Thin-walled microvessels in human coronary atherosclerotic plaques show incomplete endothelial junctions relevance of compromised structural integrity for intraplaque microvascular leakage.

OBJECTIVES: This study sought 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 are 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. RESULTS: The MVD was increased in advanced plaques compared with 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 (ECs) were abnormal, with membrane blebs, intracytoplasmic vacuoles, open EC-EC junctions, and basement membrane detachment. Leukocyte infiltration was frequently observed by electron microscopy, and confirmed by CD45RO and CD68 immunohistochemistry. CONCLUSIONS: The 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.

Vasculin, a novel vascular protein differentially expressed in human atherogenesis.

Recent suppressive subtractive hybridization analysis on human atherosclerotic plaque-derived RNA revealed genes upregulated in plaques with a thrombus versus stable plaques. Clone SSH6, containing part of a putative open reading frame of an unknown protein, was further investigated. Full-length cDNA, coding for a 473-amino acid (aa) protein, was identified in a vascular smooth muscle cell (SMC) cDNA library. Bioinformatics suggested the presence of multiple SSH6 variants due to alternative splicing of exon 3. Multiple-tissue Northern blot analysis demonstrated a differential expression pattern of these variants, as a ubiquitously expressed SSH6 mRNA missing exon 3, was detected apart from a putative vascular SMC-specific form containing exon 3. Western blot analysis indicated a ubiquitous 35-kDa protein (SSH6-beta), in addition to a 45-kDa protein (vasculin), detected in the vascular wall and in plasma. Analysis of arteries displaying various stages of atherosclerosis indicated that the vasculin/SSH6-beta ratio increases throughout atherogenesis. Immunohistochemical analysis demonstrated cytoplasmic expression of SSH6 gene products in macrophages, endothelial cells, and SMCs. In summary, we identified a novel mRNA/protein, vasculin, in the arterial wall and plasma. The regulated expression of vasculin in plaques suggests a role in atherogenesis. Moreover, its presence in plasma opens perspectives for vasculin as a marker for atherosclerosis.