Shear Forces Induced Platelet Clearance Is a New Mechanism of Thrombocytopenia.

BACKGROUND: Thrombocytopenia has been consistently described in patients with extracorporeal membrane oxygenation (ECMO) and associated with poor outcome. However, the prevalence and underlying mechanisms remain largely unknown, and a device-related role of ECMO in thrombocytopenia has been hypothesized. This study aims to investigate the mechanisms underlying thrombocytopenia in ECMO patients. METHODS: In a prospective cohort of 107 ECMO patients, we investigated platelet count, functions, and glycoprotein shedding. In an ex vivo mock circulatory ECMO loop, we assessed platelet responses and VWF (von Willebrand factor)-GP Ibα (glycoprotein Ibα) interactions at low- and high-flow rates, in the presence or absence of red blood cells. The clearance of human platelets subjected or not to ex vivo perfusion was studied using an in vivo transfusion model in NOD/SCID (nonobese diabetic/severe combined Immunodeficient) mice. RESULTS: In ECMO patients, we observed a time-dependent decrease in platelet count starting 1 hour after device onset, with a mean drop of 7%, 35%, and 41% at 1, 24, and 48 hours post-ECMO initiation (P=0.00013, P<0.0001, and P<0.0001, respectively), regardless of the type of ECMO. This drop in platelet count was associated with a decrease in platelet GP Ibα expression (before: 47.8±9.1 versus 24 hours post-ECMO: 42.3±8.9 mean fluorescence intensity; P=0.002) and an increase in soluble GP Ibα plasma levels (before: 5.6±3.3 versus 24 hours post-ECMO: 10.8±4.1 µg/mL; P<0.0001). GP Ibα shedding was also observed ex vivo and was unaffected by (1) red blood cells, (2) the coagulation potential, (3) an antibody blocking VWF-GP Ibα interaction, (4) an antibody limiting VWF degradation, and (5) supraphysiological VWF plasma concentrations. In contrast, GP Ibα shedding was dependent on rheological conditions, with a 2.8-fold increase at high- versus low-flow rates. Platelets perfused at high-flow rates before being transfused to immunodeficient mice were eliminated faster in vivo with an accelerated clearance of GP Ibα-negative versus GP Ibα-positive platelets. CONCLUSIONS: ECMO-associated shear forces induce GP Ibα shedding and thrombocytopenia due to faster clearance of GP Ibα-negative platelets. Inhibiting GP Ibα shedding could represent an approach to reduce thrombocytopenia during ECMO.

Vascular Endothelial Damage in the Pathogenesis of Organ Injury in Severe COVID-19.

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Neutrophil extracellular traps (NETs) infiltrate haematoma and surrounding brain tissue after intracerebral haemorrhage: A post-mortem study.

AIMS: Because of their prothrombotic and neuroinflammatory effects, neutrophils and neutrophil extracellular traps (NETs) represent interesting therapeutic targets for spontaneous intracerebral haemorrhage (sICH). We investigated the presence, spatial and temporal distribution of NETs in a human sICH post-mortem study. METHODS: From 2005 to 2019, all sICH patients who came to autopsy within the first month after stroke were included and grouped according to the timing of death: 72 h, 4-7 days, 8-15 days and >15 days after ICH onset. Paraffin-embedded tissue was extracted from four strategic areas: haematoma, peri-haematomal area, ipsilateral surrounding brain tissue and a control contralateral area. Myeloperoxidase and histone H3 citrulline were immunolabelled to detect neutrophils and NETs respectively. RESULTS: Neutrophils were present in the brains of the 14 cases (4 men, median age: 78 years) and NETs were found in 7/14 cases. Both neutrophils and NETs were detected within the haematoma but also in the surrounding tissue. The appearance of neutrophils and NETs was time-dependent, following a two-wave pattern: during the first 72 h and between 8 and 15 days after ICH onset. Qualitative examination showed that neutrophils and NETs were mainly located around dense fibrin fibres within the haematoma. CONCLUSIONS: These observations provide evidence for NETs infiltration in the brain of patients who die from sICH. NETs might interact with early haemostasis within the haematoma core, and with the surrounding neuroinflammatory response. These findings open research perspectives for NETs in the treatment of sICH injuries.

Texture parameters of R2* maps are correlated with iron concentration and red blood cells count in clot analogs: A 7-T micro-MRI study.

BACKGROUND AND PURPOSE: Previous studies have suggested that mechanical revascularization in acute ischemic stroke (AIS) patients could be affected by clot histology. In this 7-T micro-MRI study, we used R2* relaxometry of clot analogs to evaluate the relationship between texture parameters of R2* maps and clot constituents. MATERIALS AND METHODS: Twelve AIS clot analogs were experimentally generated to obtain a wide range of red blood cell concentrations. All clots underwent a MR acquisition using a 7-T micro-MR system. A 3D multi-echo gradient-echo sequence was performed and R2* maps were generated. First order and second order statistics of R2* histograms within the clots were calculated. Iron concentration in clots was measured using absorption spectrometry and red blood cell count (RBC) was obtained by histopathological analysis. RESULTS: RBC count was strongly correlated with iron concentration within clots (r=0.87, P<.001). Higher RBC count and iron concentration were significantly correlated with first order parameters including: (a) global positive shift of the R2* histogram with higher '10th percentile', 'median', 'mean' and '90th percentile'; (b) increase of the global magnitude of voxel values with higher 'total energy' and 'root mean squared'; (c) greater uniformity of the voxel values with higher 'uniformity' and lower 'entropy'. Second order statistical parameters confirmed that higher RBC count and iron concentration correlated with (a) greater concentration of high gray-level values in the image; (b) more "coarse" texture of R2* maps. CONCLUSIONS: Texture analysis of MRI-R2* maps can accurately estimate the red blood cell count and iron content of AIS clot analogs.

Von Willebrand Factor Multimers during Transcatheter Aortic-Valve Replacement.

BACKGROUND: Postprocedural aortic regurgitation occurs in 10 to 20% of patients undergoing transcatheter aortic-valve replacement (TAVR) for aortic stenosis. We hypothesized that assessment of defects in high-molecular-weight (HMW) multimers of von Willebrand factor or point-of-care assessment of hemostasis could be used to monitor aortic regurgitation during TAVR. METHODS: We enrolled 183 patients undergoing TAVR. Patients with aortic regurgitation after the initial implantation, as identified by means of transesophageal echocardiography, underwent additional balloon dilation to correct aortic regurgitation. HMW multimers and the closure time with adenosine diphosphate (CT-ADP), a point-of-care measure of hemostasis, were assessed at baseline and 5 minutes after each step of the procedure. Mortality was evaluated at 1 year. A second cohort (201 patients) was studied to validate the use of CT-ADP in order to identify patients with aortic regurgitation. RESULTS: After the initial implantation, HMW multimers normalized in patients without aortic regurgitation (137 patients). Among the 46 patients with aortic regurgitation, normalization occurred in 20 patients in whom additional balloon dilation was successful but did not occur in the 26 patients with persistent aortic regurgitation. A similar sequence of changes was observed with CT-ADP. A CT-ADP value of more than 180 seconds had sensitivity, specificity, and negative predictive value of 92.3%, 92.4%, and 98.6%, respectively, for aortic regurgitation, with similar results in the validation cohort. Multivariable analyses showed that the values for HMW multimers and CT-ADP at the end of TAVR were each associated with mortality at 1 year. CONCLUSIONS: The presence of HMW-multimer defects and a high value for a point-of-care hemostatic test, the CT-ADP, were each predictive of the presence of aortic regurgitation after TAVR and were associated with higher mortality 1 year after the procedure. (Funded by Lille 2 University and others; ClinicalTrials.gov number, NCT02628509.).

Von Willebrand factor as a biological sensor of blood flow to monitor percutaneous aortic valve interventions.

RATIONALE: Percutaneous aortic valve procedures are a major breakthrough in the management of patients with aortic stenosis. Residual gradient and residual aortic regurgitation are major predictors of midterm and long-term outcome after percutaneous aortic valve procedures. We hypothesized that (1) induction/recovery of high molecular weight (HMW) multimers of von Willebrand factor defect could be instantaneous after acute changes in blood flow, (2) a bedside point-of-care assay (platelet function analyzer-closure time adenine DI-phosphate [PFA-CADP]), reflecting HMW multimers changes, could be used to monitor in real-time percutaneous aortic valve procedures. OBJECTIVE: To investigate the time course of HMW multimers changes in models and patients with instantaneous induction/reversal of pathological high shear and its related bedside assessment. METHODS AND RESULTS: We investigated the time course of the induction/recovery of HMW multimers defects under instantaneous changes in shear stress in an aortic stenosis rabbit model and in patients undergoing implantation of a continuous flow left ventricular assist device. We further investigated the recovery of HMW multimers and monitored these changes with PFA-CADP in aortic stenosis patients undergoing transcatheter aortic valve implantation or balloon valvuloplasty. Experiments in the aortic stenosis rabbit model and in left ventricular assist device patients demonstrated that induction/recovery of HMW multimers occurs within 5 minutes. Transcatheter aortic valve implantation patients experienced an acute decrease in shear stress and a recovery of HMW multimers within minutes of implantation which was sustained overtime. In patients with residual high shear or with residual aortic regurgitation, no recovery of HMW multimers was observed. PFA-CADP profiles mimicked HMW multimers recovery both in transcatheter aortic valve implantation patients without aortic regurgitation (correction) and transcatheter aortic valve implantation patients with aortic regurgitation or balloon valvuloplasty patients (no correction). CONCLUSIONS: These results demonstrate that variations in von Willebrand factor multimeric pattern are highly dynamic, occurring within minutes after changes in blood flow. It also demonstrates that PFA-CADP can evaluate in real time the results of transcatheter aortic valve procedures.

Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells.

BACKGROUND: Aortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. METHODS: We first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. RESULTS: Circulating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell-conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell-conditioned media from control aortic valve donors. CONCLUSIONS: These findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.

Inflammatory markers and auto-Abs to type I IFNs in COVID-19 convalescent plasma cohort study.

BACKGROUND: COVID-19 convalescent plasma (CCP) contains neutralising anti-SARS-CoV-2 antibodies that may be useful as COVID-19 passive immunotherapy in patients at risk of developing severe disease. Such plasma from convalescent patients may also have additional immune-modulatory properties when transfused to COVID-19 patients. METHODS: CCP (n = 766) was compared to non-convalescent control plasma (n = 166) for soluble inflammatory markers, ex-vivo inflammatory bioactivity on endothelial cells, neutralising auto-Abs to type I IFNs and reported adverse events in the recipients. FINDINGS: CCP exhibited a statistically significant increase in IL-6 and TNF-alpha levels (0.531 ± 0.04 vs 0.271 ± 0.04; (95% confidence interval [CI], 0.07371-0.4446; p = 0.0061) and 0.900 ± 0.07 vs 0.283 ± 0.07 pg/mL; (95% [CI], 0.3097-0.9202; p = 0.0000829) and lower IL-10 (0.731 ± 0.07 vs 1.22 ± 0.19 pg/mL; (95% [CI], -0.8180 to -0.1633; p = 0.0034) levels than control plasma. Neutralising auto-Abs against type I IFNs were detected in 14/766 (1.8%) CCPs and were not associated with reported adverse events when transfused. Inflammatory markers and bioactivity in CCP with or without auto-Abs, or in CCP whether or not linked to adverse events in transfused patients, did not differ to a statistically significant extent. INTERPRETATION: Overall, CCP exhibited moderately increased inflammatory markers compared to the control plasma with no discernible differences in ex-vivo bioactivity. Auto-Abs to type I IFNs detected in a small fraction of CCP were not associated with reported adverse events or differences in inflammatory markers. Additional studies, including careful clinical evaluation of patients treated with CCP, are required in order to further define the clinical relevance of these findings. FUNDING: French National Blood Service-EFS, the Association "Les Amis de Rémi" Savigneux, France, the "Fondation pour la Recherche Médicale (Medical Research Foundation)-REACTing 2020".

Thrombus formation during ECMO: Insights from a detailed histological analysis of thrombus composition.

OBJECTIVES: Intra-device thrombosis remains one of the most common complications during extracorporeal membrane oxygenation (ECMO). Despite anticoagulation, approximately 35% of patients develop thrombi in the membrane oxygenator, pump heads, or tubing. The aim of this study was to describe the molecular and cellular features of ECMO thrombi and to study the main drivers of thrombus formation at different sites in the ECMO circuits. APPROACH AND RESULTS: Thrombi (n = 85) were collected immediately after veno-arterial-(VA)-ECMO circuit removal from 25 patients: 23 thrombi from the pump, 25 from the oxygenator, and 37 from the tubing. Quantitative histological analysis was performed for the amount of red blood cells (RBCs), platelets, fibrin, von Willebrand factor (VWF), leukocytes, and citrullinated histone H3 (H3Cit). ECMO thrombi consist of a heterogenous composition with fibrin and VWF being the major thrombus components. A clustering analysis of the four major histological parameters identified two typical thrombus types: RBC-rich and RBC-poor/fibrin-rich thrombi with no significant differences in VWF and platelet content. Thrombus composition was not associated with the thrombus location, except for higher amounts of H3Cit that were found in pump and oxygenator thrombi compared to tubing samples. We observed higher blood leukocyte count and lactate dehydrogenase levels in patients with fibrin-rich thrombi. CONCLUSION: We found that thrombus composition is heterogenous, independent of their location, consisting of two types: RBC-rich and a fibrin-rich types. We also found that NETs play a minor role. These findings are important to improve current anticoagulation strategies in ECMO.

Time Course of Reendothelialization with Polyzene-F Nanocoated Cobra PzF™ Coronary Stent on Rabbit Iliac Arteries.

PURPOSE: Evaluation of reendothelialization with a new thin struts cobalt chromium alloy stent coated with a nano-layer of Polyzene™-F (PzF) in a rabbit iliac artery model. METHODS: Fifteen stented external rabbit iliac arteries were harvested at Day 7 for electron microscopy analysis following Cobra PzF stents implantation to assess reendothelialization and compare to historical data. Ten additional rabbits were used to assess time course of reendothelialization at 3 and 5 days. RESULTS: At Day 7, almost complete coverage of endothelial cells was observed with a coverage of 99.54 ±â€¯0.25% of the stented area. No thrombus area was noted. At Day 3, more than half of examined pieces was reendothelialized and reached 78.30 ±â€¯3.7% at Day 5 (p < .01 between each group). All stents were well expanded against the arterial wall and no struts were mal-apposed. CONCLUSIONS: Reendothelialization was rapid and complete at Day 7. This is the fastest reendothelization process after stenting in this model. No stent occlusion was observed.

Inhibition of tissue factor-factor VIIa proteolytic activity blunts hepatic metastasis in colorectal cancer.

BACKGROUND: Expression of the principal initiator of coagulation, tissue factor (TF), by colorectal cancer (CRC) cells is involved in tumoral angiogenesis and metastasis progression, after binding of factor VIIa (FVIIa) to TF and generation of TF-FVIIa activity. We thus hypothesized that inhibition of the TF pathway by active site-blocked FVIIa (FFR-FVIIa) may prevent the development of hepatic metastasis in CRC. METHODS: Rat tumoral cells (DHDK12 proB cells) expressing high levels of TF were injected in the portal vein in syngenic BDIX rats. Rats received intraperitoneal injection of either FFR-FVIIa, from d 3 to d 7 (adjuvant treatment) (n = 19), or solvent buffer (n = 18) (control group). Additionally, cancer cells were infused subcutaneously in 20 other rats, which were assigned to FFR-FVIIa adjuvant treatment (n = 10), or buffer treatment (n = 10). Macroscopic and histological analysis was performed at d 14. RESULTS: In the control group, infusion of cancer cells resulted in development of macroscopic hepatic tumors in 17/18 rats. In the adjuvant FFR-FVIIa group, macroscopic hepatic tumors were visible on the liver surface in 3/19 rats (P = 0.002 versus control). All rats with subcutaneous injection of proB cells exhibited macroscopic tumors, with no significant difference between the control and the treated ones. CONCLUSION: Inhibition of the proteolytic activity of TF-FVIIa complex blunted hematogenous hepatic metastasis, suggesting that TF-FVIIa is a relevant target for the prevention of hepatic metastasis in CRC. TF-blocking agents should be investigated as adjuvant treatment in this setting.

Arterial Pulsatility and Circulating von Willebrand Factor in Patients on Mechanical Circulatory Support.

BACKGROUND: The main risk factor for bleeding in patients with continuous-flow mechanical circulatory support (CF-MCS) is the acquired von Willebrand factor (VWF) defect related to the high shear-stress forces developed by these devices. Although a higher bleeding rate has been reported in CF-MCS recipients who had reduced pulsatility, the relation between pulsatility and the VWF defect has never been studied. OBJECTIVES: The purpose of this study was to investigate the relation between pulsatility and VWF under CF-MCS. METHODS: We assessed the effect of 2 CF-MCS on VWF multimer degradation in a mock circulatory loop (model 1). Using these devices, we investigated in a dose-effect model (model 2) 3 levels of pulsatility in 3 groups of swine. In a cross-over model (model 3), we studied the effects of sequential changes of pulsatility on VWF. We reported the evolution of VWF multimerization in a patient undergoing serial CF-MCS and/or pulsatile-MCS. RESULTS: We demonstrated the proteolytic degradation of VWF multimers by high shear CF-MCS in a circulatory loop without pulsatility. We observed both in swine models and in a patient that the magnitude of the VWF degradation is modulated by the pulsatility level in the high shear-stress level condition, and that the restoration of pulsatility is a trigger for the endothelial release of VWF. CONCLUSIONS: We demonstrated that the VWF defect reflects the balance between degradation induced by the shear stress and the endothelial release of new VWF triggered by the pulsatility. This modulation of VWF levels could explain the relationship between pulsatility and bleeding observed in CF-MCS recipients. Preservation of pulsatility may be a new target to improve clinical outcomes of patients.

Leptin induces osteoblast differentiation of human valvular interstitial cells via the Akt and ERK pathways.

AIMS: Calcific aortic valve disease (CAVD) affects 2-6% of the population over 65 years, and age, gender, smoking, overweight, dyslipidemia, diabetes contribute to the development of this disease. CAVD results, in part, from the osteoblast differentiation of human valvular interstitial cells (VICs). This study aims to elucidate the effects of leptin on osteoblast phenotype of VICs and the signalling pathways involved. METHODS: Patients who underwent aortic valve replacement for CAVD (n = 43) were included in this study. Patients with coronary artery disease (CAD) without CAVD (n = 129) were used as controls. RESULTS: Patients with CAVD had higher serum leptin concentrations than CAD patients (p = 0.002). Leptin was found in calcific aortic valves, with higher concentrations in calcified versus non-calcified zones (p = 0.01). Chronic leptin stimulation of human VICs enhanced alkaline phosphatase (ALP) activity and ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. Moreover, inhibiting Akt or ERK during leptin stimulation lowered the expression of osteoblast markers in VIC. CONCLUSIONS: Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.

Activated protein C increases sensitivity to vasoconstriction in rabbit Escherichia coli endotoxin-induced shock.

INTRODUCTION: The aim of this study was to investigate the effects of activated protein C (aPC) on vascular function, endothelial injury, and haemostasis in a rabbit endotoxin-induced shock model. METHOD: This study included 22 male New Zealand rabbits weighing 2.5 to 3 kg each. In vitro vascular reactivity, endothelium CD31-PECAM1 immunohistochemistry, plasma coagulation factors and monocyte tissue factor (TF) expression were performed 5 days (D5) after onset of endotoxic shock (initiated by 0.5 mg/kg intravenous bolus of Escherichia coli lipopolysaccharide (LPS)) with or without treatment with aPC injected as an intravenous 2 mg/kg bolus 1 hour after LPS (LPS+aPC group and LPS group, respectively). RESULTS: LPS decreased the sensitivity to phenylephrine (PE) in aortic rings without endothelium (E-) when compared to E- rings from the control group (p < 0.05). This was abolished by NG-nitro-L-arginine methyl ester and not observed in E- rings from aPC-treated rabbits. Although aPC failed to decrease monocyte TF expression in endotoxinic animals at D5, aPC treatment restored the endothelium-dependent sensitivity in response to PE (2.0 +/- 0.2 muM in rings with endothelium (E+) versus 1.0 +/- 0.2 microM in E- rings (p < 0.05) in the LPS+aPC group versus 2.4 +/- 0.3 microM in E+ rings versus 2.2 +/- 0.2 microM in E- rings (p value not significant), in the LPS group). Endotoxin-induced de-endothelialisation was reduced by aPC at D5 (28.5 +/- 2.3% in the LPS+aPC group versus 40.4 +/- 2.4% in the LPS group, p < 0.05). CONCLUSION: These data indicate that aPC increased the sensitivity to a vasoconstrictor agent (PE) associated with restoration of endothelial modulation, and protected against endothelial histological injury in endotoxin-induced shock. It failed to inhibit TF expression at D5 after LPS injection.

Dietary lipid lowering modifies plaque phenotype in rabbit atheroma after angioplasty: a potential role of tissue factor.

BACKGROUND: Tissue factor (TF), the main initiator of blood coagulation, is involved in cellular migration and angiogenesis processes. TF is expressed strongly in lipid-rich plaques and probably plays an important role in the thrombotic complications of plaque rupture. This study analyzes the effect of dietary lipid lowering on TF expression and cellular modifications in angioplasty-induced rabbit plaque rupture. METHODS AND RESULTS: After experimental plaque rupture by balloon angioplasty in atheromatous rabbits, animals were assigned a 0.2% or a 2% cholesterol diet, and the TF content of arterial wall and the associated histological modifications were analyzed after 4 weeks. Early effects of lipid lowering were observed: The increase of TF expression in the vascular wall was stronger in the 2% than in the 0.2% cholesterol diet group (iliac arteries: 1226+/-308 versus 72+/-29 mU TF/g artery, P<0.005). Immunohistochemistry indicated that TF expression was associated with sprout of neovessels, which was more pronounced in the 2% than in the 0.2% cholesterol group. CONCLUSIONS: This study shows that dietary lipid lowering decreases the thrombotic potential of ruptured atherosclerotic plaques through TF decrease. Moreover, high TF expression is associated with marked angiogenesis in the vascular wall, which is reduced by lipid lowering. These results provide further arguments for strong dietary lipid lowering to reduce plaque instability and thrombogenicity.

Pretreatment with peroxysome proliferator-activated receptor alpha agonist fenofibrate protects endothelium in rabbit Escherichia coli endotoxin-induced shock.

OBJECTIVE: To investigate the effects of fenofibrate, an activator of peroxysome proliferator activated receptor (PPAR) alpha, on vascular endothelium and on hemostasis in a rabbit endotoxic shock model. DESIGN AND SETTING: Prospective laboratory study in a university laboratory. SUBJECTS: 36 male New Zealand rabbits weighing 2.5-3 kg. INTERVENTIONS: We determined in vitro vascular reactivity, endothelium CD31-platelet/endothelial cell adhesion molecule (PECAM) 1 immunohistochemistry, plasma coagulation factors, and monocyte tissue factor expression 5 days after onset of endotoxic shock (0.5 mg/kg intravenous bolus, Escherichia coli lipopolysaccharide) with or without treatment by fenofibrate (mixed in the chow at a concentration of 0.5%) for 15 days before lipopolysaccharide injection and 5 days afterward. MEASUREMENTS AND RESULTS: Metabolic acidosis and coagulation activation confirmed presence of shock. Fenofibrate decreased monocyte tissue factor expression. It improved endothelial-dependent relaxation at 5 days (Emax=68.2+/-3.3%, vs. 44.2+/-2.5% in the non-treated group). Endotoxin-induced deendothelialization was significantly decreased by fenofibrate at 5 days (8.5+/-1.3% vs. 19.2+/-3.1% in the nontreated group) . CONCLUSIONS: These data indicate for the first time that fenofibrate, an activator of PPAR-alpha, inhibits monocyte tissue factor expression and protects against endothelial dysfunction and histological injury in endotoxin-induced shock.

M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes.

This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1ß (IL-1ß) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects.

Effects of the angiotensin-converting enzyme inhibitor perindopril on endothelial injury and hemostasis in rabbit endotoxic shock.

OBJECTIVE: To assess the effects of the angiotensin-converting enzyme (ACE) inhibitor (ACEI) perindopril on prolonged endothelial cell dysfunction in a rabbit endotoxic model. DESIGN: Randomized, controlled, interventional trial. SETTING: University animal laboratory. SUBJECTS: A total of 65 male New Zealand White rabbits, randomly assigned to one of eight groups. INTERVENTIONS: Endotoxic shock was induced by a single lipopolysaccharide (LPS, serotype O55:B5) bolus (0.5 mg.kg(-1), i.v., Escherichia coli endotoxin). Coagulation factors and expression of monocyte tissue factor (TF) were determined by functional assay. Endothelium-dependent vascular relaxation was assessed by in vitro vascular reactivity. Immunohistochemical staining (CD31) was performed to assess endothelial injury of the abdominal aorta. These parameters were studied 5 days (D5) after the onset of endotoxic shock. Rabbits were randomized to receive perindopril (1 mg kg(-1) day(-1) orally) alone, or with N(G)-nitro-L-arginine methyl ester (L-NAME; 15 mg kg(-1) day(-1) orally), or L-NAME alone initiated 7 days before the onset of endotoxic shock and maintained for 5 days afterward. MEASUREMENTS AND RESULTS: Perindopril prevented altered endothelium-dependent relaxation to acetylcholine induced by LPS injection (E(max)=75.6+/-3.7 vs 42.3+/-9.4% in LPS group, p<0.05). This effect was inhibited by co-treatment with L-NAME. Perindopril had no effect on either LPS-induced endothelial histological injury or monocyte TF expression. CONCLUSION: These data suggest that perindopril can prevent endothelial dysfunction in endotoxin-induced shock through an NO-dependent mechanism.

Alterations in neutrophil production and function at an early stage in the high-fructose rat model of metabolic syndrome.

BACKGROUND: Although neutrophils are crucially involved in inflammation, they have received only little attention in metabolic syndrome (MetS). We hypothesized that neutrophil infiltration into adipose tissue (AT) may occur at an early stage of MetS, in association with modulation of major functions of neutrophils and of their bone marrow production. METHODS: Fifty-six male Sprague-Dawley rats were fed regular (control rats (CRs)) or high-fructose (60%; fructose-fed rats (FFRs)) diets. After 6 weeks, metabolic parameters were measured. Distribution of neutrophils into AT was investigated by immunohistochemistry. Function of circulating neutrophils (activation, reactive oxygen species production, phagocytosis, and apoptosis) was determined by flow cytometry. Granulopoiesis was evaluated by measuring the number and survival characteristics of neutrophil progenitors using bone marrow culture assays and flow cytometry. RESULTS: Compared with the CR group, the FFR group developed MetS (i.e., arterial hypertension, hypertriglyceridemia, fasting hyperglycemia, and greater intra-abdominal AT volume) and presented higher neutrophil infiltration into AT. At resting state, no significant difference for circulating neutrophil functions was observed between the 2 groups. In contrast, circulating neutrophils from the FFR group exhibited higher responses to phorbol-12-myristate-13-acetate for all studied functions, compared with the CR group, suggesting that early MetS induces neutrophil priming. In parallel, a diminished clonal capacity and an increased apoptosis in bone marrow-derived granulocyte progenitors and neutrophil precursors were observed in the FFR group compared with the CR group. CONCLUSIONS: These results provide evidence of an increased infiltration into intra-abdominal AT and modified production, function, and phenotype of neutrophils at an early stage of high-fructose diet-induced MetS.