W.M. Lee

Rapid production of advanced atherosclerosis in swine by a combination of endothelial injury and cholesterol feeding

Abstract The aim of the current study was to find out whether a combination of a balloon-endothelial-cell-denudation procedure and cholesterol feeding would result in more rapid growth of atherosclerotic lesions in the abdominal aorta of swine than if either were used alone. The results far exceeded our expectation. The two procedures appear to act synergistically. In the first 2 or 3 mo lesions are patchy and scattered but by 6 mo they become confluent so that practically the entire abdominal aorta is covered with thick lipid-rich atherosclerotic lesions. The lesions produced by 6 mo have many of the characteristics of advanced human lesions.

Advanced Coronary Atherosclerosis in Swine Produced by Combination of Balloon-Catheter Injury and Cholesterol Feeding

Abstract There has been a constant demand for an animal model of advanced coronary atherosclerosis produced in a short period. It was known that mild atherosclerosis could be produced in various vascular beds by high cholesterol diets. However, it requires considerable time to produce advanced atherosclerotic lesions by diets alone. The current study was designed to investigate effects of combination of a balloon-denudation procedure and high cholesterol diet in swine. We could produce advanced atherosclerotic lesions in coronary arteries in cholesterol-fed swine within a short period of time by inserting a balloon-catheter via carotid artery into the coronary artery, inflating it so as to distend the lumen, and pulling it back quickly, which resulted in extensive denudation of the endothelium of coronary arteries. An atherosclerosis-like lesion develops at the site with eventual narrowing of the lumen leading to myocardial ischemia, myocardial infarction, and occasionally sudden death, thus resembling in many aspects human coronary artery disease. Among 22 swine studied, 10 had severe atherosclerosis with virtual occlusion of proximal portion of either or both coronary arteries and developed myocardial infarction within 2–3 months. Seven of these died suddenly, probably due to arrhythmia. This model should be appropriate for studies where advanced coronary atherosclerosis with its complications is needed.

DISTRIBUTION OF INTIMAL SMOOTH MUSCLE CELL MASSES AND THEIR RELATIONSHIP TO EARLY ATHEROSCLEROSIS IN THE ABDOMINAL AORTAS OF YOUNG SWINE

In the abdominal aortas of young mash-fed swine, intimal cell masses (pads, cushions) are located predominantly away from blood vessel orifices. They are found scattered throughout the aorta but nevertheless have a definite pattern of distribution. In the distal one half of the abdominal aorta, they are more frequent in the ventral quandrant than in the dorsal or either lateral quadrant. In the proximal half, intimal cell masses are more frequent in the dorsal quadrant. When experimental atherosclerosis is induced in the abdominal aortas of young swine by either a hypercholesterolemic diet or by aortic ballooning followed by a hypercholesterolemic diet, the distribution of early lesions is similar. The lesions are found predominantly in quadrants where intimal cell masses were found to be most frequent in the control group of swine. The results suggest that most of the lesions, though not necessarily all, arose from pre-existing intimal cell masses beneath the aortic surface.

Population dynamics of arterial cells during atherogenesis: VII. Comparison of loss of endothelial cells over abdominal aortic intimal cellular masses (ICM) with that over non-ICM areas in swine fed a hyperlipidemic diet for 60 days☆

Abstract One of the earlies events in the development of an intimal atherosclerotic lesion may be an alteration in the endothelial cell barrier, allowing the entry of injurious or mitogenic substances, resulting in proliferation of arterial smooth muscle cells. Intimal smooth muscle cell proliferation in atherosclerosis appears to begin in intimal cellular masses (ICM), which are normally occurring intimal structures made up of 2 to 10 layers of smooth muscle cells found predominantly in relation to branching points of arteries. If endothelial cell loss is a precursor of overt atherosclerotic lesions, the loss should be greater over ICM than over areas showing no ICM. This study presents evidence suggesting that in swine the endothelial cell barrier is compromised over intimal cell masses more than in non-ICM regions prior to development of overt atherosclerotic lesions. The method used was to label swine aortic endothelium and ICM using tritiated thymidine. All swine were then placed on a low-fat, low-cholesterol diet for 15 days, at which time five baseline animals were sacrificed; the remaining five were placed on a hyperlipidemic (HL) diet and sacrificed 60 days later. The rate of endothelial cell growth, division patterns, and loss of labeled endothelial cells in the HL swine was determined by comparing the findings in this group with the baseline group. Smooth muscle cells of ICM and non-ICM medial regions were similar in regard to labeling indices in the baseline swine and in regard to division patterns in 60-day HL diet swine, except one which was an active lesion. None of the remaining ICM were active lesions and thus were suitable for study of the covering endothelial cells in what can be assumed to be the prodromal stage of lesion development. The approximate area of the abdominal aorta occupied by intimal cell mass was considerably more than we had anticipated, averaging more than 20%. The labeling indices and endothelial cell division patterns over ICM and non-ICM in the baseline swine were similar. In contrast, the labeling index was lower, and grain count changes indicated more endothelial cell divisions over ICM areas than over non-ICM areas in the hypercholesterolemic swine. Calculation showed that the percentage of endothelial cell loss over ICM in the HL swine was more than twice as great as endothelial cell loss over non-ICM areas. The results of this study suggest the possibility that in the assumed prodromal phase of experimental aortic atherosclerosis in young swine, the endothelial cell barrier may be compromised selectively over areas of predilection for the future development of atherosclerotic lesions. This occurs before there is any evidence of focal increase in intimal smooth muscle cell proliferation, which we regard as the earliest detectable stage of lesion development.

Population dynamics of arterial smooth muscle cells: V. Cell proliferation and cell death during initial 3 months in atherosclerotic lesions induced in swine by hypercholesterolemic diet and intimal trauma

Abstract The sine qua non of the lesion of atherosclerosis in man and experimental animals is excessive focal accumulation of modified smooth muscle cells in the intima of arteries. In advanced stages extensive necrosis with accumulation of lipid-rich debris is a prominent feature. In swine fed hypercholesterolemic (HC) diets focal atherosclerotic lesions are produced, but they progress slowly and do not develop frank necrosis until they have been on HC diet for many months or even years. The atherosclerotic process can be greatly accelerated by traumatizing the arterial intima with a ballon-catheter in addition to feeding the HC diet. Within a few months thick atherosclerotic lesions with extensive necrosis and calcification can be produced. In regard to arterial cell population dynamics, we showed in the first part of the current study that under the specified experimental conditions, lesions produced in young swine by balloon-catheter trauma and HC diet have at least four features in common with lesions produced in young swine by HC diet alone. (1) Multiple cells are involved in the initiation of the lesions (i.e., they are not monoclonal in origin). (2) The cells do not divide in a completely random fashion. (3) The division pattern is consistent with polyclonal origin with considerable heterogeneity as regards growth rate. (4) Most and perhaps all lesion cells were in the dividing population during the periods that were studied. The differences observed between development of lesions produced by HC diet alone and those produced by HC diet plus ballon-catheter intimal trauma were quantitative and not qualitative, i.e., the lesions produced by the latter procedure had a greater rate of cell multiplication and proceeded more rapidly to the necrotic stage than did those produced by HC diet alone. In the second part of the current study, we have devised a method for calculating cell deaths in the atherosclerotic lesions produced by HC diet plus intimal trauma. In the period 60–97 days on HC diet which was studied we found that circa 40% of the lesion cells died during the perireplicative and/or perimitotic period of the cell cycle. The high cell death rate was offset in part by a high cell birth rate. As indicated by tritiated thymidine ( 3 HTdR) labeling indices, approximately four times as many cells were synthesizing DNA at the time of observation as in the adjacent normal appearing media. Obviously the rate of growth of the atherosclerotic lesions that were studied was determined by the balance between excessive cell birth and death rates.

Alterations in population dynamics of arterial smooth muscle cells during atherogenesis. IV. Evidence for a polyclonal origin of hypercholesterolemic diet-induced atherosclerotic lesions in young swine.

Abstract The purpose of this study was to determine whether atherosclerotic lesions developing in young swine fed hypercholesterolemic (HC) diets were monoclonal or polyclonal in origin. The approach involved labeling arterial cells with tritiated thymidine prior to feeding the HC diet. Baseline swine were sacrificed and radioautography studies carried out to determine baseline labeling indices and grain number distributions in media and in intimal cellular masses (cushions). Remaining swine were then fed an HC diet for 30–60 days before sacrificing and making the same type determinations as with the baseline swine on putative active lesions and media. If a lesion arose from a single cell (or multiple unlabeled cells) labeling indices would be expected to be greatly reduced without a change in grain number patterns. This was not observed in any of the 14 putative lesions that were studied. If the lesion arose from divisions by multiple cells including labeled cells we would expect the proportion of cells with a high number of grains to be decreased and those with low numbers to be increased since with division half the isotope goes to each daughter cell and this is reflected in the grain count. This occurred in nine of the putative lesions, indicating multicellular origin. In five there was neither change in grain number distributions nor reduction in labeling indices. Hence, these showed no division activity during the period of study; they were classified as inactive lesions or pre-existing cushions and provide no pertinent information for this study. The final conclusion was that all active lesions observed were polyclonal in origin. Further information was obtained by mathematical analysis on number of divisions made by the originally labeled cells; and considerable heterogeneity was observed, with some not dividing at all and others with progeny going through as many as four divisions.

Experimental model for study of “sudden death” from ventricular fibrillation or asystole

Sudden death as a result of cardiac arrhythmias can be induced in swine by combining precordial X-irradiation and an atherogenic diet. Hypothyroidism was not essential to the rapid induction of disease. We monitored the electrical activity of the heart during the terminal episode in 18 swine; 13 electrocardiograms showed ventricular fibrillation and 5 showed ventricular asystole. All 18 animals had advanced coronary atherosclerosis, and 14 had anatomically demonstrable myocardial infarcts. In a correlated separate experiment, ventricular fibrillation threshold was significantly lower in atherosclerotic swine. Infusion of lidocaine increased the fibrillation threshold 4-fold in these animals. This animal model appears to be useful for other investigations related to cardiac arrhythmias.

Effects of methyl prednisolone and colchicine on the development of aortic atherosclerosis in swine

The effect of methyl prednisolone and colchicine on the development of both the early proliferative and advanced atherosclerotic lesion in swine aorta was studied. In order to accelerate the development of atherosclerosis, the abdominal aortic endothelium was partially denuded by a balloon before the animals were placed on either a moderate or severe hypercholesterolemic diet. Neither drug in either dietary group inhibited the development of atherosclerosis. Swine receiving methyl prednisolone and severe hypercholesterolemic diet actually had a significantly greater number of the advanced necrotic lesions and more arterial calcification than the group receiving the atherogenic diet alone. In addition, the thoracic aorta of swine receiving the moderate hypercholesterolemic diet and methyl prednisolone showed larger amounts of lipid than did the non-drug fed control group. In swine receiving the moderate hypercholesterolemic diet, methyl prednisolone significantly raised serum cholesterol levels. Colchicine only slightly worsened the atherosclerosis in swine aorta and had no effect on serum cholesterol levels.

Partial suppression by pyridinolcarbamate of growth and necrosis of atherosclerotic lesions in swine subjected to an atherogenic regimen that produces advanced lesions.

Abstract Conflicting results have been reported in the literature on the effects of pyridinol-carbamate on the prevention of experimentally induced atherosclerosis in rabbits. In the current study the effects of pyridinolcarbamate have been tested in a swine model in which advanced atherosclerosis is produced by a combination of balloon-intimal trauma and a hyperlipidemic diet. Results were compared with those in two reference groups of swine subjected to the same atherogenic regimen—one left untreated by drugs and the other treated with hydroxyurea which is one of the antimetabolites used in cancer chemotherapy. In both drug-treated groups the intimal surface involved by atherosclerotic lesions, and the lesion area expressed as a ratio to the total medial area were significantly less than those of the untreated control group. Both drugs also significantly reduced the number of necrotic atheromatous lesions as compared to the untreated group. The mechanism of action of pyridinolcarbamate in our experimental condition is not clear at present. The demonstration of a beneficial effect of a relatively non-toxic drug such as pyridinolcarbamate seems to us to warrant carrying out further “in depth” study of this drug.

Evidence for a polyclonal origin and proliferative heterogeneity of atherosclerotic lesions induced by dietary cholesterol in young swine.

Very little is known with certainty about the origin and mode of progression of atherosclerotic lesions in man. However, there are a few conclusions that can be drawn that we believe have a reasonably high probability of being valid. These conclusions are based in large part on studies in experimental animals, but on most points there is indirect evidence from man to support them. This forum seems to us an appropriate one for summarizing these conclusions. 1. A major inciting factor is a diet high in lipids, especially in cholesterol (HC diet).lP3 Injury to the arterial wall (chemical, immunologic, or mechanical, including hemodynamic) is an important factor but appears to require an HC diet for its full e f f e ~ t . ~ ~ 2. Prior to detectable development of focal atherosclerotic lesions and soon after initiation of an inciting HC diet, there is a modest activation of arterial cells (both endothelial and medial), distributed widely and sparsely, and detected by modest but significant increases in mitotic index and incorporation of tracer thymidine into cellular DNA.7-10 3. Detectable atherogenesis begins with proliferation of smooth muscle cells (SMC), which leads to the formation of small focal accumulations of cells in the previously sparsely populated intimal region of the artery W ~ I I . ~ . ~ Such foci may sometimes be sufficiently elevated for gross detection; frequently, they are discernible only in histologic sections. Some early foci tend to accumulate lipids and may be detected grossly with the aid of fat stains. 4. E v e n t ~ a l l y l ~ ~ ~ one finds large masses of cells, sometimes confluent, that exhibit signs of tissue differentiation o r scar formation (deposition of extracellular materials, such as collagen, elastin, and mucopolysaccharides) and signs of extensive cell death (necrosis with accumulation of lipid-rich debris and calcium). Only a mammoth review could do justice to a recital of what we do not know about atherogenesis, of what has been reported but either not confirmed or in some quarters denied, or of what is probably true but perhaps trivial. Here, we should like to develop a particular theme. The sequence of the numbered paragraphs above may correspond to a causal sequence, but the evidence for this possibility is inconclusive. It is clearly not true experimentally that the initiation of the process as in step 2 necessarily results in the occurrence of all subsequent effects, even if the initial inciting agent is withdrawn.