M. Carmen Martinez

Shed Membrane Particles From T Lymphocytes Impair Endothelial Function and Regulate Endothelial Protein Expression

Background—Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation. The present study was designed to dissect the effects evoked by T lymphocyte–derived MPs on vascular function. Methods and Results—MPs were produced by treatment of the human lymphoid CEM T cell line with actinomycin D or phytohemagglutinin. Incubation of mouse aortic rings with 30 nmol/L MPs resulted in a time-dependent impairment of acetylcholine-induced relaxation of precontracted vessels, with a maximal reduction after 24 hours. MPs also impaired shear stress–induced dilatation of mouse small mesenteric arteries by affecting the nitric oxide (NO) and prostacyclin but not the endothelium-derived hyperpolarizing factor components of the response. However, neither alteration of calcium signaling in response to agonists nor reduction of cyclooxygenase-1 expression accounted for the impairment of the NO and prostacyclin components of the endothelial response. The effect of MPs was rather because of a decrease in expression of endothelial NO synthase and an overexpression of caveolin-1. Furthermore, lymphocyte-derived MPs from diabetic patients or in vivo circulating MPs from either diabetic or HIV-infected patients reduced endothelial NO synthase expression. Finally, the effects of MPs on endothelial cells were not driven through CD11a/CD18 adhesion molecules or the Fas/FasL pathway. Conclusions—MPs from T cells induce endothelial dysfunction in both conductance and resistance arteries by alteration of NO and prostacyclin pathways. MPs regulate protein expression for endothelial NO synthase and caveolin-1. These data contribute to a better understanding of the deleterious effects of enhanced circulating MPs observed in disorders with cardiovascular or immune complications.

Sonic hedgehog carried by microparticles corrects endothelial injury through nitric oxide release

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation. Among the candidate proteins harbored by MPs, we recently showed that sonic hedgehog (Shh) is present in MPs generated from activated/apoptotic human T lymphocytes [Martinez et al., Blood (2006) vol. 108, 3012–3020]. We show here that Shh carried by MPs induces nitric oxide (NO) release from endothe‐lial cells, triggers changes in the expression and phos‐phorylation of enzymes related to the NO pathway, and decreases production of reactive oxygen species. When PI3‐kinase and ERK signaling were specifically inhibited, the effects of MPs were reversed. In vivo injection of MPs in mice was also able to improve endothelial function by increasing NO release, and it reversed endothelial dysfunction after ischemia/reperfusion. Silencing the effects of Shh with cyclopamine, a specific inhibitor of Shh, or siRNA, an inhibitor of the Shh receptor Patched, strongly reduced production of NO elicited by MPs. Taken together, we propose that the biological message carried by MPs harboring Shh may represent a new therapeutic approach against endothe‐lial dysfunction during acute severe endothelial injury.—Agouni A., Mostefai, H. A., Porro, C., Carusio, N., Favre, J., Richard, V., Henrion, D., Martinez, M. C., Andriantsitohaina R. Sonic hedgehog carried by micro‐particles corrects endothelial injury through nitric oxide release. FASEB J. 21, 2735–2741 (2007)

Molecular mechanisms of the cardiovascular protective effects of polyphenols.

Epidemiological studies have reported a greater reduction in cardiovascular risk and metabolic disorders associated with diets rich in polyphenols. The antioxidant effects of polyphenols are attributed to the regulation of redox enzymes by reducing reactive oxygen species production from mitochondria, NADPH oxidases and uncoupled endothelial NO synthase in addition to also up-regulating multiple antioxidant enzymes. Although data supporting the effects of polyphenols in reducing oxidative stress are promising, several studies have suggested additional mechanisms in the health benefits of polyphenols. Polyphenols from red wine increase endothelial NO production leading to endothelium-dependent relaxation in conditions such as hypertension, stroke or the metabolic syndrome. Numerous molecules contained in fruits and vegetables can activate sirtuins to increase lifespan and silence metabolic and physiological disturbances associated with endothelial NO dysfunction. Although intracellular pathways involved in the endothelial effects of polyphenols are partially described, the molecular targets of these polyphenols are not completely elucidated. We review the novel aspects of polyphenols on several targets that could trigger the health benefits of polyphenols in conditions such as metabolic and cardiovascular disturbances.

Reactive Nitrogen Species: Molecular Mechanisms and Potential Significance in Health and Disease

Reactive nitrogen species (RNS) are various nitric oxide-derived compounds, including nitroxyl anion, nitrosonium cation, higher oxides of nitrogen, S-nitrosothiols, and dinitrosyl iron complexes. RNS have been recognized as playing a crucial role in the physiologic regulation of many, if not all, living cells, such as smooth muscle cells, cardiomyocytes, platelets, and nervous and juxtaglomerular cells. They possess pleiotropic properties on cellular targets after both posttranslational modifications and interactions with reactive oxygen species. Elevated levels of RNS have been implicated in cell injury and death by inducing nitrosative stress. The aim of this comprehensive review is to address the mechanisms of formation and removal of RNS, highlighting their potential cellular targets: lipids, DNA, and proteins. The specific importance of RNS and their paradoxic effects, depending on their local concentration under physiologic conditions, is underscored. An increasing number of compounds that modulate RNS processing or targets are being identified. Such compounds are now undergoing preclinical and clinical evaluations in the treatment of pathologies associated with RNS-induced cellular damage. Future research should help to elucidate the involvement of RNS in the therapeutic effect of drugs used to treat neurodegenerative, cardiovascular, metabolic, and inflammatory diseases and cancer.

Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis

Epidemiological studies have suggested that moderate consumption of natural dietary polyphenolic compounds might reduce the risk of cardiovascular disease and also protect against cancer. The present study investigates the effects of delphinidin, an anthocyanin present in red wine, on bovine aortic endothelial cells apoptosis. Based on flow cytometry, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling analysis and detection of mitochondrial cytochrome c release, we show that delphinidin (10−2 g l−1) alone had no effect either on necrosis or on apoptosis, but it significantly reduced apoptosis elicited by actinomycin D (1 μg ml−1, 24 h) and 7β‐hydroxycholesterol (10 μg ml−1, 18 h). The protective effect of delphinidin was abolished by inhibitors of nitric oxide‐synthase (NOS) (L‐NA, 100 μM and SMT, 100 μM), guanylyl cyclase (ODQ, 100 μM) and MAP kinase (PD98059, 30 μM). Western blot analysis and protein detection by confocal microscopy demonstrate that the antiapoptotic effect of delphinidin was associated with an increased endothelial NOS expression mediated by a MAP kinase pathway. Finally, delphinidin alone had no effect on cytosolic‐free calcium ([Ca2+]i), but normalized the changes in [Ca2+]i produced by actinomycin D towards the control values, suggesting that the antiapoptotic effect of delphinidin is associated with the maintenance of [Ca2+]i in the physiological range. All of the observed effects of delphinidin may preserve endothelium integrity, the alteration of which lead to pathologies including cardiovascular diseases, such as atherosclerosis, and is often associated with cancers. In conclusion, the protective effect of delphinidin against endothelial cell apoptosis contributes to understand the potential benefits of a consumption rich in polyphenols.

Mechanisms of the noxious inflammatory cycle in cystic fibrosis

Multiple evidences indicate that inflammation is an event occurring prior to infection in patients with cystic fibrosis. The self-perpetuating inflammatory cycle may play a pathogenic part in this disease. The role of the NF-κB pathway in enhanced production of inflammatory mediators is well documented. The pathophysiologic mechanisms through which the intrinsic inflammatory response develops remain unclear. The unfolded mutated protein cystic fibrosis transmembrane conductance regulator (CFTRΔF508), accounting for this pathology, is retained in the endoplasmic reticulum (ER), induces a stress, and modifies calcium homeostasis. Furthermore, CFTR is implicated in the transport of glutathione, the major antioxidant element in cells. CFTR mutations can alter redox homeostasis and induce an oxidative stress. The disturbance of the redox balance may evoke NF-κB activation and, in addition, promote apoptosis. In this review, we examine the hypotheses of the integrated pathogenic processes leading to the intrinsic inflammatory response in cystic fibrosis.

Regulation of Phosphatidylserine Transbilayer Redistribution by Store-operated Ca2+ Entry ROLE OF ACTIN CYTOSKELETON

The phosphatidylserine transmembrane redistribution at the cell surface is one of the early characteristics of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as the phosphatidylserine-dependent procoagulant response of platelets after appropriate activation. Although an increase in cytoplasmic Ca2+ is essential to trigger the remodeling of the plasma membrane, little is known about intracellular signals leading to phosphatidylserine externalization. Here, the role of store-operated Ca2+ entry on phosphatidylserine exposure was investigated in human erythroleukemia HEL cells, a pluripotent lineage with megakaryoblastic properties. Ca2+ entry inhibitors (SKF-96365, LaCl3, and miconazole) inhibited store-operated Ca2+ entry in A23187- or thapsigargin-stimulated cells and reduced the degree of phosphatidylserine externalization concomitantly, providing evidence for a close link between the two processes. In cells pretreated with cytochalasin D, an agent that disrupts the microfilament network of the cytoskeleton, store-operated Ca2+ entry and phosphatidylserine externalization at the cell surface were inhibited. In a context where most of the key actors remain to be identified, these results provide evidence for the implication of both store-operated Ca2+ entry and cytoskeleton architectural organization in the regulation of phosphatidylserine transbilayer migration.

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.

Upregulation of Proinflammatory Proteins Through NF-κB Pathway by Shed Membrane Microparticles Results in Vascular Hyporeactivity

Objective—Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation, including apoptosis. The present study was designed in dissecting the effects evoked by microparticles on vascular reactivity. Methods and Results—Microparticles from either apoptotic T lymphocytic cells or from plasma of diabetic patients with vascular complications induced vascular hyporeactivity in response to vasoconstrictor agents in mouse aorta. Hyporeactivity was reversed by nitric oxide (NO) synthase plus cyclooxygenase-2 inhibitors, and associated with an increased production of vasodilatory products such as NO and prostacyclin. Microparticles induced an upregulation of proinflammatory protein expressions, inducible NO-synthase and cyclooxygenase-2, mainly in the medial layer of the vessels as evidenced by immunochemical staining. In addition, microparticles evoke NF-&kgr;B activation probably through the interaction with the Fas/Fas Ligand pathway. Finally, in vivo treatment of mice with lymphocyte-derived MPs induces vascular hyporeactivity, which was reversed by the combination of NO and cyclooxygenase-2 inhibitors. Conclusion—These data provide a rationale to explain the paracrine role of microparticles as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases.

Detrimental hemodynamic and inflammatory effects of microparticles originating from septic rats.

Objective:Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation and might be potentially involved in the pathophysiology of septic shock. This study was designed to assess the effects of MPs from septic origin on the systemic hemodynamics as well as on the inflammatory, oxidative, and nitrosative stresses. Design:A prospective, randomized, controlled experimental study with repeated measurements. Setting:Investigational animal laboratory. Subjects:Forty healthy rats were randomly allocated to three groups: 10 animals inoculated with MPs isolated from control rats (cMPs), 15 animals inoculated with MPs isolated from sham rats (shMPs), and 15 animals inoculated with MPs isolated from rats with peritonitis (sMPs). Interventions:Rats were anesthetized, mechanically ventilated, and infused with the same amount of cMPs, shMPs, or sMPs. We measured the heart rate, mean arterial pressure, carotid artery, and portal vein blood flows. Hemodynamic parameters were recorded during 7 hours, and then animals were killed. Aorta and heart were harvested for further in vitro tissue analyses. Measurements and Main Results:1) The cellular origin (phenotype) but not the circulating concentration of MPs was different in septic rats, characterized by a significant increase in leukocyte-derived MPs. 2) sMPs but not cMPs or shMPs decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. 3) Rats inoculated with sMPs exhibited an increase in superoxide ion production and nuclear factor kappa B activity, overexpression of inducible nitric oxide synthase with subsequent nitric oxide overproduction and decrease in endothelial nitric oxide synthase activation. Conclusions:Rats with sepsis induced by peritonitis exhibited a specific phenotype of MPs. Inoculation of sMPs in healthy rats reproduced hemodynamic, septic inflammatory patterns, associated with oxidative and nitrosative stresses.