Age-related changes in plaque composition: a study in patients suffering from carotid artery stenosis.

OBJECTIVE: The extent of atherosclerotic plaque burden and the incidence of atherosclerosis-related cardiovascular events accelerate with increasing age. The composition of the plaque is associated with plaque thrombosis and acute coronary occlusion. Surprisingly, however, the relation between advancing age and atherosclerotic plaque composition is still unclear. In the present study, we investigated the association between plaque characteristics and advancing age in a population of patients with haemodynamically significant carotid artery stenosis. METHODS: Patients (N=383), ages 39-89 years, underwent carotid endarterectomy (CEA). Morphometric analysis was performed on the dissected atherosclerotic plaques to study the prevalence of fibrous and atheromatous plaques. Picro sirius red, haematoxylin eosin, alfa actin and CD68 stainings were performed to investigate the extent of collagen, calcification, smooth muscle cells and macrophages in carotid plaques, respectively. The presence of metalloproteinases-2 and -9 was assessed by ELISA. RESULTS: With aging, a decrease in fibrous plaques and an increase in atheromatous plaques were observed. This was accompanied by an age-associated decrease in smooth muscle cell content in carotid plaques. Macrophage content slightly increased with age. In addition, total matrix metalloprotease (MMP)-2 was negatively and MMP-9 positively related with age. Differences in plaque phenotype were most prominent for the youngest age quartile compared with older age quartiles. CONCLUSIONS: With increasing age, the morphology of atherosclerotic plaques from patients with carotid artery stenosis changes. Plaques become more atheromatous and contain less smooth muscle cells with increasing age. Local inflammation and MMP-9 levels slightly increased with age in plaques obtained from patients suffering from haemodynamically significant advanced atherosclerotic lesions.     

Fluorescence spectroscopic and histochemical analysis using hematoporphyrin as a microenvironmental probe for atherosclerotic change in the human aorta.

Hematoporphyrin (HP) was used as a microenvironmental fluorescent probe to investigate the development of human atherosclerotic plaques. We compared the site of HP accumulation with changes in the HP fluorescence spectrum in atheromatous plaques and in a liposome control model, using a confocal laser scanning microscope equipped with a photonic multi-channel analyzer linked to a fluorescence spectrometer. Wavelength shifts of the two peaks of HP fluorescence (F1, 620 nm; and F2, 640-690 nm) were monitored, and the integrated F2/F1 ratio was calculated as a measure of HP fluorescence. The F1 peak is characteristic of a predominantly aqueous site. The ratio reflects selective changes in the distribution and overcrowding of HP molecules in the limited space of the artificial membrane model. Compared with a normal artery, the atherosclerotic lesions showed an increase in the area of HP fluorescence, increased fluorescence intensity, a red shift of the HP fluorescence spectrum, and an increased F2/F1 ratio. The F2/F1 ratio of the membranous structures was markedly greater in the cores of the fibrous plaques than in the other plaques. The F1 peak showed increased intensity in the atheromatous plaque core, whereas in hydrophilic fibrous regions, such as the cap of the plaque, the intensity of the F1 peak was lower than in the core region. HP aggregation was observed in damaged cells and in water surrounded by lipid in the atheromatous core. Using HP as a probe allowed us to determine not only the ionization or polarity of each region in the atherosclerotic plaques but also to detect the separation and fusion of the lipid bilayer or micelle lipids, as well as damage to the cellular membranes and cholesterol enrichment. These findings suggest that HP is useful for detecting clinically important changes in atherosclerotic lesions, to lipid-rich, unstable, and vulnerable plaques, which are closely associated with cardiovascular events.     

C-reactive protein immunohistochemical localization in normal and atherosclerotic human aortas

We examined immunohistochemically 104 formaldehyde-fixed, paraffin-embedded, human autopsy aortic specimens for C-reactive protein (CRP) presence and localization. In addition, we correlated immunoreactivity with a spectrum of the following histologic categories: normal aorta, fatty streak, atheromatous plaque, and fibrous plaque. Using appropriate controls, we confirmed CRP immunoreactivity in 3.3% of normal specimens, 75% of fatty streaks, 90.2% of atheromatous plaques, and 64.6% of fibrous plaques. Immunoreactivity in fatty streaks was located around collections of foam cells. Immunoreactivity in atheromatous plaques was in a bandlike distribution corresponding to the pale-staining insudative zone frequently seen in such lesions. The correlation and localization of CRP immunoreactivity in atherosclerotic lesions presented here suggests a functional role for CRP in the pathogenesis of atherosclerosis. Our results encourage efforts to determine more precisely the physiologic contributions of CRP to the development and exacerbation of atherosclerosis.     

256层CT对颈动脉粥样硬化斑块成分及性质的分析

目的　探讨256层螺旋CT 血管造影（CT angiography,CTA）评价颈动脉粥样硬化斑块的价值。 方法　 对234例拟诊颈动脉硬化狭窄的患者行头颈部CTA检查, 根据颈动脉有无斑块分为病变组和正常对照组,通过CT值分析斑块成分和性质。 结果　病变组266支动脉,其中脂肪斑块组51支、纤维斑块组26支、钙化斑块组67支、混合斑块组122支;正常组163支。脂肪斑块组脑梗死出现率明显大于纤维斑块组和钙化斑块组（P＜0.01）;脂肪合并钙化斑块组脑梗死出现率明显大于纤维合并钙化斑块组（P＜0.01）;含脂肪斑块组溃疡出现率大于含纤维斑块组（P＜0.05）。 结论　256层螺旋CT能够分析颈动脉斑块的成分、性质,初步评价颈动脉斑块的稳定性。     

Changes in generation of superoxide anion-radical by phagocytic and other cells of atherosclerotic plaques at different stages of human atherosclerosis

We investigated changes in generation of free radicals of oxygen including superoxide anion-radical (SAR) of oxygen by phagocytes and other cells of atherosclerotic plaques resected in operations of aortofemoral or aortoiliac bypass grafting and plastic vascular surgery of 38 patients with obliterating atherosclerosis obliterans of the lower extremities. We found some regularities in changes of SAR generation by phagocyting cells of atherosclerotic plaques at different morphological stages of their formation. Generation of the radicals progressively increased at the stage of fibrous plaques (1.56 times vs control), increased still more at the stage of their atheromatous alterations (2.3 times) and noticeably decreased at the stage of their calcinosis. Simultaneously, there was intensification of SAR formation by non-phagocyting cells of atherosclerotic plaques under their stimulation by NADPH-H (in fibrous and atheromatous plaques 1.3 and 2.0 times, respectively). On the contrary, at calcinosis stage NADPH-stimulated radical generation by these cells reduced.     

Pathological mechanisms of erythrocyte-induced vulnerability of atherosclerotic plaques

Erythrocytes are considered a new culprit contributing to atherosclerosis. Plaques with intraplaque hemorrhage are prone to new plaque hemorrhage, which may not only stimulate the progression of atherosclerosis but also promote the transition from a stable to an unstable lesion. However, the role of erythrocytes in inducing the vulnerability of plaque with intraplaque hemorrhage and the possible mechanism involved are not well understood. Recently, increased cholesterol level from erythrocytes was reported to expand the lipid core of plaque. As well, heme, iron and phospholipids derived from erythrocytes trigger peroxidization in vitro, which is strongly associated with the progression of atherosclerosis. We speculate that erythrocytes trapped in plaque may induce vulnerability of atherosclerotic plaques not only by accumulating lipids but also by promoting peroxidization within plaques, thereby expanding the lipid core, increasing the infiltration of inflammatory cells and attenuating the fibrous cap of plaques. This proposition may provide clues into the development of novel treatments to increase the stability of atherosclerotic plaques.     

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。     

Heterogeneity of smooth muscle cells in atheromatous plaque of human aorta.

This study was undertaken to investigate the expression of cytoskeletal proteins and the ultrastructure of cells in normal intima and atheromatous plaque of human aorta. It has been established, using double-labeling immunofluorescence, that smooth muscle cells (SMC) in normal aortic intima contain myosin, vimentin, and alpha-actin but do not react with antibodies against desmin. In contrast, 7 of 28 atherosclerotic plaques contained many cells expressing desmin in addition to the other cytoskeletal proteins characteristic of normal intima SMC. These cells were localized predominantly in the plaque cap and had the ultrastructural features of modulated SMC, ie, well-developed endoplasmic reticulum and Golgi apparatus. Besides, some cells in the 13 atherosclerotic plaques proved to be myosin, alpha actin, and desmin negative but contained vimentin and actin as revealed by fluorescent phalloidin. These cells were found in the immediate proximity of atheromatous material and reacted with a monoclonal antibody specific to SMC surface protein (11G10) but not with monoclonal anti-muscle actin (HHF35) and anti-macrophage (HAM56) antibodies. Electron microscopy of this plaque zone revealed that the cytoplasm of these cells was filled with rough endoplasmic reticulum and a developed Golgi complex. At the same time, a certain proportion of cells in this region retained morphologic features of differentiated SMC such as the presence of a basal lamina and myofilament bundles. The revealed peculiarities of cytoskeletal protein expression and the ultrastructure of cells in human aortic atherosclerotic plaques may be explained by a phenotypic modulation of vascular SMC.     

Determining the temperature distribution of swine aorta with simulated atheromatous plaque under pulsed laser irradiation: an experimental attempt to detect the vulnerability of atherosclerosis

We developed a method to determine the temperature distribution of swine aortas with simulated atheromatous plaques in order to measure the temperature of atherosclerotic lesions. The inflammation associated with temperature elevation is considered to be one of the aggravating mechanisms of atherosclerosis resulting in fissuring or rupture of atheromatous plaques. The temperature distribution of plaques covered by fibrous caps cannot be measured by conventional thermistors. Indocyanine green (ICG) solution was injected into the subintima of swine aorta to simulate the light absorption coefficient of human atheromatous plaques. The temperature distribution was calculated from measured temperature changes of the aortic intima under pulsed laser irradiation. The aorta was heated from the adventitial side with a halogen lamp to simulate the temperature elevation derived from inflammation. The temperature distribution of the aorta was obtained by solving the heat transfer equation using the surface layer thickness (corresponding to the fibrous cap thickness). The surface layer thickness can be calculated using the following working formula: D( µm)=1363-398 &#150 T +35 &#150 T 2, where s s &#150 T s denotes intimal surface temperature change under pulsed laser irradiation. The calculated temperature of the ICG layer (corresponding to the atheromatous core) correlated well with the measured temperature (r=0.97, p<0.0001).     

Smooth muscle immaturity in the carotid arterial neointima as a prognostic marker for systemic atherogenic cardiovascular events in the Asian male

Although immaturity of neointimal smooth muscle cells (SMCs) in coronary arteries has recently been demonstrated to be associated with acute coronary syndrome, the carotid arterial counterpart has not been investigated. We hypothesized that the same investigation of carotid endarterectomy specimens might contribute to living patients. Carotid endarterectomy specimens from 33 Asian males who underwent a 5-year follow-up were examined. Age, atherosclerotic risk factors, and percentage stenosis were investigated. Histologically, the fibrous cap/lipid core ratio was measured. Maturation of SMCs was assessed by the h-caldesmon/smooth muscle actin (SMA) ratio by immunohistochemistry in 3 different regions (luminal, medial, and opposite side of lipid core) in the neointima. Associations of these factors with preoperative symptoms along with postoperative systemic atherogenic cardiovascular events were analyzed. It was revealed that fibrous cap/lipid core ratio was significantly lower in symptomatic than in asymptomatic patients, while the h-caldesmon/SMA ratio was significantly lower in patients with than without postoperative systemic atherogenic cardiovascular events by the Student’s t-test (P<0.05). Logistic regression model demonstrated that younger age and a lower h-caldesmon/SMA ratio were associated with postoperative systemic atherogenic cardiovascular events (P<0.05). This result was not different when 3 different regions were each analyzed instead. Immaturity of neointimal SMCs shown by a lower h-caldesmon/SMA ratio by immunohistochemistry was associated with systemic atherogenic cardiovascular events. Thus, this finding may be predictive of these events after carotid endarterectomy. Uniform results among different neointimal regions suggest that immaturity of neointimal SMCs causes plaque instability and does not occur secondarily to plaque instability.     

高脂饲料喂养加静脉注射小牛血清白蛋白建立兔动脉粥样硬化模型**

背景：目前单纯饲喂高脂饲料建立动脉粥样硬化狭窄模型较常见。 目的：采用高脂饲料喂养加静脉注射小牛血清白蛋白建立兔动脉粥样硬化模型。 方法：分别单纯高脂饮食、高脂饲料+脂肪乳灌胃以及高脂饲料+小牛血清白蛋白3种不同的方法喂养兔构建动脉硬化模型,设立正常对照组,予普通饲料喂养。 结果与结论：各组兔血清胆固醇、三酰甘油、低密度脂蛋白和高密度脂蛋白水平较正常对照组显著升高(P < 0.01)。单纯高脂组可出现高脂血症,未发现典型的动脉粥样硬化病变;高脂+脂肪乳灌胃组可形成纤维斑块;高脂+小牛血清白蛋白组可形成较成熟的动脉粥样硬化斑块。结果证实,高脂饲料加静脉注射小牛血清白蛋白可形成较成熟的动脉粥样硬化斑块,可成功建立动脉粥样硬化兔模型。关键词：动脉粥样硬化;小牛血清白蛋白;高脂饲料;兔;动物模型;组织工程 doi:10.3969/j.issn.1673-8225.2012.20.018     

Th17细胞对动脉粥样硬化斑块易损性和斑块破裂的影响研究

目的 探讨Th17细胞及其主要效应性细胞因子IL-17A对动脉粥样硬化斑块易损性和斑块破裂的作用。方法 在Apoe-/-小鼠的颈动脉套置缩窄性套管并通过药物联合作用的方法建立小鼠动脉粥样硬化斑块破裂模型。将20只Apoe-/-小鼠进行颈动脉套管,套管后继续进行高脂饲料喂养,然后分为药物联合刺激的干预组和生理盐水对照组,每组10只,分别于套管后7周和15周处死小鼠进行检测。流式细胞术检测斑块破裂后Th17细胞的变化,ELISA检测细胞因子IL-17A的变化。同上方法制备斑块破裂模型,将30只模型小鼠分为IL-17A处理组和对照组。IL-17A处理组外源性给Apoe-/-小鼠腹腔注射IL-17A,对照组腹腔注射生理盐水,处理后2、5、7周检测对斑块大小、脂质沉积和胶原含量的影响,免疫组化染色检测巨噬细胞和平滑肌细胞在斑块局部的表达,评价IL-17A对斑块稳定性及斑块破裂的作用。结果 在动脉套管后7周,此时仅有斑块内出血时,干预组小鼠脾脏中Th17细胞的比例高于对照组（P＜0.05）;在动脉套管后15周,此时有明显斑块破裂时,两组小鼠脾脏中Th17细胞的比例统计学意义显著（P＜0.01）。对Th17细胞关键细胞因子IL-17A的检测发现,动脉套管后7周干预组小鼠与对照组相比,血清中IL-17A的水平无统计学意义,而15周干预组小鼠与对照组小鼠比较,血清中IL-17A的水平升高,统计学意义显著（P＜0.01）。外源性IL-17A处理5周可增加斑块易损性,7周更为明显。表现为处理7周斑块面积较对照组增大（P＜0.05）,但纤维帽面积减小（P＜0.05）,脂核面积增大（P＜0.05）,帽/核比值降低（P＜0.01）。对斑块破裂的研究发现,干预组5周出现埋入式纤维帽斑块,7周出现埋入式纤维帽斑块例数增多。结论 Th17细胞及IL-17A具有促进动脉粥样斑块易损性和斑块破裂的作用。     

ADAM-10 could mediate cleavage of N-cadherin promoting apoptosis in human atherosclerotic lesions leading to vulnerable plaque: A morphological and immunohistochemical study

Atherosclerosis remains a major cause of mortality. Whereas the histopathological progression of atherosclerotic lesions is well documented, much less is known about the development of unstable or vulnerable plaque, which can rupture leading to thrombus, luminal occlusion and infarct. Apoptosis in the fibrous cap, which is rich in vascular smooth muscle cells (VSMCs) and macrophages, and its subsequent weakening or erosion seems to be an important regulator of plaque stability. The aim of our study was to improve our knowledge on the biological mechanisms that cause plaque instability in order to develop new therapies to maintain atherosclerotic plaque stability and avoid its rupture. In our study, we collected surgical specimens from atherosclerotic plaques in the right or left internal carotid artery of 62 patients with evident clinical symptoms. Histopathology and histochemistry were performed on wax-embedded sections. Immunohistochemical localization of caspase-3, N-cadherin and ADAM-10 was undertaken in order to highlight links between apoptosis, as expressed by caspase-3 immunostaining, and possible roles of N-cadherin, a cell-cell junction protein in VSMCs and macrophages that provides a pro-survival signal reducing apoptosis, and ADAM-10, a “disintegrin and metalloproteases” that is able to cleave N-cadherin in glioblastomas. Our results showed that when apoptosis, expressed by caspase-3 immunostaining, increased in the fibrous cap, rich in VSMCs and macrophages, the expression of N-cadherin decreased. The decreased N-cadherin expression, in turn, was linked to increased ADAM-10 expression. This study shows that apoptotic events are probably involved in the vulnerability of atherosclerotic plaque.     

Dual-wavelength multifrequency photothermal wave imaging combined with optical coherence tomography for macrophage and lipid detection in atherosclerotic plaques using gold nanoparticles

The objective of this study was to assess the ability of combined photothermal wave (PTW) imaging and optical coherence tomography (OCT) to detect, and further characterize the distribution of macrophages (having taken up plasmonic gold nanorose as a contrast agent) and lipid deposits in atherosclerotic plaques. Aortas with atherosclerotic plaques were harvested from nine male New Zealand white rabbits divided into nanorose- and saline-injected groups and were imaged by dual-wavelength (800 and 1210 nm) multifrequency (0.1, 1 and 4 Hz) PTW imaging in combination with OCT. Amplitude PTW images suggest that lateral and depth distribution of nanorose-loaded macrophages (confirmed by two-photon luminescence microscopy and RAM-11 macrophage stain) and lipid deposits can be identified at selected modulation frequencies. Radiometric temperature increase and modulation amplitude of superficial nanoroses in response to 4 Hz laser irradiation (800 nm) were significantly higher than native plaque (P<0.001). Amplitude PTW images (4 Hz) were merged into a coregistered OCT image, suggesting that superficial nanorose-loaded macrophages are distributed at shoulders on the upstream side of atherosclerotic plaques (P<0.001) at edges of lipid deposits. Results suggest that combined PTW-OCT imaging can simultaneously reveal plaque structure and composition, permitting characterization of nanorose-loaded macrophages and lipid deposits in atherosclerotic plaques.     

人体冠状动脉粥样硬化斑块中氧化低密度脂蛋白和新生血管与斑块稳定性的关系

目的探讨氧化低密度脂蛋白(oxLDL)在人体冠脉稳定斑块和不稳定斑块内的分布、与血管新生之间的关系及其在不稳定斑块形成过程中的可能作用。方法选取经临床和病理解剖检查确诊的急性冠脉综合征(ACS)病例进行免疫组织化学SABC法染色并通过计算机图像分析系统进行量化分析。结果(1)不稳定斑块组内各部位oxLDL的聚集程度均明显高于稳定斑块组。oxLDL主要分布于斑块的肩部,纤维帽及基底部相对较少。oxLDL阳性面积比(‰)分别为肩部:不稳定斑块组20.43±3.12,稳定斑块组17.65±4.22;基底部:不稳定斑块组5.65±1.65,稳定斑块组3.22±1.02;纤维帽:不稳定斑块组4.77±2.03,稳定斑块组2.80±0.22。oxLDL是脂质核心的主要组成部分。(2)斑块肩部Ⅷ因子与oxLDL的阳性面积比呈明显正相关(r=0.8247,P=0.000)。结论不稳定斑块组较稳定斑块组oxLDL的聚集量高,尤其是肩部oxLDL的分布明显高于稳定组,提示斑块内oxLDL含量的多少及分布区域是影响斑块稳定性的重要因素。oxLDL通过促进炎细胞经新生血管在斑块局部聚集而影响斑块的稳定性。     

Phenotypic variants of the smooth-muscle cells in an atheromatous plaque of the human aorta

The authors investigated the expression of cytoskeletal proteins and the ultrastructure of cells in normal intima and atheromatous plaque of human aorta. It has been established, using double immunofluorescent method and a set of antibodies that intimal smooth muscle cells /SMC/ of normal aorta express myosin, vimentin, alpha-actin and actin but not desmin. In seven out of 28 atherosclerotic plaques the cells contained desmin and all other SMC cytoskeletal proteins were found. These cells had the ultrastructural features of SMC, i.e. well-developed endoplasmic reticulum and Golgi apparatus. Besides, some cells in 13 atherosclerotic plaques proved to be myosin-, alpha-actin- and desmin-negative. The cells were stained with monoclonal antibodies specific to SMC but not with macrophage-specific antibody. Ultrastructurally, the cytoplasm of the cells was filled with rough endoplasmic reticulum and a developed Golgi complex, but a certain portion of the cells retained basal lamina and myofilament bundles. The peculiarities of cytoskeletal protein in expression and ultrastructure of cells in human aortic atherosclerotic plaques may be explained by a phenotypic modulation of vascular SMC.     

Macrophage differentiation in atherosclerosis. An in situ immunohistochemical analysis in humans.

The differentiation of macrophages present in diffuse intimal thickening, fatty streaks, and atheromatous plaques, was analyzed with immunohistochemical methods, using segments of aorta, coronary, and carotid arteries obtained at autopsy. Various differentiation antigens were studied with the monoclonal antibodies anti-HLA-DR, EBM-11, Leu M3, OKM1, and OKM5. Adjacent sections were stained for lipids (oil red O) and lysosomal activity (acid phosphatase). Almost all macrophages identified with the pan-macrophage antibody EBM-11, also stained with the anti-HLA-DR antibody. Diffuse intimal thickening showed a predominance of Leu M3+ cells; fatty streaks also showed OKM1+ and OKM5+ macrophages. Classical atheromatous plaques showed a gradual shift in phenotypic expression towards the center of the lesion. Cells in the superficial layers were positive only with Leu M3, deeper localized cells showed double expression of Leu M3 and OKM1 or double expression of OKM1 and OKM5. Cells that were localized adjacent to the atheromatous debris stained only with OKM5. The phenotypic changes occurred in parallel with an increase in both fat uptake and lysosomal activity of the macrophages. This shift in phenotypic expression suggests a process of differentiation and maturation of the macrophages involved. The results indicate that macrophages within the arterial intima are activated and mature towards cells that express receptors for adhesion proteins and complement during the development of atherosclerotic plaques. This may imply that the macrophages involved in lipid metabolism also have a potential to act as effector cells in a chronic inflammatory process, and thus, may contribute to the progression of an atherosclerotic plaque. Functional studies of macrophage subpopulations are needed to verify this hypothesis.     

Human intracranial atherosclerosis

Summary Morphology of atheromatous plaques from human intracranial arteries was examined by light and electron microscopy. Overall morphology of lesions did not differ from those reported for other arterial beds. In the presence of an intact elastic membrane, lipid cores were localized only to the intima and covered with a fibromuscular cap. Lipid-filled cells at the periphery resembled smooth muscle cells or blood monocytes, but within the atheromatous core took the form of macrophages filled with lipid crystals and variable-density lipid droplets. Calcium crystallized spherules were localized to the surface of translucent lipid crystals which themselves were seen to coalesce with large amorphous lipid droplets. This study together with previous studies on cerebral arteries suggest that fatty streak lesions may undergo transformation to atheromatous plaques. Similarity in structure of atherosclerotic plaques between cerebral and other arterial beds suggests that documented differences in susceptibility to atheroma of various vascular beds in relation to age cannot be resolved by morphological studies alone.     

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.