Activation of leukocytes in vivo produces marked constriction of large arteries in atherosclerotic, but not in normal, monkeys. We tested the hypotheses that vasoconstrictor responses to activated leukocytes in vivo may be abnormal during hypercholesterolemia before the development of atherosclerotic lesions and that responses may return to normal after the regression of atherosclerosis. Leukocytes were activated by injection of the chemotactic peptide formylmethionine-leucine-phenylalanine (fMLP) into the blood-perfused hind limb of four groups of cynomolgus monkeys: monkeys fed a normal diet (normal group, n=18), monkeys fed an atherogenic diet for 3-4 months (hypercholesterolemic group, n=6), monkeys fed an atherogenic diet for 20 months (atherosclerotic group, n= 19), and monkeys fed an atherogenic diet for 18 months, followed by a normal diet for 20 months (regression group, n= 14). Baseline resistance of large arteries was 1.5±0.2 (mean±SEM), 2.0±0.66 3.5±0.4 (p<0.05 versus normal), and 1.7±0.2 mm Hg/ml/min per 100 g tissue for the normal, hypercholesterolemic, atherosclerotic, and regression groups, respectively. Injection of fMLP did not change resistance of large arteries in normal or hypercholesterolemic monkeys. Injection of fMLP increased resistance of large arteries by 3.0±0.7 mm Hg/ml/min per 100 g tissue in atherosclerotic monkeys and by 1.3±0.4 mm Hg/ml/min per 100 g tissue in regression monkeys (p<0.05 versus atherosclerotic and normal). Thus, abnormal vasoconstriction in response to activation of leukocytes persists, but to a lesser extent, after regression. In contrast, vasoconstrictor responses to serotonin, which were potentiated in atherosclerotic monkeys, were normal after regression. In summary, hypercholesterolemia without arterial lesions does not result in abnormal vascular responses to activation of leukocytes. Abnormal constrictor responses of atherosclerotic arteries to activation of leukocytes are improved but not abolished after regression of atherosclerosis. (Circulation Research 1992;70:423-429)

In monkeys with early and advanced atherosclerosis, we examined responses to the three major vasoactive agonists that are released when platelets aggregate. Measurements were obtained in normal cynomolgus monkeys and in monkeys fed an atherogenic diet for 4+1, 9±1, and 19±1 months (mean± SEM). Morphometry of femoral and iliac arteries indicated that 4 months of atherogenic diet produced only slight intimal thickening, 9 months produced early lesions, and 19 months produced approximately 5-10 -fold greater intimal proliferation than did 9 months of atherogenic diet. Serotonin and adenosine 5'-diphosphate (ADP), which are endotheliumdependent agonists, and adenosine and phenylephrine, which are endothelium-independent agonists, were injected intra-arterially into the perfused hind limb. Thromboxane A2 analogue U46619 also was studied. Vasoconstrictor responses to serotonin were potentiated, and vasodilator responses to ADP were impaired by early and advanced atherosclerosis. In contrast, vasoconstrictor responses to phenylephrine and vasodilator responses to adenosine were similar in all groups. Vasoconstrictor responses to U46619 were potentiated by advanced atherosclerosis. Thus, vascular responses to serotonin, ADP, and thromboxane A2 are altered by atherosclerosis in a direction that would favor vasoconstriction when platelets aggregate. Furthermore, because responses to endothelium-dependent agonists are altered, these data suggest that endothelium is dysfunctional in early atherosclerosis. These findings may explain, in part, the propensity for exaggerated vasoconstriction even in arteries with minimal atherosclerotic lesions. (Circulation 1989;79:698-705)

When we think of atherosclerosis as a morphologic process, several images come to mind. We see an angiopathy that affects the intimal coat of certain arteries, chiefly the aorta and the first three orders of its branches (1,2). We see an intimal process that is hyperplastic as well as infiltrative, that starts from inconspicuous precursor lesions (3) and progresses to a predominantly fibrous plaque stage (4). With plaque growth, we see narrowing of the vascular lumen. Finally, we see progression of luminal narrowing to the point of hemodynamic compromise, causing tissue ischemia or necrosis with ensuing symptoms or death.

In this study hemodynamic and morphometric consequences of atherosclerosis were examined in cynomolgus monkeys. We tested the hypothesis that atherosclerosis augments cerebral vasoconstrictor responses to serotonin. We studied 8 normal and 8 atherosclerotic monkeys, which were fed an atherogenic diet for 17 months. Morphometric studies indicated marked intimal proliferation of extracranial carotid arteries, with only modest reduction in the vascular lumen, as atherosclerotic lesions were displaced outward. Cerebral blood flow was measured with microspheres and microvascular pressure was measured with a micropipette in pial arteries approximately 350 microns diameter. Intracarotid infusion of serotonin reduced microvascular pressure, which indicates constriction of large arteries upstream, but cerebral blood flow did not decrease. Serotonin produced a 2-fold greater reduction in cerebral microvascular pressure in atherosclerotic monkeys than in normal monkeys. Intracarotid histamine increased flow and hypocapnia reduced flow in both normal and atherosclerotic monkeys, without altering cerebral microvascular pressure. We conclude: First, atherosclerosis potentiates constrictor responses to serotonin in large cerebral arteries. Because platelets release serotonin when they aggregate, augmentation of responses by atherosclerosis may have implications for cerebral vascular responses during aggregation of platelets at carotid lesions. Second, despite marked proliferation of intima, atherosclerotic lesions are displaced outward during a prestenotic phase of the disease, so that the lumen is relatively well preserved.

There is currently no accepted approach for intraoperative evaluation of the technical adequacy of coronary artery bypass graft anastomoses. High-frequency epicardial echocardiography performed intraoperatively could assess coronary artery bypass graft anastomoses by providing on-line short-axis (cross-sectional) and longitudinal two-dimensional images of the vessels. To validate measurements of anastomoses with high-frequency epicardial echocardiography, luminal diameter determined by high-frequency epicardial echocardiography was compared with that determined histologically after perfusion fixation in 12 dogs studied after coronary artery bypass grafting. Technical errors were deliberately created in some grafts. The results of these animal validation studies showed that maximum luminal diameter of the anastomosis by high-frequency epicardial echocardiography correlated well with histologic measurements (r = .92; high-frequency epicardial echocardiography = 0.8 histology + 0.3). All deliberately created technical errors were detected by an independent observer using high-frequency epicardial echocardiography. After completion of the animal studies, we demonstrated the clinical applicability of this approach in 12 patients. Fifteen coronary artery bypass graft anastomoses were examined intraoperatively with high-frequency epicardial echocardiography. The measured maximum luminal diameter of the anastomosis was greater than the maximum luminal diameter of the native artery, as expected, in all end-to-side anastomoses. However, the maximum luminal diameter of the side-to-side anastomoses was equal to or slightly less than that of the native artery. In this initial patient group, minor technical errors were noted in two of 15 graft anastomoses. In conclusion, high-frequency epicardial echocardiography can accurately measure coronary arterial bypass graft anastomoses and has potential for intraoperative detection of technical errors and inadequacies.

The purpose of this study was to determine the accuracy of a new high frequency echocardiographic technique for the quantitative assessment of coronary artery luminal and wall dimensions. In 32 open chest animals, high frequency echocardiographic measurements (echo) of luminal diameter correlated well with in vitro histologic measurements (Histo) (r = 0.86; high frequency echo = 0.89 Histo + 0.79) (range 1.7 to 5.8 mm). Similar results were found in the evaluation of five human autopsy hearts studied in vitro. Coronary artery wall thickness measurements in human autopsy hearts showed a good correlation with high frequency echocardiographic measurements (r = 0.86; high frequency echo = 0.65 Histo + 0.24) (range 0.3 to 0.8 mm). In eight open chest calves, high frequency echocardiographic measurements of total vessel diameter correlated well with sonomicrometer measurements (Sono) (r = 0.94; high frequency echo = 1.03 Sono + 0.4) (range 2.1 to 5.3 mm). Inter- and intraobserver variability measurements of high frequency echocardiographic measurements demonstrated excellent reproducibility (r = 0.95, interobserver variability for wall thickness; r = 0.97, inter-observer variability for luminal diameter; n = 10 postmortem human coronary arteries). In conclusion, high frequency echocardiography is an accurate and reproducible method of measuring coronary luminal and wall geometry and may be a potentially useful tool for in vivo coronary artery evaluation in patients.

Macrophages provide an important way for cholesteryl esters to accumulate in tissues in pathologic amounts. We studied cholesteryl ester metabolism in thioglycollate-induced peritoneal macrophages obtained from normocholesterolemic and hypercholesterolemic rabbits. The macrophage preparations from normocholes-terolemic rabbit (MN cells) had 26 nmol esterified cholesterol/mg cellular protein, incorporated 1 nmol of labeled oleate into cholesteryloleate/2 h per mg cellular protein and had an acyl-coenzyme A: cholesterol acyltransferase activity of 22 pmol cholesterylpalmitate formed/min per mg protein in isolated membranes. The macrophage preparations from hypercholesterolemic rabbits (MHC cells) contained a 12-fold greater mass of cholesteryl ester, had an 8-times higher rate of formation of cholesteryloleate, and had 3-times more acyl-coenzyme A: cholesterol acyltransferase activity in the isolated membranes. When a cholesterol acceptor (10% fetal bovine serum or 10 mg of lipid-free fetal bovine serum protein) was added to the culture medium of rabbit MHC cells, the MHC cells retained more than 70% of their cholesteryl esters after 48 h of incubation. In contrast, when a cholesterol acceptor (10% fetal bovine serum) was added to the medium of thioglycollate-induced, cholesterol-enriched macrophages from mice, the mice macrophages retained only 19% of their cholesteryl esters after 48 h of incubation. The limited capacity of rabbit macrophages to release unesterified cholesterol from stored cytoplasmic cholesteryl esters to an exogenous acceptor may be related to the propensity of rabbits to develop atherosclerotic lesions.

1.1. Plasma carnitine, cholesterol, and triglycerides were measured over a 13-week period in male cynomolgus monkeys (M. fascicularis) that were fed a control diet (Purina Monkey Chow, n = 5) and a semisynthetic hypercholesterolemic diet (n = 15).2.2. Plasma cholesterol levels rose from 100 ± 5 to 743 ± 50 mg/dl in the cholesterol-fed group during the 13-week period but remained below 133 ± 13 mg/dl in the control group.3.3. Plasma triglyceride levels tended to be lower in the cholesterol-fed group, particularly at 4 week (28 ± 3 vs 42 ± 7 mg/dl, P < 0.05).4.4. Plasma carnitine levels rose from 43 ± 4 to 53 ± 5 nmol/ml within two weeks in the cholesterol-fed group and remained above control values for the duration of the study.5.5. Carnitine levels were significantly higher in the carotid arteries of cholesterol-fed animals relative to control (224 ± 25 vs 109 ± 15 pmol/mmin situ length, P < 0.01). Higher mean values of carnitine were also found in iliac, subclavian, and coronary arteries of cholesterol-fed animals but not in femoral arteries.

Numerous studies of coronary obstructive lesions in patients by angiographic and pathologic techniques have concluded that the severity of obstruction is seriously underestimated by angiography. It has generally been assumed that morphologic examination of an undistended coronary vessel is a reasonable reference standard. To test this assumption, three methods of examining coronary arteries were compared in cynomolgus monkeys with diet-induced coronary atherosclerosis. Total and regional myocardial perfusion during maximal coronary vasodilation were measured, postmortem coronary arteriography was performed with contrast medium injected at systemic pressure and the undistended coronary vessels were extended pathologically. In these atherosclerotic monkeys, there were widespread intimai changes in the epicardial arteries ranging from fatty streaks to large plaques with medial atrophy; morphometric studies showed a mean (± standard error of the mean) luminal stenosis of 57 ± 3 percent (range 38 to 76). Postmortem coronary arteriograms showed no evidence of localized obstruction. Under resting conditions, global and transmural left ventricular perfusion were normal. During maximal coronary vasodilation, minimal coronary vascular resistance was higher (p <0.07) in these monkeys with atherosclerosis than in normal control monkeys (0.21 ± 0.04 versus 0.13 ± 0.07 mm Hg/ml × ml/min × 100 g). No evidence of localized perfusion deficits was observed in either group of monkeys under control conditions or during maximal coronary vasodilation.

This chapter reviews the current state of knowledge on optimal levels of blood lipids based on research in the laboratory, including work with human tissues, cells, and lipoproteins, and investigations using experimental animal models of atherosclerosis. It presents a summary of report of the laboratory–experimental section examined in the workshop. The workshop examined the experimental results on optimal plasma lipid and lipoprotein profile that are expected to favor prevention, retardation, and/or regression of clinically important atherosclerosis. It examined many of the nutritional variables that appear to influence the development of atherosclerosis, including the amount of food fat and the degree of saturation or randomization of fatty acids, the type and amount of dietary fiber, the presence of dietary saponins, the amount and source of dietary protein, the amount of lysine in the diet, and the type of dietary carbohydrate. All of these dietary factors seem to mediate their effects by affecting serum low-density lipoproteins (LDL) levels or the high-density lipoproteins (HDL)/LDL ratios.

All regression studies conform to a general model of intervention (Fig. 1). As new experimental settings are used to explore the limits of regressive change, some interventions will doubtless have only minimal or no favorable effects, and the possibility of lesion worsening is always present. In this presentation, however, we are not concerned with factors that might tend to override all regressive effects. Our focus is on the sequences of change that may occur in studies in which regression has been unequivocally established.