Guy Lesèche

Shed Membrane Microparticles With Procoagulant Potential in Human Atherosclerotic Plaques A Role for Apoptosis in Plaque Thrombogenicity

BACKGROUND The specific role of apoptosis in human atherosclerosis remains unknown. During apoptotic cell death, phosphatidylserine exposure on the cell surface confers a high tissue-factor (TF)-dependent procoagulant activity. METHODS AND RESULTS In this study, we examined the role of apoptotic cell death in the promotion of plaque thrombogenicity. TF expression and its relation to apoptosis was analyzed in 16 human atherosclerotic plaques by the use of immunohistochemical techniques. The presence of shed membrane apoptotic microparticles was analyzed in extracts from 6 human atherosclerotic plaques and 3 underlying arterial walls. The microparticles were captured by annexin V and their amounts estimated with respect to their phospholipid content by use of a prothrombinase assay. The prothrombogenic potential of the microparticles was further assessed by the measurement of total and microparticle-dependent TF activity in the extracts. The cell origin of the microparticles was determined after capture by specific antibodies. We were able to detect marked TF expression in the plaques in close proximity to apoptotic cells and debris, suggesting a potential interaction between TF and the apoptotic cell surfaces. High levels of shed membrane apoptotic microparticles were detected in extracts from atherosclerotic plaques but not in the underlying arterial walls (29.5+/-3.7 nmol/L phosphatidylserine equivalent versus 1.3+/-0.4 nmol/L, respectively, P<0.02). The microparticles were mainly of monocytic and lymphocytic origin and retained 97+/-2% of total TF activity, indicating a direct causal relationship between shed membrane microparticles and procoagulant activity of plaque extracts. CONCLUSIONS These results indicate that shed membrane microparticles with procoagulant potential are produced in human atherosclerotic plaques. Apoptosis could be a critical determinant of plaque thrombogenicity after plaque rupture.

A transient ischaemic attack clinic with round-the-clock access (SOS-TIA): feasibility and effects

BACKGROUND Diagnosis and treatment of cerebral and retinal transient ischaemic attacks (TIAs) are often delayed by the lack of immediate access to a dedicated TIA clinic. We evaluated the effects of rapid assessment of patients with TIA on clinical decision making, length of hospital stay, and subsequent stroke rates. METHODS We set up SOS-TIA, a hospital clinic with 24-h access. Patients were admitted if they had sudden retinal or cerebral focal symptoms judged to relate to ischaemia and if they made a total recovery. Assessment, which included neurological, arterial, and cardiac imaging, was within 4 h of admission. A leaflet about TIA with a toll-free telephone number for SOS-TIA was sent to 15 000 family doctors, cardiologists, neurologists, and ophthalmologists in Paris and its administrative region. Endpoints were stroke within 90 days, and stroke, myocardial infarction, and vascular death within 1 year. FINDINGS Between January, 2003, and December, 2005, we admitted 1085 patients with suspected TIA; 574 (53%) were seen within 24 h of symptom onset. 701 (65%) patients had confirmed TIA or minor stroke, and 144 (13%) had possible TIA. 108 (17%) of the 643 patients with confirmed TIA had brain tissue damage. Median duration of symptoms was 15 min (IQR 5-75 min). Of the patients with confirmed or possible TIA, all started a stroke prevention programme, 43 (5%) had urgent carotid revascularisation, and 44 (5%) were treated for atrial fibrillation with anticoagulants. 808 (74%) of all patients seen were sent home on the same day. The 90-day stroke rate was 1.24% (95% CI 0.72-2.12), whereas the rate predicted from ABCD(2) scores was 5.96%. INTERPRETATION Use of TIA clinics with 24-h access and immediate initiation of preventive treatment might greatly reduce length of hospital stay and risk of stroke compared with expected risk.

Relation Between Endothelial Cell Apoptosis and Blood Flow Direction in Human Atherosclerotic Plaques

BACKGROUND Blood flow characteristics influence endothelial cell apoptosis. However, little is known about the occurrence of endothelial cell apoptosis in human atherosclerosis and its relation to blood flow. METHODS AND RESULTS A total of 42 human carotid atherosclerotic plaques were retrieved by endarterectomy; they were examined in the longitudinal axial direction. Plaques were included in this study when upstream and downstream parts were clearly visible, occlusion was absent, and immunostaining for luminal endothelium was present all along the plaque. Using these criteria, 13 plaques were processed for further immunohistochemical studies (using anti-CD31, anti-Ki-67, and anti-splicing factor antibodies) and in situ detection of apoptosis (terminal dUTP nick end-labeling and ligase assay). Eight plaques showed > or =1 apoptotic endothelial cell at the luminal surface. Quantitative analysis of endothelial cell apoptosis in these plaques showed a systematic preferential occurrence of apoptosis in the downstream parts of plaques, where low flow and low shear stress prevail, in comparison with the upstream parts (18.8+/-3.3% versus 2.7+/-1.2%, respectively, P<0.001). Endothelial cell apoptosis was barely detectable in plaque microvessels. CONCLUSIONS Our results suggest that in vivo local shear stress influences luminal endothelial cell apoptosis and may be a major determinant of plaque erosion and thrombosis.

Expression of Interleukin-10 in Advanced Human Atherosclerotic Plaques: Relation to Inducible Nitric Oxide Synthase Expression and Cell Death

Inflammation is a major feature of human atherosclerosis and is central to development and progression of the disease. A variety of proinflammatory cytokines are expressed in the atherosclerotic plaque and may modulate extracellular matrix remodeling, cell proliferation, and cell death. Little is known, however, about the expression and potential role of anti-inflammatory cytokines in human atherosclerosis. Interleukin-10 (IL-10) is a major anti-inflammatory cytokine whose expression and potential effects in advanced human atherosclerotic plaques have not been evaluated. We studied 21 advanced human atherosclerotic plaques. IL-10 expression was analyzed by use of reverse transcription-polymerase chain reaction and immunohistochemical techniques. Inducible nitric oxide synthase expression was assessed by using immunohistochemistry, and cell death was determined by use of the TUNEL method. Reverse transcription-polymerase chain reaction identified IL-10 mRNA in 12 of 17 atherosclerotic plaques. Immunohistochemical staining of serial sections and double staining identified immunoreactive IL-10 mainly in macrophages, as well as in smooth muscle cells. Consistent with its anti-inflammatory properties, high levels of IL-10 expression were associated with significant decrease in inducible nitric oxide synthase expression (P<0.0001) and cell death (P<0. 0001). Hence, IL-10, a potent anti-inflammatory cytokine, is expressed in a substantial number of advanced human atherosclerotic plaques and might contribute to the modulation of the local inflammatory response and protect from excessive cell death in the plaque.

Lactadherin Deficiency Leads to Apoptotic Cell Accumulation and Accelerated Atherosclerosis in Mice

Background— Atherosclerosis is an immunoinflammatory disease; however, the key factors responsible for the maintenance of immune regulation in a proinflammatory milieu are poorly understood. Methods and Results— Here, we show that milk fat globule-EGF factor 8 (Mfge8, also known as lactadherin) is expressed in normal and atherosclerotic human arteries and is involved in phagocytic clearance of apoptotic cells by peritoneal macrophages. Disruption of bone marrow–derived Mfge8 in a murine model of atherosclerosis leads to substantial accumulation of apoptotic debris both systemically and within the developing lipid lesions. The accumulation of apoptotic material is associated with a reduction in interleukin-10 in the spleen but an increase in interferon-γ production in both the spleen and the atherosclerotic arteries. In addition, we report a dendritic cell-dependent alteration of natural regulatory T-cell function in the absence of Mfge8. These events are associated with a marked acceleration of atherosclerosis. Conclusions— Lack of Mfge8 in bone marrow–derived cells enhances the accumulation of apoptotic cell corpses in atherosclerosis and alters the protective immune response, which leads to an acceleration of plaque development.

p47phox-Dependent NADPH Oxidase Regulates Flow-Induced Vascular Remodeling

Chronic alterations in blood flow elicit an adaptive response that tends to normalize shear stress, involving nitric oxide (NO) and matrix metalloproteinases (MMPs). To evaluate the role of NADPH oxidase in this process, we developed a new model of mouse arteriovenous fistula (AVF) connecting the right common carotid artery (RCCA) with the jugular vein, which does not affect blood pressure. Mice deficient for gp91phox and p47phox subunits of NADPH and wild-type controls were used. AVF greatly increased RCCA blood flow (0.78±0.12 to 4.71±0.78 mL/min; P<0.01), producing an abrupt rise in shear stress (35±1 to 261±17 dynes/cm2; P<0.01) within 24 hours. RCCA diameter (460±14 &mgr;m) gradually enlarged 1 and 3 weeks after AVF (534±14 &mgr;m and 627±19 &mgr;m; P<0.01), reducing shear stress (173±13 and 106±10 dynes/cm2, respectively). In gp91phox−/− mice, changes in RCCA caliber and shear stress matched controls. However, p47phox−/− mouse RCCAs enlarged only marginally, such that shear stress remained high (199±8 dynes/cm2 at 3 weeks). Likewise, remodeling was minimal in endothelial NO synthase (eNOS)−/− mice. In both control and gp91phox−/− animals, reactive oxygen species (ROS) production and MMP induction was enhanced by AVF, whereas in p47phox−/− and eNOS−/− mice such response was negligible. Similarly, nitrotyrosine staining, indicating peroxynitrite formation, was more pronounced in control and gp91phox−/− mice than in p47phox−/− and eNOS−/− mice. Hence, shear stress induces vascular NADPH oxidase comprising p47phox but not gp91phox. Generated ROS interact with NO to produce peroxynitrite, which in turn activates MMPs, facilitating vessel remodeling. Our study provides the first evidence that ROS play a fundamental role in flow-induced vascular enlargement.

Survival benefit of lung transplantation for patients with idiopathic pulmonary fibrosis

OBJECTIVE Although lung transplantation is viewed as an acceptable option for patients with end-stage idiopathic pulmonary fibrosis, the survival benefit of this approach is still debated. This study examined whether there was a survival benefit of lung transplantation in a cohort of patients referred to our transplant center with a diagnosis of idiopathic pulmonary fibrosis according to American Thoracic Society criteria. METHODS Forty-six patients accepted for lung transplantation during a 12-year period with a diagnosis of idiopathic pulmonary fibrosis form the basis of this study. Survival benefit offered by lung transplantation was assessed using Cox proportional-hazards modeling, with patients on a waiting list as the control group. RESULTS Twenty-eight patients underwent lung transplantation (27 single and 1 double), 16 patients died while waiting, and 2 patients remained on the active waiting list. Diagnosis of idiopathic pulmonary fibrosis was made on histologic examination of the explanted lung or lung biopsy before lung transplantation. There was a pattern of usual interstitial pneumonia in 31 cases (67%). The 15 remaining patients fulfilled all American Thoracic Society criteria for idiopathic pulmonary fibrosis. The median waiting time for organs was 51 days. Survival after lung transplantation was 79.4% at 1 year, 63.5% at 2 years, and 39% at 5 years. The multivariable analysis showed that lung transplantation reduced the risk of death by 75% (95% confidence interval, 8%-86%; P =.03) after adjustment on potential confounding variables. CONCLUSIONS Lung transplantation is effective in improving the survival of selected patients affected by idiopathic pulmonary fibrosis.

The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis

Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1, 738 +/- 274 vs. 1,002 +/- 107 pmol/ micromol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.

Microparticles From Human Atherosclerotic Plaques Promote Endothelial ICAM-1–Dependent Monocyte Adhesion and Transendothelial Migration

Rationale and Objective: Membrane-shed submicron microparticles (MPs) released following cell activation or apoptosis accumulate in atherosclerotic plaques, where they stimulate endothelial proliferation and neovessel formation. The aim of the study was to assess whether or not MPs isolated from human atherosclerotic plaques contribute to increased endothelial adhesion molecules expression and monocyte recruitment. Method and Results: Human umbilical vein and coronary artery endothelial cells were exposed to MPs isolated from endarterectomy specimens (n=62) and characterized by externalized phosphatidylserine. Endothelial exposure to plaque, but not circulating, MPs increased ICAM-1 levels in a concentration-dependant manner (3.4-fold increase) without affecting ICAM-1 mRNA levels. Plaque MPs harbored ICAM-1 and transferred this adhesion molecule to endothelial cell membrane in a phosphatidylserine-dependent manner. MP-borne ICAM-1 was functionally integrated into cell membrane as demonstrated by the increased ERK1/2 phosphorylation following ICAM-1 ligation. Plaque MPs stimulated endothelial monocyte adhesion both in culture and in isolated perfused mouse carotid. This effect was also observed under flow condition and was prevented by anti–LFA-1 and anti–ICAM-1 neutralizing antibodies. MPs isolated from symptomatic plaques were more potent in stimulating monocyte adhesion than MPs from asymptomatic patients. Plaque MPs did not affect the release of interleukin-6, interleukin-8, or MCP-1, nor the expression of VCAM-1 and E-selectin. Conclusion: These results demonstrate that MPs isolated from human atherosclerotic plaques transfer ICAM-1 to endothelial cells to recruit inflammatory cells and suggest that plaque MPs promote atherosclerotic plaque progression.

CD40 ligand+ microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization

OBJECTIVES Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques. BACKGROUND Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis. METHODS Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin. RESULTS Plaque MPs increased both endothelial proliferation assessed by (3)H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice. CONCLUSIONS These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.