Expression of intercellular adhesion molecule-1 in atherosclerotic plaques.

Immunohistochemistry of human atherosclerotic arteries demonstrates expression of the intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, macrophages, and smooth muscle cells of the plaques. Normal arterial endothelial cells and intimal smooth muscle outside plaques give weaker or negative reactions; these differ from the strong endothelial expression in small vessels. Quantitative color-image analysis of the endothelial layer shows increased expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques. Endothelial expression of ICAM-1 may be involved in the recruitment of monocytes to the lesion, as suggested by its role in the entry of leukocytes, including monocytes, into foci of inflammation. Collaboration with other mechanisms, particularly chemoattractant factors, may be important for this effect. ICAM-1 enhanced monocyte recruitment is a potential mechanism for the growth of an atherosclerotic plaque.     

Angiogenesis in human coronary atherosclerotic plaques.

Neovascularization in the walls of coronary arteries is associated with the presence of atherosclerotic plaque. The mechanisms responsible for the formation of these intraplaque microvessels are not understood. The purpose of this study is to examine the prevalence of endothelial cell replication in plaque microvessels. Two hundred and one primary and restenotic coronary atherectomy specimens were analyzed for the presence of microvessels and proliferation as reflected by positive immunolabeling for Ulex agglutinin and the proliferating cell nuclear antigen, respectively. In primary but not restenotic specimens, proliferation of any cell type was associated with the detection of microvessels on the same slide. However, intraplaque microvessels were more commonly found in restenotic compared to primary specimens (P = 0.004). Twelve highly vascularized specimens with evidence of replication were subjected to detailed histomorphological and quantitative image analyses. At 200 x, the most vascular optical field of each slide was identified and consistently included plaque macrophages. Total slide endothelial cell replication indices for these specimens varied, but in some instances were remarkably elevated (eg, 43.5%). The role of intraplaque angiogenesis may be analogous to that of tumor or wound angiogenesis and be important in development and progression of coronary artery lesions and restenosis.     

Molecular interactions in human atherosclerotic plaques.

Most plasma proteins appear to be present in intima at concentrations that are a linear function of molecular weight and concentration in the plasma. Thus low density lipoprotein (LDL) (molecular weight, 2 X 10(6)) has the greatest retention relative to its plasma concentration, whereas the relative retention of albumin is only 15% of the relative retention of LDL. This gives rise to the concept that "whole plasma" crosses endothelium, and the steady state concentrations reflect rates of egress of the macromolecules, which in turn depend on molecular sieving. Fibrinogen is a major plasma protein in intima in addition to LDL and albumin, and there are also substantial amounts of the protease inhibitors alpha2-macroglobulin and alpha1-antitrypsin. Intima also contains insoluble derivatives of plasma--extracellular cholesterol, both free and esterified, and fibrin. The balances of intact LDL/"deposited" cholesterol and of fibrinogen/fibrin are closely linked with intimal morphology. Fibrinogen and electrophoretically mobile LDL are increased about threefold in gelatinous lesions, whereas there are only slight rises in fibrin and deposited cholesterol. In the deep layers of fibrous plaques, fibrin is increased fivefold and cholesterol up to thirtyfold. In these lipid-rich layers, LDL is rapidly lost on incubation of tissue samples, but in some gelatinous lesions it first increases and only decreases on longer incubation, suggesting release of a previously immobilized lipoprotein fraction. This immobilized lipoprotein was investigated by subjecting tissue samples to immunoelectrophoresis to remove mobile LDL and tissue enzymes, followed by treatment of the tissue with enzyme and measurement of the lipoprotein released on fresh immunoelectrophoresis plates. Plasmin or a crude collagenase released large amounts of lipoprotein from samples of amorphous atheroma lipid. For all samples the amount of lipoprotein released was highly correlated with the accumulation of deposited cholesterol, suggesting that immobilization of LDL may be an intermediate step in the irreversible deposition of extracellular cholesterol. Plasmin is highly effective in releasing immobilized lipoprotein, and the concentration of immobilized lipoprotein is significantly correlated with the concentration of insoluble fibrin, suggesting that the lipoprotein may in some way be immobilized by fibrin.     

Clonal architecture of normal and atherosclerotic aorta: implications for atherogenesis and vascular development.

X chromosome inactivation studies indicate that smooth muscle cells in human atherosclerosis are monoclonal. Monoclonality could arise either by 1) proliferation of a single cell in the adult intima, eg, by selection or mutation, or 2) proliferation of many cells within a large, pre-existing clonal patch that formed during development. To determine whether clonal expansion occurs concomitantly with plaque growth or as part of normal development, X chromosome inactivation patterns were mapped in microdissected samples of aortic smooth muscle, using the human androgen receptor locus as a marker. As expected, 43% of plaque samples were skewed toward one X chromosome, indicating a monoclonal population. Surprisingly, 25% of normal medial samples and 31% of diffuse intimal thickening samples also were skewed toward one X chromosome, indicating a relatively large patch size. Furthermore, 30% of diffuse intimal thickening and 22% of medial samples showed contiguous regions of 4 mm skewed to the same allele, showing that patch length often exceeded 4 mm. Intima and overlying media typically were skewed to the same allele (73% concordance), suggesting common cells of origin. Because patch size is large in normal arteries, X-inactivation analysis cannot discriminate between a monoclonal and a polyclonal origin of plaque smooth muscle cells. We propose that human arteries grow by expanding coherent smooth muscle clones, with little mixing of adjacent clones. Determining whether plaques arise by clonal expansion will require other approaches, such as analysis of somatic mutations; the finding of large X-inactivation patches raises the possibility that plaques arise from a pre-existing (developmental) clone.     

Hyperspectral imaging of atherosclerotic plaques in vitro

Vulnerable plaques constitute a risk for serious heart problems, and are difficult to identify using existing methods. Hyperspectral imaging combines spectral- and spatial information, providing new possibilities for precise optical characterization of atherosclerotic lesions. Hyperspectral data were collected from excised aorta samples (n = 11) using both white-light and ultraviolet illumination. Single lesions (n = 42) were chosen for further investigation, and classified according to histological findings. The corresponding hyperspectral images were characterized using statistical image analysis tools (minimum noise fraction, K-means clustering, principal component analysis) and evaluation of reflectance/fluorescence spectra. Image analysis combined with histology revealed the complexity and heterogeneity of aortic plaques. Plaque features such as lipids and calcifications could be identified from the hyperspectral images. Most of the advanced lesions had a central region surrounded by an outer rim or shoulder-region of the plaque, which is considered a weak spot in vulnerable lesions. These features could be identified in both the white-light and fluorescence data. Hyperspectral imaging was shown to be a promising tool for detection and characterization of advanced atherosclerotic plaques in vitro. Hyperspectral imaging provides more diagnostic information about the heterogeneity of the lesions than conventional single point spectroscopic measurements.     

Polyclonal origin of T lymphocytes in human atherosclerotic plaques.

The human atherosclerotic plaque contains large numbers of T lymphocytes and macrophages; this indicates that immune and inflammatory mechanisms may be important factors in the pathogenesis of atherosclerosis. A significant proportion of the T lymphocytes express activation markers, which suggests that they may be stimulated by local antigens, proliferate in response to such antigens, and secrete lymphokines that could serve as paracrine factors in the arterial tissue. However, it is not known, whether plaque T lymphocytes constitute a homogeneous population of clonally proliferating cells or represent a heterogeneous mixture of cells with different immunologic specificities. Clonally-derived T lymphocytes can be identified since they carry identical T cell antigen receptor (TCR) genes. These genes rearrange during T cell ontogeny resulting in TCR genes and TCR proteins that are unique for each T cell clone. We have now employed TCR gene analysis to T lymphocytes derived from atherosclerotic plaques in order to determine their clonal composition. T lymphocytes were isolated from carotid endarterectomy samples and cultured after limiting dilution cell cloning. TCR genes were analyzed by Southern blotting using probes for the TCR gamma (J gamma 2) and TCR beta (C beta 1) genes. TCR gene rearrangement patterns were totally heterogeneous, indicating that plaque T lymphocytes constitute a polyclonal population of cells. This suggests that the T cells are either recruited to the plaque in an activated state or activated locally by mechanisms that do not lead to clonal proliferation.     

HMGA1 proteins in human atherosclerotic plaques

One of the major characteristics of atherosclerosis is the migration of smooth muscle cells (SMC) from the tunica media to the intima, caused by alterations in the environment, e.g. mechanical, chemical, or immunologic injuries of the arterial walls. A group of molecules that may act as a main regulator of SMC phenotype switching is formed by the so-called HMGA1 high-mobility group proteins. One target gene of the HMGA1 protein, playing a major role in the development of atherosclerotic lesions, is CD44. The expression of CD44 is regulated by IL-1beta, but binding of HMGA1 potentiates the transactivation of the CD44 promoter. In this study, the HMGA1 expression of human atherosclerotic plaque samples was examined. Compared to the non-active components, all major components of the well-developed atherosclerotic plaques showed strong positivity of the high-mobility group protein HMGA1 in their activated areas, e.g. neointimal SMCs, macrophages, newly built blood vessels. This report is the first to describe HMGA1 as one of the first mediators in the development of human atherosclerotic plaques.     

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.     

Implications of the monoclonal character of human atherosclerotic plaques.

Evidence for the monoclonal nature of human atherosclerotic plaques is reviewed. Eighty percent of discrete raised atherosclerotic plaques are of single phenotype. Interpretations alternative to single-cell orgin, based on patch size, selection due to linked genes, or repetitive sampling do not seem to explain the apparent monoclonality. Search for carriers in serum of mutagens, such as may be present in cigarette smoke, show them to be the lipoproteins, and the presence and possible role of intrinsic mutagens, e.g., cholesterol-alpha-oxide, are presented. The possible role for other factors implied by the monoclonal hypothesis, e.g., the mechanism by which estrogen therapy may increase coronary attacks, is discussed.     

Bradykinin and angiotensin immunoreactivity in atherosclerotic plaques of human aortas.

Atherosclerotic human aortas were dissected post-mortem. Extracts of plaques and adjacent intima were examined for the presence of immunoreactive angiotensin and bradykinin by radioimmunoassays. All plaques and intimas contained bradykinin immunoreactivity with the two regions having roughly equal amounts. The immunoreactivity was susceptible to proteolysis and passed through a filter that excluded molecules heavier than 10,000 daltons. Angiotensin immunoreactivity was found in 10 plaques and 14 intimal specimens in roughly equal amounts. Bioassays on rat uteri suggested the presence of other smooth muscle activators in the extracts. The amounts of vasoactive peptides bore no relationship to the patient's age, degree of atherosclerosis, time between death and dissection, or amount of lipid in the plaques. Thus, while vasoactive peptides might be involved in atherosclerosis, we could find no evidence of their selective accumulation by gross atherosclerotic lesions.     

Role of monocytes in atherogenesis

This review focuses on the role of monocytes in the early phase of atherogenesis, before foam cell formation. An emerging consensus underscores the importance of the cellular inflammatory system in atherogenesis. Initiation of the process apparently hinges on accumulating low-density lipoproteins (LDL) undergoing oxidation and glycation, providing stimuli for the release of monocyte attracting chemokines and for the upregulation of endothelial adhesive molecules. These conditions favor monocyte transmigration to the intima, where chemically modified, aggregated, or proteoglycan- or antibody-complexed LDL may be endocytotically internalized via scavenger receptors present on the emergent macrophage surface. The differentiating monocytes in concert with T lymphocytes exert a modulating effect on lipoproteins. These events propagate a series of reactions entailing generation of lipid peroxides and expression of chemokines, adhesion molecules, cytokines, and growth factors, thereby sustaining an ongoing inflammatory process leading ultimately to lesion formation. New data emerging from studies using transgenic animals, notably mice, have provided novel insights into many of the cellular interactions and signaling mechanisms involving monocytes/macrophages in the atherogenic processes. A number of these studies, focusing on mechanisms for monocyte activation and the roles of adhesive molecules, chemokines, cytokines and growth factors, are addressed in this review.     

Apolipoprotein A1-derived amyloid in human aortic atherosclerotic plaques.

Amyloid deposits in the aortic intima are very common in association with atherosclerosis and aging. In the present study, a major fibril protein purified from amyloid present in human atherosclerotic plaques was shown to be a 69-amino acid N-terminal fragment of apolipoprotein AI. Although senile form of localized apolipoprotein AI-derived amyloidosis has recently been documented in pulmonary vessels of dogs, this is the first example of a localized human amyloid derived from this apolipoprotein.     

HIV-1 Tat increases the adhesion of monocytes and T-cells to the endothelium in vitro and in vivo: implications for AIDS-associated vasculopathy

HIV-1-infected patients exhibit severe damages of the aortic endothelium, develop angioproliferative lesions such as Kaposi's sarcoma (KS), and have an increased risk of cardiovascular diseases and atherosclerosis. An increased adhesion of leukocytes to the endothelium is a common pathogenic parameter of AIDS-associated vascular diseases. Here we show that the HIV-1 Tat protein, a regulatory protein of HIV-1 released by infected cells, and TNF-alpha, a cytokine increased in sera and tissues of HIV-1-infected patients, activate synergistically the adhesion of leukocytes to endothelial cells both in vitro and in vivo. This effect is selectively mediated by HIV-1 Tat, since HIV-1 Nef, another HIV-1 regulatory protein, and the HIV-1 envelope protein gp41, had no effect. In vitro adhesion assays with PBMC and quantitative cell type analysis of adherent cells by FACS demonstrated that HIV-1 Tat selectively activates the adhesion of T-cells and monocytes but not of B-cells. Intravital microscopic studies in mice confirmed the synergistic activity of HIV-1 Tat and TNF-alpha on leukocyte adhesion to the endothelium in vivo. These data indicate that HIV-1 Tat in cooperation with TNF-alpha may contribute to the vascular damage and cardiovascular diseases observed in AIDS patients but also to the prominent extravasation of T-cells and monocytes which is a key process in the formation and progression of KS lesions.     

Electron microscopic features of lipoprotein-glycosaminoglycan complexes from human atherosclerotic plaques.

Lipoprotein-glycosaminoglycan (GAG) complexes were isolated from fibrous plaque lesions of human aortas and examined by electron microscopy. After fractionation by gel chromatography and ultracentrifugation, both very low density lopoprotein-GAG and low density lopoprotein-GAG complexes showed particles which were mainly 1,000 to 2,000 A in diameter. Occasional large aggregations 3,000 to 10,000 A in diameter were seen after gel filtration and in the very low density lipoprotein fraction of complexes. In general, the complexes were larger than serum very low density lipoprotein and low density lipoprotein, although serum very low density lipoprotein particles ranged from 250 to 2,000 A. In vitro low density lipoprotein-heparin complexes consisted of spherical particles generally 1,000 to 2,000 A in diameter formed by the aggregation and coalescing of smaller low density liproprotein particles in the presence of heparin and Ca2+. These observations support a concept that GAG of the aortic intimal "ground substance" sequester certain serum lipoproteins in a manner similar to in vitro complexing of lipoproteins and GAG in the presence of Ca2+.     

Lactate dehydrogenase (LDH) isozymes of human atherosclerotic plaques.

We have been searching for additional markers to explore differences between the smooth muscle cells of human atherosclerotic fibrous plaques and their putative cells of origin in an aortic media and intima. Lactate dehydrogenase (LDH) isozyme analysis was performed on samples of human fibrous plaques selected by gross and microscopic criteria, and significant shifts in M4/M2H2 LDH isozyme ratios were found, relative to the underlying media and adjacent intima specimens. These changes are in the same direction seen in neoplastic tissues in vitro and in vivo and are probably not secondary to positional factors, inflammatory changes, or degenerative changes. The significance of these findings in relation to the monoclonal hypothesis of atherosclerosis is discussed.     

Heterogeneity of human macrophages in culture and in atherosclerotic plaques

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.     

Origins of human atherosclerotic plaques. The role of altered gene expression

The dramatic rise and equally dramatic fall in the mortality from coronary heart disease in the 20th century is only partly explained. This article reviews the development of our ideas concerning possible pathways other than lipids that might play a role in the development of human atherosclerosis alone or in combination with one or more of the usually considered risk factors. In some instances, such as that of cigarette smoking, the proposed concept regarding genetic alterations in vascular smooth muscle suggests a mechanism for development of at least some of the lesions. Recent studies have shown that an aberration in platelet-derived growth factor gene expression is unlikely to be a factor in proliferation of smooth-muscle cells. Aberrant expression of other oncogenes or some as yet unknown virus remain as possible explanations of some of the incidence of atherosclerosis and its consequent coronary heart disease.     

MnSOD expression in human atherosclerotic plaques: an immunohistochemical and ultrastructural study

BackgroundOver the last decades, the role of oxidative stress in atherosclerosis has become well established, and considerable effort has been made to understand the mechanism of action of free radicals within the cardiovascular system. Conversely, relatively little attention has been directed towards the characterization of the antioxidant status of the arterial wall under disease state. Among the antioxidant enzymes, the manganese-dependent and mitochondria-specific isoform of SOD (MnSOD) represents the first line of defense against superoxide radicals attack. To date, the pathological significance of MnSOD in atherosclerosis is still unclear with conflicting data published.MethodsIn the present study, we used immunohistochemical techniques at the light and electron microscopy level in combination with biochemical assays to localize and characterize the activity and expression profiles of MnSOD in healthy and atherosclerotic human aorta.ResultsMnSOD has been found to be highly expressed in the atherosclerotic plaques where specifically localized to the foam cells of the lipid-rich regions but not to other (nonfoamy) cell types. No ultrastructural evidence of apoptosis, such as chromatin condensation and membrane blebbing, has been observed in MnSOD-expressing cells. The up-regulation of MnSOD at the protein level has been associated with a parallel, significant increase of its catalytic activity.ConclusionsOur data demonstrate that MnSOD is not negatively regulated by the prooxidative and proinflammatory environment of the plaque and evidence a regional and cellular selectivity of MnSOD protein expression under disease state. We suggest that MnSOD induction might represent a protective response against the cytotoxic effect of oxidized low-density lipoprotein.     

ICAM-1-mediated adhesion of peripheral blood monocytes to the maternal surface of placental syncytiotrophoblasts: implications for placental villitis.

Accumulation of maternal monocytes in the villous/intervillous space (villitis) is associated with increased risk of perinatal morbidity and mortality and may initiate in utero transmission of cell-associated infectious agents such cytomegalovirus and HIV-1. We have developed an in vitro model of trophoblast syncytialization and have investigated the adhesive interactions between this tissue and peripheral blood monocytes. We show that monocytes strongly adhere to cultured syncytiotrophoblasts (STs) and that treatment with the inflammatory cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin-1 alpha greatly increase the number bound. Pretreatment of STs with these cytokines upregulated apical expression of intercellular cell adhesion molecule (ICAM)-1 but not E-or L-selection, ICAM-2 or -3, or various integrins. ICAM-1 expression was cytokine concentration dependent, significantly increased within 6 hours of treatment, peaked after 24 hours, and remained undiminished for 48 hours after cytokine removal from the cultures. Adhesion of monocytes to STs was inhibited > 80% by antibody to ICAM-1 or its cognate ligand LFA-1. ICAM-1 was detected immunohistochemically only in rare foci on intact term placental villi. These results suggest that villous trophoblast expression of ICAM-1 occurs only during an immune inflammatory reaction and that aberrant expression of this molecule may be an important pathological feature in those immunoinflammatory disorders of the placenta characterized by an excessive accumulation of leukocytes in the intervillous/villous space such as spontaneous abortion, perinatal hematogenous infections, and villitis of unknown etiology.