The role of platelets and von Willebrand factor in the procoagulant phenotype of inflammatory bowel disease.

OBJECTIVE: Although the risk for thrombosis is well documented for inflammatory bowel disease (IBD) patients, the underlying pathological mechanism seems to be different from other thrombotic conditions. Deciphering the actors responsible for the increased risk of thrombosis in IBD would help to improve management of this frequent complication. DESIGN: We studied the interplay between platelets, coagulation, and von Willebrand factor (VWF) in 193 IBD patients and in experimental models (acute and chronic) of colitis in wild-type and VWF-deficient mice. RESULTS: We found a platelet-dependent increase in thrombin generation in IBD patients and in our mouse model of colitis. Agglutinated platelets were present in the blood of patients and mice. Interestingly, we observed not only a significant increase in total VWF antigen, but we were able to detect the presence of active VWF (VWF in its platelet-binding conformation; 3.2±2.7µg/ml) in the plasma of 30% of all IBD patients. In healthy controls, active VWF levels were below 0.3µg/ml. This led us to further explore experimental colitis in VWF-deficient mice and we observed that these mice were protected against the procoagulant state triggered by the colitis. Unexpectedly, these mice also manifested a significant worsening of colitis severity both in acute and chronic models. CONCLUSION: Platelets and VWF (including its active form) appear to be central players in the procoagulant phenotype in IBD. We observed that the role of VWF in hemostasis differs from its role in colic tissue healing, potentially opening new therapeutic avenues for a life-threatening complication in IBD patients.

Smooth muscle αv integrins regulate vascular fibrosis via CD109 downregulation of TGF-ß signalling.

Aims: αv integrins are implicated in fibrosis in a number of organs through their ability to activate TGF-ß. However their role in vascular fibrosis and collagen accumulation is only partially understood. Here we have used αv conditional knockout mice and cell lines to determine how αv contributes to vascular smooth muscle cell (VSMC) function in vascular fibrosis and the role of TGF-ß in that process. Methods and results: Angiotensin II (Ang II) treatment causes upregulation of αv and ß3 expression in the vessel wall, associated with increased collagen deposition. We found that deletion of αv integrin subunit from VSMCs (αv SMKO) protected mice against angiotensin II-induced collagen production and assembly. Transcriptomic analysis of the vessel wall in αv SMKO mice and controls identified a significant reduction in expression of fibrosis and related genes in αv SMKO mice. In contrast, αv SMKO mice showed prolonged expression of CD109, which is known to affect TGF-ß signalling. Using cultured mouse and human VSMCs, we showed that overexpression of CD109 phenocopied knockdown of αv integrin, attenuating collagen expression, TGF-ß activation, and Smad2/3 signalling in response to angiotensin II or TGF-ß stimulation. CD109 and TGF-ß receptor were internalized in early endosomes. Conclusion: We identify a role for VSMC αv integrin in vascular fibrosis and show that αv acts in concert with CD109 to regulate TGF-ß signalling.

Circulating Endothelial Cells are Associated with Thromboembolic Events in Patients with Antiphospholipid Antibodies.

BACKGROUND: Endothelial damage has been described in antiphospholipid antibody (aPL)-positive patients. However, it is uncertain whether circulating endothelial cells (CECs)-which are released when endothelial injury occurs-can be a marker of patients at high risk for thrombosis. METHODS: Ninety-seven patients with aPL and/or systemic lupus erythematosus (SLE) were included. CECs were determined by an automated CellSearch system. We also assayed plasma levels of tissue factor-bearing extracellular vesicles (TF+/EVs) and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) as markers of endothelial dysfunction/damage. RESULTS: Patients' mean age was 46.1 ± 13.9 years, 77 were women. Thirty-seven had SLE and 75 patients were suffering from antiphospholipid syndrome. Thirty-seven percent of patients presented a medical history of arterial thrombosis and 46% a history of venous thromboembolism (VTE). Thirteen patients had increased levels of CECs (>20/mL), with a mean CEC level of 48.3 ± 21.3 per mL. In univariate analysis, patients with obesity or medical history of myocardial infarction (MI), VTE, or nephropathy had a significant increased CEC level. In multivariate analysis, obesity (odds ratio [OR] = 6.07, 95% confidence interval [CI]: 1.42-25.94), VTE (OR = 7.59 [95% CI: 1.38-41.66]), and MI (OR = 5.5 [95% CI: 1.1-26.6)] were independently and significantly associated with elevated CECs. We also identified significant correlations between CECs and other markers of endothelial dysfunction: sTREM-1 and TF+/EVs. CONCLUSION: This study demonstrated that endothelial injury assessed by the levels of CECs was associated with thromboembolic events in patients with aPL and/or autoimmune diseases.

Monocytic Cell Adhesion to Oxidised Ligands: Relevance to Cardiovascular Disease.

Atherosclerosis, the major cause of vascular disease, is an inflammatory process driven by entry of blood monocytes into the arterial wall. LDL normally enters the wall, and stimulates monocyte adhesion by forming oxidation products such as oxidised phospholipids (oxPLs) and malondialdehyde. Adhesion molecules that bind monocytes to the wall permit traffic of these cells. CD14 is a monocyte surface receptor, a cofactor with TLR4 forming a complex that binds oxidised phospholipids and induces inflammatory changes in the cells, but data have been limited for monocyte adhesion. Here, we show that under static conditions, CD14 and TLR4 are implicated in adhesion of monocytes to solid phase oxidised LDL (oxLDL), and also that oxPL and malondialdehyde (MDA) adducts are involved in adhesion to oxLDL. Similarly, monocytes bound to heat shock protein 60 (HSP60), but this could be through contaminating lipopolysaccharide. Immunohistochemistry on atherosclerotic human arteries demonstrated increased endothelial MDA adducts and HSP60, but endothelial oxPL was not detected. We propose that monocytes could bind to MDA in endothelial cells, inducing atherosclerosis. Monocytes and platelets synergized in binding to oxLDL, forming aggregates; if this occurs at the arterial surface, they could precipitate thrombosis. These interactions could be targeted by cyclodextrins and oxidised phospholipid analogues for therapy.

The ß 1 -Adrenergic Receptor Contributes to Sepsis-Induced Immunosuppression Through Modulation of Regulatory T-Cell Inhibitory Function.

OBJECTIVES: Although cardiovascular benefits of ß 1 -adrenergic receptor blockade have been described in sepsis, little is known about its impact on the adaptive immune response, specifically CD4 T cells. Herein, we study the effects of ß 1 -adrenergic receptor modulation on CD4 T-cell function in a murine model of sepsis. DESIGN: Experimental study. SETTING: University laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: High-grade sepsis was induced by cecal ligation and puncture in wild-type mice (ß 1+/+ ) with or without esmolol (a selective ß 1 -adrenergic receptor blocker) or in ß 1 -adrenergic receptor knockout mice (ß 1-/- ). At 18 hours after surgery, echocardiography was performed with blood and spleen collected to analyze lymphocyte function. MEASUREMENTS AND MAIN RESULTS: At 18 hours, ß 1+/+ cecal ligation and puncture mice exhibited characteristics of high-grade sepsis and three surrogate markers of immunosuppression, namely decreased splenic CD4 T cells, reduced CD4 T-cell proliferation, and increased regulatory T lymphocyte cell proportions. Pharmacologic and genetic ß 1 -adrenergic receptor blockade reversed the impact of sepsis on CD4 T and regulatory T lymphocyte proportions and maintained CD4 T-cell proliferative capacity. ß 1 -adrenergic receptor blocked cecal ligation and puncture mice also exhibited a global decrease in both pro- and anti-inflammatory mediators and improved in vivo cardiovascular efficiency with maintained cardiac power index despite the expected decrease in heart rate. CONCLUSIONS: ß 1 -adrenergic receptor activation enhances regulatory T lymphocyte inhibitory function and thus contributes to sepsis-induced immunosuppression. This can be attenuated by ß 1 -adrenergic receptor blockade, suggesting a potential immunoregulatory role for this therapy in the management of sepsis.

Long-Term Overconsumption of Fat and Sugar Causes a Partially Reversible Pre-inflammatory Bowel Disease State.

Nutrition appears to be an important environmental factor involved in the onset of inflammatory bowel diseases (IBD) through yet poorly understood biological mechanisms. Most studies focused on fat content in high caloric diets, while refined sugars represent up to 40% of caloric intake within industrialized countries and contribute to the growing epidemics of inflammatory diseases. Herein we aim to better understand the impact of a high-fat-high-sucrose diet on intestinal homeostasis in healthy conditions and the subsequent colitis risk. We investigated the early events and the potential reversibility of high caloric diet-induced damage in mice before experimental colitis. C57BL/6 mice were fed with a high-fat or high-fat high-sucrose or control diet before experimental colitis. In healthy mice, a high-fat high-sucrose diet induces a pre-IBD state characterized by gut microbiota dysbiosis with a total depletion of bacteria belonging to Barnesiella that is associated with subclinical endoscopic lesions. An overall down-regulation of the colonic transcriptome converged with broadly decreased immune cell populations in the mesenteric lymph nodes leading to the inability to respond to tissue injury. Such in-vivo effects on microbiome and transcriptome were partially restored when returning to normal chow. Long-term consumption of diet enriched in sucrose and fat predisposes mice to colitis. This enhanced risk is preceded by gut microbiota dysbiosis and transcriptional reprogramming of colonic genes related to IBD. Importantly, diet-induced transcriptome and microbiome disturbances are partially reversible after switching back to normal chow with persistent sequelae that may contribute to IBD predisposition in the general population.

Elastase and exacerbation of neutrophil innate immunity are involved in multi-visceral manifestations of COVID-19.

BACKGROUND: Many arguments suggest that neutrophils could play a prominent role in COVID-19. However, the role of key components of neutrophil innate immunity in severe forms of COVID-19 has deserved insufficient attention. We aimed to evaluate the involvement of neutrophil elastase, histone-DNA, and DNases in systemic and multi-organ manifestations of COVID-19. METHODS: We performed a multicenter study of markers of neutrophil innate immunity in 155 cases consecutively recruited in a screening center, local hospitals, and two regional university hospitals. The cases were evaluated according to clinical and biological markers of severity and multi-organ manifestations and compared to 35 healthy controls. RESULTS: Blood neutrophil elastase, histone-DNA, myeloperoxidase-DNA, and free dsDNA were dramatically increased, and DNase activity was decreased by 10-fold, compared with controls. Neutrophil elastase and histone-DNA were associated with intensive care admission, body temperature, lung damage, and markers of cardiovascular outcomes, renal failure, and increased interleukin-6 (IL-6), IL-8, and CXCR2. Neutrophil elastase was an independent predictor of the computed tomography score of COVID-19 lung damage and the number of affected organs, in multivariate analyses. The increased blood concentrations of NE and neutrophil extracellular traps were related to exacerbation of neutrophil stimulation through IL-8 and CXCR2 increased concentrations and increased serum DAMPs, and to impaired degradation of NETs as a consequence of the dramatic decrease in blood DNase activity. CONCLUSION: Our results point out the key role of neutrophil innate immunity exacerbation in COVID-19. Neutrophil elastase and DNase could be potential biomarkers and therapeutic targets of severe systemic manifestations of COVID-19.

Endothelial-driven increase in plasma thrombin generation characterising a new hypercoagulable phenotype in acute heart failure.

BACKGROUND: Subjects with heart failure (HF) are at higher risk of developing thrombosis. We investigated whether endothelium activation and inflammation induce a prothrombotic biological profile in patients with acute decompensated HF (ADHF) and sinus rhythm. METHODS: Our prospective study included 34 ADHF patients, 30 patients with stable chronic HF (CHF) and 30 control inpatients without HF. In vitro thrombin generation and its downregulation by activated protein C (APC) was monitored by calibrated automated thrombography at hospital admission, at the day of discharge and after discharge, following at least six weeks of clinical stability. Circulating endothelium-derived extracellular vesicles (eEVs) were quantified by flow cytometry and nucleosomes by ELISA. RESULTS: Thrombin generation is increased and APC sensitivity is decreased independently of platelets in ADHF at admission compared to controls (p < 0.01). Thrombin generation was also increased in CHF but only in the presence of platelets. Plasma markers of endothelium activation (von Willebrand factor, factor VIII, procoagulant eEVs and circulating nucleosomes) and the ability of plasmas to induce neutrophil extracellular trap formation in control neutrophils are elevated in ADHF at admission compared to controls (p < 0.001). In-hospital prothrombotic changes in ADHF improved significantly at the post-discharge time-point. Circulating nucleosomes were positively correlated with APC sensitivity (p = 0.013) and annexin-V-positive eEVs (p = 0.004). CONCLUSIONS: This proof-of-concept study identified an endothelial-driven hypercoagulable phenotype at the acute phase of decompensated HF contrasting with the platelet-dependent prothrombotic state in CHF. These results highlighted a cross-talk between circulating eEVs and nucleosomes, procoagulant factors and impairment of the APC anticoagulant activity in ADHF.

Implication of Free Fatty Acids in Thrombin Generation and Fibrinolysis in Vascular Inflammation in Zucker Rats and Evolution with Aging.

Background: The metabolic syndrome (MetS) and aging are associated with modifications in blood coagulation factors, vascular inflammation, and increased risk of thrombosis. Objectives: Our aim was to determine concomitant changes in thrombin generation in the blood compartment and at the surface of vascular smooth muscle cells (VSMCs) and its interplay with adipokines, free fatty acids (FFA), and metalloproteinases (MMPs) in obese Zucker rats that share features of the human MetS. Methods: Obese and age-matched lean Zucker rats were compared at 25 and 80 weeks of age. Thrombin generation was assessed by calibrated automated thrombography (CAT). Results: Endogenous thrombin potential (ETP) was increased in obese rats independent of platelets and age. Clot half-lysis time was delayed with obesity and age. Interleukin (IL)-1ß and IL-13 were increased with obesity and age respectively. Addition of exogenous fibrinogen, leptin, linoleic, or palmitic acid increased thrombin generation in plasma whereas adiponectin had an opposite effect. ETP was increased at the surface of VSMCs from obese rats and addition of exogenous palmitic acid further enhanced ETP values. Gelatinase activity was increased in aorta at both ages in obese rats and MMP-2 activity was increased in VSMCs from obese rats. Conclusions: Our study demonstrated in MetS an early prothrombotic phenotype of the blood compartment reinforced by procoagulant properties of dedifferentiated and inflammatory VSMCs. Mechanisms involved (1) increased fibrinogen and impaired fibrinolysis and (2) increased saturated fatty acids responsible for additive procoagulant effects. Whether specifically targeting this hypercoagulability using direct thrombin inhibitors would improve outcome in MetS is worth investigating.

Moderate Hypothermia Improves Cardiac and Vascular Function in a Pig Model of Ischemic Cardiogenic Shock Treated With Veno-Arterial ECMO.

Cardiogenic shock (CS) patients treated with extracorporeal membrane oxygenation (ECMO) have severe cardiac failure, associated with ischemia-reperfusion. The use of moderate hypothermia during ischemia-reperfusion syndrome is supported by experimental data. We therefore studied the effects of moderate hypothermia on cardiac and vascular function in pig ischemic CS treated with veno-arterial extracorporeal membrane oxygenation (VA-ECMO). CS was induced in 12 anesthetized pigs by coronary ligation. After 1 h of CS, VA-ECMO was initiated and pigs were randomized to normothermia (38°C) or moderate hypothermia (34°C) during 8 h. Intrinsic cardiac function was measured using a left ventricular conductance catheter. At the end of the experiment, tissues were harvested for Western blotting. ECMO associated with norepinephrine infusion and volume resuscitation increased mean arterial pressure, mixed venous oxygen saturation as well as carotid, renal, and coronary blood flow without any differences between normothermia and hypothermia. Hypothermia was associated with less fluid and less norepinephrine infusion, lower lactate level, and higher urinary output. Vascular reactivity was superior in hypothermia comparatively to normothermia as expressed using norepinephrine dose-response curves. Pressure development during isovolumic contraction, left ventricular ejection fraction, and prerecruitable stroke work index were higher in the hypothermia group. There were no differences between normothermia and hypothermia with regard to carotid and mesenteric protein expression for iNOs, eNOS, and phospho AKt/AKt measured at the end of the experimentation. The incidence of surgical bleeding and coagulation disorders was the same in both groups. In conclusion, moderate and rapid hypothermia improves hemodynamics and cardiac and vascular function in a pig model of ischemic CS treated with ECMO.

Effects of low doses of esmolol on cardiac and vascular function in experimental septic shock.

BACKGROUND: Administration of a selective ß1-blocker, such as esmolol, in human septic shock has demonstrated cardiovascular protective effects related to heart rate reduction. Certain experimental data also indicate that esmolol exerts systemic anti-inflammatory and beneficial effects on vascular tone. Thus, the present study aimed to determine whether a non-chronotropic dose of esmolol maintains its protective cardiovascular and anti-inflammatory effects in experimental septic shock. METHODS: Four hours after cecal ligation and puncture (CLP), Wistar male rats were randomly allocated to the following groups (n = 8): CLP, CLP + E-1 (esmolol: 1 mg.kg-1.h-1), CLP + E-5 (esmolol: 5 mg.kg-1.h-1), CLP + E-18 (esmolol: 18 mg.kg-1.h-1). An additional eight rats underwent sham operation. All rats received a continuous infusion of saline, analgesic and antibiotics 4 hours after the surgery. Assessment at 18 hours included in vivo cardiac function assessed by echocardiography and ex vivo vasoreactivity assessed by myography. Circulating cytokine levels (IL-6 and IL-10) were measured by ELISA. Cardiac and vascular protein expressions of p-NF-κB, IκBα, iNOS, p-AKT/AKT and p-eNOS/eNOS were assessed by western blotting. RESULTS: CLP induced tachycardia, hypotension, cardiac output reduction, hyperlactatemia and vascular hypo-responsiveness to vasopressors. Compared to CLP animals, heart rate was unchanged in CLP + E-1 and CLP + E-5 but was reduced in CLP + E-18. Stroke volume, cardiac output, mean arterial pressure and lactatemia were improved in CLP + E-1 and CLP + E-5, while vascular responsiveness to phenylephrine was only improved in CLP + E-5 and CLP + E-18. Plasma IL-6 levels were decreased in all esmolol groups. p-NF-κB was decreased in both cardiac and vascular tissues in CLP + E-5 and CLP + E-18. CONCLUSION: In experimental septic shock, low doses of esmolol still improved cardiac function and vasoreactivity. These benefits appear to be associated with a modulation of inflammatory pathways.

If Channel Inhibition With Ivabradine Does Not Improve Cardiac and Vascular Function in Experimental Septic Shock.

OBJECTIVE: Previous studies have suggested that lowering heart rate (HR) by selective ß1-blockers improves sepsis-induced cardiac and vascular dysfunction primarily by decreasing proinflammatory pathways. However, the impact of isolated heart rate reduction (HRR) on hemodynamics and inflammatory pathways remains unknown. The present study was designed to assess the effects of HRR by ivabradine, an If channel inhibitor, on cardiovascular function and inflammatory pathways in peritonitis-induced septic shock in rats. DESIGN: Randomized animal study. SETTING: University research laboratory. INTERVENTIONS: Four hours after cecal ligation and puncture (CLP), Wistar rats were randomly allocated to the following groups: CLP (n = 8) and CLP + ivabradine (n = 8, administered per os 4 h after the surgery). Another eight Wistar male rats underwent sham operation. All rats received a continuous infusion of saline (10 mL kg h), analgesic (nalbuphine: 0.2 mg kg h), and antibiotics (imipenem and cilastatin sodium: 10 mg kg) 4 h after the surgery. Assessment at 18 h included hemodynamics, in vivo cardiac function by echocardiography, and ex vivo vasoreactivity by myography. Circulating cytokine levels (TNF-α, IL-6, and IL-10) were measured by ELISA, whereas cardiac and vascular protein expressions of NF-κB/IκBα/iNOS and Akt/eNOS were assessed by Western blotting. RESULTS: Compared with sham animals, CLP induced tachycardia, hypotension, decreased cardiac output, hyperlactatemia, and vascular hyporesponsiveness to vasopressors. Compared with the CLP group, adjunction of ivabradine decreased the HR without any impact on blood pressure, lactatemia, or vascular responsiveness to vasopressors. Adjunction of ivabradine to CLP rats had no impact on TNF-α, IL-6, and IL-10 cytokines, or on the protein expression levels of phosphorylated forms of NF-κB, Akt, eNOS, and degradation of IκBα in cardiac and vascular tissues. CONCLUSION: Isolated HRR by ivabradine in an experimental model of septic shock does not appear to be associated with any effect on the tested parameters of cardiac function or on vascular responsiveness to vasopressors. Moreover, in this setting, ivabradine does not alter the circulating levels of selected pro/anti-inflammatory cytokines or cardiac and vascular NF-κB/IκBα protein expression levels.

Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1ß, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive.

ß1-Adrenergic Inhibition Improves Cardiac and Vascular Function in Experimental Septic Shock.

OBJECTIVE: Preliminary experimental data suggest that selective ß1-blockers may improve ex vivo cardiac function in animal sepsis. Currently, the effects of esmolol on in vivo cardiac function and on vascular function are unknown. The present study was designed to examine the effects of the ß1-selective blocker esmolol on myocardial and vascular function in peritonitis-induced septic rats and to explore the inflammatory pathways involved in this process. DESIGN: Randomized animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Four hours after cecal ligation and puncture, Wistar rats were randomly allocated to the following groups: control, esmolol, norepinephrine (started at 18 hr after the surgery), and esmolol (started at 4 hr after the surgery) + norepinephrine (started at 18 hr after the surgery). Assessment at 18 hours after surgery was focused on cardiac contractility and vascular ex vivo function. Cardiac and vascular protein expressions of nuclear factor κB and endothelial nitric oxide synthase/Akt/inducible nitric oxide synthase pathways were assessed by Western blotting. MEASUREMENTS AND MAIN RESULTS: When compared with sham-operated animals, cecal ligation and puncture animals developed hypotension, cardiac depression, and vascular hyporesponsiveness to vasopressor treatment. Esmolol infusion increased cardiac contractility and restored mesenteric vasoreactivity. This effect was associated with a decrease in nuclear factor κB activation, an increase in Akt and endothelial nitric oxide synthase phosphorylation, and a decrease in inducible nitric oxide synthase expression both at the cardiac and vessel level. Esmolol infusion was also associated with an up-regulation in α1-vascular adrenoreceptors. CONCLUSION: Adjunction of selective ß1-blockade to standard septic shock management enhances intrinsic cardiac contractility and vascular responsiveness to catecholamines. These protective cardiovascular effects are likely predominantly attributed to the anti-inflammatory effect of esmolol.

Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries.

OBJECTIVE: The hypothesis that hypertension induces a hypercoagulable state arises from the complications associated with hypertension: stroke and myocardial infarction. Here, we determine whether hypertension causes changes in the thrombin-generating capacity of the vascular wall. APPROACH AND RESULTS: We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aortic rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared with equivalent rings from Wistar. This increase occurred in 12- but not 5-week-old rats and was prevented by an angiotensin II-converting enzyme inhibitor, indicating that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher on aortic smooth muscle cells (SMCs) from SHR than on those from Wistar. Exposure of negatively charged phospholipids was higher on SHR than on Wistar rings, as well as on cultured SMCs. Tissue factor activity was higher in SHR SMCs. Twelve-week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries, and the resulting occlusive thrombi were disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombin-induced proliferation than Wistar SMCs. This effect was totally abolished by a protease-activated receptor 1 inhibitor. CONCLUSIONS: The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which was sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MR(SMKO)), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MR(SMKO) mice, contractions induced by potassium chloride and calcium (Ca(2+)) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca(2+) suggests that the VSMC-specific deficiency of the MR modifies VSM Ca(2+) signaling but without altering the intracellular Ca(2+) store handling. The relaxation induced by acetylcholine is not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MR(SMKO) mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca(2+) signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca(2+) signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.

Assessment of the early stage of cardiac remodeling of spontaneously hypertensive heart failure rats using the quantitative 3-dimensional analysis provided by acipimox-enhanced FDG-PET.

Spontaneously hypertensive heart failure rats (SHHF) appear to constitute an original model for analyzing the evolution of the metabolic syndrome towards heart failure. This study aimed to characterize early cardiac dysfunction and remodeling in SHHF rats: (1) as compared with spontaneously hypertensive rats (SHR) and with a control group of Kyoto rats (WKY), and (2) by using the 3-dimensional quantitative analysis provided by acipimox-enhanced positron emission tomography (PET) with (18)F-fluorodesoxyglucose (FDG). Left ventricular (LV) ejection fraction (EF) and volume were quantified by automatic software on the FDG-PET images recorded in SHR (n = 20), SHHF (n = 18) and WKY-rats (n = 19) at ages 3 or 10 months old. Arterial blood pressure was determined by cardiac catheterization and cardiac fibrosis was quantified after sacrifice. Blood pressure was similarly elevated in SHR and SHHF rats (respective systolic blood pressures at 10-months: 199 ± 39 vs. 205 ± 2 mmHg), but SHHF rats had higher body mass than SHR rats (at 10-months, 630 ± 36 vs. 413 ± 27 g, p < 0.05) and higher blood levels of cholesterol and of triglycerides. At 3 months, cardiac parameters did not show significant differences between groups but at 10-months, SHHF and SHR rats exhibited an enhancement in myocardial mass and fibrosis associated with a clear decline in LV-EF (SHHF: 46 ± 6 %; SHR: 47 ± 5 %) as compared with WKY (56 ± 6 %, p < 0.01 for both comparisons). Cardiac remodeling of SHHF rats was clearly observable by FDG-PET from the age of 10-months, but in a similar way to that observed for SHR rats, suggesting a predominant role of hypertension.

Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice.

Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 µg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

Cyclic stretch-induced thrombin generation by rat vascular smooth muscle cells is mediated by the integrin αvß3 pathway.

AIMS: Vascular smooth muscle cell (VSMC) phenotypic modulation plays a pivotal role in atherothrombotic diseases. Thrombin generation at the surface of VSMCs and activation of integrin mechanotransduction pathways represent potential mechanisms. Here, we examine whether mechanical stretch increases thrombin generation on cultured rat aortic VSMCs. METHODS AND RESULTS: The integrin α(v)ß(3) antagonist peptide (cRGDPV) dose-dependently decreased thrombin generation without stretch. Static stretch (5%, 1 Hz) failed to modify the thrombin-forming capacity of VSMCs, whereas 10% cyclic stretch during 60 and 360 min enhanced integrin α(v)ß(3) expression and thrombin generation at the surface of VSMCs by 30% without inducing apoptosis. Cyclic stretch also stimulated Src phosphorylation, cleavage of talin, and binding of prothrombin to VSMCs. Upregulation of α(v)ß(3) expression, Src phosphorylation, and enhanced thrombin generation by cyclic stretch were abolished by cRGDPV and silencing RNA (siRNA) against α(v) as well as by selective inhibition of integrin α(v)ß(3) inside-out signalling by a talin-siRNA. Complete abolition of stretch-induced VSMC-supported thrombin generation by the RGT peptide, which disrupts the interaction of Src with the ß(3) cytoplasmic tail, demonstrates the link between outside-in pathways involving ß(3)-Src interaction and thrombin activity dependent on inside-out signalling. CONCLUSION: These data show that the contribution of cyclic stretch to VSMC-supported thrombin generation is driven by the integrin α(v)ß(3) signalling pathway and suggest a role for pulsatility-induced intramural thrombin in VSMC-dependent vascular remodelling.

Increased microparticle production and impaired microvascular endothelial function in aldosterone-salt-treated rats: protective effects of polyphenols.

We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h(-1)), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg(-1).day(-1)), or spironolactone (30 mg.kg(-1).day(-1)) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.