Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

冠状动脉斑块中炎性细胞和平滑肌细胞的定量及其与斑块稳定 …

目的 探讨稳定及不稳定斑块中巨细胞、Ｔ淋巴细胞及平滑肌细胞数量的差异及其与斑块稳定性的关系。方法 将急性心肌梗死死亡病例的尸检冠状动脉完整剥离后固定、脱钙、连续取材并常规切片及ＨＥ染色,按有无脂质中心及其大小将斑块分为稳定斑块（斑块无或仅有较小的脂质中心）及不稳定斑块（脂质中心超过斑块的４０％）。选取２种斑块各１６３个,用ＣＤ６８、肌动蛋白、ＵＣＨＬ－１抗体分别对巨噬细胞、平滑肌细胞、Ｔ淋巴细胞进     

冠状动脉斑块中炎性细胞和平滑肌细胞的定量及其与斑块稳定性的关系

目的　探讨稳定及不稳定斑块中巨噬细胞、Ｔ淋巴细胞及平滑肌细胞数量的差异及其与斑块稳定性的关系。方法　将急性心肌梗死死亡病例的尸检冠状动脉完整剥离后固定、脱钙、连续取材并常规切片及ＨＥ染色,按有无脂质中心及其大小将斑块分为稳定斑块（ 斑块无或仅有较小的脂质中心） 及不稳定斑块（ 脂质中心超过斑块的４０ ％ ）。选取２ 种斑块各１６３ 个,用ＣＤ６８ 、肌动蛋白、ＵＣＨＬ－ １ 抗体分别对巨噬细胞、平滑肌细胞、Ｔ淋巴细胞进行免疫组化染色,并通过计算机图像分析系统对２ 种斑块纤维帽中的３ 种细胞进行定量。结果　不稳定斑块纤维帽中巨噬细胞及Ｔ淋巴细胞的面积百分比分别为８９．６％ 和３２．５％ ,稳定斑块分别为１８．３％ 和７．２ ％ ,差异均具有显著性（ Ｐ＜０．０１） 。而平滑肌细胞在稳定斑块纤维帽中的面积百分比明显大于不稳定斑块,分别为６８．４ ％ 和２１ ．８％ ,差异具有显著性（Ｐ＜ ０．０５） 。结论　稳定斑块与不稳定斑块不仅脂质中心大小不同,其纤维帽中炎细胞和平滑肌细胞的数量也不同,粥样坏死中心越大,其表面纤维帽中的巨噬细胞、Ｔ淋巴细胞就越多,而平滑肌细胞则越少。脂质中心大小和纤维帽中炎细胞数量是决定斑块稳定性的主要因素。     

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.     

Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques.

Cell proliferation, an important mechanism of atherosclerotic plaque growth, occurs among smooth muscle, inflammatory cell, and other cell types. We have identified different topographical patterns of cell proliferation in human carotid plaques, based on cell type. Cell proliferation was determined with an antibody to the proliferating cell nuclear antigen (PCNA), combined with cell type-specific antibodies. Despite low levels of overall proliferative activity, the intima displayed more proliferative activity than the underlying media (1.61 +/- 0.35% in intima versus 0.05 +/- 0.03% in media; P < 0.01). The preponderant proliferative cell type in the intima was the monocyte/macrophage (46.0% of PCNA-positive cells), with a minority being smooth muscle alpha-actin-positive (9.7%), microvascular endothelial (14.3%), and T cells (13.1%). Smooth muscle cells were the dominant proliferating cell type in the media (44.4% of PCNA-positive cells versus 20% endothelial cells, 13.0% monocyte/macrophages, and 14.3% T cells). Within the plaque, foam-cell-rich regions mostly displayed proliferation among macrophages (66.5%), whereas in vascularized fields PCNA positivity was almost equally shared by endothelial cells (23.8%), monocyte/macrophages (26.3%), smooth muscle alpha-actin-positive cells (14.0%), and to a lesser extent, T cells (8.2%). Logistic and linear regression analyses also demonstrated that location in foam-cell-rich regions was a significant predictor of proliferation only among monocyte/macrophages, whereas location in vascularized regions was a good predictor of PCNA positivity among both inflammatory and noninflammatory cells. These different patterns of cell type proliferation suggest possibly different distributions of putative responsible growth regulatory factors in human atherosclerosis.     

Genetic loss of Gas6 induces plaque stability in experimental atherosclerosis

The growth arrest-specific gene 6 (Gas6) plays a role in pro-atherogenic processes such as endothelial and leukocyte activation, smooth muscle cell migration and thrombosis, but its role in atherosclerosis remains uninvestigated. Here, we report that Gas6 is expressed in all stages of human and mouse atherosclerosis, in plaque endothelial cells, smooth muscle cells and macrophages. Gas6 expression is most abundant in lesions containing high amounts of macrophages, ie thin fibrous cap atheroma and ruptured plaque. Genetic loss of Gas6 does not affect the number and size of initial and advanced plaques in ApoE(-/-) mice, but alters its plaque composition. Compared to Gas6(+/+): ApoE(-/-) mice, initial and advanced plaques of Gas6(-/-): ApoE(-/-) mice contained more smooth muscle cells and more collagen and developed smaller lipid cores, while the expression of TGFbeta was increased. In addition, fewer macrophages were found in advanced plaques of Gas6(-/-): ApoE(-/-) mice. Hence, loss of Gas6 promotes the formation of more stable atherosclerotic lesions by increasing plaque fibrosis and by attenuating plaque inflammation. These findings identify a role for Gas6 in plaque composition and stability.     

T-cell subpopulations and macrophages in stable and unstable coronary atherosclerotic plaques

Unstable atherosclerotic plaques are a cause of acute myocardium infarction. Because peripheral blood mononuclear cells are often present in atherosclerotic plaques, we've examined T-cells (CD4, CD8) and macrophages (CD68) in the different areas of atherosclerotic plaques. The cells were counted individually in the center, shoulder at the bottom and in the cap of plaque. All types of studied cells prevailed in the unstable plaque cap than in the stable one (p < 0.05). CD4 and CD68 cells dominated in the shoulder of atherosclerotic plaque (p < 0.05). The difference between the numbers of macrophages at the bottom or in the center of stable and unstable plaques was statistically insignificant (p > 0.05). Prevalence of peripheral blood mononuclears in the cap and at the periphery of unstable plaques points their participation in the development of atherosclerotic plaque instability.     

The relationship between pre-existing subendothelial smooth muscle cell accumulations and foam cell lesions in cholesterol-fed rabbits

We investigated whether pre-existing subendothelial smooth muscle cell (SMC) accumulations in cholesterol-fed rabbits are transformed into foam cell plaques. Twenty-four rabbits received a standard diet supplemented with 2% cholesterol for 4 or 8 weeks. Six rabbits received a supplement of 0.3% cholesterol for 35 weeks. The aorta and other systemic and pulmonary vessels were studied by immunohistochemistry for smooth muscle cells SMC (-SMC actin), macrophages (RAM11), cell replication (proliferating cell nuclear antigen) and endothelial cells (von Willebrand factor; vWF). Initially the foam cell plaques were composed exclusively of foam cells of macrophage origin (MFC). In more advanced lesions SMC and collagen fibres were also present, leading to a fibrous transformation of the plaque. Cell replication was mainly located in the MFC. The endothelial cells covering the plaques showed an increased immunoreactivity for vWF which was also deposited in the interstitium between the FC. Pre-existing subendothelial SMC did not transform into FC. The newly formed FC plaques remained clearly separated from the pre-existing subendothelial SMC. The development of the plaques can be attributed not only to monocyte recruitment but also to macrophage multiplication.     

Expression of pentraxin 3 (PTX3) in human atherosclerotic lesions

Pentraxin 3 (PTX3) and C-reactive protein (CRP) are members of the pentraxin superfamily. PTX3 expression is induced in response to inflammatory signals, and is produced at sites of inflammation by several types of cell, primarily monocytes/macrophages, dendritic cells (DCs), endothelial cells, smooth muscle cells (SMCs), and fibroblasts, but is not produced by hepatocytes, which are a major source of CRP. The aim of our study was to investigate the expression pattern of PTX3 in human atherosclerotic lesions using a novel monoclonal antibody against PTX3. We examined coronary arterial thrombi containing an atherosclerotic plaque component removed from patients with acute myocardial infarction and human aortic tissues with various degrees of atherosclerosis sampled from autopsy cases. Immunohistochemical study of paraffin and frozen sections indicated that macrophages, mainly foam cells, expressed PTX3 in advanced atherosclerotic lesions. Interestingly, we also clearly observed PTX3-positive neutrophils infiltrating into atherosclerotic plaques, suggesting that PTX3 derived from neutrophils as well as macrophages plays an important role in atherogenesis.     

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.     

Monoclonality of smooth muscle cells in human atherosclerosis.

Atherosclerotic plaques contain a large monoclonal population of cells. Monoclonality could arise by somatic mutation, selection of a pre-existing lineage, or expansion of a pre-existing (developmental) clone. To determine the monoclonal cell type in plaque and learn when monoclonality arises, we studied X chromosome inactivation patterns using methylation of the X-linked human androgen receptor gene. Assays based on polymerase chain reaction were performed on samples of known cellular composition, microdissected from histological sections of human arteries. In atherosclerotic vessels, the majority of medial samples (7/11 coronary and 2/3 aortic) showed balanced (paternal and maternal) patterns of X inactivation, indicating polyclonality. In contrast, most samples of plaque smooth muscle cells showed a single pattern of X inactivation (3/4 aortic plaques and 9/11 coronary plaques; P < 0.01 versus media), indicating that plaque smooth muscle cells are monoclonal. Samples of plaque containing inflammatory or endothelial cells showed balanced X inactivation, also demonstrating polyclonality. Multiple plaques from a given patient showed no bias toward one allele, indicating there was no X-linked selection of cells during plaque growth. To determine whether plaques might arise from pre-existing clones (large X inactivation patches), we then studied 10 normal coronaries with diffuse intimal thickening. Six of the ten coronaries showed skewed X inactivation patterns in normal media and intima, suggesting the patch size in normal arteries is surprisingly large. Thus, smooth muscle cells constitute the monoclonal population in atherosclerotic plaques. The finding that normal arteries may have large X inactivation patches raises the possibility that plaque monoclonality may arise by expanding a pre-existing clone of cells rather than generating a new clone by mutation or selection.     

稳定性,不稳定性心绞痛及急性心肌梗死病人冠状动脉病变的？…

比较稳定型心绞痛、不稳定型心绞痛及急性心肌梗死病人冠状动病变的组织学差异,以阐明其发生的病理学机制。方法选天临床诊明确的ＳＡ、ＵＡ及ＡＭＩ病人死后的尸检收脏标本,对其冠状动脉取材并做组织学及免疫组织化学观察。     

Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes

The surface distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells was investigated by freeze-fracture. Replicas of fractured smooth muscle cell plasma membrane showed caveola-free regions with few intramembrane particles, interspersed with caveola-populated areas with a higher intramembrane particle density. Electron microscopy of thin sections of quail gizzard smooth muscle revealed the regions free of caveolae to be occupied by membrane-associated dense plaques; anchoring sites for the contractile filaments of the cell. Demarcation between the caveola-populated and caveolafree regions on the relicated intramembrane surface was not clear and thus provided little information concerning the distribution of dense plaque sites. However, treatment of the smooth muscle tissue with the cholesterol-binding agents filipin or tomatin prior to freeze-fracture allowed the dense plaque sites to be easily observed as the sites remained free of the membrane deformations characteristic of these agents. The dense plaque sites consist of caveola-free oval areas juxtaposed in regular bands that traverse the long axis of the cell. The dense plaque sites on the freeze-fracture replica were confirmed by electron microscopy of thin sections of filipin-treated quail gizzard smooth muscle cells, which showed the plasma membrane associated with the dense plaques to be unaffected by the actions of filipin, whereas that of the caveola-populated region was severely deformed. The observations presented in this study provide evidence for a highly ordered distribution of dense plaques at the cell surface of intact quail gizzard smooth muscle cells and thus corroborate existing evidence for an organized substructure of smooth muscle cells.     

Arterial macrophages and regenerating endothelial cells express P-selectin in atherosclerosis-prone apolipoprotein E-deficient mice

P-selectin expression has been reported in platelets, endothelial cells, and vascular smooth muscle cells in response to vascular injury. Here, we report P-selectin expression on macrophages in the arterial wall after carotid denudation injury and spontaneous atherosclerosis in atherosclerosis-prone apoE-deficient (apoE(-/-)) mice. Double-immunofluorescence staining revealed robust P-selectin expression in macrophage-rich regions of both denudation-induced carotid neointimal lesions and innominate atherosclerotic plaques. Co-localization of P-selectin with macrophages was verified at the single cell level using double immunostaining plus 4,6-diamidino-2-phenylindole (for nuclei) counterstaining. No platelet staining was seen in association with the macrophage staining, excluding platelet contamination. Furthermore, P-selectin mRNA expression was readily detectable in macrophage-rich plaques of atherosclerotic innominate arteries and blood monocyte-derived macrophages from apoE(-/-) mice. Strong P-selectin expression was also seen in the areas of regenerated endothelium after arterial injury. In addition, co-localization of P-selectin with vascular smooth muscle cells was readily observed in denudation-injured carotid arteries at 7 and 14 days. We conclude that macrophages in carotid injury-induced neointimal lesions and spontaneous atherosclerotic plaques of the innominate artery acquire the ability to express P-selectin, as does regenerating endothelium. These findings provide a potential new paradigm in macrophage-mediated vascular inflammation, atherosclerosis, and neointimal hyperplasia after arterial injury.     

Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions.

The authors have performed immunocytochemical investigations of the distribution of various cell types in human atherosclerotic plaques using monoclonal antibodies specific to smooth muscle cells (CGA7 [Gown et al, J Cell Biol 1985, 100:807-813] and HHF35 [Tsukada et al, Am J Pathol (In press)] ); lymphocytes (T200 antigen); endothelial cells (Factor VIII and the Ulex europeus agglutinin); and macrophages, the latter with a new macrophage-specific antibody HAM56. All studies were performed on methanol-Carnoy's-fixed, paraffin-embedded tissues. In areas of grossly normal aorta, significant numbers of macrophages were noted within areas of diffuse intimal thickening. The cellular composition of the following three types of raised lesions were analyzed: fibro-fatty lesions, which, despite their gross appearance, consistent with fibrous plaques, were composed almost exclusively of macrophages and lymphocytes and almost devoid of smooth muscle cells; fibrous plaques, which were predominantly composed of smooth muscle cells displaying considerable morphologic heterogeneity and an admixture of blood-borne cells; advanced plaques, which were characterized by complex layers of smooth muscle cells and macrophages with considerable variation from region to region. Also noted were foci of medial and even intimal vascularization subjacent to the more advanced plaques. These studies demonstrate the application of monoclonal antibody technology to the study of the cellular composition of human atherosclerotic lesions.     

Angiogenesis and lymphangiogenesis and expression of lymphangiogenic factors in the atherosclerotic intima of human coronary arteries

Little information regarding the development of lymphangiogenesis in coronary atherosclerosis is available. We immunohistochemically investigated the correlation among intimal neovascularization (CD34 for angiogenesis and lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin for lymphangiogenesis), the expression of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGF-D), and the progression of atherosclerosis using 169 sections of human coronary arteries from 23 autopsy cases. The more the atherosclerosis advanced, the more often the neointimas contained newly formed blood vessels ( P < .0001). Vascular endothelial growth factor-C was expressed mostly in foamy macrophages and in some smooth muscle cells, whereas VEGF-D was abundantly expressed in both. The number of VEGF-C-expressing cells, but not that of VEGF-D-expressing cells, was increased as the lesion advanced and the number of intimal blood vessels increased ( P < .01). Lymphatic vessels were rare in the atherosclerotic intima (LYVE-1 vs CD34 = 13 vs 3955 vessels) compared with the number seen in the adventitia (LYVE-1 vs CD34 = 360 vs 6921 vessels). The current study suggests that VEGF-C, but not VEGF-D, may contribute to plaque progression and be a regulator for angiogenesis rather than lymphangiogenesis in coronary atherosclerotic intimas. Imbalance of angiogenesis and lymphangiogenesis may be a factor contributing to sustained inflammatory reaction during human coronary atherogenesis.     

What's new in the pathology of atherosclerosis?

Intimal smooth muscle cell proliferation has been known to be the key event in the development of advanced lesions of atherosclerosis. Since the important role of macrophages in the lipoprotein metabolism has been detected, however, current interest focuses on the macrophage reaction in the arterial wall. Animal experiments have shown that blood monocytes become attached to certain endothelial areas and enter the intima, where they are transformed to macrophages. Subendothelial infiltration of monocytes is the earliest cellular event in the formation of fatty streaks. Transformed to macrophages, they incorporate LDL by receptor-mediated endocytosis and are thus transformed to foam cells. The majority of foam cells in the atherosclerotic plaque is derived from macrophages. Furthermore, the importance of macrophages in the regulation of the lipoprotein metabolism and cholesterol homeostasis is increasingly attributed to their secretory capacities. It has been shown in vitro that they can secrete apolipoprotein E which associates with cholesterol and HDL to form a lipoprotein complex, which targets resecreted cholesterol to the liver cells. Recently, apolipoprotein E secretion of macrophages has also been demonstrated immunohistologically in the human atherosclerotic plaque. Cell culture investigations revealed that macrophages secrete different growth factors for fibroblasts and smooth muscle cells. So they are probably able, among other factors, to initiate smooth muscle cell proliferation in the intima. While smooth muscle cell proliferation and matrix components in the arterial wall had occupied the center of interest in previous investigations, the current focus on the cellular reactions of endothelial cells and monocytes/macrophages, especially in the early stages of atherosclerotic plaque formation, seems well justified.     

内质网应激中IRE1级联反应对动脉粥样硬化的作用

正1概述内质网(endoplasmic reticulum,ER)蛋白质折叠在生理上是至关重要的,它的破坏导致内质网应激(endoplasmic reticulum stress,ERS)触发动脉粥样硬化(atherosclerosis,AS)发生发展。未折叠蛋白反应(unfolded protein response,UPR)是目前研究最为透彻的ERS信号通路,一定程度的UPR有利于维持     

Cannabinoid receptor and N-acyl phosphatidylethanolamine phospholipase D--evidence for altered expression in multiple sclerosis

Cannabinoids have been shown to have a beneficial effect in both animal models of multiple sclerosis (MS) and human disease, although the mechanisms of action are unclear. We examined expression of the major cannabinoid receptors [(CBRs) cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)] and a key enzyme involved in synthesis of the endocannabinoid anandamide [N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] in autopsy brain samples from patients with MS. CB1 was expressed in neurons, injured axons, oligodendrocytes, macrophages/microglia, some astrocytes, endothelial cells, smooth muscle cells and pericytes. CB2 and NAPE-PLD were localized to cerebral endothelial cells, pericytes, smooth muscle cells, astrocytes and macrophages/microglia. NAPE-PLD immunoreactivity was also seen in neurons. Endothelial CB2 expression was greatest in chronic inactive plaques, and in areas was seen in segments of endothelium where the endothelial expression of adhesion molecules (VCAM-1 and ICAM-1) was focally undetectable, and was often expressed in areas of blood-brain barrier damage. Vascular density was increased in chronic active plaques and normal-appearing white matter compared with controls. These data support findings from animal models which suggest a role for the endocannabinoid system in the MS, particularly in the regulation of endothelial leukocyte adhesion and the cellular response to injury.     

Development of atherosclerotic plaque with endothelial disruption in Watanabe heritable hyperlipidemic rabbit aortas.

To better understand the morphogenesis of atherosclerotic plaque, we evaluated temporal distribution of leukocytes, macrophages, foam cells, vascular smooth muscle cells, and subendothelial lipid in Watanabe heritable hyperlipedimic (WHHL) rabbit aortas. Aortas of WHHL (n=20) and New Zealand White (NZW, controls; n=8) rabbits were perfusion fixed at 1, 3, 6, and 12 months of age. At initial gross evaluation of lipid distribution, we identified aortic areas at high risk for lesion development. In WHHL rabbits, the lipid-positive portion of high-risk areas increased from 3% at 1 month to 50% at 12 months; during the same period, adherent cell count increased from <1 leukocyte and monocyte/mm(2) to 25 leukocytes, 44 monocytes, and 10 foam cells/mm(2). Controls showed no increase over time in lipid-positive areas or cellular adherence to the endothelium. One-month-old WHHL rabbit aortas had scattered lipid-positive cells in the intima (primarily branch points). Immunostaining of these areas did not show rabbit macrophages (RAM antibody) but were actin positive. Occasionally, platelets and monocytes adhered to the endothelial surface. By age 3 months, well-defined fatty streaks/atherosclerotic plaques had RAM-positive cells within foam cell core, along core margins, and in focal clusters in the fibrous cap and subendothelium. By age 12 months, isolated RAM-positive cells were on the endothelial surface, and surface morphology showed endothelial cell disruption foci containing clusters of macrophages and foam cells. Our results indicate that lipid accumulation (extra- and intracellular) is important in the early development of atherosclerotic lesions; a corresponding, slower accumulation of adherent cells on the lesion surface promotes lipid conversion from fatty streak to plaque.