Variability in subjective review of umbilical cord blood colony forming unit assay.

BACKGROUND: The goal of this study was to quantitatively evaluate the reproducibility of current manual counting methods of colony forming units (CFUs) from umbilical cord blood samples METHODS: Fresh and reconstituted frozen cells from 10 cord blood samples were cultured under standard conditions. The number of BFU-Es, CFU-GMs, and CFU-GEMMs were counted by three expert reviewers using the standard microscope method and manually traced CFUs on digital images of cell cultures. RESULTS: The mean colony count based on the traced digital images was 82 (22% CV) and 52 (15% CV) for the fresh and frozen samples, respectively. This was significantly greater than that observed using the microscope, 61 (13% CV) for fresh and 43 (16% CV) for frozen. The difference was mainly due to the reviewers observing more CFU-GMs in the digital images than through the microscope review. All three reviewers agreed on the presence of a colony 72% of the time based on the digital review in both fresh and frozen samples. Reviewer agreement with respect to colony type in the fresh samples was 38% (22%CV), 25% (51%CV), and 6% (115%CV) for BFU-Es, CFU-GMs, and CFU-GEMMs, respectively. Reviewer agreement increased for BFU-Es and CFU-GMs in the frozen samples where fewer colonies were present. CONCLUSIONS: Although this study showed marked variability between reviewers, the analysis of manually traced digital images has the potential to improve inter-observer variation when compared to current methods by identifying features that lead to discrepancies in colony counting and providing cases with consensus results. © 2016 International Clinical Cytometry Society.

Atherosclerosis in youth.

BACKGROUND: Coronary heart disease (CHD) and the related diseases due to atherosclerosis continue to be major public health problems in the industrialized countries and are likely to become serious problems in the developing countries. Treatment of end stage disease has improved, and risk factor modification has succeeded in reducing risk among adults. However, the age at which to begin risk factor control for long-range primary prevention is controversial. METHODS: A multicenter cooperative study, Pathobiological Determinants of Atheroscle-rosis in Youth (PDAY), was organized in 1985 to examine the relationship of the risk factors for adult CHD to preclinical atherosclerotic lesions in youth. Fourteen participating centers collected arteries, blood, other tissue, and data from 3,000 persons 15-34 years of age who died from external causes and were autopsied in forensic laboratories. Central laboratories evaluated atherosclerosis in the aorta and coronary arteries, measured lipoproteins and thio-cyanate (for smoking) in serum, glycohemo-globin in red blood cells (for blood glucose), thickness of small renal arteries (for hypertension), and body mass index (for obesity). The data were analyzed to determine the progression of atherosclerosis with age in both sexes and the association of the risk factors with atherosclerotic lesions. RESULTS: Raised lesions of the coronary arteries, the advanced lesions of atherosclerosis that lead directly to CHD, are associated positively with non-HDL cholesterol concentration, hypertension, obesity (in men), and blood glucose concentration; and inversely, with HDL cholesterol concentration. Smoking affects predominantly the abdominal aorta. CONCLUSIONS: These results suggest that long-range prevention of CHD should begin in adolescence or at least in young adulthood with control of the major established risk factors for adult CHD.

Bioequivalent or nonbioequivalent?

The current tests for bioequivalence are based on assumptions that are not valid in general; this paper shows why it is necessary to use a method that does not depend upon assumptions that cannot, and need not, be proved in general.

Statistical comparison of dissolution curves.

The purpose of this investigation was to develop statistical procedures to determine if two sets of dissolution curves could have come from the same population of curves. The f(2)statistic developed by the Food and Drug Administration, FDA, has been shown to have many limitations and is too liberal in concluding similarity between dissolution profiles. The procedure currently used by the FDA involves computing the mean amount dissolved at each time and then comparing the two mean curves. This approach ignores all of the variability within sets of profiles, which, from a statistical viewpoint, is a serious limitation. This investigation presents three different statistics for comparison of dissolution curves with associated decision rules and power functions. These three statistics are extensions of existing procedures: (1) an extension of the Mann--Whitney test which compares the variability within each set of profiles and between the two sets; (2) an extension of the Kolmogorov--Smirnov D statistic which compares three empirical cumulative distribution functions; and (3) an adaptation of the well known chi-squared test. A computer program, which includes the algorithm for each of the three statistics and varying sample sizes, is also available.

Origin of atherosclerosis in childhood and adolescence.

Atherosclerosis begins in childhood as deposits of cholesterol and its esters, referred to as fatty streaks, in the intima of large muscular arteries. In some persons and at certain arterial sites, more lipid accumulates and is covered by a fibromuscular cap to form a fibrous plaque. Further changes in fibrous plaques render them vulnerable to rupture, an event that precipitates occlusive thrombosis and clinically manifest disease (sudden cardiac death, myocardial infarction, stroke, or peripheral arterial disease). In adults, elevated non-HDL-cholesterol concentrations, low HDL-cholesterol concentrations, hypertension, smoking, diabetes, and obesity are associated with advanced atherosclerotic lesions and increased risk of clinically manifest atherosclerotic disease. Control of these risk factors is the major strategy for preventing atherosclerotic disease. To determine whether these risk factors also are associated with early atherosclerosis in young persons, we examined arteries and tissue from approximately 3000 autopsied persons aged 15-34 y who died of accidental injury, homicide, or suicide. The extent of both fatty streaks and raised lesions (fibrous plaques and other advanced lesions) in the right coronary artery and in the abdominal aorta was associated positively with non-HDL-cholesterol concentration, hypertension, impaired glucose tolerance, and obesity and associated negatively with HDL-cholesterol concentration. Atherosclerosis of the abdominal aorta also was associated positively with smoking. These observations indicate that long-range prevention of atherosclerosis and its sequelae by control of the risk factors for adult coronary artery disease should begin in adolescence and young adulthood.

Association of Coronary Heart Disease Risk Factors with microscopic qualities of coronary atherosclerosis in youth.

BACKGROUND: This study examined whether atherosclerosis in young people is associated with the risk factors for clinical coronary heart disease (CHD). Methods and Results-Histological sections of left anterior descending coronary arteries (LADs) from 760 autopsied 15- to 34-year-old victims of accidents, homicides, and suicides were graded according to the American Heart Association (AHA) system and computerized morphometry. Risk factors (dyslipoproteinemia, smoking, hypertension, obesity, impaired glucose tolerance) were assessed by postmortem measurements. Approximately 2% of 15- to 19-year-old men and 20% of 30- to 34-year-old men had AHA grade 4 or 5 (advanced) lesions. No 15- to 19-year-old women had grade 4 or 5 lesions; 8% of 30- to 34-year-old women had such lesions. Approximately 19% of 30- to 34-year-old men and 8% of 30- to 34-year-old women had atherosclerotic stenosis > or =40% in the LAD. AHA grade 2 or 3 lesions (fatty streaks), grade 4 or 5 lesions, and stenosis > or =40% were associated with non-HDL cholesterol > or =4.14 mmol/L (160 mg/dL). AHA grade 2 or 3 lesions were associated with HDL cholesterol <0.91 mmol/L (35 mg/dL) and smoking. AHA grade 4 or 5 lesions were associated with obesity (body mass index > or =30 kg/m(2)) and hypertension (mean arterial pressure > or =110 mm Hg). CONCLUSIONS: -Young Americans have a high prevalence of advanced atherosclerotic coronary artery plaques with qualities indicating vulnerability to rupture. Early atherosclerosis is influenced by the risk factors for clinical CHD. Long-range prevention of CHD must begin in adolescence or young adulthood.

Intersession repeatability of macular thickness measurements with the Humphrey 2000 OCT.

PURPOSE: This study was designed to determine intersession repeatability of measurements of macular thickness made with a commercially available optical coherence tomography (OCT) system. The images that can be routinely acquired with the commercial instrument differ significantly in quality from the images in the literature, which have mostly been acquired on prototype systems. METHODS: Multiple OCT images of the nasal macula were obtained from the right eye during three independent measuring sessions, using the Humphrey 2000 OCT system (Humphrey, San Leandro, CA). Twenty-six volunteers with no history of ocular disease participated in this investigation. Eyes in all subjects were undilated during scan acquisition. Scans were horizontal, 3 mm long, and through the fovea. Five scans were used from each session, for a total of 15 scans per subject. Retinal boundaries were automatically detected using custom software. Statistical software was used to calculate intersession and intrasession repeatability. Manual correction was performed on the automatically detected boundaries, and a second analysis was performed using these boundaries. RESULTS: When no manual correction of boundaries was performed, there were no significant effects between different sessions (P = 0.529) or between different scans within the same session (P = 0.509). Average retinal thickness was found to be 274 +/- 17 microm for a 1-mm long region 0.75 mm from the fovea. Individual scan averages differed from overall patient averages by 0 +/- 4.3 microm (99% confidence interval, 11.2 microm). CONCLUSIONS: OCT measurements of macular thickness made with the Humphrey 2000 OCT system are repeatable over different sessions with an expected variation of less than 11 microm (99% confidence interval).

Vascular mechanics of the coronary artery.

UNLABELLED: This paper describes our research into the vascular mechanics of the coronary artery and plaque. The three sections describe the determination of arterial mechanical properties using intravascular ultrasound (IVUS), a constitutive relation for the arterial wall, and finite element method (FEM) models of the arterial wall and atheroma. METHODS: Inflation testing of porcine left anterior descending coronary arteries was conducted. The changes in the vessel geometry were monitored using IVUS, and intracoronary pressure was recorded using a pressure transducer. The creep and quasistatic stress/strain responses were determined. A Standard Linear Solid (SLS) was modified to reproduce the non-linear elastic behavior of the arterial wall. This Standard Non-linear Solid (SNS) was implemented into an axisymetric thick-walled cylinder numerical model. Finite element analysis models were created for five age groups and four levels of stenosis using the Pathobiological Determinants of Atherosclerosis Youth (PDAY) database. RESULTS: The arteries exhibited non-linear elastic behavior. The total tissue creep strain was epsilon creep = 0.082 +/- 0.018 mm/mm. The numerical model could reproduce both the non-linearity of the porcine data and time dependent behavior of the arterial wall found in the literature with a correlation coefficient of 0.985. Increasing age had a strong positive correlation with the shoulder stress level, (r = 0.95). The 30% stenosis had the highest shoulder stress due to the combination of a fully formed lipid pool and a thin cap. CONCLUSIONS: Studying the solid mechanics of the arterial wall and the atheroma provide important insights into the mechanisms involved in plaque rupture.

Effects of coronary heart disease risk factors on atherosclerosis of selected regions of the aorta and right coronary artery. PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth.

We examined topographic distributions of atherosclerosis and their relation to risk factors for adult coronary heart disease in right coronary arteries and abdominal aortas of more than 2000 autopsied persons 15 through 34 years of age. We digitized images of Sudan IV-stained fatty streaks and of manually outlined raised lesions and computed the percent surface area involved by each lesion in each of 6 regions of each artery. In abdominal aortas of 15- to 24-year-old persons, fatty streaks involve an elongated oval area on the dorsolateral intimal surface and another oval area in the middle third of the ventral surface. Raised lesions in 25- to 34-year-old persons involve an oval area in the distal third of the dorsolateral intimal surface. In other areas of the abdominal aortas of older persons, fatty streaks occur but raised lesions are rare. In the right coronary arteries of 15- to 24-year-old persons, fatty streaks are most frequent on the myocardial aspect of the first 2 cm. Raised lesions follow a similar pattern in 25- to 34-year-old persons. High non-HDL cholesterol and low HDL cholesterol concentrations are associated with more extensive fatty streaks and raised lesions in all regions of both arteries. Smoking is associated with more extensive fatty streaks and raised lesions of the abdominal aorta, particularly in the dorsolateral region of the distal third of the abdominal aorta. Hypertension is not associated with fatty streaks in whites or blacks but is associated with more extensive raised lesions in blacks. Risk factor effects on arterial regions that are vulnerable to lesions are approximately 25% greater than risk factor effects assessed over entire arterial segments. These risk factor effects on vulnerable sites emphasize the need for risk factor control during adolescence and young adulthood to prevent or delay the progression of atherosclerosis.

LCAT modulates atherogenic plasma lipoproteins and the extent of atherosclerosis only in the presence of normal LDL receptors in transgenic rabbits.

Elevated low density lipoprotein cholesterol (LDL-C) and reduced high density lipoprotein cholesterol (HDL-C) concentrations are independent risk factors for coronary heart disease. We have previously demonstrated that overexpression of an enzyme with a well established role in HDL metabolism, lecithin:cholesterol acyltransferase (LCAT), in New Zealand White rabbits not only raises HDL-C concentrations but reduces those of LDL-C as well, ultimately preventing diet-induced atherosclerosis. In the present study, the human LCAT gene (hLCAT) was introduced into LDL receptor (LDLr)-deficient (Watanabe heritable hyperlipidemic) rabbits to (1) investigate the role of the LDLr pathway in the hLCAT-mediated reductions of LDL-C and (2) determine the influence of hLCAT overexpression on atherosclerosis susceptibility in an animal model of familial hypercholesterolemia. Heterozygosity or homozygosity for the LDLr defect was determined by polymerase chain reaction, and 3 groups of hLCAT-transgenic (hLCAT+) rabbits that differed in LDLr status were established: (1) LDLr wild-type (LDLr+/+), (2) LDLr heterozygotes (LDLr+/-), and (3) LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared with those of nontransgenic (hLCAT-) rabbits of the same LDLr status. Plasma HDL-C concentrations were significantly elevated in the hLCAT+ animals of each LDLr status. However, LDL-C levels were significantly reduced only in hLCAT+/LDLr+/+ and hLCAT+/LDLr+/- rabbits but not in hLCAT+/LDLr-/- rabbits (405+/-14 versus 392+/-31 mg/dL). Metabolic studies revealed that the fractional catabolic rate (FCR, d(-1)) of LDL apolipoprotein (apo) B-100 was increased in hLCAT+/LDLr+/+ (26+/-4 versus 5+/-0) and hLCAT+/LDLr+/- (4+/-1 versus 1+/-0) rabbits, whereas the FCR of LDL apoB-100 in both groups of LDLr-/- rabbits was nearly identical (0.16+/-0.02 versus 0.15+/-0.02). Consistently, neither aortic lipid concentrations nor the extent of aortic atherosclerosis was significantly different between hLCAT+/LDLr-/- and hLCAT-/LDLr-/- rabbits. Significant correlations were observed between the percent of aortic atherosclerosis and both LDL-C (r=0.985) and LDL apoB-100 FCR (-0.745), as well as between LDL-C and LDL apoB-100 FCR (-0.866). These data are the first to establish that LCAT modulates LDL metabolism via the LDLr pathway, ultimately influencing atherosclerosis susceptibility. Moreover, LCAT's antiatherogenic effect requires only a single functional LDLr allele, identifying LCAT as an attractive gene therapy candidate for the majority of dyslipoproteinemic patients.

Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention from the Pathobiological Determinants of Atherosclerosis in Youth Study.

CONTEXT: Atherosclerosis, the underlying cause of coronary heart disease, has been shown to be present even in young adults. OBJECTIVE: To document the extent and severity of atherosclerosis in adolescents and young adults in the United States. DESIGN AND SETTING: The Pathobiological Determinants of Atherosclerosis in Youth Study, a multi-institutional autopsy study conducted in US medical centers. Subjects A total of 2876 study subjects, between 15 and 34 years old, black and white, men and women, who died of external causes and underwent autopsy between June 1, 1987, and August 31, 1994. MAIN OUTCOME MEASURES: Extent, prevalence, and topography of atherosclerotic lesions. RESULTS: Intimal lesions appeared in all the aortas and more than half of the right coronary arteries of the youngest age group (15-19 years) and increased in prevalence and extent with age through the oldest age group (30-34 years). Fatty streaks were more extensive in black subjects than in white subjects, but raised lesions did not differ between blacks and whites. Raised lesions in the aortas of women and men were similar, but raised lesions in the right coronary arteries of women were less than those of men. The prevalence of total lesions was lower in the right coronary artery than in the aorta, but the proportion of raised lesions among total lesions was higher in the right coronary artery than in the aorta. CONCLUSIONS: Atherosclerosis begins in youth. Fatty streaks and clinically significant raised lesions increase rapidly in prevalence and extent during the 15- to 34-year age span. Primary prevention of atherosclerosis, as contrasted with primary prevention of clinically manifest atherosclerotic disease, must begin in childhood or adolescence.

Relation of a postmortem renal index of hypertension to atherosclerosis and coronary artery size in young men and women. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group.

In a cooperative multicenter study, the Pathobiological Determinants of Atherosclerosis in Youth, we measured atherosclerosis of the aorta and right coronary artery (RCA) in 2403 black and white men and women 15 through 34 years of age who died of external causes and were autopsied in forensic laboratories. We measured the diameter of the opened, flattened, and fixed RCA and the diameter, intimal thickness, intimal cross-sectional area, medial thickness, and medial cross-sectional area of the pressure-perfused, fixed left anterior descending (LAD) coronary artery. Using the ratio of intimal thickness to outer diameter of the small renal arteries to predict mean arterial pressure during life, we classified the cases as normotensive (mean arterial pressure < 110 mm Hg) or hypertensive (mean arterial pressure > or = 110 mm Hg). The prevalence of hypertension by age, sex, and race corresponded closely with that measured in a survey of the living population. Hypertension had little or no effect on fatty streaks. Hypertension was associated with more extensive raised lesions in the abdominal aortas and RCAs of blacks > 20 years of age and in the RCAs of whites > 25 years of age. At all ages, women had less extensive raised lesions in the RCAs than did men, but the effect of hypertension on raised lesions was similar to that in men. Adjustment for serum lipoprotein cholesterol levels and smoking in a subset of cases yielded results similar to those obtained without adjustment. Hypertension was associated with larger diameters of the RCA and LAD coronary artery and with larger cross-sectional intimal and medial areas of the LAD coronary artery. Hypertension augments atherosclerosis in both men and women primarily by accelerating the conversion of fatty streaks to raised lesions beginning in the third decade of life, and the effect of hypertension increases with age.

Age-related development of atherosclerotic plaque stress: a population-based finite-element analysis.

BACKGROUND: In order to identify those age-related factors in the development of coronary atherosclerosis that would affect the stability of the plaque system, we have developed idealized, finite-element, cross-sectional models of the arterial wall and associated lesions, derived from population-based data. METHODS: The physical development and morphology of coronary plaques was documented in the Pathobiological Determinants of Atherosclerosis in Youth histological study. Using this database, finite-element analysis models were created for five age groups (15-19, 20-24, 25-29 and 30-34 years) and for the 25 largest lesions. Cosmos (Structural Research, Inc., Los Angeles, California, USA) was used to create and analyze the models. RESULTS: The area of greatest stress shifted from the intima opposite the lesion in the 15-19 years age group to the edge of the cap and adjacent healthy tissue in the later age groups. Increasing age had a strong positive correlation with the shoulder stress level (r = 0.95) and the per cent stenosis correlated well with shoulder stress (r = 0.99, P < 0.002). Increasing the cap stiffness from a soft cap to a fibrous cap in the 30-34 year age group model resulted in a localized increase in shoulder surface stress by 10%. A calcified cap increased this shoulder surface stress by 30%. CONCLUSIONS: This finite-element analysis of the population-based data shows that the increase in stress appears to be closely related to the impaired load-bearing capability of the lipid pool that develops with age. The shoulder area of the lesion has been shown to be the location of most of the plaque fractures.

Altered compliance and residual strain precede angiographically detectable early atherosclerosis in low-density lipoprotein receptor deficiency.

BACKGROUND: This study was performed to detect changes in vascular biomechanical properties early in atherogenesis. METHODS AND RESULTS: Age- and weight-matched LDL-receptor deficient Watanabe hypercholesterolemic male rabbits (Group I: n = 11) and normal rabbits (Group II: n = 11) were studied. Fasting plasma lipoprotein concentrations, aortic angiography and intravascular ultrasound, in vivo aortic compliance evaluation, ex vivo aortic residual strain measurements, aortic lipid content and histopathology were determined. Plasma cholesterol was increased 9.8 fold and aortic cholesterol content was increased from 20 to 43 fold in Group I compared to Group II, respectively (P < .00005). Angiography revealed no stenoses in either group, whereas intravascular ultrasound and histological studies of Group I showed small circumferential plaques with < 10% cross-sectional area involvement. The residual strain in Group I was significantly increased in the ascending thoracic aorta (22.1 +/- 6.9% versus 10.4 +/- 3.2% in Group II, P < .0001), descending thoracic aorta (15.7 +/- 7.2% versus 4.8 +/- 1.3% in Group II, P < .0001), and abdominal aorta (18.0 +/- 4.8% versus 8.3 +/- 6.3% in Group II, P < .005). Changes in residual strain were inversely correlated with the aortic cholesterol content in the ascending thoracic aorta (r = -.72; P = -.001), descending thoracic aorta (r = -.95; P < .001), and abdominal aorta (r = -.51; P = .019). CONCLUSIONS: Early atherosclerosis in LDL-receptor deficient rabbits, undetectable by angiography yet observed by intravascular ultrasound imaging and histology, is associated with marked changes in ex vivo residual strain. Alterations in vascular biomechanical properties, associated with changes in cholesterol content, may have physiologic consequences and may be useful in detecting and quantitating early atherosclerosis.

Hypercholesterolemia in the rabbit induced by feeding graded amounts of low-level cholesterol. Methodological considerations regarding individual variability in response to dietary cholesterol and development of lesion type.

While a number of studies have presented detailed examinations of lesion development in the cholesterol-fed rabbit, individual variability in response to cholesterol feeding and type of lesion produced relative to the degree of cholesterol exposure is not well defined. This study analyzed such critical parameters in an attempt to further characterize the model and establish a baseline for future testing of treatments targeted at limiting atherosclerosis. For these experiments, male New Zealand White rabbits were fed atherogenic diets consisting of 0.05%, 0.10%, 0.15%, 0.20%, or 0.25% cholesterol dissolved in 6% peanut oil for 31 to 32 weeks. Raising dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in total plasma cholesterol (TPC) exposure (area under plasma cholesterol versus time curve), whereas further increases in cholesterol intake resulted in an exponential four- to fivefold increase in TPC exposure. Regression analysis of TPC exposure with aortic sudanophilia demonstrated a threshold of approximately 5000 cholesterol weeks; below this limit lesions were minimal, and above this value the degree of plaque correlated with TPC exposure. Furthermore, a wide biological variability occurred among rabbits with respect to individual responsiveness to dietary cholesterol. In the aorta, various types of plaques, from fatty streaks to atheromatous lesions, were observed, depending on the degree of cholesterol intake. Diets consisting of < 0.15% cholesterol resulted in the development of fatty streak lesions, while transitional lesions and atheromatous plaques were mostly found with higher cholesterol feeding. Coronary artery atherosclerosis was present in > 50% of animals fed diets > or = 0.15% cholesterol. Despite the level of TPC exposure, coronary lesions in epicardial vessels were generally the fibrous type, whereas intramyocardial arteries demonstrated predominantly intimal foam cells. In conclusion, by adjusting dietary cholesterol intake and selecting rabbits with a similar responsiveness to cholesterol, the overall cholesterol exposure can be more closely controlled to minimize the inherent individual variability among animals in this model. The nature of the target lesion must also be carefully considered, because the efficacy of some treatments may depend on the type of atherosclerotic plaque.

High resolution MR imaging and localization of laser-induced thermal injury in the vascular wall.

With the increasing use of lasers in surgical procedures, there is need for a noninvasive imaging modality to monitor laser-tissue interactions. MRI can be used readily for imaging human anatomy and holds the potential to map laser-induced thermal injury. This study investigates high resolution T1-weighted MR imaging of human aorta samples (in vitro) that have been damaged thermally using an argon ion laser and the corresponding histology. High resolution T1-weighted MR images (voxel size, .156 x .156 x .700 mm) clearly detected residual thermal injury as areas of bright signal intensity. Effective localization of thermal injury was achieved by subtraction of preinjury and postinjury slices with pseudocoloring of positive and negative differences. The results may serve as a basis from which to guide future in vivo studies.

Macrophage-mediated 15-lipoxygenase expression protects against atherosclerosis development.

Oxidative modification of LDL increases its atherogenicity, and 15-lipoxygenase (15-LO) has been implicated in the process. To address this issue, we generated transgenic rabbits that expressed 15-LO in a macrophage-specific manner and studied their susceptibility to atherosclerosis development when they were fed a high-fat, high-cholesterol (HFHC) diet (Teklad 0533 rabbit diet 7009 with 10% corn oil and 0.25% cholesterol) for 13.5 wk. Transgenic and nontransgenic rabbits developed similar degrees of hypercholesterolemia and had similar levels of triglyceride, VLDL, LDL, and HDL. Quantitative morphometric analysis of the aortic atherosclerosis indicated that the transgenic animals (n = 19) had significantly smaller lesion areas (9.8+/-6.5%, mean+/-SD) than their littermate controls (n = 14, 17.8+/-15.0%) (P < 0.05). In a subgroup (n = 9) of transgenic rabbits that received the HFHC diet plus the antioxidant N',N '-diphenyl-phenylenediamine (1%), the extent of lesion involvement (9.8+/-7.5%) did not differ from the subgroup (n = 10) that received the regular HFHC diet (9.7+/-5.9%). Since the results were unexpected, we repeated the experiments. Again, we found that the nontransgenic littermates (n = 12) had more extensive lesions (11.6+/-10.6%) than the transgenic rabbits (n = 13; 9.5+/-7.8%), although the difference was not significant. In a third set of experiments, we crossed 15-LO transgenic rabbits with Watanabe heritable hyperlipidemic (WHHL) rabbits and found that the lesion area in the 15-LO transgenic/heterozygous WHHL rabbits (n = 14) was only about one third (7.7+/-5.7%) that found in nontransgenic heterozygous WHHL littermate controls (n = 11, 20.7+/-19.4%) (P < 0.05). These data suggest that overexpression of 15-LO in monocytes/macrophages protects against lipid deposition in the vessel wall during early atherogenesis in these rabbit models of atherosclerosis.

Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis.

Lecithin:cholesterol acyltransferase (LCAT) is a key plasma enzyme in cholesterol and high density lipoprotein (HDL) metabolism. Transgenic rabbits overexpressing human LCAT had 15-fold greater plasma LCAT activity that nontransgenic control rabbits. This degree of overexpression was associated with a 6.7-fold increase in the plasma HDL cholesterol concentration in LCAT transgenic rabbits. On a 0.3% cholesterol diet, the HDL cholesterol concentrations increased from 24 +/- 1 to 39 +/- 3 mg/dl in nontransgenic control rabbits (n = 10; P < 0.05) and increased from 161 +/- 5 to 200 +/- 21 mg/dl (P < 0.001) in the LCAT transgenic rabbits (n = 9). Although the baseline non-HDL concentrations of control (4 +/- 3 mg/dl) and transgenic rabbits (18 +/- 4 mg/dl) were similar, the cholesterol-rich diet raised the non-HDL cholesterol concentrations, reflecting the atherogenic very low density, intermediate density, and low density lipoprotein particles observed by gel filtration chromatography. The non-HDL cholesterol rose to 509 +/- 57 mg/dl in controls compared with only 196 +/- 14 mg/dl in the LCAT transgenic rabbits (P < 0.005). The differences in the plasma lipoprotein response to a cholesterol-rich diet observed in the transgenic rabbits paralleled the susceptibility to developing aortic atherosclerosis. Compared with nontransgenic controls, LCAT transgenic rabbits were protected from diet-induced atherosclerosis with significant reductions determined by both quantitative planimetry (-86%; P < 0.003) and quantitative immunohistochemistry (-93%; P < 0.009). Our results establish the importance of LCAT in the metabolism of both HDL and apolipoprotein B-containing lipoprotein particles with cholesterol feeding and the response to diet-induced atherosclerosis. In addition, these findings identify LCAT as a new target for therapy to prevent atherosclerosis.

Effect of age, race, body surface area, heart weight and atherosclerosis on coronary artery dimensions in young males.

Compensatory arterial enlargement in response to atherosclerosis has been demonstrated for the left main coronary artery. Only limited data is available on the interaction of patient characteristics and atherosclerosis with coronary artery dimensions. The purpose of the present study was to evaluate the influence of age, race, body habitus, heart weight and atherosclerosis on coronary artery dimensions of young males. Hearts from 137 young men (age 32 +/- 8 years; 78 black, 59 white) with unnatural deaths (homicide, suicide, accident, drug overdose) were perfusion-fixed, and histologic sections were obtained from the left main, proximal left anterior descending and left circumflex coronary arteries. Computerized planimetry was performed on Movat stained sections. Multiple regression analysis was used to evaluate the relative contribution of plaque size, age, race, heart weight and body surface area on coronary dimensions and compensatory enlargement in response to atherosclerosis. In the left anterior descending and left main coronary arteries, black race, body surface area and age were independent predictors of increased lumen area. In the left circumflex, age was a predictor of lumen area. Plaque area, black race and body surface area independently predicted increased area enclosed by the internal elastic lamina area. There was compensatory enlargement of internal elastic lamina with increasing plaque size in both races in the three arteries, but the percent luminal stenosis was greater in whites due to smaller artery size. Luminal narrowing did not develop until plaques occupied 30% of internal elastic lamina area. Among a population of young men with non-cardiac deaths, blacks have larger lumen and area enclosed by internal elastic lamina than whites. Age and body surface area are major determinants of lumen areas, and compensatory arterial enlargement was seen in all examined arteries in the present study.

Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation.

Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.