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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.

Background-Interleukin (IL)-18 is a potent proinflammatory cytokine with potential atherogenic properties. Its expression and role in atherosclerosis, however, are unknown. Methods and Results-In the present study, we examined stable and unstable human carotid atherosclerotic plaques retrieved by endarterectomy for the presence of IL-18 using reverse transcription-polymerase chain reaction (PCR), Western blot, and immunohistochemical techniques. IL-18 was highly expressed in the atherosclerotic plaques compared with control normal arteries and was localized mainly in plaque macrophages. IL-18 receptor was also upregulated in plaque macrophages and endothelial cells, suggesting potential biological effects. To examine the role of IL-18 in atherosclerosis, we determined the relation between IL-18 mRNA expression and signs of plaque instability using real-time quantitative PCR. Interestingly, significantly higher levels of IL-18 mRNA were found in symptomatic (unstable) plaques than asymptomatic (stable)

These results demonstrate that microparticles are more abundant and more thrombogenic in human atherosclerotic plaques than in plasma. The different cellular origins of plaque and plasma microparticles might explain the increased thrombogenic activity of plaque microparticles.

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.

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)26؍‬ 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. Key Words: microparticle Ⅲ ICAM-1 Ⅲ adhesion Ⅲ monocyte Ⅲ microvesicle A therosclerosis is a chronic inflammatory disease characterized by the accumulation of leukocytes, lipids, and fibrous tissue in the intima of arteries. 1 In atherosclerotic plaques, endothelial cells express elevated amounts of adhesion molecules such as selectins (P-selectin and E-selectin) and intercellular (ICAM-1) and vascular (VCAM-1) adhesion molecules at their surface. 1,2 Cytokines and chemokines are also secreted in excess by activated vascular cells in this context. These conditions favor the recruitment and the accumulation of monocytes and lymphocytes in the intima of vessels. 1,2 Human atherosclerotic plaques contain large amounts of microparticles (MPs), which are submicron membrane vesicles released following cell activation or apoptosis. [3][4][5] MPs harbor at their surface most of the membrane-associated proteins of the cells they stem from and are characterized by the loss of plasma membrane asymmetry resulting in the exposure of phosphatidylserine on their outer leaflet. 6,7 MPs isolated from human atherosclerotic lesions are highly thrombogenic and originate from multiple cells, including macrophages, lymphocytes, erythrocytes, and smooth muscle and endothelial cells. 4,5 MPs are no longer taken as innocent bystanders because several studies point out that MPs generated in vitro from cultured cells can affect several cellular functions, including inflammatory responses. 8 -15 However,...

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.

Abstract-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 (0.78Ϯ0.12 to 4.71Ϯ0.78 mL/min; PϽ0.01), producing an abrupt rise in shear stress (35Ϯ1 to 261Ϯ17 dynes/cm 2 ; PϽ0.01) within 24 hours. RCCA diameter (460Ϯ14 m) gradually enlarged 1 and 3 weeks after AVF (534Ϯ14 m and 627Ϯ19 m; PϽ0.01), reducing shear stress (173Ϯ13 and 106Ϯ10 dynes/cm 2 , respectively). In gp91phox Ϫ/Ϫ mice, changes in RCCA caliber and shear stress matched controls. However, p47phoxϪ/Ϫ mouse RCCAs enlarged only marginally, such that shear stress remained high (199Ϯ8 dynes/cm 2 at 3 weeks). Likewise, remodeling was minimal in endothelial NO synthase (eNOS) Ϫ/Ϫ mice. In both control and gp91phox Ϫ/Ϫ animals, reactive oxygen species (ROS) production and MMP induction was enhanced by AVF, whereas in p47phox Ϫ/Ϫ and eNOS Ϫ/Ϫ mice such response was negligible. Similarly, nitrotyrosine staining, indicating peroxynitrite formation, was more pronounced in control and gp91phoxϪ/Ϫ mice than in p47phox Ϫ/Ϫ and eNOS Ϫ/Ϫ 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. (Circ Res. 2005;97:533-540.)Key Words: extracellular matrix Ⅲ metalloproteinases Ⅲ reactive oxygen species Ⅲ shear stress C hanges in blood flow drive both acute and long-term compensatory responses in the vascular wall that tend to normalize wall shear stress. In the case of persistent increases in flow, adaptive remodeling of the vessel involves the reorganization of cellular and extracellular components. This is best exemplified in models of arteriovenous fistula (AVF), where steep increases in flow result in extensive arterial enlargement through restructuring of the extracellular matrix and modulation of vascular cell synthetic capacity. 1 One of the striking characteristics of the arterial wall proximal to an AVF is extensive tearing and fragmentation of the internal elastic lamina (IEL) 2,3 which augments arterial distensibility, leading to enhanced vessel diameter. Matrix metalloproteinases (MMPs) are likely instigators of IEL degradation in the vessel wall. MMP-2 and MMP-9 are upregulated shortly after AVF construction, and heightened activity of these enzymes persists until shear stress is normalized. 4,5 Furthermore, several studies have reported that MMP inhibition diminishes flow-mediated arterial enlargement in rat 4,6 and rabbit 5 AVF mod...