelial cells with MPs decreased NO production that was associated with overexpression and phosphorylation of endothelial NO synthase (eNOS). Also, MPs enhanced expression of caveolin-1 and decreased its phosphorylation. Microparticles enhanced ROS by a mechanism sensitive to xanthine oxidase and P-IκBα inhibitors. PI3K inhibition reduced the effects of MPs on eNOS, but not on caveolin-1, whereas it enhanced the effects of MPs on ROS production. Microparticles stimulated ERK1/2 phosphorylation via a PI3K-depedent mechanism. Inhibition of MEK reversed eNOS phosphorylation but had no effect on ROS production induced by MPs. In vivo injection of MPs in mice impaired endothelial function. In summary, MPs activate pathways related to NO and ROS productions through PI3K, xanthine oxidase, and NF-κB pathways. These data underscore the pleiotropic effects of MPs on NO and ROS, leading to an increase oxidative stress that may account for the deleterious effects of MPs on endothelial function