Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi.

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s-1) and high shear (1690 s-1) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA-PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.

Paradoxical Effect of Nonalcoholic Red Wine Polyphenol Extract, Provinols™, in the Regulation of Cyclooxygenases in Vessels from Zucker Fatty Rats (fa/fa).

The aim of this work was to study the vascular effects of dietary supplementation of a nonalcoholic red wine polyphenol extract, Provinols, in Zucker fatty (ZF) obese rats. ZF or lean rats received diet supplemented or not with Provinols for 8 weeks. Vasoconstriction in response to phenylephrine (Phe) was then assessed in small mesenteric arteries (SMA) and the aorta with emphasis on the contribution of cyclooxygenases (COX). Although no difference in vasoconstriction was observed between ZF and lean rats both in SMA and the aorta, Provinols affected the contribution of COX-derived vasoconstrictor agents. The nonselective COX inhibitor, indomethacin, reduced vasoconstriction in vessels from both groups; however, lower efficacy was observed in Provinols-treated rats. This was associated with a reduction in thromboxane-A2 and 8-isoprostane release. The selective COX-2 inhibitor, NS398, reduced to the same extent vasoconstriction in aortas from ZF and Provinols-treated ZF rats. However, NS398 reduced response to Phe only in SMA from ZF rats. This was associated with a reduction in 8-isoprostane and prostaglandin-E release. Paradoxically, Provinols decreased COX-2 expression in the aorta, while it increased its expression in SMA. We provide here evidence of a subtle and paradoxical regulation of COX pathway by Provinols vessels from obese rats to maintain vascular tone within a physiological range.

Pharmacological modulation of procoagulant microparticles improves haemodynamic dysfunction during septic shock in rats.

Circulating microparticles play a pro-inflammatory and procoagulant detrimental role in the vascular dysfunction of septic shock. It was the objective of this study to investigate mechanisms by which a pharmacological modulation of microparticles could affect vascular dysfunction in a rat model of septic shock. Septic or sham rats were treated by activated protein C (aPC) and resuscitated during 4 hours. Their microparticles were harvested and inoculated to another set of healthy recipient rats. Haemodynamic parameters were monitored, circulating total procoagulant microparticles assessed by prothrombinase assay, and their cell origin characterised. Mesenteric resistance arteries, aorta and heart were harvested for western blotting analysis. We found that a) the amount and phenotype of circulating microparticles were altered in septic rats with an enhanced endothelial, leucocyte and platelet contribution; b) aPC treatment significantly reduced the generation of leucocyte microparticles and norepinephrine requirements to reach the mean arterial pressure target in septic rats; c) Microparticles from untreated septic rats, but not from aPC-treated ones, significantly reduced the healthy recipients' mean arterial pressure; d) Microparticle thromboxane content and aPC activity were significantly increased in aPC-treated septic rats. In inoculated naïve recipients, microparticles from aPC-treated septic rats prompted reduced NF-κB and cyclooxygenase-2 arterial activation, blunted the generation of pro-inflammatory iNOS and secondarily increased platelet and endothelial microparticles. In conclusion, in this septic shock model, increased circulating levels of procoagulant microparticles led to negative haemodynamic outcomes. Pharmacological treatment by aPC modified the cell origin and levels of circulating microparticles, thereby limiting vascular inflammation and favouring haemodynamic improvement.

Interleukin-10 controls the protective effects of circulating microparticles from patients with septic shock on tissue-engineered vascular media.

During sepsis, inflammation can be orchestrated by the interaction between circulating and vascular cells that, under activation, release MPs (microparticles). Previously, we reported that increased circulating MPs in patients with sepsis play a pivotal role in ex vivo vascular function suggesting that they are protective against vascular hyporeactivity. The present study was designed to investigate the effects of MPs from patients with sepsis on the contractile response of TEVM (tissue-engineered vascular media). TEVM that were composed only of a media layer were produced by tissue engineering from human arterial SMCs (smooth muscle cells) isolated from umbilical cords. TEVM was incubated with MPs isolated from whole blood of 16 patients with sepsis. TEVM were incubated for 24 h with MPs and used for the study of vascular contraction, direct measurements of NO and O2- (superoxide anion) production by EPR and quantification of mRNA cytokine expression. MPs from patients with sepsis increased contraction induced by histamine in TEVM. This effect was not associated with inflammation, neither linked to the activation of NF-κB (nuclear factor κB) pathway nor to the increase in iNOS (inducible NO synthase) and COX (cyclo-oxygenase)-2 expression. In contrast, mRNA expression of IL (interleukin)-10 was enhanced. Then, we investigated the effect of IL-10 on vascular hyporeactivity induced by LPS (lipopolysaccharide). Although IL-10 treatment did not modify the contractile response in TEVM by itself, this interleukin restored contraction in LPS-treated TEVM. In addition, IL-10 treatment both prevented vascular hyporeactivity induced by LPS injection in mice and improved survival of LPS-injected mice. These findings show an association between the capacity of MPs from patients with sepsis to restore vascular hyporeactivity induced by LPS and their ability to increase IL-10 in the tissue-engineered blood vessel model.

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.

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.

Red wine polyphenols prevent metabolic and cardiovascular alterations associated with obesity in Zucker fatty rats (Fa/Fa).

BACKGROUND: Obesity is associated with increased risks for development of cardiovascular diseases. Epidemiological studies report an inverse association between dietary flavonoid consumption and mortality from cardiovascular diseases. We studied the potential beneficial effects of dietary supplementation of red wine polyphenol extract, Provinols, on obesity-associated alterations with respect to metabolic disturbances and cardiovascular functions in Zucker fatty (ZF) rats. METHODOLOGY/PRINCIPAL FINDINGS: ZF rats or their lean littermates received normal diet or supplemented with Provinols for 8 weeks. Provinols improved glucose metabolism by reducing plasma glucose and fructosamine in ZF rats. Moreover, it reduced circulating triglycerides and total cholesterol as well as LDL-cholesterol in ZF rats. Echocardiography measurements demonstrated that Provinols improved cardiac performance as evidenced by an increase in left ventricular fractional shortening and cardiac output associated with decreased peripheral arterial resistances in ZF rats. Regarding vascular function, Provinols corrected endothelial dysfunction in aortas from ZF rats by improving endothelium-dependent relaxation in response to acetylcholine (Ach). Provinols enhanced NO bioavailability resulting from increased nitric oxide (NO) production through enhanced endothelial NO-synthase (eNOS) activity and reduced superoxide anion release via decreased expression of NADPH oxidase membrane sub-unit, Nox-1. In small mesenteric arteries, although Provinols did not affect the endothelium-dependent response to Ach; it enhanced the endothelial-derived hyperpolarizing factor component of the response. CONCLUSIONS/SIGNIFICANCE: Use of red wine polyphenols may be a potential mechanism for prevention of cardiovascular and metabolic alterations associated with obesity.

Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome.

Microparticles are membrane vesicles that are released during cell activation and apoptosis. Elevated levels of microparticles occur in many cardiovascular diseases; therefore, we characterized circulating microparticles from both metabolic syndrome (MS) patients and healthy patients. We evaluated microparticle effects on endothelial function; however, links between circulating microparticles and endothelial dysfunction have not yet been demonstrated. Circulating microparticles and their cellular origins were examined by flow cytometry of blood samples from patients and healthy subjects. Microparticles were used either to treat human endothelial cells in vitro or to assess endothelium function in mice after intravenous injection. MS patients had increased circulating levels of microparticles compared with healthy patients, including microparticles from platelet, endothelial, erythrocyte, and procoagulant origins. In vitro treatment of endothelial cells with microparticles from MS patients reduced both nitric oxide (NO) and superoxide anion production, resulting in protein tyrosine nitration. These effects were associated with enhanced phosphorylation of endothelial NO synthase at the site of inhibition. The reduction of O2(-) was linked to both reduced expression of p47 phox of NADPH oxidase and overexpression of extracellular superoxide dismutase. The decrease in NO production was triggered by nonplatelet-derived microparticles. In vivo injection of MS microparticles into mice impaired endothelium-dependent relaxation and decreased endothelial NO synthase expression. These data provide evidence that circulating microparticles from MS patients influence endothelial dysfunction.

Circulating microparticles from patients with septic shock exert protective role in vascular function.

RATIONALE: Sepsis is an archetypal condition with molecular links between inflammation and coagulation. Both events can be orchestrated by the interaction between circulating and vascular cells that under activation release microparticles. OBJECTIVES: We characterized circulating microparticles from both nonseptic subjects and patients with septic shock and evaluated their contribution to vascular function. METHODS: Circulating microparticles and their cell origin were measured in blood from 36 patients with septic shock and 18 nonseptic subjects by flow cytometry. Microparticles were then injected intravenously into mice and vascular reactivity was assessed in aorta. Expression and activity of enzymes involved in nitric oxide (NO) and cyclooxygenase metabolite production were analyzed. MEASUREMENTS AND MAIN RESULTS: Circulating levels of microparticles and platelet- and endothelial-derived microparticles were increased in septic patients. Surprisingly, septic microparticles enhanced the sensitivity of contraction of mouse aorta in response to serotonin. Interestingly, septic microparticles enhanced the contraction of aorta from lipopolysaccharide-treated mice. This effect was linked neither to increased calcium entry nor to Rho kinase inhibitor-sensitive mechanisms. In addition, the effect of septic microparticles was not modified either by NO-synthase or cyclooxygenase-2 inhibitors, and was not associated with NO or O2- overproduction. The nonselective cyclooxygenase-2 inhibitor indomethacin reduced, and the specific thromboxane A2 antagonist SQ-29548 abolished, aortic contraction in mice treated with nonseptic and septic microparticles. The effect of septic microparticles was associated with increased thromboxane A2 production, and was sensitive to a selective thromboxane A2 antagonist. CONCLUSIONS: We provide evidence that increased circulating microparticles are protective against vascular hyporeactivity accounting for hypotension in patients with septic shock.