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.     

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.     

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.     

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.     

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.     

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.     

Difference in the cell proliferation and colony-forming ability of normal human T lymphocytes.

Cell transfer experiments were carried out with phytohaemagglutinin-induced normal human T lymphocyte colonies after 2--10 days of primary colony growth. The cells gave a cloning efficiency of 15% after 2 days of incubation and this decreased to 0.2% with the progressive growth of the colonies. The primary colonies had a proliferative capacity to give about 240 cells after 10 days of incubation, but only contained 0.5 to one cell per colony that could form a new colony. This number of colony-forming cells per colony did not change with an increase in colony size. These results indicate that colonies maintained the same number of colony-forming cells that they started with and that the other cells could proliferate but not form colonies. It is suggested that this ability to distinguish between colony-forming and proliferative T cells may be useful for determining specific deficiencie in either cell type in various diseases.     

The expression of vascular dendritic cells in human atherosclerotic carotid plaques

Atherosclerosis is currently considered a chronic inflammatory disease, and evidence is accumulating for a role of the immune system in the progression of atherosclerosis. Dendritic cells are specialized antigen-presenting cells with the unique ability to initiate a primary immune response to certain antigens by the activation of naive T-lymphocytes. Although dendritic cells are well known to be important in the development of different diseases, studies of vascular dendritic cells in atherosclerosis are rare, and their role is not clearly understood. Therefore, we investigated the immunohistochemical expression of vascular dendritic cells in atherosclerotic plaques. Between April 2003 and December 2005, carotid endarterectomy was performed in 26 consecutive patients, and 27 carotid plaque specimens were analyzed. We investigated the immunohistochemical expression of vascular dendritic cells in human carotid plaques by measuring the signal intensity of fascin-positive cells using an image analyzer. In addition, these immunohistochemical results were related to clinical data. The highest signal intensity of dendritic cells was found in plaque shoulders, and the mean signal intensity of dendritic cells was significantly higher in complicated than in uncomplicated plaques (P = .0029). Moreover, the mean signal intensity of dendritic cells in plaques from symptomatic patients was significantly elevated compared with plaques from asymptomatic patients (P = .0004). Although atherosclerotic plaque instability is determined by multiple factors, the immune and inflammatory pathways play a particularly important role. Dendritic cells play a role in atherosclerosis, and the present study suggests that the expression of dendritic cells in human carotid arteries may be strongly associated with the occurrence of ischemic stroke.     

Active targeting of early and mid-stage atherosclerotic plaques using self-assembled peptide amphiphile micelles

Inflammatory cell adhesion molecules expressed by endothelial cells on the luminal surface of atherosclerotic plaques, such as vascular cell adhesion molecule-1 (VCAM-1), provide a rational target for diagnostic and therapeutic delivery vehicles. Therefore, the potential of using spherical, self-assembled micelles synthesized from VCAM-1 targeted peptide amphiphile molecules was examined for the ability to specifically bind to both early and mid-stage atherosclerotic plaques. In vitro, cells incubated with VCAM-1 targeted and dye-labeled micelles show enhanced fluorescence signal as compared to cells incubated with a PEG micelle control. In vivo, VCAM-1 targeted and Cy7-labeled peptide amphiphile micelles were shown to specifically accumulate at atherosclerotic plaques in both early and mid-stage ApoE −/− mice through co-localization of Cy7 signal with anti-VCAM-1 antibody staining in fixed tissue. No specific accumulation was observed with a PEG micelle control. Histological analysis of excised tissue provided evidence for the in vivo biocompatibility of these micelle formulations as no tissue damage was observed. These results demonstrate that VCAM-1 targeted micelles have potential as a platform for targeted drug delivery to multiple stages of atherosclerotic plaque formation due to their established specificity and safety.     

Smooth muscle homeostasis in human atherosclerotic plaques through interleukin 15 signalling

Interleukin (IL)-15 is a cytokine that has a broad tissue distribution and is important in maintaining homeostasis of cells and stability of tissues. When II-15 is also expressed by vascular smooth muscle cells (SMC), which are the dominant type of cells in most atherosclerotic plaques, it could be important in maintaining plaque tissue integrity and hence resistance of plaques towards development of clinically relevant complications such as plaque rupture and thrombosis. In this study, IL-15 and IL-15Rα in vitro expression by coronary artery SMC was investigated using RT -PCR and FACS analysis. Immunohistochemistry was used to study in situ expression of IL-15 and IL-15R by SMC of human carotid artery atherosclerotic plaques. Multiplex ligand-dependent probe amplification (MLPA) was used to investigate the mRNA expression of 40 pro- and anti inflammatory genes after stimulating coronary SMC with IL-15. We found that atherosclerotic SMC express both IL-15 and its receptor IL-15R, and TNF-γ and TNF-α enhance IL-15R expression in cultured SMC. MLPA studies on SMC revealed enhanced expression of PDGF beta mRNA after IL15 stimulation. In conclusion, our data suggest that IL-15 may contribute to atherosclerotic plaque integrity by stimulation of smooth muscle cells, probably in a PDGF dependent fashion.     

Apolipoprotein A-1-derived amyloid in atherosclerotic plaques of the human aorta

Previous studies have shown that the amyloid localized to the aortic intima may be a biochemical entity different from other forms of localized amyloid. The amyloid fibril protein in one patient studied consisted of an N-terminal fragment of apolipoprotein A-1 (apo A-1). Since this patient was later shown to carry a missense mutation in the apo A-1 gene, leading to a deletion at position 107 of the mature protein, the question remained whether wild-type apo A-1 is amyloidogenic. In autopsy specimens from the thoracic aorta from 69 individuals, intimal atherosclerotic plaque-related amyloid was present in 11 cases (16%) and amyloid outside plaques in 37 cases (54%). The immunoreactivity of amyloid localized to the aortic intima was evaluated with the aid of antisera against N-terminal segments of apo A-1. The amyloid in association with atherosclerotic plaques was positively labelled by immunohistochemistry. The amyloid fibril protein from one patient, previously shown not to carry any mutation in the apo A-1 gene, was purified and shown by amino acid sequence analysis to be of apo A-1 nature. The result shows that wild-type apo A-1 is amyloidogenic and gives rise to a common localized form of amyloid associated with atherosclerosis.     

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.