Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P ‫؍‬ .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis. (Blood. 2009; 114:1276-1279) IntroductionDirect evidence from autopsy studies and phlebography, as well as circumstantial evidence such as the correlation between frequency of deep venous thrombosis and the number of valves in individuals, have established the venous valvular sinus as a frequent location of thrombosis initiation. [1][2][3][4] This phenomenon has been attributed to stasis, one of the components of Virchow triad. In the 1960s, contrast media was shown to linger in valve sinuses an average of 27 minutes postvenography. 5 Valvular sinus stasis has also been associated with hypoxia and increased hematocrit, 6 as well as preventing the efflux of activated clotting factors and influx of clotting factor inhibitors, constituting a potentially hypercoagulable microenvironment, another component of Virchow triad. Stasis alone, however, is not a sufficient explanation for the propensity of thrombi to form in deep venous valve sinuses because, for example, prolonged periods of stasis associated with sleep are not associated with thrombus formation. Accordingly, there must be other factors interacting with stasis in the generation of venous valvular thrombi.In recent years, attention has been focused on the importance of endothelial heterogeneity in different vascular beds. 7,8 Venous endothelium manifests multiple distinct phenotypes in different organs such as the kidney and liver. Gene expression microarray studies have shown that endothelial cells from macro-versus microvascular beds, from arteries versus veins, and from different organs have distinctly different and characteristic gene expression profiles. 9 In response to local changes in flow, shear stress, or oxygenation, endothelial cells often adapt by increasing or decreasing expression of critical cell-surface and cytoplasmic proteins. 10 Thus, we hypothesized that valvular sinus endothelium would maintain a thromboresistant phenotype with the expression of the anticoagulant proteins thrombomodulin (TM) and endothelial protein C receptor (EPCR) u...

Objective-Group V secretory phospholipase A 2 (GV sPLA 2 ) has been detected in both human and mouse atherosclerotic lesions. This enzyme has potent hydrolytic activity towards phosphatidylcholine-containing substrates, including lipoprotein particles. Numerous studies in vitro indicate that hydrolysis of high density lipoproteins (HDL) and low density lipoproteins (LDL) by GV sPLA 2 leads to the formation of atherogenic particles and potentially proinflammatory lipid mediators. However, there is no direct evidence that this enzyme promotes atherogenic processes in vivo. Methods and Results-We performed gain-of-function and loss-of-function studies to investigate the role of GV sPLA 2 in atherogenesis in LDL receptor-deficient mice. Compared with control mice, animals overexpressing GV sPLA 2 by retrovirus-mediated gene transfer had a 2.7 fold increase in lesion area in the ascending region of the aortic root. Increased atherosclerosis was associated with an increase in lesional collagen deposition in the same region. Mice deficient in bone marrow-derived GV sPLA 2 had a 36% reduction in atherosclerosis in the aortic arch/thoracic aorta. Conclusions-Our data in mouse models provide the first in vivo evidence that GV sPLA 2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease. Key Words: Group V secretory phospholipase A 2 Ⅲ atherosclerosis Ⅲ retrovirus-mediated gene transfer Ⅲ bone marrow transplantation T he secretory phospholipase A 2 (sPLA 2 ) family of enzymes hydrolyze the fatty acid esterified at the sn-2 position of glycerophospholipids. 1 Of the 10 sPLA 2 s described in mammals, Group IIA (GIIA), Group V (GV), and Group X (GX) sPLA 2 have been detected in human and/or mouse atherosclerotic lesions. [2][3][4] These enzymes have been proposed to exert multiple proatherogenic effects in the arterial wall. Phospholipid hydrolysis by sPLA 2 generates potentially bioactive lipids, namely free fatty acids and lysophospholipids, which may promote various proinflammatory processes. Hydrolysis by either GV or GX sPLA 2 markedly reduces the capacity of HDL to promote cellular cholesterol efflux from lipid-loaded macrophages. 5 Hydrolysis of LDL by sPLA 2 in vitro results in an increased affinity for extracellular matrix proteoglycans and promotes LDL aggregation. 3,6 When incubated with mouse peritoneal macrophages, LDL hydrolyzed by either GV or GX sPLA 2 induces foam cell formation. 2,3 Thus, in vitro studies suggest that sPLA 2 s could promote atherogenesis by increasing the retention of LDL particles in the subendothelium and by generating potent inducers of macrophage foam cells. See page 445In this study, we directly tested the hypothesis that GV sPLA 2 promotes atherosclerosis in vivo. Using both gain-offunction and loss-of-function approaches, we demonstrate for the first time that bone marrow-derived GV sPLA 2 contributes to atherogenesis in LDL receptor-deficient mice. Methods Generation of Retroviral VectorsR...

S U M M A R YRupture of vulnerable atheroma often underlies acute coronary syndromes. Vulnerable plaques exhibit a paucity of vascular smooth muscle cells (VSMCs) in the cap. Therefore, decreased VSMC migration into the neointima may predispose to vulnerability. The balance between cell surface plasminogen activator activity and its inhibition [mediated primarily by plasminogen activator inhibitor type 1 (PAI-1)] modulates migration of diverse types of cells. We sought to determine whether increased expression of PAI-1 would decrease migration of VSMCs in vitro and neointimal cellularity in vivo in apolipoprotein E knockout (ApoE Ϫ / Ϫ ) mice fed a high-fat diet. Increased vessel wall expression of PAI-1 in transgenic mice was induced with the SM22 ␣ promoter. VSMC migration through Matrigel in vitro was quantified with laser scanning cytometry. Expression of PAI-1 was increased threefold in the aortic wall of SM22-PAI transgene-positive mice. Neointimal cellularity of vascular lesions was decreased by 26% ( p ϭ 0.01; n ϭ 5 each) in ApoE Ϫ / Ϫ mice with the SM22-PAI transgene compared with ApoE Ϫ / Ϫ mice. VSMCs explanted from transgenepositive mice exhibited twofold greater expression of PAI-1 and their migration was attenuated by 27% ( p ϭ 0.03). Accordingly, increased expression of PAI-1 protein by VSMCs reduces their migration in vitro and their contribution to neointimal cellularity in vivo. A therogenesis is a multifactorial and prolonged process (Ross 1993). Atherosclerotic plaques particularly prone to rupture, so-called vulnerable plaques, are pivotal in the genesis of acute coronary syndromes (Davies and Thomas 1985;Falk et al. 1995;Libby 1995;Sobel 1999;Sobel et al. 2003). Such plaques have a relative paucity of vascular smooth muscle cells (VSMCs), thin fibrous caps, lipid-laden cores, and high ratios of lipid and extracellular matrix (ECM) to VSMCs. Macrophages and T-lymphocytes, especially in shoulder regions of vulnerable plaques and implicated in plaque rupture (Libby 1995), are relatively prominent cellular constituents, whereas VSMCs are not (Davies and Thomas 1985;Falk et al. 1995;Libby 1995;Sobel 1999;Sobel et al. 2003).We have hypothesized that limitation of activation of intramural plasminogen activators by their primary inhibitor, plasminogen activator inhibitor type 1 (PAI-1), is one factor predisposing to evolution of vulnerable plaques by promoting or retarding neointimal migration of VSMCs, leading to a paucity of such cells and consequent elaboration of thin rather than thick fibrous plaques (Sobel 1999;Sobel et al. 2003). The present study was performed to determine whether decreased migration of VSMCs, characterized in vitro, would ensue if VSMC expression of PAI-1 were increased with the use of the promoter region of the VSMC gene SM-22 ␣ . In addition, we determined whether neointimal cellularity would be diminished in vivo by overexpression of VSMC PAI-1 during the evolution of atheroma in apolipoprotein E (ApoE)-deficient mice. A specific objective was to characterize the effect...

To quantitatively characterize contributions of major constituents to the composition of a given atherosclerotic plaque, we have developed an approach employing immunohistochemistry, confocal scanning laser microscopy, and computer-assisted image analysis. The method developed permits identification of plaques that are particularly vulnerable to rupture and elucidation of the nature of the composition of a given plaque, as well as the extent of luminal encroachment. Thus, it should be useful in experimental animals and ultimately in patients in delineating compositional changes in response to potentially deleterious genetic and environmentally induced factors and to potentially therapeutic interventions designed to diminish plaque vulnerability.