Marilyn P. Wadsworth

Group V Secretory Phospholipase A2 Promotes Atherosclerosis Evidence From Genetically Altered Mice

Objective—Group V secretory phospholipase A2 (GV sPLA2) 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 sPLA2 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 sPLA2 in atherogenesis in LDL receptor–deficient mice. Compared with control mice, animals overexpressing GV sPLA2 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 sPLA2 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 sPLA2 contributes to atherosclerotic processes, and draw attention to this enzyme as an attractive target for the treatment of atherosclerotic disease.

Valves of the deep venous system: an overlooked risk factor

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.

Attenuation of Neointimal Vascular Smooth Muscle Cellularity in Atheroma by Plasminogen Activator Inhibitor Type 1 (PAI-1)

Rupture 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 apolipo-protein E knockout (ApoE−-/–) mice fed a high-fat diet. Increased vessel wall expression of PAI-1 in transgenic mice was induced with the SM22α 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=0.01; n=5 each) in ApoE−-/– mice with the SM22-PAI transgene compared with ApoE−-/– mice. VSMCs explanted from transgene-positive mice exhibited twofold greater expression of PAI-1 and their migration was attenuated by 27% (p=0.03). Accordingly, increased expression of PAI-1 protein by VSMCs reduces their migration in vitro and their contribution to neointimal cellularity in vivo.

Improved quantitative characterization of atherosclerotic plaque composition with immunohistochemistry, confocal fluorescence microscopy, and computer-assisted image analysis

Abstract 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.

Attenuation of Accumulation of Neointimal Lipid by Pioglitazone in Mice Genetically Deficient in Insulin Receptor Substrate-2 and Apolipoprotein E

Rupture of vulnerable atherosclerotic plaques that are characterized by extensive neointimal accumulation of lipid is a cause of acute coronary syndromes. To identify whether insulin resistance alters atherogenesis, we characterized the composition of atherosclerotic lesions in the proximal aortas in mice deficient in apolipoprotein E (ApoE−/-) and in ApoE−/- mice in which insulin resistance was intensified by a concomitant heterozygous deficiency in insulin receptor substrate type 2 (IRS2+/- ApoE−/- mice). In addition, we characterized the effect of an insulin sensitizer, pioglitazone, on the atherogenesis in IRS2+/- ApoE−/- mice. The extent of the aortic intima occupied by lesion was increased in the IRS2+/- ApoE−/- compared with ApoE−/- mice (79 ± 3% compared with 68 ± 8%, p<0.05). Treatment with pioglitazone decreased the neointimal content of lipid in 20-week-old mice from 50 ± 6% to 30 ± 7%, p=0.005 and decreased the cellularity reflected by the multisection cross-sectional areas of lesions comprising cells in atheroma from 24 ± 1% to 19 ± 3%, p=0.018. Accordingly, genetically induced intensification of insulin resistance increases atheroma formation. Furthermore, attenuation of insulin resistance by treatment with pioglitazone decreases accumulation of lipid in the neointima.

Localization of CD44 at the Invasive Margin of Glioblastomas by Immunoelectron Microscopy

Glioblastoma multiforme is a highly invasive primary brain tumor, which is known to strongly express the CD44 cell adhesion receptor. A number of experimental studies suggest that the interaction of this receptor with extracellular matrix (ECM) proteins such as hyaluronic acid may in part mediate human glioma cell adhesion and invasion of brain tissue. Although the expression of CD44 and its spliced variants in brain tumors have been extensively studied, there have been no reports localizing its expression to the invasive margin of the tumor. The authors used immunoelectron microscopy to investigate the expression patterns of CD44 in an in vitro organotypic invasion assay. Tumor spheroids initiated from the U373 MG human glioblastoma line were confronted with fetal rat brain aggregates in a spheroid coculture system. The CD44 expression appeared at the interface between glioblastoma tumor spheroids and brain tissue, as well as in the spheroid itself. CD44 immunoreactivity was not detectable in mature 21-day fetal brain aggregates. The findings provide direct evidence that CD44 is expressed at the confrontational invasive border between glioblastomas and brain tissue, further supporting its role in glioma cell-ECM recognition and attachment.

Imaging aspects of cardiovascular disease at the cell and molecular level.

Cell and molecular imaging has a long and distinguished history. Erythrocytes were visualized microscopically by van Leeuwenhoek in 1674, and microscope technology has evolved mightily since the first single-lens instruments, and now incorporates many types that do not use photons of light for image formation. The combination of these instruments with preparations stained with histochemical and immunohistochemical markers has revolutionized imaging by allowing the biochemical identification of components at subcellular resolution. The field of cardiovascular disease has benefited greatly from these advances for the characterization of disease etiologies. In this review, we will highlight and summarize the use of microscopy imaging systems, including light microscopy, electron microscopy, confocal scanning laser microscopy, laser scanning cytometry, laser microdissection, and atomic force microscopy in conjunction with a variety of histochemical techniques in studies aimed at understanding mechanisms underlying cardiovascular diseases at the cell and molecular level.

Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm.

Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores - ranging in emission from 435 to 665 nm - applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent.

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries.

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.

Cell adhesion molecule 1 (CADM1) is ubiquitously present in the endothelium and smooth muscle cells of the human macro- and micro-vasculature

Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin cell adhesion molecule family. Recently, we identified CADM1 to be a novel risk factor for venous thrombosis in a large, protein C deficient, thrombophilic family and showed, for the first time, the expression of CADM1 in endothelial cells (Hasstedt et al. in Blood 114:3084–3091, 2009). To further investigate its role in venous thrombosis, as well as other vasculopathies, we undertook a systematic confocal microscopic investigation for the presence of CADM1 in the vasculature of 28 different human tissues. Paraffin embedded tissue sections were dual immunostained with an antibody against CADM1, together with an antibody against either von Willebrand factor (to identify endothelial cells), or α-smooth muscle actin (to identify smooth muscle cells). The results showed that CADM1 was ubiquitously present in endothelial cells and smooth muscle cells in the vasculature from all 28 tissues, though its representation in the various classes of vessels was tissue dependent.