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AimsIgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression.Methods and ResultsThrough histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably,in vitrothe IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysedin vivoin ApoE-/-mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression.ConclusionsOur data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.

International audience; High-density lipoproteins (HDLs) display endothelial protective effects. We tested the role of SR-BI, an HDL receptor expressed by endothelial cells, in the neuroprotective effects of HDLs using an experimental model of acute ischemic stroke. After transient intraluminal middle cerebral artery occlusion (tMCAO), control and endothelial SR-BI deficient mice were intravenously injected by HDLs or saline. Infarct volume and blood-brain barrier (BBB) breakdown were assessed 24 h post tMCAO. The potential of HDLs and the role of SR-BI to maintain the BBB integrity was assessed by using a human cellular model of BBB (hCMEC/D3 cell line) subjected to oxygen-glucose deprivation (OGD). HDL therapy limited the infarct volume and the BBB leakage in control mice relative to saline injection. Interestingly, these neuroprotective effects were thwarted by the deletion of SR-BI in endothelial cells and preserved in mice deficient for SR-BI in myeloid cells. In vitro studies revealed that HDLs can preserve the integrity of the BBB in OGD conditions, and that this effect was reduced by the SR-BI inhibitor, BLT-1. The protection of BBB integrity plays a pivotal role in HDL therapy of acute ischemic stroke. Our results show that this effect is partially mediated by the HDL receptor, SR-BI expressed by endothelial cells.

Embolic events from vegetations are commonly accepted as the main mechanism involved in neurologic complications of infective endocarditis. The pathophysiology may imply other phenomena, including vasculitis. We aimed to define the cerebral lesion spectrum in an infective endocarditis rat model.Experimental model of Staphylococcus aureus or Enterococcus faecalis infective endocarditis. Neurologic lesions observed in the infective endocarditis model were compared with three other conditions, namely bacteremia, nonbacterial thrombotic endocarditis, and healthy controls.Research laboratory of a university hospital.Male Wistar rats.Brain MRI, neuropathology, immunohistochemistry for astrocyte and microglia, and bacterial studies on brain tissue were used to characterize neurologic lesions.In the infective endocarditis group, MRI revealed at least one cerebral lesion in 12 of 23 rats (52%), including brain infarctions (n = 9/23, 39%) and cerebral microbleeds (n = 8/23, 35%). In the infective endocarditis group, neuropathology revealed brain infarctions (n = 12/23, 52%), microhemorrhages (n = 10/23, 44%), and inflammatory processes (i.e., cell infiltrates including abscesses, vasculitis, meningoencephalitis, and/or ependymitis; n = 11/23, 48%). In the bacteremia group, MRI studies were normal and neuropathology revealed only hemorrhages (n = 2/11, 18%). Neuropathologic patterns observed in the nonbacterial thrombotic endocarditis group were similar to those observed in the infective endocarditis group. Immunochemistry revealed higher microglial activation in the infective endocarditis group (n = 11/23, 48%), when compared with the bacteremia (n = 1/11, 9%; p = 0.03) and nonbacterial thrombotic endocarditis groups (n = 0/7, 0%; p = 0.02).This original model of infective endocarditis recapitulates the neurologic lesion spectrum observed in humans and suggests synergistic mechanisms involved, including thromboembolism and cerebral vasculitis, promoted by a systemic bacteremia-mediated inflammation.

Background and Purpose— Previous experimental studies found that the infusion of human purified nascent HDL (high-density lipoprotein) significantly reduced infarct volume and hemorrhagic transformation rate by decreasing neutrophil recruitment. ApoA1-M (apolipoprotein A1-Milano) is a natural variant of human ApoA1 that confers protection against atherosclerosis. Recombinant ApoA1-M has been formulated as a complex with phospholipids to mimic the properties of nascent HDL. The aim of this study was to assess the impact of intravenous ApoA1-M in a transient middle cerebral artery occlusion stroke model in rats. Methods— In a first experiment, rats were subjected to 120-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused immediately or 4 hours after occlusion. In a second experiment, rats were subjected to 240-minute transient middle cerebral artery occlusion and intravenous ApoA1-M was infused with or without recombinant tPA (tissue-type plasminogen activator) immediately after recanalization. Primary outcome criteria were the infarct volume and hemorrhagic transformation rate measured at 24 hours. Platelets, coagulation, and neutrophil activation biomarkers were measured in brain homogenates and plasma. Additional in vitro experiments studied the effects of ApoA1-M on platelet aggregation and platelet-neutrophil interactions. Results— The infusion of ApoA1-M immediately or 4 hours after 120-minute transient middle cerebral artery occlusion significantly reduced the infarct volume compared with saline ( P =0.034 and P =0.036, respectively). Compared with tPA alone, co-administration of ApoA1-M and tPA showed similar rates of hemorrhagic transformation. ApoA1-M had no significant inhibition effect on neutrophil activation biomarkers. Platelet activation was slightly decreased in rats treated with ApoA1-M compared with saline. In vitro, the incubation of human and rat platelet-rich plasma with ApoA1-M significantly reduced ADP-induced platelet aggregation ( P =0.001 and P =0.02, respectively). Conclusions— ApoA1-Milano significantly decreased the infarct volume through an inhibition of platelet aggregation but did not reduce hemorrhagic transformation and neutrophils activation as expected after previous experimental studies with nascent HDL. Visual Overview— An online visual overview is available for this article.

La cavité buccale est l’une des voies majeures des contaminations environnementales connues pour être impliquées dans de nombreuses maladies chroniques via l’alimentation, les médications ou même la respiration. D’autres facteurs peuvent également influer sur l’environnement oral, certains endogènes, comme le microbiote, les variations hormonales, la salive, d’autres exogènes, comme les biomatériaux dentaires et les agents pathogènes. Cette synthèse fait le point sur l’état des connaissances, les questions et controverses sur les facteurs environnementaux courants au contact de la sphère orale impliqués dans les maladies de la cavité orale diagnostiquées chez l’adulte telles que les cancers des voies aéro-digestives supérieures, les ostéonécroses des mâchoires, et les parodontites, ces dernières pouvant d’ailleurs être directement liées à des pathologies systémiques comme les accidents vasculaires cérébraux, la maladie d’Alzheimer ou la maladie de Crohn notamment. La caractérisation des impacts environnementaux sur le microbiote oral, la salive, l’émail dentaire peut servir de marqueur pronostic précoce des maladies diagnostiquées ultérieurement, en lien avec ces expositions.

The oral cavity is one of the main route for environmental contaminations associated to many chronic diseases via alimentation, medications and respiration. Other factors may also impact the oral environment, some of them are endogenous, like microbiota, hormones and saliva, and others are exogenous, like dental materials and pathogens.La sphère orale, cible et marqueur de l’exposition environnementale - II. Maladies diagnostiquées chez l’adulte.La cavité buccale est l’une des voies majeures des contaminations environnementales connues pour être impliquées dans de nombreuses maladies chroniques via l’alimentation, les médications ou même la respiration. D’autres facteurs peuvent également influer sur l’environnement oral, certains endogènes, comme le microbiote, les variations hormonales, la salive, d’autres exogènes, comme les biomatériaux dentaires et les agents pathogènes. Cette synthèse fait le point sur l’état des connaissances, les questions et controverses sur les facteurs environnementaux courants au contact de la sphère orale impliqués dans les maladies de la cavité orale diagnostiquées chez l’adulte telles que les cancers des voies aéro-digestives supérieures, les ostéonécroses des mâchoires, et les parodontites, ces dernières pouvant d’ailleurs être directement liées à des pathologies systémiques comme les accidents vasculaires cérébraux, la maladie d’Alzheimer ou la maladie de Crohn notamment. La caractérisation des impacts environnementaux sur le microbiote oral, la salive, l’émail dentaire peut servir de marqueur pronostic précoce des maladies diagnostiquées ultérieurement, en lien avec ces expositions.

Human abdominal aortic aneurysm (AAA) pathophysiology is not yet completely understood. In conductance arteries, the insoluble extracellular matrix, synthesized by vascular smooth muscle cells, assumes the function of withstanding the intraluminal arterial blood pressure. Progressive loss of this function through extracellular matrix proteolysis is a main feature of AAAs. As most patients are now treated via endovascular approaches, surgical AAA specimens have become rare. Animal models provide valuable complementary insights into AAA pathophysiology. Current experimental AAA models involve induction of intraluminal dilation (nondissecting AAAs) or a contained intramural rupture (dissecting models). Although the ideal model should reproduce the histological characteristics and natural history of the human disease, none of the currently available animal models perfectly do so. Experimental models try to represent the main pathophysiological determinants of AAAs: genetic or acquired defects in extracellular matrix, loss of vascular smooth muscle cells, and innate or adaptive immune response. Nevertheless, most models are characterized by aneurysmal stabilization and healing after a few weeks because of cessation of the initial stimulus. Recent studies have focused on ways to optimize existing models to allow continuous aneurysmal growth. This review aims to discuss the relevance and recent advances of current animal AAA models. Visual Overview— An online visual overview is available for this article.

Establishing a prognosis at hospital admission after stroke is a major challenge. Inflammatory processes, hemostasis, vascular injury, and tissue remodeling are all involved in the early response to stroke. This study analyzes whether 22 selected biomarkers, sampled at admission, predict clinical outcomes in 153 stroke patients treated by thrombolysis and mechanical endovascular treatment (MET). Biomarkers were related to hemostasis (u-plasminogen activator/urokinase (uPA/urokinase), serpin E1/PAI-1, serpin C1/antithrombin-III, kallikrein 6/neurosin, alpha 2-macroglobulin), inflammation[myloperoxidase (MPO), chemokine ligand 2/monocyte chemoattractant protein-1 chemokine (CCL2/MCP-1), adiponectin, resistin, cell-free DNA (cDNA), CD40 Ligand (CD40L)], endothelium activation (Vascular cell adhesion protein 1 (VCAM-1) intercellular adhesion molecule 1 (ICAM-1), platelet endothelial cell adhesion molecule 1 (CD31/PECAM-1)], and tissue remodeling (total cathepsin S, osteopontin, cystatin C, neuropilin-1, matrix metallopeptidase 2 (MMP-2), matrix metallopeptidase 3 (MMP-3), matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 13 (MMP-13)]. Correlations between their levels and excellent neurological improvement (ENI) at 24 h and good outcomes (mRS 0&ndash, 2) at three months were tested. Osteopontin and favorable outcomes reached the significance level (p = 0.008), the adjusted OR per SD increase in log-transformed osteopontin was 0.34 (95%CI, 0.18&ndash, 0.62). The relationship between total cathepsin S and MPO with ENI, was borderline of significance (p = 0.064), the adjusted OR per SD increase in log-transformed of total cathepsin S and MPO was 0.54 (95%CI, 0.35&ndash, 0.81) and 0.51 (95%CI, 0.32&ndash, 0.80), respectively. In conclusion, osteopontin levels predicted three-month favorable outcomes, supporting the use of this biomarker as a complement of clinical and radiological parameters for predicting stroke prognosis.

SummaryClinical and experimental studies have highlighted the potential implication of periondontal bacteria contamination in the pathogenesis of abdominal aortic aneurysms (AAA). In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDLs) display multiple functions, including anti-inflammatory and lipopolysaccharide scavenging properties. Low plasma levels of HDL-cholesterol have been reported in AAA patients. We tested the effect of a HDL therapy in Sprague-Dawley rat model of AAA, obtained by intraluminal elastase infusion followed by repeated injections of Porphyromonas gingivalis (Pg). HDLs, isolated by ultracentrifugation of plasma from healthy human volunteers, were co-injected intravenously (10 mg/kg) with Pg (1.107 Colony Forming Unit) one, eight and 15 days after elastase perfusion. Rats were sacrificed one week after the last injection. Our results show that Pg injections promote the formation of a persistent neutrophil-rich thrombus associated with increased aortic diameter in this AAA model. HDLs significantly reduced the increased AAA diameter induced by Pg. Histology showed the onset of a healing process in the Pg/HDL group. HDL injections also reduced neutrophil activation in Pg-injected rats associated with decreased cytokine levels in conditioned media and plasma. Scintigraphic analysis showed an intense uptake of 99mTc-HDL by the AAA suggesting that HDLs could exert their beneficial effect by acting directly on the thrombus components. HDL supplementation may therefore constitute a new therapeutic tool for AAA treatment.Supplementary Material to this article is available online at www.thrombosis-online.com.

Background The authors recently found that a CD31 agonist peptide reaches macrophages in injured aortas and exerts beneficial effects on apolipoprotein E-knockout (Apo E−/−) mice subjected to angiotensin (Ang) II infusion, a model of experimental acute aortic dissection and intramural hematoma (ADIM). Objectives The purpose of this study was to evaluate the therapeutic potential of a drug-suitable agonist peptide in experimental ADIM. Methods P8RI, a retro-inverso sequence of the best candidate identified by functional in vitro screening of a peptide library, passed an absorption, distribution, metabolism, excretion and toxicology analysis. Apo E−/− mice (male, 28-week-old) implanted with Ang II-releasing pumps received P8RI (2.5 mg/kg/day) or vehicle from day 14 (n = 10/group). Leukocytes were analyzed by flow cytometry. Healing features of human and mouse dissected aortic segments were assessed by histology and immunofluorescence. The effect of CD31 on macrophages was evaluated using cells from CD31−/− mice and P8RI, in vitro. Results Human and experimental ADIM were characterized by the infiltration of proinflammatory macrophages. The absence of CD31 enhanced the proinflammatory polarization of macrophages, whereas the CD31 agonist P8RI favored reparative macrophages both in vitro and in vivo. The administration of P8RI after the occurrence of ADIM prevented aneurysmal transformation by promoting the resolution of intramural hematoma and the production of collagen in dissected aortas in vivo, associated with enrichment of M2 macrophages at the site of injury. Conclusions CD31 signaling promotes the switching of proinflammatory macrophages to the reparative phenotype and favors the healing of experimental dissected aortas. Treatment with a drug-suitable CD31 agonist may facilitate the clinical management of ADIM.

Background CD31 is a key transmembrane neutrophil immunoregulatory receptor. Mesenteric ischemia/reperfusion-induced neutrophil activation leads to a massive cleavage and shedding of the most extracellular domains of CD31 into plasma, enhancing the deleterious effect of neutrophil activation. We have evaluated the preventive therapeutic potential of an engineered synthetic octapeptide (P8RI), which restores the inhibitory intracellular signaling of cleaved CD31, in an experimental model of acute mesenteric ischemia/reperfusion. Methods In a randomized, controlled, and experimenter-blinded preclinical study, mesenteric ischemia/reperfusion (I/R) was induced in Wistar rats by superior mesenteric artery occlusion for 30 min followed by 4 h of reperfusion. Three groups of rats were compared: I/R + saline perfusion (I/R controls group, n = 7), I/R + preventive P8RI perfusion (P8RI group, n = 7), and sham-operated rats + saline perfusion (sham group, n = 7). Results Compared with I/R controls, P8RI perfusion significantly decreased intestinal ischemia/reperfusion injury (Chiu’s score, P = 0.01; epithelial area, P = 0.001) and bacterial translocation (plasma Escherichia coli DNA, P = 0.04) and could limit intestinal bleeding (P = 0.09). P8RI decreased neutrophil activation assessed by matrix metalloproteinase-9 release in plasma (P

Background and Purpose— Neutrophil Extracellular Traps (NETs) are DNA extracellular networks decorated with histones and granular proteins produced by activated neutrophils. NETs have been identified as major triggers and structural factors of thrombosis. A recent study designated extracellular DNA threads from NETs as a potential therapeutic target for improving tissue-type plasminogen activator (tPA)-induced thrombolysis in acute coronary syndrome. The aim of this study was to assess the presence of NETs in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) and their impact on tPA-induced thrombolysis. Methods— We analyzed thrombi from 108 AIS patients treated with endovascular therapy. Thrombi were characterized by hematoxylin/eosin staining, immunostaining, and ex vivo enzymatic assay. Additionally, we assessed ex vivo the impact of deoxyribonuclease 1 (DNAse 1) on thrombolysis of AIS thrombi. Results— Histological analysis revealed that NETs contributed to the composition of all AIS thrombi especially in their outer layers. Quantitative measurement of thrombus NETs content was not associated with clinical outcome or AIS pathogenesis but correlated significantly with endovascular therapy procedure length and device number of passes. Ex vivo, recombinant DNAse 1 accelerated tPA-induced thrombolysis, whereas DNAse 1 alone was ineffective. Conclusions— This study suggests that thrombus NETs content may be responsible for reperfusion resistance, including mechanical or pharmacological approaches with intravenous tPA, irrespectively of their etiology. The efficacy of a strategy involving an administration of DNAse 1 in addition to tPA should be explored in the setting of AIS. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT02907736.

Background and Purpose— Admission hyperglycemia is associated with a poor outcome in acute ischemic stroke. How hyperglycemia impacts the pathophysiology of acute ischemic stroke remains largely unknown. We investigated how preexisting hyperglycemia increases ischemia/reperfusion cerebral injury. Methods— Normoglycemic and streptozotocin-treated hyperglycemic rats were subjected to transient middle cerebral artery occlusion. Infarct growth and brain perfusion were assessed by magnetic resonance imaging. Markers of platelet, coagulation, and neutrophil activation were measured in brain homogenates and plasma. Downstream microvascular thromboinflammation (DMT) was investigated by intravital microscopy. Results— Hyperglycemic rats had an increased infarct volume with an increased blood–brain barrier disruption and hemorrhagic transformation rate compared with normoglycemic rats. Magnetic resonance imaging scans revealed that hyperglycemia enhanced and accelerated lesion growth and was associated with hemorrhagic transformation originating from territories that were still not completely reperfused at 1 hour after middle cerebral artery recanalization. Intravital microscopy and analysis of brain homogenates showed that DMT began immediately after middle cerebral artery occlusion and was exacerbated by hyperglycemia. Measurement of plasma serotonin and matrix metalloproteinase-9 indicated that platelets and neutrophils were preactivated in hyperglycemic rats. Neutrophils from hyperglycemic diabetic patients showed increased adhesion to endothelial cells as compared with neutrophils from normoglycemic donors in flow chamber experiments. Conclusions— We show that hyperglycemia primes the thromboinflammatory cascade, thus, amplifying middle cerebral artery occlusion–induced DMT. DMT exacerbation in hyperglycemic rats impaired reperfusion and precipitated neurovascular damage, blood–brain barrier disruption, and hemorrhagic transformation. Our results designate DMT as a possible target for reduction of the deleterious impact of hyperglycemia in acute ischemic stroke.

Introduction CD31 is a mechanosensory receptor and recent studies in CD31 knockout mice have suggested a protective role for CD31 in condition of altered mechanic stress such as in heart failure. Objective To directly assess the impact of CD31 signaling on the outcome of heart failure (HF) with both reduced and preserved ejection fraction (HFrEF and HFpEF, respectively). Method Permanent ligature of the proximal left coronary in C57Bl6 mice was used for modeling experimental HFrEF. Five days after surgery, mice displaying a myocardial infarction involving > 30% of the left ventricle (LV) at echocardiography were randomized to receive either a CD31 agonist peptide (P8RI, 2.5 mg/kg) or placebo by subcutaneous continuous infusion (osmotic pumps). Low dose angiotensin II infusion was applied to apolipoprotein E knockout (ApoE KO) mice as a model of HEpEF. The osmotic pumps delivered angiotensin II (200 μg/Kg/day) alone or together with P8RI. The Ejection Fraction (EF%) and the E/E’ ratio were evaluated by echocardiography. Results In the HFrEF model, the daily treatment with the CD31 agonist peptide starting from the 5th day after the myocardial infarction, improved both the EF% (68 ± 3% vs. 47 ± 4%, P = 0.05) evaluated 30 days later. Also, the LV filling pressure was improved by the treatment, as reflected by the E/E’ ratio (24.6 ± 2.4% vs. 49.7 ± 7.5, P = 0.05). An even more striking beneficial effect was observed in the model of HEpEF, in which the CD31 agonist treatment prevented the development of the diastolic LV dysfunction induced by the low dose of angiotensin II infusion as determined by the variation of the E/E’ ratio at the end of the treatment period (6 ± 7 in P8RI treated mice vs. 37 ± 8 in controls, P Conclusion CD31 agonist drugs can contribute to improve function of the heart in experimental conditions of myocardial mechanic stress and may represent a novel therapeutic strategy for heart failure with both reduced and preserved ejection fraction.

Aims Abdominal aortic aneurysm (AAA) is a particular form of atherothrombotic disease characterized by the dilation of the aortic wall and the presence of an intraluminal thrombus (ILT). The objective of the present study was to evaluate the pro-oxidant properties of the ILT and to characterize the anti-oxidant capacity of high-density lipoproteins (HDLs). Methods and results Our results show that ILT, adventitia, and plasma from AAA patients contained high concentrations of lipid and protein oxidation products. Mediators produced within or released by the thrombus and the adventitia were shown to induce reactive oxygen species (ROS) production by cultured aortic smooth muscle cells (AoSMCs) and to trigger the onset of apoptosis (an increase in mitochondrial membrane potential). Iron chelation limited these effects. Both concentration and functionality of HDLs were altered in AAA patients. Plasma levels of Apo A-I were lower, and small HDL subclasses were decreased in AAA patients. Circulating HDLs in AAA patients displayed an impaired capacity to inhibit copper-induced low-density lipoprotein oxidation and AoSMC ROS production. Western blot analyses of HDLs demonstrated that myeloperoxidase is associated with HDL particles in AAA patients. Conclusion ILT and adventitia are a source of pro-oxidant products, in particular haemoglobin, which may impact on the wall stability/rupture in AAA. In addition, HDLs from AAA patients exhibit an impaired anti-oxidant activity. In this context, restoring HDL functionality may represent a new therapeutic option in AAA.

High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity.

Background and Purpose— We have previously reported that intravenous injection of high-density lipoproteins (HDLs) was neuroprotective in an embolic stroke model. We hypothesized that HDL vasculoprotective actions on the blood–brain barrier (BBB) may decrease hemorrhagic transformation-associated with tissue plasminogen activator (tPA) administration in acute stroke. Methods— We used tPA alone or in combination with HDLs in vivo in 2 models of focal middle cerebral artery occlusion (MCAO) (embolic and 4-hour monofilament MCAO) and in vitro in a model of BBB. Sprague–Dawley rats were submitted to MCAO, n=12 per group. The rats were then randomly injected with tPA (10 mg/kg) or saline with or without human plasma purified-HDL (10 mg/kg). The therapeutic effects of HDL and BBB integrity were assessed blindly 24 hours later. The integrity of the BBB was also tested using an in vitro model of human cerebral endothelial cells under oxygen–glucose deprivation. Results— tPA-treated groups had significantly higher mortality and rate of hemorrhagic transformation at 24 hours in both MCAO models. Cotreatment with HDL significantly reduced stroke-induced mortality versus tPA alone (by 42% in filament MCAO, P =0.009; by 73% in embolic MCAO, P =0.05) and tPA-induced intracerebral parenchymal hematoma (by 92% in filament MCAO, by 100% in embolic MCAO; P P Conclusions— HDL injection decreased tPA-induced hemorrhagic transformation in rat models of MCAO. Both in vivo and in vitro results support the vasculoprotective action of HDLs on BBB under ischemic conditions.

Background and Purpose— High-density lipoprotein (HDL) levels are inversely associated with stroke incidence, suggesting a protective effect. Using a rat model, we tested the hypothesis that HDL exerts direct vasculo-/neuroprotective effects when administered during the acute phase of embolic stroke. Methods— After embolic occlusion, Sprague-Dawley rats were randomly treated intravenously with purified HDL versus saline immediately (2, 10 mg/kg) or 3 or 5 hours (10 mg/kg) after stroke. The effects of HDL were assessed blindly 24 hours later by evaluating neurological deficit score and measuring the infarct volume and blood–brain barrier breakdown. Protease activities and neutrophil infiltration were also evaluated. Results— HDL injection immediately after stroke (10 mg/kg) reduced by 68% the mortality at 24 hours ( P =0.015). HDL administration immediately or at 3 or 5 hours after stroke also reduced cerebral infarct volume by 74%, 68%, and 70.7%, respectively ( P =0.0003, P =0.011, and P =0.019; n=17 per group). The neurological deficit at 24 hours in the HDL-treated group was decreased versus the saline-treated group ( P =0.015). Ischemia-induced blood–brain barrier breakdown was significantly reduced in HDL-treated rats versus controls ( P =0.0045). Neuroprotective effects of HDL were associated with decreased neutrophil recruitment in the infarct area ( P =0.0027) accompanied by reduced matrix metalloproteinase gelatinase activity. Immunostaining showed that HDL was associated with endothelial and glial cells, and also that intercellular adhesion molecule-1 expression was decreased in vessels within the infarct area. Conclusions— Administration of HDL is neuroprotective when performed up to 5 hours after experimental stroke. This effect may be attributed to the ability of HDL to protect the blood–brain barrier and limit neutrophil recruitment.

Background and aim In type 2 diabetes, the interplay between cells and inflammatory mediators up-regulates CD36 expression in macrophages. The aim of this work was to investigate advanced glycation end products (AGE)-induced CD36 expression and its regulation by insulin in aortic vascular smooth muscle cells (VSMCs) from Goto–Kakisaki (GK) rats, a non-obese insulin model of both insulin resistance and type 2 diabetes. The context of overexpression of CD36 in aortas was also evaluated. Methods and results VSMCs were isolated and cultured from the aortas of GK rats and non-diabetic rats. The expression of proteins was evaluated by Western blot. The aortic production of superoxide anion (O 2 ·− ) was measured by luminescence on isolated tissue. AGEs and advanced oxidation protein products (AOPPs) were determined in plasma by fluorescence spectroscopy and spectrophotometry, respectively. AGE receptor (RAGE), NF-κB, and CD36 protein expression as well as O 2 ·− production were higher in GK aortas than in control aortas, and AGEs and AOPPs were higher in GK plasma. In VSMCs from non-diabetic rats, insulin was able to reduce (10nM) or suppress (100nM) the protein overexpression of CD36 induced by AGEs–BSA. In contrast, in VSMCs from GK rats, insulin was unable to reduce AGEs–BSA-induced CD36 overexpression. Conclusions The results suggest an overexpression of CD36 in VSMCs from GK rats and impaired control by insulin. In the context of increased plasma AGEs, aortic RAGE overexpression and increased oxidative stress markers, the data are compatible with an AGEs induced CD36 overexpression in diabetes.

Background— The atheromodulating activity of B cells during the development of atherosclerosis is well documented, but the mechanisms by which these cells are regulated have not been investigated. Methods and Results— Here, we analyzed the contribution of Qa-1–restricted CD8 + regulatory T cells to the control of the T follicular helper–germinal center B-cell axis during atherogenesis. Genetic disruption of CD8 + regulatory T cell function in atherosclerosis-prone apolipoprotein E knockout mice resulted in overactivation of this axis in secondary lymphoid organs, led to the increased development of tertiary lymphoid organs in the aorta, and enhanced disease development. In contrast, restoring control of the T follicular helper–germinal center B-cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of tertiary lymphoid organs. Moreover, analyses of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of T follicular helper cells within tertiary lymphoid organs. Conclusions— This study is the first to demonstrate that the T follicular helper–germinal center B-cell axis is proatherogenic and that CD8 + regulatory T cells control the germinal center reaction in both secondary and tertiary lymphoid organs. Therefore, disrupting this axis represents an innovative therapeutic approach.

Reactive oxygen species formation by phagocytes and subsequent modifications of vascular wall are involved in the early step of human atherogenesis. This study looked for the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors on NADPH oxidase-dependent superoxide anion production in THP-1 cells, a monocyte-derived cell line, and on the translocation of p21 Rac 2 and p67. A 30-min incubation with simvastatin (50 micro M ) inhibited phorbol 12-myristate 13-acetate-induced superoxide anion production by monocytes (32%) and a maximum inhibition was obtained at 3 h of incubation (69.5%). In addition, after 3 h of incubation a dose-dependent inhibition was obtained in the range 10-50 micro M of simvastatin with a median inhibitory concentration of 36 +/- 2.3 micro M Mevalonic acid (100 and 300 micro M ) and geranylgeraniol (100 micro M ) totally prevented the simvastatin-induced inhibitory effect of superoxide production by monocytes whereas farnesyl PP (100 micro M ) partially prevented (50%) this effect. In addition, simvastatin inhibited the translocation of p21 rac 2 and p67, suggesting that geranylgeranylation is required for NADPH oxidase activation. In another set of experiments, the rank order of potency of different statins on NADPH oxidase was determined (pravastatin < cerivastatin < lovastatin < fluvastatin < simvastatin). In conclusion, inhibition of superoxide formation by HMG CoA reductase inhibitors is highly suitable to prevent or limit the oxidative stress involved in the atherosclerosis process.

The influence of the HMG-CoA reductase inhibitor simvastatin was assessed on the cardiovascular alterations and production of free radicals associated with chronic angiotensin II (Ang II) infusion. Simvastatin (60 mg/kg per day PO) or placebo were given concomitantly for 10 days in Sprague-Dawley rats infused with Ang II (200 ng/kg per minute SC, osmotic pump). In addition, simvastatin or placebo was also given in vehicle-infused rats. Tail-cuff pressure and albuminuria were measured before and at the end of the treatment period. Cardiac weight, carotid structure, production of reactive oxygen species (ROS, by chemiluminescence) by polymorphonuclear leukocytes and aortic wall as well as protein and lipid oxidation products were determined at the end of the study. Ang II increased tail-cuff pressure by 56±12 mm Hg and simvastatin blunted the development of hypertension by ≈70% (19±5 mm Hg). Increases in heart weight index and carotid cross-sectional area induced by Ang II were obliterated by simvastatin (3.18±0.09 versus 3.46±0.11 mg/g body wt and 0.125±0.010 versus 0.177±0.010 mm2, respectively). The Ang II–induced increases in leukocyte and aortic production of ROS as well as protein and lipid oxidation products were prevented by simvastatin. No effect of simvastatin was detected in non–Ang II–infused rats. These results indicate that simvastatin prevented the development of hypertension and cardiovascular hypertrophy together with inhibition of the induced angiotensin II production of ROS. Therefore, inhibition of HMG CoA reductase by statins may have a beneficial effect on cardiovascular alterations through its antioxidant action in experimental Ang II–dependent hypertension.

Background Experimental atherosclerosis is characterized by the formation of tertiary lymphoid structures (TLOs) within the adventitial layer, which involves the chemokine-expressing aortic smooth muscle cells (SMCs). TLOs have also been described around human atherothrombotic arteries but the mechanisms of their formation remain poorly investigated. Herein, we tested whether human vascular SMCs play the role of chemokine-expressing cells that would trigger the formation of TLOs in atherothrombotic arteries. Results We first characterized, by flow cytometry and immunofluorescence analysis, the prevalence and cell composition of TLOs in human abdominal aneurysms of the aorta (AAAs), an evolutive form of atherothrombosis. Chemotaxis experiments revealed that the conditioned medium from AAA tissues recruited significantly more B and T lymphocytes than the conditioned medium from control (N-AAA) tissues. This was associated with an increase in the concentration of CXCL13, CXCL16, CCL19, CCL20, and CCL21 chemokines in the conditioned medium from AAA tissues. Immunofluorescence analysis of AAA cryosections revealed that α-SMA-positive SMCs were the main contributors to the chemokine production. These results were confirmed by RT-qPCR assays where we found that primary vascular SMCs from AAA tissues expressed significantly more chemokines than SMCs from N-AAA. Finally, in vitro experiments demonstrated that the inflammatory cytokines found to be increased in the conditioned medium from AAA were able to trigger the production of chemokines by primary SMCs. Conclusion Together, these results suggest that human vascular SMCs in atherothrombotic arteries, in response to inflammatory signals, are converted into chemokine-expressing cells that trigger the recruitment of immune cells and the formation of aortic TLOs.

SummaryIron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter 5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 1990(1) in a rat, then described in mice(2). Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Intraplaque hemorrhages are mainly related to inward neoangiogenesis, initiated from the adventitia by lipid-dependent outwardly convected signals, and by the immaturity of these neovessels, allowing leaks and hemorrhages. Repeated intraplaque hemorrhages play a major role in the evolution of thrombotic occlusive disease, similar to the role of intraluminal thrombus in the progression of abdominal aortic aneurysm toward rupture. Red blood cells (RBCs) are an important source of unesterified cholesterol, because their membranes are particularly cholesterol rich. This unesterified cholesterol is rapidly organized in cholesterol crystals, highly toxic for cell and membranes. Oxidized cholesterol and LDL provoke the irreversible covalent aggregation of proteins, including hemoglobin, forming ceroids, which are also highly toxic. Hemoglobin play a major role of prooxydant molecules in this context, by its ability to release heme and iron, the main catalyser of oxidative reaction. In the context of type 2 diabetes, the oxidative potential of intraplaque-free hemoglobin play a predominant role in the progression of atherothrombotic disease toward clinical expression. Associated to RBC, intraplaque hemorrhages convey leukocyte, mainly neutrophils in human, and plasma zymogen that are the main source of proteases, including coagulation proteases, activation of the fibrinolytique system, release of leukocyte serine proteases and cathepsins and activation of MMPs. These proteases concentrate in the hemorrhagic/necrotic core rendered plaque highly vulnerable. An adaptive immune response takes place in the adventitia, in regard of hemorrhagic plaques, in relation to outwardly convected oxidized or proteolyzed neoantigens, and chemokinic signals. Finally, intraplaque hemorrhages and thrombi are the site of weak pathogen entrapment, which promote all these oxydative and proteolytic phenomenons.

PurposeNeutrophils have been shown to be involved in all stages of human and experimental abdominal aortic aneurysm (AAA) development. The initial processes of neutrophil rolling and trapping in the intraluminal thrombus (ILT) are mediated mainly by P-selectin expressed by activated platelets. In the present study, we propose to evaluate the beneficial effect of fucoidan, a competitive binding agent of P-selectin, on aneurysmal growth in a rat model of aortic aneurysm with neutrophil enrichment of the ILT induced by repeated episodes of weak bacteremia.MethodsSixty Lewis rats with experimental AAAs, developed from decellularized aortic xenografts, were divided into four groups. Two groups were used as controls: group fucoidan control (FC) was treated with 200 mg of fucoidan (F) delivered by 2 mL, 4-week osmotic pumps placed intraperitoneally before closing the abdomen, and group C received saline instead of fucoidan. Two more groups were injected weekly with Porphyromonas gingivalis (P. gingivalis [Pg]): group F+Pg received 200 mg of intraperitoneal fucoidan and group Pg received saline. AAAs were harvested after 4 weeks and peripheral blood was sampled at that time. Cell-free DNA (cf-DNA) and myeloperoxydase (MPO) antigen concentrations were determined in plasma and in AAA-conditioned media. Histology and P-selectin immunostaining were performed on AAA tissue samples.ResultsComparing rats injected with Pg, those receiving fucoidan presented reduced aneurysmal diameter. Histologic analysis of AAAs showed that fucoidan reduced the ILT thickness in Pg-injected rats, with fewer trapped neutrophils, and with signs of a healing process, as observed in control group C. Immunohistological analysis revealed a substantial decrease in P-selectin immunostaining at the luminal surface of aneurysms in fucoidan-treated rats compared to the other groups, suggesting an interaction between fucoidan and P-selectin. A significant decrease in MPO concentrations in both plasma and conditioned medium was induced by fucoidan treatment in Pg-injected rats, reflecting a pacification of the ILT biological activity. This effect was associated with a reduction in neutrophil activation and apoptosis, reflected by a significant decrease in cf-DNA concentration in both plasma and conditioned medium of fucoidan-treated rats.ConclusionsOur results suggest that fucoidan has a beneficial effect on experimental aneurysmal degeneration by decreasing neutrophil activation in the ILT enhanced by weak pathogen contamination. This effect seems to be related to its interaction with P-selectin, which may decrease the trapping of neutrophils into the ILT. Fucoidan could represent a therapeutic option in AAAs to decrease the neutrophil activation involved in the degenerative process of aneurysmal expansion and rupture.Clinical RelevanceIn the present study, we propose to evaluate the beneficial effect of fucoidan, a competitive binding agent of P-selectin, on aneurysmal growth in a rat model of aortic aneurysm with neutrophil enrichment of the intraluminal thrombus induced by repeated episodes of weak bacteremia. Our results suggest that fucoidan has a beneficial effect on experimental aneurysmal degeneration enhanced by weak pathogen contamination by decreasing the trapping of neutrophils and the release of proteolytic enzymes into the intraluminal thrombus. Fucoidan could, therefore, represent a therapeutic option in abdominal aortic aneurysm to decrease the degenerative process leading to aneurysmal expansion and rupture.

Aims CD163 is a macrophage receptor for haemoglobin–haptoglobin (Hb–Hp) complexes, responsible for the clearance of haemoglobin. We hypothesized that production of soluble CD163 (sCD163) may be due to proleolytic shedding of membrane CD163 by neutrophil elastase, reported to be increased in culprit atherosclerotic plaques. We analysed the relationship between CD163 solubilization and elastase in vitro, in macrophage culture, ex vivo in human atherosclerotic plaque samples, and in vivo , in plasma of patients with coronary artery disease. Methods and results Neutrophil elastase was shown to enhance CD163 shedding and to decrease the uptake of Hb–Hp complexes by cultured macrophages. In addition, cultured carotid endarterectomy samples showing features of intraplaque haemorrhage released more sCD163 and elastase/α1-antitrypsin (α1-AT) complexes than non-haemorrhagic plaques ( n = 44). Plasma levels of sCD163 and neutrophil elastase (complexed with α1-AT) were measured in patients with an acute coronary syndrome (ACS, n = 42), stable angina pectoris (SAP, n = 28), or normal coronary angiograms without subclinical atherosclerosis ( n = 21). Acute coronary syndrome patients had higher sCD163 and elastase/α1-AT complexes plasma concentrations than subjects without coronary atherosclerosis. Circulating sCD163 and elastase/α1-AT complexes were positively correlated in patients with ACS ( r = 0.56, P < 0.0002) and SAP ( r = 0.62, P < 0.0005). Conclusion Our results suggest that neutrophil elastase promotes CD163 shedding, resulting in a decreased clearance of Hb by macrophages, which may favour plaque destabilization. This may be reflected by increased plasma levels of sCD163 and elastase/α1-AT complexes which are positively correlated in patients with coronary artery disease.

Objective— In the search of novel biomarkers of abdominal aortic aneurysm (AAA) progression, proteins released by intraluminal thrombus (ILT) were analyzed by a differential proteomic approach. Methods and Results— Different layers (luminal/abluminal) of the ILT of AAA were incubated, and the proteins released were analyzed by 2-dimensional difference in-gel electrophoresis. Several differentially expressed proteins involved in main AAA pathological mechanisms (proteolysis, oxidative stress, and thrombosis) were identified by mass spectrometry. Among the proteins identified, peroxiredoxin-1 (PRX-1) was more released by the luminal layer compared with the abluminal layer of the ILT, which was further validated by Western blot, ELISA, and immunohistochemistry. We demonstrated increased PRX-1 serum levels in AAA patients compared with healthy subjects and also positive correlation among PRX-1 and AAA diameter, plasmin-antiplasmin, and myeloperoxidase levels. Finally, a prospective study revealed a positive correlation between PRX-1 serum levels and AAA expansion rate. Moreover, the combination of PRX-1 and AAA size had significantly additive value in predicting growth. Conclusion— Several proteins associated with AAA pathogenesis have been identified by a proteomic approach in ILT-conditioned medium. Among them, PRX-1 serum levels are increased in AAA patients and correlate with AAA size and growth rate, suggesting the potential use of PRX-1 as a biomarker for AAA evolution.

The Notch pathway is involved in the regulation of the migratory/proliferative phenotype acquired by vascular smooth muscle cells (VSMCs) in the pro-inflammatory context of vascular diseases. Here, we investigated whether docosahexaenoic acid (DHA), a polyunsaturated, omega-3 fatty acid, could reduce fibrinolytic/matrix-metalloproteinase (MMP) activity and whether this reduction occurs through the modulation of Notch signaling. Rat VSMCs were transdifferentiated with interleukin-1beta and then treated with DHA. Migration/proliferation was determined by performing a wound healing assay and measuring MMP-2/-9 activity, type 1 plasminogen activator inhibitor levels, and the expression of these proteins. The involvement of Notch in regulating the fibrinolytic/MMP system was evidenced using Notch pathway inhibitors and the forced expression of Notch1 and Notch3 intracellular domains. DHA significantly decreased VSMC migration/proliferation induced by interleukin-1beta as well as fibrinolytic/MMP activity. Prevention of Notch1 target gene transcription enhanced the interleukin-1beta effects on MMPs and on migration, whereas Notch3 intracellular domain overexpression reduced these effects. Finally, DHA increased Notch3 expression, Hes-1 transcription (a Notch target gene), and enhanced gamma-secretase complex activity. These results suggest that inhibition of the Notch pathway participates in the transition of VSMCs toward a migratory phenotype. These results also suggest that the beneficial inhibitory effects of DHA on fibrinolytic/MMP activity are related in part to the effects of DHA on the expression of Notch pathway components, providing new insight into the mechanisms by which omega-3 fatty acids prevent cardiovascular diseases.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (TOFMS) allows for rapid differential proteomic analysis of various experimental conditions. In the first step of retention chromatography, the proteome to be analyzed is fractionated based on the biochemical properties of the proteins or peptides of which it is composed. TOF-MS separates the proteins according to their molecular mass and charge (m/z in which conditions are optimized to obtain single charged proteins: z = 1). The most discriminating conditions are used to validate differential peaks on a larger number of samples. Once the biomarker is statistically validated, its identification is performed by reproducing the chromatographic conditions that are appropriate for its retention on spin-columns, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsin digestion of the band of interest, and subsequent peptide mapping after MS. It may be necessary to determine part of the amino acid sequence of the protein of interest to confirm its identity.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (TOFMS) allows for rapid differential proteomic analysis of various experimental conditions. In the first step of retention chromatography, the proteome to be analyzed is fractionated based on the biochemical properties of the proteins or peptides of which it is composed. TOF-MS separates the proteins according to their molecular mass and charge (m/z in which conditions are optimized to obtain single charged proteins: z = 1). The most discriminating conditions are used to validate differential peaks on a larger number of samples. Once the biomarker is statistically validated, its identification is performed by reproducing the chromatographic conditions that are appropriate for its retention on spin-columns, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsin digestion of the band of interest, and subsequent peptide mapping after MS. It may be necessary to determine part of the amino acid sequence of the protein of interest to confirm its identity.

During the past decade, hemodialysis (HD)-induced inflammation has been linked to the development of long-term morbidity in end-stage renal disease (ESRD) patients on regular renal replacement therapy. Because interleukins and anaphylatoxins produced during HD sessions are potent activators for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, an example of an enzyme that is responsible for overproduction of reactive oxygen species (ROS), this may constitute a link between leukocyte activation and cell or organ toxicity. Oxidative stress, which results from an imbalance between oxidant production and antioxidant defense mechanisms, has been documented in ESRD patients using lipid and/or protein oxidative markers. Characterization of HD-induced oxidative stress has included identification of potential activators for NADPH oxidase. Uremia per se could prime phagocyte oxidative burst. HD, far from improving the oxidative status, results in an enhancement of ROS owing to hemoincompatibility of the dialysis system, hemoreactivity of the membrane, and trace amounts of endotoxins in the dialysate. In addition, the HD process is associated with an impairment in antioxidant mechanisms. The resulting oxidative stress has been implicated in long-term complications including anemia, amyloidosis, accelerated atherosclerosis, and malnutrition. Prevention of oxidative stress in HD might focus on improving the hemocompatibility of the dialysis system, supplementation of deficient patients with antioxidants, and modulation of NADPH oxidase by pharmacologic approaches.