Michael E. Rosenfeld

A Definition of Advanced Types of Atherosclerotic Lesions and a Histological Classification of Atherosclerosis A Report From the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association

This report is the continuation of two earlier reports that defined human arterial intima and precursors of advanced atherosclerotic lesions in humans. This report describes the characteristic components and pathogenic mechanisms of the various advanced atherosclerotic lesions. These, with the earlier definitions of precursor lesions, led to the histological classification of human atherosclerotic lesions found in the second part of this report. The Committee on Vascular Lesions also attempted to correlate the appearance of lesions noted in clinical imaging studies with histological lesion types and corresponding clinical syndromes. In the histological classification, lesions are designated by Roman numerals, which indicate the usual sequence of lesion progression. The initial (type 1) lesion contains enough atherogenic lipoprotein to elicit an increase in macrophages and formation of scattered macrophage foam cells. As in subsequent lesion types, the changes are more marked in locations of arteries with adaptive intimal thickening. (Adaptive thickenings, which are present at constant locations in everyone from birth, do not obstruct the lumen and represent adaptations to local mechanical forces). Type II lesions consist primarily of layers of macrophage foam cells and lipid-laden smooth muscle cells and include lesions grossly designated as fatty streaks. Type III is the intermediate stage between type II and type IV (atheroma, a lesion that is potentially symptom-producing). In addition to the lipid-laden cells of type II, type III lesions contain scattered collections of extracellular lipid droplets and particles that disrupt the coherence of some intimal smooth muscle cells. This extracellular lipid is the immediate precursor of the larger, confluent, and more disruptive core of extracellular lipid that characterizes type IV lesions. Beginning around the fourth decade of life, lesions that usually have a lipid core may also contain thick layers of fibrous connective tissue (type V lesion) and/or fissure, hematoma, and thrombus (type VI lesion). Some type V lesions are largely calcified (type Vb), and some consist mainly of fibrous connective tissue and little or no accumulated lipid or calcium (type Vc).

Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein.

Increasing evidence indicates that low density lipoprotein (LDL) has to be modified to induce foam cell formation. One such modification, oxidation of LDL, generates a number of highly reactive short chain-length aldehydic fragments of oxidized fatty acids capable of conjugating with lysine residues of apoprotein B. By immunizing animals with homologous malondialdehyde-modified LDL (MDA-LDL), 4-hydroxynonenal-LDL (4-HNE-LDL), and Cu+(+)-oxidized LDL, we developed polyvalent and monoclonal antibodies against three epitopes found in oxidatively modified LDL. The present article characterizes an antiserum and monoclonal antibody (MAL-2 and MDA2, respectively) specific for MDA-lysine, and an antiserum and monoclonal antibody (HNE-6 and NA59, respectively) specific for 4-HNE-lysine. In addition, a monoclonal antibody (OLF4-3C10) was developed against an as yet undefined epitope generated during Cu++ oxidation of LDL. With these antibodies, we demonstrated that MDA-lysine and 4-HNE-lysine adducts develop on apo-lipoprotein B during copper-induced oxidation of LDL in vitro. The application of these antibodies for immunocytochemical demonstration of oxidized lipoproteins in atherosclerotic lesions of progressive severity is described in the companion article. These antibodies should prove useful in studying the role of oxidatively modified lipoproteins as well as other oxidatively modified proteins in atherogenesis.

Distribution of oxidation specific lipid-protein adducts and apolipoprotein B in atherosclerotic lesions of varying severity from WHHL rabbits.

Antisera and monoclonal antibodies generated against autologous malondialdehyde-conjugated low density lipoprotein (MDA-LDL), 4-hydroxynonenal conjugated LDL (4-HNE-LDL), and the protein fragments of apoprotein B resulting from the copper oxidation of LDL, as well as antibodies against apoprotein B, were used to immunostain atherosclerotic lesions of varying severity from Watanabe heritable hyperlipemic rabbits. In macrophage-rich fatty streaks and transitional lesions, all of the antibodies recognizing oxidation specific epitopes exhibited predominantly cell-associated staining in particulate and annular patterns. This is in contrast to the limited, extracellular, diffuse staining obtained with the antibodies recognizing apoprotein B. In more advanced lesions containing areas with reduced numbers of cells, there was increased extracellular, diffuse staining with the antibodies against oxidation specific epitopes and co-localization with apoprotein B. In addition, there were annular staining patterns associated with the necrotic core and increased staining of intimal and medial smooth muscle cells. We interpret these data as suggesting that in areas of lesions rich in macrophages, LDL is oxidized and taken up by the cells. In more advanced lesions that are relatively devoid of macrophages, both native and oxidized LDL, as well as oxidation products released from dead and decaying cells, are trapped in the matrix, out of reach of those cells capable of accumulating oxidized LDL.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Advanced Atherosclerotic Lesions in the Innominate Artery of the ApoE Knockout Mouse

Abstract—Most previous studies of atherosclerosis in hyperlipidemic mouse models have focused their investigations on lesions within the aorta or aortic sinus in young animals. None of these studies has demonstrated clinically significant advanced lesions. We previously mapped the distribution of lesions throughout the arterial tree of apolipoprotein E knockout (apoE−/−) mice between the ages of 24 and 60 weeks. We found that the innominate artery, a small vessel connecting the aortic arch to the right subclavian and right carotid artery, exhibits a highly consistent rate of lesion progression and develops a narrowed vessel characterized by atrophic media and perivascular inflammation. The present study reports the characteristics of advanced lesions in the innominate artery of apoE−/− mice aged 42 to 60 weeks. In animals aged 42 to 54 weeks, there is a very high frequency of intraplaque hemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap. By 60 weeks of age, the lesions are characterized by the presence of collagen-rich fibrofatty nodules often flanked by lateral xanthomas. The processes underlying these changes in the innominate artery of older apoE−/− mice could well be a model for the critical processes leading to the breakdown and healing of the human atherosclerotic plaque.

Fatty streak initiation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits.

Morphologic and immunocytochemical studies were conducted to determine the sequence of cellular interactions that occur during the initiation of the fatty streak in the aorta of Watanabe Heritable Hyperlipemic rabbits and comparably hypercholesterolemic fat-fed rabbits. Watanabe rabbits from 3.5 weeks gestation to 2 months of age and fat-fed rabbits from 1 week to 2 months duration of hypercholesterolemia were compared utilizing light microscopic and scanning and transmission microscopic techniques. In both groups of animals, the earliest detectable events were an increase in monocyte adherence and subendothelial migration followed by formation of a single layer of intimal macrophage-derived foam cells. Immunocytochemical studies using macrophage-specific and muscle-actin-specific monoclonal antibodies support the morphologic data which suggests that the early fatty streak in both the Watanabe and fat-fed rabbits is predominantly composed of macrophage-derived foam cells. Thus, the absence of functional low density lipoprotein receptors in the Watanabe rabbit and differences in the distribution of cholesterol among the lipoproteins in the Watanabe and fat-fed rabbits do not appear to alter the initial responses of the cells of the artery wall to chronic hypercholesterolemia.

Macrophage and smooth muscle cell proliferation in atherosclerotic lesions of WHHL and comparably hypercholesterolemic fat-fed rabbits.

Lesions of atherosclerosis were analyzed at varying stages of development in Watanabe heritable hyperlipidemic (WHHL) and comparably hypercholesterolemic fat-fed (FF) rabbits for the capacity to incorporate thymidine into proliferating cells within both the intima and the underlying media. An identical inverse relationship between the intimal/medial ratio (which reflects both lesion size and severity) and the labeling index was observed for both the WHHL and FF rabbits. Analysis of the spatial distribution of the labeled cells within each lesion revealed the highest rates of thymidine incorporation in cells situated within the superficial areas and lateral margins of the lesions. Up to 12% of the labeled cells were foam cells, which were predominantly located immediately beneath the endothelium and within the lateral margins. Some labeled macrophages were also observed within the necrotic core of advanced lesions. There were no differences in the labeling indexes at differing sites in the aorta, providing lesions of comparable size were compared. Simultaneous thymidine autoradiography and immunostaining with cell type-specific monoclonal antibodies revealed that approximately 30% of the labeled cells were macrophages and 45% were smooth muscle cells in advanced lesions from both WHHL and FF rabbits.

Chlamydia pneumoniae Infection Accelerates the Progression of Atherosclerosis in Apolipoprotein E—Deficient Mice

Accumulating evidence supports an association between Chlamydia pneumoniae infection and atherosclerosis. To determine whether there is a causal relationship, the effects of chronic infection with C. pneumoniae on the development of atherosclerosis in apolipoprotein E (apoE)-deficient mice were evaluated. Eight-week-old male apoE-deficient mice were inoculated intranasally with C. pneumoniae three times, at 8, 9, and 10 weeks of age. The combined area of atherosclerotic lesions in the lesser curvature of the aortic arch was measured en face by computer-assisted morphometry. The lesion area was 2.4-fold greater (P=.05) at 16 weeks of age and 1.6-fold greater (P=.05) at 20 weeks of age in infected mice than in control mice. There were no differences in total plasma cholesterol levels between groups. This study demonstrates that C. pneumoniae infection accelerates the progression of atherosclerosis in the aortic arch of apoE-deficient mice.

A definition of the intima of human arteries and of its atherosclerosis-prone regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.

T his report is a concise review of current knowledge of the structure and function of the intima of the aorta and the major distributing arteries. The main purpose of the review is to delineate normal arterial intima from atherosclerotic lesions and, in particular, to distinguish physiological adaptations from atherosclerotic increases in intimal thickness. To characterize normal intima, including the adaptive intimal thickenings, some of which represent locations in which atherosclerotic lesions are prone to develop, the structure, composition, and functions of the arterial intima in young people as well as in laboratory animals not subjected to known atherogenic stimuli are reviewed. This report on arterial intima is the first in a series of four. The second report will review and define initial, fatty streak, and intermediate types of atherosclerotic lesions, and the third report will review all types of advanced (i.e., potentially clinical and clinical) lesions. The overall objective is to define arterial intima and all types of atherosclerotic lesions, and then to postulate, in a fourth and final report, a valid and up-to-date pathobiological nomenclature and classification of atherosclerotic lesions.