Experimental Induction of Atheroarteriosclerosis by the Synergy of Allergic injury to Arteries and Lipid-Rich Diet: II. Effect of Repeatedly Injected Foreign Protein in Rabbits Fed a Lipid-Rich, Cholesterol-Poor Diet

Rabbits fed a lipid-rich, cholesterol-poor diet and given concomitant injections of foreign protein, over a period as long as 17 months, developed in their coronary arteries both a) proliferative fibromuscular intimal thickening closely resembling the diffuse intimal thickening that commonly occurs in coronary arteries of man, and b) fatty-proliferative fibromuscular intimal thickening that closely resembles coronary atherosclerosis in man. In contrast, rabbits of another group that were concurrently fed the same diet for as long as 22 months without injections of foreign protein developed changes in arteries of their hearts that resemble neither coronary atherosclerosis nor diffuse intimal thickening in man. Fatty-proliferative changes in aortas of the first group of rabbits are strikingly greater and more closely resemble human aortic atherosclerosis than those in the latter group. In the course of the experiments, the average serum cholesterol was not significantly different in the two groups of rabbits. It was approximately 200 to 250 mg%, which is the average serum cholesterol in adult humans in the United States. These experiments support the hypothesis that the synergy of arterial injury, in particular immunologic injury, and a diet rich in lipid can lead to atherosclerosis in man.     

Experimental Induction of Atheroarteriosclerosis by the synergy of Allergic Injury to Arteries and Lipid-Rich Diet: III. The Role of Earlier Acquired Fibromuscular Intimal Thickening in the Pathogenesis of Later Developing Atherosclerosis

Clinicopathologic evidence suggests that diffuse intimal thickening, a type of arteriosclerosis without manifest lipid deposit, may predispose to later developing atherosclerosis in man. This hypothesis was tested in the following experiments. Injury to coronary arteries of rabbits was induced by immunologic means, and arterial lesions were allowed to heal for many weeks. One group of animals was then sacrificed, and in their coronary arteries were found numerous fibromuscular intimal lesions closely resembling diffuse intimal thickening in man. The remaining rabbits were fed a cholesterol-supplemented diet for 80 days and then sacrificed. Fibromuscular intimal lesions of coronary arteries were found in these rabbits also. However, approximately two-thirds of these lesions were found to contain lipid, and many closely resembled coronary atherosclerosis in man. Further analysis of the data indicates that the atherosclerotic lesions in the rabbits evolved from immunologically induced fibromuscular intimal lesions which later and preferentially accumulated lipid in the presence of hypercholesterolemia. Results of these experiments suggest that in man fibromuscular intimal lesions, and in particular diffuse intimal thickening, acquired earlier in life can later accumulate lipid preferentially and thus redispose to atherosclerosis.     

The importance of a substantial elastic lamina subjacent to the endothelium in limiting the progression of atherosclerotic changes

This study examines the hypothesis that progressive intimal thickening and atherosclerosis in the larger pulsatile arteries arise from failure to maintain, subjacent to the endothelial cells, a substantial elastin membrane, a component which has been shown to be of special structural significance. The internal thoracic arteries of 293 subjects of all ages up to 60 years were compared histologically with the anterior descending coronary arteries of the same individuals by light- and electronmicroscopy and immunoperoxidase staining for macromolecules. The internal thoracic arteries usually developed a new robust reduplicated internal elastic lamina at an early age, no further intimal thickening, and no significant entry of lipid or cells to the intima. The coronary arteries showed areas of rapid intimal thickening with poor and incomplete reduplicated internal elastic laminae, entry of lipid, macrophages, and other cells to the intima. The reduplicated internal elastic laminae appeared to be formed primarily by the endothelial cells themselves. An elastin membrane subjacent to the endothelial cells appears to be essential. It provides a secure attachment for the cells and a barrier to the entry of macromolecules and cells to the intima. Its absence is associated with progressive intimal thickening and atherosclerosis.     

Preliminary and early stages of atherosclerosis in childhood

According to the unified theory of atherosclerosis, endothelial cell injury and lipid infiltration play an important role in atherogenesis. Newborn babies may suffer endothelial cell damage, as may be detected by electron microscopy. Connective tissue elements are occasionally abundant already in newborns. Chondroitin sulfate A and C increase with age. The children may exhibit continuous accumulation of cholesterol esters in the intima of coronary arteries. Cholesteryl ester fatty acid composition, along with age, tends to approach that of serum low-density lipoproteins. Fatty streaks appear in coronary arteries in puberty, and fibrous plaques are recordable beyond the age of 20 years. The topography of myo-intimal thickenings, fatty streaks, and fibrous plaques is similar to complicated atherosclerotic lesions. Even newborn babies have obstructive myo-intimal thickenings in their coronary arteries. One fifth of all infants under one week of age suffer 20% stenosis, with percentile manifestation of stenosis in the arterial cross-section being established as ratio of intimal area to luminal area of a dilated coronary artery multiplied by 100. Occasionally, the intima is very thick, in our series initiating up to 57% of all narrowing. There are probably noxious factors which temporarily damage the endothelial cells and initiate a rapid, partially reversible thickening reaction. Some of this response of the intima to exogenous stimuli might be genetically determined. A thickened intima is susceptible to lipid deposition and atherosclerosis.     

Pathology of myocardial infarction,coronary artery disease,plaque disruption,and the vulnerable atherosclerotic plaque

Despite scientific advances, cardiovascular disease remains the leading cause of death in developed countries. The pathologic process responsible for the majority of this mortality is atherosclerosis. Human atherosclerosis is characterized by the transition of arteries through distinct pathologic stages. Initially, there is vascular wall activation, characterized by the formation of a smooth muscle cell rich intimal hyperplasia/thickening. The thickened intima promotes the lipid and macrophage accumulation characteristic of atherosclerosis. In some patients, the atherosclerotic plaque becomes disrupted stimulating formation of luminal thrombus and acute clinical events. Understanding the pathology of such vulnerable plaques has been a challenging and controversial area of investigation. Recent prospective longitudinal imaging studies of human coronary arteries have confirmed earlier pathologic observations reporting pathologic features that predispose to acute events in some patients include plaques with a thin fibrous cap overlying a large lipid-rich necrotic core as well as plaques with severe stenosis.     

Nogo‐B expression,in arterial intima,is impeded in the early stages of atherosclerosis in humans

Nogo‐B (Reticulon 4B) is considered to be a novel vascular marker, which may have a protective role in injury‐induced neointima formation and atherosclerosis. Nogo A/B is found to be crucial for monocyte/macrophage recruitment in acute inflammation and it is expressed in CD68 + macrophages. We hypothesize that macrophage infiltration in atherosclerosis is not dependent on Nogo‐B expression in arterial wall. We have assessed Nogo‐B expression and macrophage accumulation in the iliac arteries of healthy organ donors and organ donors with cardiovascular risk factors. Paraffin sections of 66 iliac arteries, from 44 deceased organ donors (17 women and 27 men), were studied. The healthy and cardiovascular risk (CVR) subgroups were created. With regard to staging of the atherosclerotic process, the thickness of arterial intima was measured in digitalized images of H+E stained tissue sections. Immunohistochemical reactions (Nogo‐B and CD68) were carried out in all arteries (66 samples). Western blotting (WB‐19 samples) and real‐time PCR (27 samples) were performed on selected arteries. Significantly higher Nogo‐B expression was demonstrated in the intima of the healthy subjects' subgroup, using immunohistochemistry. WB and real‐time PCR revealed a trend toward lower Nogo‐B expression in the adventitia of the CVR subgroup. Furthermore, the thickness of the intima was found to negatively correlate with the expression of Nogo‐B in the intima and media (r = ?0.32; p < 0.05; r = ?0.32; p < 0.05). Macrophage infiltrates were more prominent in intima of CVR subjects (0.65 vs 3.52 a.u.; p < 0.01). Macrophage density in intima increased with atherosclerosis progression (r = 0.37; p < 0.01). CD68 macrophages density in adventitia was lower in CVR arteries than in healthy arteries. The expression of Nogo‐B, in arterial intima, is impeded in the early stages of atherosclerosis. Accumulation of arterial intimal CD68 macrophages has been shown to progress; however, the overall macrophage density in the adventitia is reduced in arteries shown to have intimal thickening. Macrophage infiltration is not accompanied by Nogo‐B expression in atherosclerotic arteries.     

Atherosclerosis in Macaca mulatta: Histopathological,morphometric, and histochemical studies in aorta and coronary arteries of spontaneous and induced atherosclerosis

A comparative study of morphology, size, and histochemistry of the intimal lesions in aorta and coronary arteries of spontaneously occurring and cholesterol-induced atherosclerosis in rhesus monkeys has been carried out. A group of 30 normal monkeys was also investigated. Spontaneous atherosclerosis was noted in 10 of 55 adult monkeys autopsied serially; fatty streaks or atheroma in the aorta was noted in seven, fibrous plaque was noted in two, and diffuse intimal thickening was observed on one animal only. The coronary arteries showed fibrous intimal thickening without lipid in 8 of these 10 monkeys. There was fair to heavy deposition of acid mucopolysaccharides in the thickened intima along with proliferation of myointimal cells and collagen fibers. In the seven monkeys which were fed an atherogenic diet for 6 months, the aorta showed fatty streaking and atheroma in all animals. The coronary arteries also showed a variable degree of atherosclerosis but the lipid in the thickened intima was not marked. The atherosclerotic plaque height was not significantly greater than that in the spontaneous disease. These differences between spontaneously occurring and cholesterol-induced atherosclerosis in monkeys tend to indicate that the basic mechanism of lesion formation in the two states may be different.     

Atherosclerosis and aging. A morphometric study on arterial lesions of elderly and very elderly necropsy subjects

A histomorphometric study was performed on arterial wall lesions of different arterial regions (arch, thoracic, abdominal parts of the aorta; right and left common and internal carotid arteries; coronary arteries; and basilar and middle cerebral arteries), collected from 108 elderly and very elderly (greater than 90 years of age) subjects who underwent necropsy. Lumen stenosis percentage, mean intimal thickening, and mean thickness of the media were measured by means of a computerized system using a manual input and graphic printout; statistical tests were performed using variance and regression analysis. Results showed that the arterial wall lesions in patients over 60 years of age tend to be stabilized in all regions studied except for the cerebral region, where the lesions tend to have a continuous progression related to age, even in a group of subjects over 90 years of age.     

Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis

BackgroundAtherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia. Advanced coronary lesions with lipid cores are associated with adventitial inflammation, but the early inflammatory process in human coronary adventitia is largely unknown. We hypothesized that adventitial inflammatory cell infiltration accompanies the early stages of atherogenesis in human coronary arteries, and it is synchronous with the inflammatory process in the intima.MethodsCoronary artery samples were obtained from 111 forensic autopsy cases aged from 7 to 25 years. Adventitial and intimal macrophages, T lymphocytes and B lymphocytes, and intimal microvessels were detected by immunohistochemical methods and quantified by computerized image analysis. Body height, weight, waist circumference, and the size of mesenteric and omental fat depots were measured.ResultsAdventitial densities of macrophages and T lymphocytes were significantly higher in arteries showing intimal xanthomas than in cases with only scattered intimal macrophages. The xanthoma group also had significantly higher body mass index and larger visceral fat depots. Highest densities of all adventitial cell types were seen in intermediate lesions and fibroatheromas. There were significant positive correlations between intimal and adventitial densities of T cells and B cells in the groups with or without intimal xanthomas, but the positive correlation between intimal and adventitial macrophages was significant only in the group without xanthomas.ConclusionsAdventitial immune-inflammatory cell accumulation accompanies the early stages of coronary atherogenesis in young individuals, and lymphocyte accumulation seems to be synchronous in the intima and adventitia. Macrophage accumulation is also synchronous before xanthomas are seen.     

Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase.

Apolipoprotein E (apo E) mediates both lipid accumulation by and removal from cells and may be secreted by both macrophages and smooth muscle cells in vitro, but its cellular source in atherosclerotic plaques is not known. Lipoprotein lipase (LPL) also enhances cell lipid accumulation and is synthesized by macrophage foam cells in atherosclerotic plaques. To determine the cellular source of apo E in human coronary atherosclerotic lesions and its relationship to LPL synthesis, in situ hybridization and immunohistochemistry were performed on 12 atherosclerotic plaques and six nondiseased coronary artery segments from 10 cardiac transplant recipients. Apo E messenger RNA was localized to both non-foam cell and foam cell macrophages in plaques, but not to other cell types, and was not detected in nonatherosclerotic arteries. Half of the regions with non-foam cell macrophages expressed neither apo E nor LPL messenger RNA, whereas 86% of macrophage foam cell-containing regions contained both messenger RNAs. Polyclonal antisera raised against human apo E localized apo E protein to the surface of macrophages and surrounding matrix in plaques but not in control coronary segments. An LPL-specific monoclonal antibody demonstrated that, similar to apo E, LPL protein on foam cell and non-foam cell macrophages was detected in atherosclerotic lesions, but LPL was also localized to intimal muscle smooth muscle cells and was not distributed as widely in association with matrix as was apo E. The expression of both apo E and LPL in atherosclerotic lesions but not in normal intima suggest that these molecules play a role in lipid metabolism in atherosclerosis.     

Theoretical study of the effect of local flow disturbances on the concentration of low-density lipoproteins at the luminal surface of end-to-end anastomosed vessels

To elucidate the mechanisms of localisation of intimal hyperplasia in anastomosed arteries, the effects of flow disturbances on the transport of lowdensity lipoproteins (LDLs) from the flowing blood to the wall of end-to-end anastomosed arteries, with and without a moderate stenosis, were studied theoretically by means of a computer simulation under the condition of steady flow. In an artery with moderate stenosis at the anastomotic junction and intimal thickening distal to it, we found that, owing to the water-permeable nature of the arterial wall, the surface concentration of LDL was elevated up to 20% higher than that of the bulk flow distal to the stenosis, where a recirculation zone was formed and wall shear stresses were low. In contrast to this, no significant elevation of surface concentration of LDLs occurred in another anastomosed vessel in which no stenosis was formed and no intimal thickening was observed. These results suggest that flowdependent concentration polarisation of LDLs plays a causative role in the localisation of anastomotic intimal hyperplasia in the human arterial system by locally elevating the surface concentration of LDLs, thus augmenting their uptake by endothelial cells.     

CORONARY ATHEROSCLEROSIS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AT AUTOPSY

Using a computed image analyzer, coronary arteries from 50 autopsied patients with systemic lupus erythematosus (SLE) were examined on the three vessels (RCA, LAD, LCX) and compared with those of age-matched controls. The intima of coronary artery was significantly thickened much more in the case of SLE than in the case of age-matched controls. This was statistically significant (p<0.01). Hypertension and glomerulonephritis did not but corticosteroid therapy had an influence on the development of intimal thickening ratio of the coronary arteries in SLE patients. The mean intimal thickening ratio of the coronary arteries in the patients with SLE and without corticosteroid therapy was larger than that of patients with corticosteroid therapy (p<0.1). It appears possible to conclude that inflammatory change of SLE itself is one of the promoting factors of coronary atherosclerosis.     

Chlamydia pneumoniae in different coronary artery segments in the young

Chlamydia pneumoniae has emerged as the most likely pathogen to have a causative role in the development and/or for progression of atherosclerosis. Evidence for this is based on epidemiological and pathological studies. In an effort to better understand the significance of finding C. pneumoniae in atheromata, we examined coronary artery segments of young adults (15-34 years) with and without atherosclerosis. Left anterior descending coronary arteries (LAD) of 74 young adults who died suddenly were examined histologically and for the presence of C. pneumoniae by immunohistochemistry. C. pneumoniae was identified in advanced lesions (Stary types III to VI) in 17 of 32 cases (53%), and in early lesions (Stary type I-II) in 8 of 37 cases (21%), mainly at the proximal segments of the LAD. C. pneumoniae was not found in the intimal and medial layer of normal-appearing coronary arteries. C. pneumoniae was detected in the adventitia in 51 (67%) coronary arteries: in 27 of normal arteries and early lesions (64%), and in 24 of atherosclerotic lesions (75%). C. pneumoniae was found most often in macrophages, less offen in smooth muscle cells. We also observed a correlation between C. pneumoniae positivity and cigarette smoking. In conclusion: C. pneumoniae may relate to the severity of atherosclerosis in young people, and it may thus initiate atherosclerotic injury or facilitate its progression with other risk factors.     

Histopathological Characteristics of Post-inflamed Coronary Arteries in Kawasaki Disease-like Vasculitis of Rabbits

Kawasaki disease (KD) is a systemic vasculitis in infants that develops predominantly in the coronary arteries. Despite the clinically transient nature of active inflammation in childhood albeit rare complications (e.g., coronary artery aneurysm), KD has recently been suggested to increase the incidence of ischemic heart diseases in young adulthood. However, little is known about the histopathology of the coronary artery long after development of the acute KD vasculitis. To address this, we conducted histological studies of rabbit coronary arteries in adolescent phase after induction of the KD-like vasculitis induced by horse serum administration. After a transmural infiltration of inflammatory cells in acute phase at day 7, the artery exhibited a gradual decrease in the number of inflammatory cells and thickening of the intima during the chronic phase up to day 90, where proteoglycans were distinctly accumulated in the intima with abundant involvement of α-smooth muscle actin (α-SMA)-positive cells, most of which accompanied expression of VCAM-1 and NF-κB. Distinct from classical atherosclerosis, inflammatory cells, e.g., macrophages, were barely detected during the chronic phase. These observations indicate that the KD-like coronary arteritis is followed by intimal thickening via accumulation of proteoglycans and proliferation of α-SMA-positive cells, reflecting aberrant coronary artery remodeling.     

Coronary atherosclerosis in patients with systemic lupus erythematosus at autopsy

Using a computed image analyser, coronary arteries from 50 autopsied patients with systemic lupus erythematosus (SLE) were examined on the three vessels (RCA, LAD, LCX) and compared with those of age-matched controls. The intima of coronary artery was significantly thickened much more in the case of SLE than in the case of age-matched controls. This was statistically significant (p less than 0.01). Hypertension and glomerulonephritis did not but corticosteroid therapy had an influence on the development of intimal thickening ratio of the coronary arteries in SLE patients. The mean intimal thickening ratio of the coronary arteries in the patients with SLE and without corticosteroid therapy was larger than that of patients with corticosteroid therapy (p less than 0.1). It appears possible to conclude that inflammatory change of SLE itself is one of the promoting factors of coronary atherosclerosis.     

Coronary atherosclerotic lesions in human immunodeficiency virus–infected patients: a histopathologic study

BackgroundStudies suggest human immunodeficiency virus–positive (HIV+) patients have an increased risk of coronary artery disease (CAD), yet little is known about the histopathology, severity, or distribution of lesions.MethodsThe coronary arteries of 66 deceased AIDS patients and 19 HIV controls (age <55) were dissected and graded for percent luminal stenosis by intimal lesions, percent of intima involved with lipid, and extent of intimal calcification on a scale of 0 to 3. Medical histories, antiretroviral therapies, and CAD risk factors were reviewed.ResultsHIV+ patients were older than controls (P=.06), and more were male (P=.02). Thirty-five percent of HIV+ patients had stenosis ≥75% of at least one artery. Compared to controls, HIV+ patients had three times greater odds of stenosis ≥75%, controlling for age and sex (one-sided P=.03). Older age and male sex were also risk factors (one-sided P<.001). HIV seropositivity was associated with increased plaque lipid content (one-sided P=.02) and calcification (one-sided P=.08). Duration of HIV infection, antiretroviral therapy, and immune status did not predict severe disease in multivariate analyses. Previously unreported patterns of dystrophic calcification were observed in HIV+ patients and older controls.ConclusionsYoung to middle-aged patients dying from advanced AIDS have atherosclerotic CAD that may result in luminal narrowing, heavy calcification, and high plaque lipid content. The pattern of disease, location of lesions, and plaque composition are typical of atherosclerosis in HIV-negative patients. No relationship between antiretroviral therapies and atherosclerosis was seen in this small study of heavily treated patients.     

A comparison of stent-induced stenosis in coronary and peripheral arteries

BACKGROUND AND OBJECTIVES: Restenosis is a complication of interventional procedures such as angioplasty and stenting, often limiting the success of these procedures. Knowledge regarding the relative behaviour of different arteries after these procedures is limited, despite the extensive use of different vascular models. Although the results from studies using different vessels are analysed to predict the behaviour of coronary arteries and other vasculature, direct controlled comparisons between different arteries are necessary for a better understanding of the differential response to restenosis. METHODS: This study examines the response to stenting in coronary and internal iliac arteries as characterised by intimal hyperplasia and restenosis. In a swine model of in-stent stenosis, coronary arteries exhibited higher levels of intimal hyperplasia and per cent stenosis than internal iliac arteries. RESULTS: After normalisation for injury score, coronary arteries were found to undergo 47% more intimal hyperplasia (p<0.05), whereas per cent stenosis normalised for injury score tended to be higher (p = 0.01). Other measurements reflecting post-stenting intimal hyperplasia (maximal intimal thickness, medial area) did not exhibit significant differences between the artery groups. CONCLUSIONS: These results show that coronary vessels are more prone to develop significant intimal hyperplasia and subsequent restenosis than internal iliac vessels. A better insight into how different arteries and arterial components behave is important in understanding and developing newer and better therapeutic measures for restenosis.     

Alterations of endothelin-converting enzyme expression in early and advanced stages of human coronary atherosclerosis

Endothelin-1 is a potent vasoconstrictor and exhibits a mitogenic activity on vascular smooth muscle cells (SMCs). Endothelin-converting enzyme (ECE) is the final key enzyme of endothelin-1 processing. We studied the immunolocalization of ECE in human coronary atherosclerotic lesions with different disease stages. Frozen sections of normal coronary arteries with diffuse intimal thickening (n=13) and those of coronary arteries with early (n=10) or advanced atherosclerotic plaques (n=13) were studied. Monoclonal antibodies used were directed against SMCs, macrophages, endothelial cells, and ECE. For the identification of cell types that express ECE, double immunostaining analysis was also used. In normal coronary arteries, ECE immunoreactivity was observed in luminal endothelial cells and medial SMCs. Early atherosclerotic plaques, which consisted predominantly of SMCs, showed enhanced ECE expression in luminal endothelial cells and intimal SMCs. In advanced atherosclerotic plaques, distinct ECE expression was found in accumulated macrophages and in endothelial cells of intraplaque microvessels, while luminal endothelial cells showed relatively weak immunoreactivity for ECE. In conclusion, the present study demonstrates that the major cell types expressing ECE within the plaques are different between early and advanced stages of human coronary atherosclerosis. Enhanced ECE expression and possible endothelin-1 generation may contribute to SMC proliferation and vasoconstriction in early atherosclerotic stages, and may promote plaque destabilization in advanced atherosclerotic stages.     

Cytofluorometric analysis of DNA content in proliferating cells of coronary arteriosclerotic lesions]

Cytofluorometric determination of DNA content was done on paraffin-embedded tissues of 19 cases of coronary arteriosclerotic lesions including fibrocellular intimal thickening lesions (FT) or atherosclerosis (AS). DNA distribution pattern of medial smooth muscle cells of coronary arteries with FT and AS was all diploid. The average proliferative index (PI) of both medial smooth muscle cells of coronary arteries with FT and AS was 4.8 +/- 0.6. DNA distribution patterns of intimal cells of coronary arteries with FT and AS were also diploid. The average PI of intimal cells of coronary arteries with FT and AS was 8.4 +/- 1.0 and 9.1 +/- 0.7, respectively. These results suggest that intimal cellular proliferation plays an important role in the development of atherosclerosis.     

TWO AUTOPSY CASES OF "PULSELESS DISEASE"

Two cases of "Pulseless Disease" are reported. The main lesions In this condition are localized to the arteries of elastic type including pulmonary artery. The chief changes in the initial stage are limited to the adventitia and outer portion of media, based on an inflammatory reaction, which in turn brings about several following changes in arterial wall; marked fibrous thickening of adventitia, destruction of media and intimal thickening. In "Pulseless Disease," the lumen of the affected artery tends to become stenosed or obstructed, but can also be dilated and partially aneurysm-like. The stenosis of the lumen is chiefly caused by the contraction of the adventitial fibrosis overcoming the pressure on the arterial wall, promoted with the intimal thickening. Dilatation of that may be caused by increased arterial pressure, which exceedes the contracting effect of the wall, and is also influenced by the duration of the disease.