Objectives: To examine the ultrastructure of microvessels in normal and atherosclerotic coronary arteries and its association with plaque phenotype. Background: Microvessels in atherosclerotic plaques are an entry point for inflammatory and red blood cells. Yet there is limited data on the ultrastructural integrity of microvessels in human atherosclerosis. Methods: Microvessel density (MVD) and ultrastructural morphology were determined in the adventitia, intima-media border, and atherosclerotic plaque of 28 coronary arteries using immunohistochemistry for endothelial cells (Ulex europeaus, CD31/CD34), basement membrane (laminin, collagen IV), and mural cells (desmin, alpha-smooth muscle (SM) actin, smoothelin, SM1, SM2, Smemb). Ultrastructural characterization of microvessel morphology was performed by electron microscopy (EM). Results: MVD was increased in advanced plaques compared to early plaques, which correlated with lesion morphology. Adventitial MVD was higher than intraplaque MVD in normal arteries and early plaques, but adventitial and intraplaque MVD were similar in advanced plaques. Although microvessel basement membranes were intact, the percentage of thin-walled microvessels was similarly low in normal and atherosclerotic adventitia, in the adventitia and the plaque, and in all plaque types. Intraplaque microvascular endothelial cells (EC) were abnormal, with membrane blebs, intracytoplasmic vacuoles, open EC-EC junctions, and basement membrane detachment. Leukocyte infiltration was frequently observed by EM, and confirmed by CD45RO and CD68 immunohistochemistry. Conclusions: MVD was associated with coronary plaque progression and morphology. Microvessels were thin-walled in normal and atherosclerotic arteries, and the compromised structural integrity of microvascular endothelium may explain the microvascular leakage responsible for intraplaque hemorrhage in advanced human coronary atherosclerosis.

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Angiotensin-converting enzyme (ACE)2 is a recently identified homologue of ACE. As ACE2 inactivates the pro-atherogenic angiotensin II, we hypothesize that ACE2 may play a protective role in atherogenesis. The spatiotemporal localization of ACE2 mRNA and protein in human vasculature and a possible association with atherogenesis were investigated using molecular histology (in situ hybridization, immunohistochemistry). Also, the ACE : ACE2 balance was investigated using enzymatic assays. ACE2 mRNA was expressed in early and advanced human carotid atherosclerotic lesions. In addition, ACE2 protein was present in human veins, non-diseased mammary arteries and atherosclerotic carotid arteries and expressed in endothelial cells, smooth muscle cells and macrophages. Quantitative analysis of immunoreactivity showed that total vessel wall expression of ACE and ACE2 was similar during all stages of atherosclerosis. The observed ACE2 protein was enzymatically active and activity was lower in the stable advanced atherosclerotic lesions, compared to early and ruptured atherosclerotic lesions. These results suggest a differential regulation of ACE2 activity during the progression of atherosclerosis and suggest that this novel molecule of the renin-angiotensin system may play a role in the pathogenesis of atherosclerosis.

Background-Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified. Methods and Results-Microarray analysis was performed on mRNA of aortic arches of ApoE Ϫ/Ϫ mice fed normal chow (NC group) or Western-type diet (WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (Ͼ2ϫ) expressed in at least 1 group compared with a common reference (ApoE

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serpin (serine proteinase inhibitor) superfamily (1-4), controls the plasminogen system at the level of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively). Because PAI-1 is the main physiological inhibitor of t-PA in plasma (5), increased levels of PAI-1 result in a hypofibrinolytic state and are correlated with various vascular disorders such as venous thrombo-embolism, coronary artery disease, myocardial infarction, and atherosclerosis (6 -9). The u-PA-inhibiting effect of PAI-1 has its main physiological implications in processes outside of the circulation (10, 11).PAI-1 is a unique serpin because of its functional and conformational flexibility (12). PAI-1, synthesized as an active molecule, converts spontaneously into a nonreactive, latent form, which can be partially reactivated by denaturing reagents (13). Additionally, a third distinct, noninhibitory form (substrate) is identified that is reactive toward its target proteinases without the formation of a stable complex (14 -16).Elucidation of the three-dimensional structure of active PAI-1 (17, 18) reveals that the N-terminal side of the reactive site loop (extended from P16 to P3Ј and including the bait peptide bond Arg 345 -Met 346 (P1-P1Ј)) is exposed and accessible for the target proteinase. The C-terminal side of the reactive site loop (P4Ј-P13Ј) forms strand s1C in ␤-sheet C.Conversion to the latent state implies the insertion of the N-terminal side of the reactive site loop into ␤-sheet A, the loss of strand s1C from ␤-sheet C, and the formation of an unusual extended loop by the C-terminal side of the reactive site loop, resulting in the distortion of the P1-P1Ј "bait" peptide bond (19). It is hypothesized that th...

Abstract-During the past 6 years, gene expression profiling of atherosclerosis has been used to identify genes and pathways relevant in vascular (patho)physiology. This review discusses some critical issues in the methodology, analysis, and interpretation of the data of gene expression studies that have made use of vascular specimens from animal models and humans. Analysis of gene expression studies has evolved toward the genome-wide expression profiling of large series of individual samples of well-characterized donors. Despite the advances in statistical and bioinformatical analysis of expression data sets, studies have not yet fully exploited the potential of gene expression data sets to obtain novel insights into the molecular mechanisms underlying atherosclerosis. To assess the potential of published expression data, we compared the data of a CC chemokine gene cluster between 18 murine and human gene expression profiling articles. Our analysis revealed that an adequate comparison is mainly hindered by the incompleteness of available data sets. The challenge for future vascular genomic profiling studies will be to further improve the experimental design, statistical, and bioinformatical analysis and to make data sets freely accessible.

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