Vascular-associated lymphoid tissue in swine (Sus scrofa)

Focal accumulations of mononuclear cells in the arterial wall of healthy humans at predilection sites for atherosclerotic lesions have been described as 'vascular-associated lymphoid tissue' (VALT). Here we investigated whether pigs (Sus scrofa), a commonly used animal model for studying cardiovascular disease, have VALT. Samples of major arteries were collected from 10 conventional crossbred pigs (age, 2 to 24 mo) and processed for routine light microscopy, immunohistochemistry, and immunofluorescence. Single or small aggregates of mononuclear cells were noted in the intima and occasionally the inner portion of the tunica media and adventitia at branching sites. The infiltrating cells were primarily CD3+CD4+ T cells, with some macrophages. No CD8+ T cells were present. Infiltrating leukocytes and overlying endothelial cells frequently expressed major histocompatibility class II molecules. Two Ossabaw pigs on low-fat diet had similar leukocytic aggregates at locations where animals of the same breed but fed a high-fat and high-cholesterol diet developed atherosclerotic lesions. Further, the densities of CD3+ T lymphocytes and in these areas were decreased in 2 sedentary and 2 exercised Ossabaw pigs on an atherogenic diet compared with conventional crossbred and Ossabaw pigs on a normal diet. This study shows that focal aggregates of lymphocytes occur in the vasculature of pigs at locations predisposed to development of atherosclerotic lesions. These cellular aggregates are similar to the structures described as VALT in human arteries and reinforce the value of the pig as a model for the study of human cardiovascular disease.     

Endogenous ferritin protects cells with iron-laden lysosomes against oxidative stress

Previous studies have shown that a variety of mammalian cell types, including macrophages, contain small amounts of redox-active iron in their lysosomes. Increases in the level of this iron pool predispose the cell to oxidative stress. Limiting the availability of intralysosomal redox-active iron could therefore represent potential cytoprotection for cells under oxidative stress.

In the present study we have shown that an initial 6 h exposure of J774 macrophages to 30 μM iron, added to the culture medium as FeCl₃, increased the lysosomal iron content and their sensitivity to H₂O₂-induced (0.25 mM for 30 min) oxidative stress. Over time (24-72 h), however, the cells were desensitized to the cytotoxic effects of H₂O₂; most likely as a consequence of both lysosomal iron exocytosis and of ferritin synthesis (demonstrated by atomic absorption spectrophotometry, autometallography, and immunohistochemistry). When the cells were exposed to a second dose of iron, their lysosomal content of iron increased again but the cells became no further sensitized to the cytotoxic effects of H₂O₂. Using the lysosomotropic weak base, acridine orange, we demonstrated that after the second exposure to iron and H₂O₂, lysosomes remained intact and were no different from control cells which were exposed to H₂O₂ but not iron.

These data suggest that the initial induction of ferritin synthesis leads to enrichment of lysosomes with ferritin via autophagocytosis. This limits the redox-availability of intralysosomal iron and, in turn, decreases the cells' sensitivity to oxidative stress. These in vitro observations could also explain why cells under pathological conditions, such as haemochromatosis, are apparently able to withstand high iron concentrations for some time in vivo.     

Transport of siderotic Kupffer cells to the lung and bronchial excretion of iron in experimental iron-overload.

In 3,5,5-trimethylhexanoylferrocene-induced iron overload of rats, three different types of iron-loaded macrophages and derivatives thereof were found in the lungs. On the basis of their localization and of their pattern of iron load it was possible to distinguish: (1) Resident macrophages, showing an alveolar localization and a moderate iron content represented by lysosomal ferritin and haemosiderin. (2) Liver-derived macrophages and giant cells, as well as fragments of them. They showed an exclusive localization in capillaries and alveolar septa, and high concentrations of free ferritin molecules in addition to polymorphous ferritin- and haemosiderin-containing siderosomes. (3) Monocyte-derived intravascular pulmonary macrophages. Initially, they contained iron only as lysosomal aggregates of ferritin and haemosiderin, as a result of phagocytosis of liver-derived macrophageal cell fragments. Later in iron overload, they also showed free ferritin molecules in the cytosol and fused intrapulmonarily to giant cells. The resident as well as the liver-derived siderotic pulmonary macrophages provide a way for iron excretion through the airways.     

Medial and adventitial macrophages are associated with expansive atherosclerotic remodeling in rabbit femoral artery

Expansive vascular remodeling is considered a feature of vulnerable plaques. Although inflammation is upregulated in the media and adventitia of atherosclerotic lesions, its contribution to expansive remodeling is unclear. We investigated this issue in injured femoral arteries of normo- and hyperlipidemic rabbits fed with a conventional (CD group; n=20) or a 0.5% cholesterol (ChD group; n=20) diet. Four weeks after balloon injury of the femoral arteries, we examined vascular wall alterations, localization of macrophages and matrix metalloproteases (MMP)-1, -2, -9, and extracellular matrix. Neointimal formation with luminal stenosis was evident in both groups, while expansive remodeling was observed only in the ChD group. Areas immunopositive for macrophages, MMP-1, -2 and -9 were larger not only in the neointima, but also in the media and/or adventitia in the injured arterial walls of the ChD, than in the CD group. Areas containing smooth muscle cells (SMCs), elastin and collagen were smaller in the injured arterial walls of the ChD group. MMP-1, -2 and -9 were mainly localized in infiltrating macrophages. MMP-2 was also found in SMCs and adventitial fibroblasts. Vasa vasorum density was significantly increased in injured arteries of ChD group than in those of CD group. These results suggest that macrophages in the media and adventitia play an important role in expansive atherosclerotic remodeling via extracellular matrix degradation and SMC reduction.     

Overexpression of transferrin receptor and ferritin related to clinical symptoms and destabilization of human carotid plaques

Accumulation of tissue iron has been implicated in development of atherosclerotic lesions mainly because of increased iron-catalyzed oxidative injury. However, it remains unknown whether cellular iron import and storage in human atheroma are related to human atheroma development. We found that transferrin receptor 1 (TfR1), a major iron importer, is highly expressed in foamy macrophages and some smooth muscle cells in intimal lesions of human carotid atheroma, mainly in cytoplasmic accumulation patterns. In 52 human carotid atherosclerotic lesions, TfR1 expression was positively correlated with macrophage infiltration, ectopic lysosomal cathepsin L, and ferritin expression. Highly expressed TfR1 and ferritin in CD68-positive macrophages were significantly associated with development and severity of human carotid plaques, smoking, and patient's symptoms. The findings suggest that pathologic macrophage iron metabolism may contribute to vulnerability of human atheroma, established risk factors, and their clinical symptoms. The cytoplasmic overexpression of TfR1 may be the result of lysosomal dysfunction and ectopic accumulation of lysosomal cathepsin L caused by atheroma-relevant lipids in atherogenesis.     

Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells

Summary

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.     

A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima

Coronary atherosclerosis still represents the major cause of mortality in western societies. Initiation of atherosclerosis occurs within the intima, where major histological and molecular changes are produced during pathogenesis. So far, proteomic analysis of the atherome plaque has been mainly tackled by the analysis of the entire tissue, which may be a challenging approach because of the great complexity of this sample in terms of layers and cell type composition. Based on this, we aimed to study the intimal proteome from the human atherosclerotic coronary artery. For this purpose, we analyzed the intimal layer from human atherosclerotic coronaries, which were isolated by laser microdissection, and compared with those from preatherosclerotic coronary and radial arteries, using a two-dimensional Differential-In-Gel-Electrophoresis (DIGE) approach. Results have pointed out 13 proteins to be altered (seven up-regulated and six down-regulated), which are implicated in the migrative capacity of vascular smooth muscle cells, extracellular matrix composition, coagulation, apoptosis, heat shock response, and intraplaque hemorrhage deposition. Among these, three proteins (annexin 4, myosin regulatory light 2, smooth muscle isoform, and ferritin light chain) constitute novel atherosclerotic coronary intima proteins, because they were not previously identified at this human coronary layer. For this reason, these novel proteins were validated by immunohistochemistry, together with hemoglobin and vimentin, in an independent cohort of arteries.     

Numbers of cells and cell proliferation in intima of different human arteries

Increased cell proliferation in early atherosclerotic lesions is recognized as an essential event of atherogenesis but the levels of cell proliferation in different stages of atherosclerotic plague formation in different types of human large arteries are still insufficiently studied. In the present work, we studied intima thickness and proliferation of newly "infiltrates" hematogenous and resident cells in atherosclerotic lesions of the carotid and coronary arteries and compared these parameters with those in the aorta, reported by us in earlier publication. Analysis of intima thickness and proliferation in grossly unaffected intima and in different types pf atherosclerotic lesions (initial lesions, fatty streaks, lipofibrous, plaques, and fibrous plaque) revealed that although there were similar tendencies in the change of the infiltration levels of hematogenous cells and proliferation in different types of arteries, there were significant quantitative differences between different types of arteries. Hematogenous cells in lipofibrous plaques of the coronary and carotid arteries were found to account for a third and almost for a half of the total cell population, respectively, while atherosclerotic lesions in the aorta, as it has been shown by us earlier, to contain no more than 15% ofhematogenous cells. This suggests that the contribution of hematogenous cells to the development of atherosclerosis in the carotid and the coronary artery appears to be more significant than that in the aorta. Despite the differences in numbers of accumulating hematogenous cells in the intima, a similar "bell-shaped" dependence of cell numbers on the lesion type, involved in the following sequence: unaffected intima-initial lesions-fatty streaks-lipofibrous plaques-fibrous plaques, was detected in the coronary and carotid arteries. The visualization of proliferating cells (PCNA-positive) in atherosclerotic and unaffected zones of the coronary and carotid arteries revealed similar patterns. The maximum numbers of PCNA-positive resident cells were identified in lipofibrous plaques. The changes in the total cell numbers were accompanied by the changes in the numbers of both proliferating resident cells and proliferating hematogenous cells.     

Plasminogen-activator of the arterial wall in occluded arteries

The plasminogen activator in 117 specimen of 20 coronary and 29 pulmonary arteries occluded completely by thrombi or emboli within the adventitia and intima was studied using TODD's histochemical method. 39 cadavers were used, 1--18 hours post mortem from subjects aged from 45 to 88 years. In occluded arteries both coronary and pulmonary the plasminogen activator activity was decreased in comparison with normal and atherosclerotic patients. In coronary and pulmonary arterial thrombi a low grade focal activity of plasminogen activator was detected. It is assumed that the decrease of plasminogen activator in the occluded human arterial wall is due to the impaired oxygen supply of the vessel wall and to the consumption of the plasminogen activator for thrombus lysis. These mechanisms are likely to influence the plasminogen activator for a certain and prolonged time, since there were no changes of fibrinolysis within the vessel wall of arteria carotis in rats where an acute thrombosis was elicited by means of an electric current.     

The number of cells and the cell proliferation in intima of various human arteries

Increased cell proliferation at the early stages of an atherosclerotic lesion is considered an important stage of development of this pathology, but the degree of the proliferation at various stages of formation of atherosclerotic plaque in various human large arteries so far has been studied insufficiently. In the present work, we studied the thickness of intima and proliferation of the newly “infiltrated” hematogenic and resident cells in atherosclerotic lesion of carotid and coronary arteries; a comparison is also made with similar results obtained on the aorta and presented in our earlier publications. Analysis of thickness of intima and of proliferation in normal intima and at various stages of atherosclerotic lesion (initial stages, lipid strips, lipofibrous plaques, fibrous plaques) showed that, in spite of similar tendencies toward changing the level of infiltration of hematogenic cells and proliferation in various types of arteries, there exist significant quantitative differences between various types of arteries. Thus, it is found that hematogenic cells in lipofibrous plaques of coronary and carotid arteries account for one-third and almost half of the total cell population, respectively, whereas the atherosclerosis-lesioned sites of the aorta, as we showed earlier, contain no more than 15% of hematogenic cells. This allows one to think that the contribution of hemopoietic cells to development of atherosclerosis in carotid and coronary arteries is greater than in the aorta. In spite of differences in the number of the hemopoietic cells accumulating in intima, an analogous bell-shaped dependence of the number of cells on the type of lesion (in the sequence normal intima-initial stages of pathology-lipid strips-lipofibrous plaques-fibrous plaques) was shown for coronary and carotid arteries. Visualization of proliferating (PCNA-positive) cells in atherosclerosed and normal (unchanged) zones of coronary and carotid arteries revealed a similar picture. The maximum number of PCNA-positive resident cells was found in lipofibrous plaques. Changes of the total number of cells were accompanied by a change in the number of proliferating resident and proliferating hematogenic cells.     

Pericoronary Adipose Tissue as Storage and Supply Site for Oxidized Low-Density Lipoprotein in Human Coronary Plaques

Objectives

It is generally believed that low-density lipoprotein enters the vascular wall from its lumen and oxidized (oxLDL), after which it plays an important role in atherosclerosis. Because voluminous epicardial adipose tissue is a risk factor for coronary events, there is a possibility that the pericoronary adipose tissue (PCAT), which is a part of epicardial adipose tissue, acts as a risk factor by supplying oxLDL to the coronary arterial wall. The present study was performed whether PCAT stores and supplies oxLDL to the coronary wall.

Methods

Localization of oxLDL in PCAT and its relation to plaque morphology were examined by immunohistochemical techniques in 27 epicardial coronary arteries excised from 9 human autopsy cases.

Results

OxLDL deposited in all PCAT of the studied cases. The percent (%) incidence of oxLDL in the intima of 25 normal segment, 19 white plaques, 15 yellow plaques without necrotic core (NC) and 10 yellow plaques with NC, was 32, 84, 93 (p<0.05 vs normal segments and yellow plaques with NC), and 30, respectively. OxLDL deposited either in dotted or diffuse pattern. Double immunohistochemical staining revealed that the dotted oxLDL was that contained in CD68(+)-macrophages. The oxLDL-containing macrophages were observed in the interstitial space but not inside of the vasa vasorum, and they traversed PCAT, adventitia, external and internal elastic laminae, suggesting their migration towards the intima. Diffuse oxLDL deposits were observed in 17 preparations, the majority of which were co-localized with the vasa vasorum in outer or in both inner and outer halves of intima, and rarely in the inner half alone.

Conclusions

The results suggested that PCAT is a supply source of oxLDL to coronary intima and acts as a risk factor for coronary events, that oxLDL increasingly deposits in the intima with plaque growth and decreases after plaque maturation, and therefore molecular therapies targeting the PCAT before plaque growth could be effective in preventing human coronary atherosclerosis.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta: Hypothetical role of ganglioside GM3 in foam cell formation

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle α-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.     

Foam cell death induced by 7beta-hydroxycholesterol is mediated by labile iron-driven oxidative injury: mechanisms underlying induction of ferritin in human atheroma

Human atherosclerotic lesions typically contain large amounts of ferritin associated with apoptotic macrophages and foam cells, although the reasons are unknown. In the present investigation, we studied the relationship between ferritin induction and occurrence of apoptosis in 7beta-hydroxycholesterol (7beta-OH)-treated monocytic cells and macrophages. We found that 7beta-OH enlarges the intracellular labile iron pool, increases formation of reactive oxygen species (ROS), and induces ferritin and cytosolic accumulation of lipid droplets, lysosomal destabilization, and apoptototic macrophage death. Since ferritin is a phase II-type protective protein, our findings suggest that ferritin upregulation here worked as an inefficient defense mechanism. Addition to the culture medium of both a membrane-permeable iron chelator 10-phenanthroline and the non-membrane-permeable iron chelators apoferritin and desferrioxamine afforded significant protection against the 7beta-OH-induced effects. Consequently, endocytosed iron compounds dramatically augmented 7beta-OH-induced cytotoxicity. We conclude that oxidized lipid 7beta-OH causes not only foam cell formation but also oxidative damage with abnormal metabolism of cellular iron. The findings suggest that modulation of iron metabolism in human atheroma may be a potential therapeutic strategy against atherosclerosis.     

Phenotype determination of anti-GM3 positive cells in atherosclerotic lesions of the human aorta. Hypothetical role of ganglioside GM3 in foam cell formation

Earlier we reported that atherosclerotic plaques contain cells which were specifically and very intensively stained with anti-GM3 antibodies although no GM3 positive cells were detected in the normal non-diseased arterial intima. Because of their lipid inclusions, GM3 positive cells in atherosclerotic lesions seemed to be foam cells but their origin needed clarification. Using an immunohistochemical technique in the present work, we showed that some of these foam cells contained CD68 antigen. However, the most intense accumulation of GM3 occurred in the areas composed of foam cells which did not stain with any cell type-specific antibodies, including antibodies to macrophages (anti-CD68) and smooth muscle cells (anti-smooth muscle alpha-actin), perhaps, because the cell type-specific antigens were lost during the transformation of intimal cells into foam cells. Ultrastructural analysis of the areas where foam cells overexpressed GM3 demonstrated that some foam cells lacked both a basal membrane and myofilaments but contained a large number of secondary lysosomes and phagolysosomes, morphological features which might indicate their macrophage origin. Other foam cells contained a few myofilaments and fragments of basal membrane around their plasmalemmal membrane, suggesting a smooth muscle cell origin. These observations indicate that accumulation of excessive amounts of GM3 occurs in different cell types transforming into foam cells. We suggest that up-regulation of GM3 synthesis in intimal cells might be an essential event in foam cell formation. Shedding of a large number of membrane-bound microvesicles from the cell surface of foam cells was observed in areas of atherosclerotic lesions corresponding to extracellular GM3 accumulation. We speculate that extracellularly localised GM3 might affect the differentiation and modification of intimal cells in atherosclerotic lesions.     

Iron storage in macrophages and endothelial cells. Histochemistry, ultrastructure, and clinical significance.

1 hour after i. v. infusion of colloidal iron in iron deficient subjects uniform phagosomal iron granules were observed in macrophages and endothelial cells of several organs. 7 to 10 days later transformation into ferritin coould be visualized in macrophages only. Now, these cells showed diffuse iron staining of the cytoplasm due to dispersed ferritin molecules. Polymorphous lysosomes contained densely packed particles from still unchanged ferric hydroxide to paracristalline ferritin. The macrophageal iron was mobilizable in few days to several weeks. The univorm lysosomal iron granules of endothelial cells disappeared after 1 to 2 years. Endothelial iron siderosis without previous i. v. iron application was a frequent finding in pernicious anaemia and iron overload of diverse origin.     

Immunolocalization of collagen types I and III in the arterial wall of the rat

Summary Collagen types I and III were located by immunofluorescence procedures in the aorta and coronary arteries of the rat. Type I collagen was most prevalent in the adventitia of the aorta with only small amounts present in the intima and media. Type III collagen appeared to be a significant component in the media of the aorta and also in the adventitia of both blood vessels. The intima and media of the coronary arteries did not stain strongly for either type I or III collagen. Neither staining procedure was altered with preincubation of the sections with hyaluronidase or chondroitinase ABC. These studies indicate that type III collagen is a major component of the adventitia which has previously not been recognized by immunohistochemical techniques, possibly due to masking of collagen staining with glycosaminoglycans.     

Identification of dendritic cells in aortic atherosclerotic lesions in rats with diet-induced hypercholesterolaemia

We have previously identified dendritic cells (DCs) in the intima of human large arteries. These vascular DCs are common in atherosclerotic lesions but their immature forms are also present in normal arterial intima. Pathophysiological studies on vascular DCs are limited because they have only been studied in human specimens obtained at operation or post-mortem. The aim of the current study was to determine whether DCs participate in the development of atherosclerotic lesions in hypercholesterolemic rats. Male Wistar rats were divided into a control (n=13) and experimental cohort (n=48). The experimental animals were fed an atherogenic diet and 1% saline, while the controls were fed standard rat cubes and water. The aortas were obtained from both groups at 10, 20, and 30 weeks following commencement of the diet. An en face immunohistochemical technique, routine section immunohistochemistry, and transmission electron microscopy were used to detect the presence of DCs in the aortas. Examination of the aortas showed that S100+ cells with dendritic cell morphology were present in the aortic intima of hypercholesterolemic rats. The S100+ DCs displayed immunopositivity for OX-62 and MHC Class II antibodies. Within various types of atherosclerotic lesions, these cells were clustered throughout the intima but were especially prominent around arterial branch-points where they co-localized with various cell types, including T-cells and macrophages. Ultrastructural analysis confirmed the presence of cells with characteristics typical of DCs. These features included the presence of a well-developed tubulovesicular system, dendritic processes, and a lack of secondary lysosomes and phagosomes. This study establishes the presence of DCs in the aortic intima of rats with diet-induced atherosclerosis. The presence of DCs in this model of experimental atherogenesis could provide a new approach to investigating the function of DCs and may help clarify the immune-inflammatory mechanisms underlying atherosclerosis.     

Natriuretic peptide system gene expression in human coronary arteries.

The natriuretic peptides (NPs) ANF, BNP, and CNP have potent anti-proliferative and anti-migratory effects on vascular smooth muscle cells (SMCs). These properties make NPs relevant to the study of human coronary atherosclerosis because vascular cell proliferation and migration are central to the pathophysiology of atherosclerosis. However, the existence and cytological distribution of NPs and their receptors in human coronary arteries remain undetermined. This has hampered the development of hypotheses regarding the possible role of NPs in human coronary disease. We determined the pattern of expression of NPs and their receptors (NPRs) in human coronary arteries with atherosclerotic lesions classified by standard histopathological criteria as fatty streak/early atherosclerotic lesions, intermediate plaques, or advanced lesions. The investigation was carried out using a combination of immunocytochemistry (ICC), in situ hybridization (ISH), and semi-quantitative polymerase chain reaction (PCR). Both by ICC and ISH, ANF was found in the intimal and medial layers of all lesions. BNP was highly expressed in advanced lesions where it was particularly evident by a strong ISH signal but weak ICC staining. CNP was demonstrable in all types of lesions, giving a strong signal by ISH and ICC. This peptide was particularly demonstrable in the endothelium, as well as in the SMCs of the intima, media, and vasa vasorum of the adventitia and in macrophages. By ISH, NPR-A was not detectable in any of the lesions but both NPR-B and NPR-C were found in the intimal and the inner medial layers. By RT-PCR, mRNA levels of all NPs tended to be increased in macroscopically diseased arteries, but only the values for BNP were significantly so. No significant changes in NPR mRNA levels were detected by PCR. In general, the signal intensity given by the NPs and their receptors by ICC or ISH appeared dependent on the type of lesion, being strongest in intermediate plaques and decreasing with increasing severity of the lesion. This study constitutes the first demonstration of NPs and NPR mRNAs in human coronary arteries and supports the existence of an autocrine/paracrine NP system that is actively modulated during the progression of atherosclerotic coronary disease. This suggests that the coronary NP system is involved in the pathobiology of intimal plaque formation in humans and may be involved in vascular remodeling.     

Changes of lysosomes in the earliest stages of the development of atherosclerosis

One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the ‘normal intima‐initial lesion‐fatty streak’ sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease.