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The impact of race (black-white) and family history of type 2 diabetes mellitus on metabolic characteristics in early life was examined in a community-based sample from Bogalusa, LA. Study subjects included offspring of type 2 diabetics (n = 53, 47% black) and nondiabetics (n = 52, 40% black), with the mean age of each group ranging from 14.2 to 15.6 years. Offspring were given a 1-hour oral glucose tolerance test. Measures of body fatness such as body weight, body-mass index (BMI; weight/height2), and triceps and subscapular thicknesses were significantly higher only in white offspring of diabetics versus nondiabetics; measures of abdominal fat (waist circumference and waist-to-hip ratio) were significantly higher among offspring of diabetics of both races. Among the measures of glucose homeostasis, basal glucose, insulin, insulin-to-C-peptide ratio (a measure of hepatic insulin extraction), insulin resistance index (derived from basal glucose and insulin levels), and glucose response after glucose challenge were higher in the offspring of diabetics of both races. The differences in insulin-to-C-peptide ratio and glucose response remained significant after adjusting for BMI; further, these two variables were independently associated with parental diabetes in both races. Waist-to-hip ratio, glucose response, C-peptide response (a measure of insulin secretion) were lower, and basal insulin-to-C-peptide ratio and postglucose suppression of free fatty acids greater in blacks versus whites, regardless of status of parental diabetes. Black-white differences in postglucose suppression of free fatty acids disappeared after adjusting for BMI. Thus, blacks and whites with parental type 2 diabetes show multiple abnormalities in parameters governing glucose homeostasis early in life, and some of these traits differ between the races, regardless of status of parental diabetes.

Proteoglycans in human coronary arteries were characterized immunohistochemically, using specific monoclonal antibodies to distinct proteoglycan types. In addition, apoB, macrophage, and arterial smooth muscle cell alpha-actin markers were localized. The expression of chondroitin sulfate proteoglycans and apoB was observed in healthy areas (operationally defined by morphology) as well as in lesions in the intima, but with greater expression in the atheromatous lesions. In healthy intima heparan sulfate proteoglycan was cell associated, but in lesions it was found also in the extracellular space. A dermatan sulfate proteoglycan of decorin type was not observed in the healthy intima but was observed in the intima with adaptive thickening especially in zones with reduced staining for smooth muscle cell alpha-actin. In atheroma (fibrous plaque with necrotic core) decorin along with alpha-actin and macrophage marker stained brightly in the extracellular regions in fibrous cap (actively progressing lesion) but was sparse in fibrous base (quiescent lesion). The observations suggest that decorin along with extracellular alpha-actin and macrophage marker may be useful for differentiating lesions that tend to progress with disease.

The distribution of circulating insulin-like growth factor-1 (IGF-1) and its relationship to blood pressure was examined in a community study of 1073 biracial (black-white) adolescents aged 11 to 18 years. Girls of both races displayed higher levels of plasma IGF-1 than did their male counterparts (P < .01), independent of age and sexual maturation. In boys, IGF-1 was correlated positively with height (r = 0.37 P < .001), weight (r = 0.26, P < .001), Tanner stage (r = 0.31, P < .001), and age (r = 0.11, P < .05). Girls, on the other hand, showed an inverse association with age (r = -0.38, P < .001) and Tanner stage (r = -0.10, P < .05). Plasma IGF-1 was correlated positively with systolic blood pressure in boys of both races (r = 0.21 to 0.25, P < .01) and with diastolic blood pressure in white boys (r = 0.18, P < .05), but not in girls of either race. Boys with elevated levels of IGF-1 (>80th percentile) showed significantly higher blood pressure levels, especially during early to middle stages of puberty. Multivariate analysis revealed that IGF-1 was associated with systolic and diastolic blood pressure, independent of age, race, sexual maturation, height, weight, and insulin in boys. These results suggest that plasma IGF-1 may contribute to the regulation of blood pressure only in males during puberty.

The association between microalbuminuria and blood pressure levels was examined in young white and black adults (n = 1131) aged 19 to 32 years. Urinary ratio of albumin (mg/L) to creatinine (mmol/L) was used as an estimation of urinary albumin excretion. Black men and women compared with their white counterparts had higher levels of blood pressure. Significantly positive correlations between urinary albumin excretion and systolic and diastolic blood pressures were observed in black men (r = 0.20 and r = 0.24, P < .01) and black women (r = 0.15 and r = 0.14, P < .05). Similar correlations of significance were not seen in the white counterparts. Systolic and diastolic blood pressure levels were significantly higher in normotensive black subjects (< 140/90 mm Hg) with increased urinary albumin excretion (> or = 90th percentile) than in those without increased urinary albumin excretion. After accounting for potential confounding by age, sex, and body mass index, blacks in the uppermost systolic and diastolic blood pressure group were 7.1 times (95% CI, 2.0 to 25.8) and 4.8 times (1.3 to 18.3), respectively, as likely to have elevated albumin/creatinine excretion as those in the lowest group. In contrast, the likelihood for elevated albumin/creatinine excretion were 0.9 times (95% CI, 0.5 to 2.2) and 1.1 times (0.5 to 2.3), respectively, in whites, which were not significant. These data suggest that a stronger association between blood pressure levels and urinary albumin excretion exists in young blacks than in whites, which supports the notion that blacks may be more susceptible to renal damage from relatively low levels of blood pressure increases.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Noting the distribution of blood values in a biracial southern community according to age, sex, and race variations will help in understanding the normative developmental changes in early life and provides background information., Methods: Complete blood counts were obtained from 3,018 free-living children ages 5-17 years from a well-defined black-white community as part of a cardiovascular risk factor screening., Results: For children ages 5-17 years, hemoglobin levels, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and red blood count increased (P < 0.01) with age. Levels of platelet count and white blood count decreased (P < 0.0005) with age. Compared with females, males between ages 12 and 17 years have (P < 0.05) 0.4 x 10(12)/liter higher red blood count, 1 g/dl higher hemoglobin, 2% higher hematocrit, 0.2 g/dl higher mean corpuscular hemoglobin concentration, 0.5 x 10(9)/liter lower white blood count, 1.4 fl lower mean corpuscular volume, 0.3 pg lower mean corpuscular hemoglobin, and 14 x 10(9)/liter lower platelet count. Compared with blacks, whites have (P < 0.05) 0.5 10(9)/liter higher white blood count, 0.05 10(12)/liter higher red blood count, 0.7 g/dl higher hemoglobin, 1.7% higher hematocrit, 2.4 fl higher mean corpuscular volume, 1 pg higher mean corpuscular hemoglobin, and 0.5 g/dl higher mean corpuscular hemoglobin concentration. A positive association was noted among blood count variables and hemoglobin with blood pressure similar to that in adults., Conclusion: Blood values differ by age, sex, and race. These differences change at maturation and should be considered when defining normal and "abnormal" blood values.

We studied the metabolism of lipoprotein-proteoglycan complexes by macrophage-derived foam cells. Foam cells were isolated from atherosclerotic rabbit aortas. ApoB-lipoprotein-proteoglycan complex was isolated from human aorta fibrous plaque lesions and LDL-proteoglycan complex was formed in vitro. Both in vitro and in vivo complexes stimulated cholesteryl ester synthesis in foam cells by a dose-dependent, saturable process that resulted in the intracellular accumulation of cholesteryl ester. Stimulation of cholesteryl ester synthesis was linear with time over a 32-h period. Polyinosinic acid inhibited the stimulation of cholesteryl ester synthesis by the complexes by 32-37%, whereas cytochalasin D only produced a 6-16% inhibition. Foam cells degraded 125I-LDL-proteoglycan complex and 125I-acetyl LDL in a saturable, dose-dependent manner. Excess unlabeled acetyl-LDL inhibited the degradation of 125I-LDL-proteoglycan complex by 52%, while LDL had no effect. Similarly, excess unlabeled complex suppressed the degradation of 125I-acetyl-LDL by 48%. Foam cells degraded 125I-methyl-LDL-proteoglycan complex to the same extent as 125I-LDL-proteoglycan complex. These results show that foam cells from atherosclerotic lesions metabolize lipoprotein-proteoglycan complexes predominantly via receptor-mediated endocytosis and consequently continue to accumulate intracellular cholesteryl ester.

Race and sex differences in aorta and coronary atherosclerotic lesions were studied in 150 persons aged 6 to 30 years. The intimal surface involvement with aorta fatty streaks was extensive, 0 to 71%, and greater in blacks than in whites (32 vs 20%, p less than 0.001). Coronary artery fatty streaks were more extensive in male than in female subjects (range 0 to 22%). Fibrous plaque lesions were present but not extensive in either the aorta (0 to 12%) or the coronary artery (0 to 24%) specimens. Lesions were more prevalent in male than in female persons, particularly white male subjects. The relation of fatty streaks to fibrous plaques was greater in the coronary vessels than in the aorta. In male subjects, aorta fatty streaks were strongly related to antemortem levels of total cholesterol, low-density lipoprotein cholesterol and ponderal index in white male subjects. Coronary artery fatty streaks in white male persons were significantly associated with serum triglycerides, very low density lipoprotein cholesterol, systolic and diastolic blood pressure and ponderal index. These results link antemortem risk factors to the development of atherosclerotic lesions and emphasize the need for preventive cardiology in early life.

1. Isomeric chondroitin sulfate proteoglycans were extracted from human, bovine, swine and rabbit aortas by 4 M guanidine-HCl and were fractionated and purified by CsCl isopycnic centrifugation, Sepharose CL-4B gel filtration, DEAE-Sepharose ion-exchange chromatography and octyl-Sepharose hydrophobic interaction chromatography. 2. The molecular size and the composition of isomeric chondroitin sulfate proteoglycans varied among species. Variations were also noted in the composition and molecular weight of constituent glycosaminoglycan chains. 3. Observations made on chondroitinase ABC and chondroitinase AC digests of proteoglycans indicate that dermatan sulfate is linked to the core proteins through chondroitin sulfates.

Hypertension was induced in young male pigs by subcutaneous implantation of deoxycorticosterone acetate in a Silastic rubber carrier. Hemodynamic variables were periodically monitored. Six to eight weeks after implantation, the animals were necropsied and aortas were dissected. Glycosaminoglycans (GAG) were isolated from aortas from hypertensive pigs and from normotensive controls. Individual glycosaminoglycans in mixtures were fractionated and quantitated by chromatography on Dowex 1 Cl- column. No differences were noted in total glycosaminoglycan concentration between hypertensive and control animal aortas; the differences in individual GAG in aorta media-adventitia between the groups were not statistically significant. The relative proportions of heparan sulfate and dermatan sulfate in aorta intima were significantly greater (p less than 0.05) in hypertensive aortas than in normotensive intima. These two glycosaminoglycans increased in hypertensive animals at the expense of chondroitin 4-sulfate and hyaluronic acid. There was no difference in chondroitin 6-sulfate between the groups. A possible explanation of changes of glycosaminoglycans in hypertension because of altered lysosomal activity is suggested.

Serum alpha-lipoprotein responses to variations in dietary cholesterol, protein, and carbohydrate were studied in different nonhuman primate species. Chimpanzee, rhesus, green, patas, squirrel and spider monkeys all showed significant interspecies differences in serum total cholesterol responses to 1.84 mg/kcal exogenous cholesterol. Dietary cholesterol significantly increased the alpha-lipoprotein cholesterol in all species except rhesus and chimpanzee. Among these species, there was no relationship between the basal serum lipoprotein profile and subsequent lipoprotein responses to dietary cholesterol. Although the level of dietary protein at 6%, 12%, and 37% of calories had no appreciable main effect on serum total cholesterol in spider monkeys, very low protein diet (6% of calories) produced a significant elevation in alpha-lipoprotein cholesterol. Serum alpha-lipoprotein responses to exogenous cholesterol (1.84 mg/kcal) was highest for the very low protein diet and lowest for low protein diet (12% of calories). Diets with high sucrose (76.5% of calories) and low saturated fat (12.5% of calories) containing no added cholesterol were tested in squirrel and spider monkeys and produced a consistent serum total cholesterol response; the alpha-lipoprotein response was significantly higher in squirrel monkeys than in spider monkeys. The above findings have implications in experimentally induced and comparative atherogenesis.

Dynamics of lipoprotein-glycosaminoglycan interactions in aortas were studied in vivo using the atherosclerotic rabbit model. Severe hypercholesterolemia and atherosclerosis were produced by relatively long-term feeding of a high cholesterol diet. [35S]Sulfate uptake by aorta was measured to assess the sulfated glycosaminoglycan metabolism while the plasma and aorta distribution of 125I-labeled LDL after intravascular injection was determined to monitor aortic LDL uptake and complex formation with glycosaminoglycans. The retention and distribution of LDL as lipoprotein-glycosaminoglycan complexes in different extracellular connective tissue elements were evaluated by extracting the tissues with saline, collagenase and elastase. Hypercholesterolemia with atherosclerosis resulted in a several-fold increase in the uptake of LDL by aorta despite a marked reduction of 125I-labeled LDL in the plasma compartment and in a significant increase in glycosaminoglycan content of aorta coupled with an increased 35S incorporation into glycosaminoglycans. Elastase-solubilized fractions from normal aortas and collagenase-solubilized fractions from atherosclerotic aortas contained maximum labeled and nonlabeled glycosaminoglycan, suggesting alterations in the make-up of fibrous structures of connective tissue matrix in atherosclerosis. Saline extraction and collagenase and elastase digestions solubilized varied proportions of lipoprotein-cholesterol and 125I-labeled LDL, thereby representing different pools of extracellular matrixbound lipoproteins. A tendency for 125I-labeled LDL to increase in collagenase- and elastase-solubilized fractions with time (4 h vs. 24 h) was noted. The occurrence of both lipoproteins and glycosaminoglycan (labeled and nonlabeled) in the ultracentrifugal floating fraction at solvent density 1.063 g/ml demonstrated that the lipoproteins solubilized by different extraction procedures occur in part as lipoprotein-glycosaminoglycan complexes. The specific activities of glycosaminoglycan in the complexes obtained by different extraction procedures differed markedly (elastase greater than collagenase greater than saline), emphasizing the presence of different pools of complexes. Thus, besides arterial cell-mediated processes, extracellular matrix components are important in affecting the retention and accumulation of LDL in atherosclerosis.

The nature of lipoprotein and elastin associated with hyaluronate in human atherosclerotic plaque tissue was studied by digesting the tissues with elastase. The elastase-solubilized lipoprotein-hyaluronate complexes, isolated by Bio-Gel A-50m column chromatography, contained 7.8 mg calcium/100 mg protein. The Sephadex G-200 chromatography of delipidated complexes yielded high (fraction I) and low (fraction II) molecular weight protein fractions. Dialysis of the complexes against 0.01% EDTA resulted in removal of fraction II. Fraction I reacted immunologically against antihuman LDL, and its amino acid composition resembled that of human apoB. Fraction II contained 5 peptide fragment and had high levels of nonpolar amino acids that are characteristic of elastin. However, these peptides also contained high levels of polar amino acids, thus resembling the plaque elastin. These findings suggest that certain regions of plaque elastin may have affinity for apoB containing lipoproteins and calcium.

A defect in Na+-K+ transport across the red cell membrane has been shown to be associated with essential hypertension. A sensitive assay system to measure active, co- and countertransport systems in erythrocytes from normotensive adults was developed. Active, co- and countertransport systems in the erythrocytes were assayed by measuring the influx of radioactive 22Na+ and 86Rb+. In the biracial (black-white) population group studied, analysis of variance of the active transport showed a significant race effect (p = 0.003). Cotransport activity showed age by race interaction (p = 0.001) and age by sex (p = 0.02). Cotransport activity was significantly higher in whites than blacks (p = 0.0001). Countertransport activity did not vary either by sex or race. Of the Spearman correlation coefficients for transport activities and blood pressure, white males showed a strong positive correlation with countertransport, whereas in black males, blood pressures showed a strong interaction with active transport. Among the transport activities, active transport showed significant interaction with countertransport activity in black males, whereas cotransport activity in whites showed a strong interaction with countertransport. The results suggest a subtle difference in Na+-K+ transport systems between blacks and whites, and these variations may be related to differences for susceptibility to essential hypertension.

Racial differences in prevalence of essential hypertension are well known. In order to explore these differences at an early age in terms of etiology, we investigated schoolchildren in an entire, biracial community. A sample of 278 children, stratified by diastolic (fourth-phase) blood pressure and specific for age, race, and sex, was reexamined 1--2 yr after initial observation for the following: (1) a physical examination and urinalysis to exclude secondary hypertension; (2) 24-hr urine sodium, potassium, plasma renin activity, and serum dopamine beta-hydroxylase; (3) 1-hr oral glucose tolerance test; and (4) heart rate and blood pressure at rest and under standarized physical stress. We found that 24-hr urine sodium was positively associated with blood pressure level as measured on the same day for the high blood pressure strata of black children. Urine potassium excretion was lower in blacks than in whites, although their intakes seemed equal. In the high blood pressure strata especially, black boys had lower renin activity than whites, and the resting-supine and stressed systolic blood pressures were higher in black boys than in any other group. In these black boys, resting and stressed systolic pressures were negatively related to plasma renin activity. On the other hand, dopamine beta-hydroxylase levels in white children were higher than in blacks for all blood pressure strata, and in the high blood pressure strata white children had higher 1-hr glucose levels and faster resting heart rates than black children. Different mechanisms may play a role in and contribute to the early stage of essential hypertension.

Free and protein-bound serum sugars and serum lipids were analyzed in 65 adult diabetic patients, 10 age-matched controls, and 24 male medical students for correlation of carbohydrate changes with the extent of retinal, renal, and cardiovascular disease. In diabetic sera, both protein-bound sugars and free mannose, fucose, and hexosamine were significantly elevated; free galactose and inositol were elevated in some diabetic patients, and essentially undetectable in sera from controls. Serum triglycerides and pre-beta-lipoproteins were also elevated in diabetics, but alpha-lipoproteins decreased. Although no specific relationships were observed with the extent of retinal and renal disease, bivariate analyses by Pearson coefficients of correlation showed correlations between levels of serum-free mannose and systolic blood pressure, free hexosamine and duration of diabetes, and serum protein-bound fucose and age. Serum triglycerides and pre-beta-lipoprotein levels correlated with insulin therapy. These are preliminary leads of laboratory studies related to carbohydrate macromolecular changes which might aid in a better understanding of the cardiovascular complications associated with diabetes.

The concept of injury as a mechanism leading to atherosclerosis has been fostered by numerous studies of initiating factors and by observation of the response of cardiovascular connective tissue, ie, cellular and extracellular matrix components. Carbohydrate-protein macromolecules of the extracellular matrix are a complex group of biologically important substances that play a crucial role in mesenchymal tissue repair following injury, a process needed to maintain arterial wall integrity. Of particular interest are the proteoglycans that enter into a variety of roles, from that of inhibiting atherosclerosis and helping to maintain fibrillar structures to that of taking part in lipid deposition in the development of atherosclerosis.

Three hundred-eighty-eight children were selected from a total community and biracial (black-white) population for a special in-depth study related to serum lipoproteins and carbohydrate metabolism. Based on two previous serum lipoprotein determinations of high and/or low beta and pre-beta-lipoprotein cholesterol, they were stratified into four groups. A glucose tolerance test was performed for fasting, 30-minute, and one-hour glucose, insulin, free fatty acids, calcium, and magnesium. Observations of height, weight, and triceps skinfold were also obtained on the children. Children in the high beta-lipoprotein cholesterol groups tended to have higher glucose levels and were more obese than the other groups, while children in the high pre-beta-lipoprotein cholesterol group tended to have high insulin levels following the glucose load. Fasting blood levels were not appreciably different in the various groups, but after the glucose load an unusually high insulin secretory response occurred in black children, especially black girls. Black girls also demonstrated somewhat lower blood sugars than the other race-sex groups. The insulin/glucose ratios were dramatically different in the black children, especially black girls. These differences were particularly noted in the groups with the high pre-beta-lipoprotein. Black children also tended to have higher insulin/free fatty acid ratios during the glucose tolerance test. These differences persisted even after adjusting for obesity. Although not significant, calcium levels consistently decreased in all groups following a glucose load. The observation of racial contrasts in glucose and insulin responses are interesting. While black girls appear to show low glucose and high insulin responses to a glucose load, low and delayed insulin response along with high glucose response occur in whites, especially white girls. Since white children have greater body fat content, these observations suggest more insulin resistance in white children. Even at low levels of obesity, subtle carbohydrate lipid metabolic aberrations are found in children having high levels of serum lipoproteins. A persistence of these conditions could contribute to accelerated atherosclerosis and subsequent coronary artery disease.

Connective tissue macromolecules, glycosaminoglycans, glycoproteins, collagen, and elastin were isolated from different parts of the respiratory system and characterized. The materials included bronchiolar tissue, gas-exchange tissue, lung pleura, and tracheal mucosa. The similarity of the macromolecular composition of lung pleura and tracheal mucosa suggests a common cellular component in these structures. The high concentration of GAG and collagen in bronchiolar tissue is consistent with the cartilagenous nature of this tissue. Particularly interesting is the high content of heparin in all pulmonary structures, a relatively greater content of hyaluronic acid in gas-exchange tissue, and a high content of heparan sulfate and dermatan sulfate in vascular tissues. Elastin also occurs as a major fibrous structure. Although the biologic role of these connective tissue macromolecules has not been established, certain functional relationships are inferred.

Proteoglycans from human atherosclerotic lesions and from uninvolved aortic intima were isolated and their composition was studied. The tissues were sequentially extracted by guanidine hydrochloride followed by hydrolysis of the tissue by elastase. Chondroitin sulfate/dermatan sulfate proteoglycans were predominant in guanidine hydrochloride extracts of the tissue. Most of the heparan sulfate proteoglycans were released from the tissue by hydrolysis with elastase. The content of proteoglycan material, measured as uronate per unit weight of wet tissue, was lower in fatty streaks and fibrous plaques than in uninvolved tissue (0.58 and 0.48 mg vs. 0.7 mg/g wet tissue). The distribution of different glycosaminoglycans in guanidine hydrochloride-extracted proteoglycans was similar among the lesions and uninvolved tissue, but varied in the elastase-hydrolyzed extracts. Gel filtration studies suggested that the major proteoglycan material, chondroitin sulfate proteoglycans, from lesions had greater molecular weight than proteoglycans from uninvolved tissue. The studies indicate that alteration in intrinsic composition and molecular size of proteoglycans occurs in atherosclerotic lesions.

Changes of arterial wall lipid and connective tissue components were studied under various regression regimes on experimental high fat-cholesterol diet induced atherosclerosis in non-human primates. Levels of plasma lipids and aorta tissue lipids were closely related, while an inverse relationship between plasma cholesterol levels was noted with the ratio of aorta tissue free/ester cholesterol. Although the total content of glycosaminoglycan increased slightly with increasing disease, changes of individual glycosaminoglycans were most closely related to the severity of aorta atherosclerosis associated with both induction and regression of disease. With increasing atherosclerosis hyaluronic acid tended to decrease, while chondroitin sulfates decreased. Heparan sulfate decreased considerably with increasing severity of atherosclerosis. Consequently, regression regimens reducing the severity reversed the glycosominoglycan changes. Withdrawal of the dietary stimulus and cholestryamine produced the greatest reversal of atherosclerosis. In contrast, D-thyroxine treatment resulted in severe aorta atherosclerosis comparable to controls receiving no treatment. The effectiveness of regression depends on favorably altering plasma lipids but without adversely affecting the arterial wall structural elements.

The influence of serum lipoprotein profile and measures of carbohydrate metabolism on erythrocyte membrane composition were examined in four groups of children (n = 356, ages 6 to 18 years) from a total biracial population whose earlier very low density (VLDL-C) or low density lipoprotein cholesterol (LDL-C) levels (or both) were in the extreme quintiles or quartiles. Erythrocyte membranes of white children contained significantly increased levels of protein (+2.0%) and phospholipid (+2.7%) and decreased levels of neutral sugars (-4.6%) when compared to black children. Membrane neutral sugars were markedly higher (+4.7%) in girls than in boys. Membrane protein was lowest in children characterized by high LDL-C and low VLDL-C. The phospholipid/protein ratio was consistently higher (+1.9%) in the low VLDL-C strata. The relationships between membrane constituents and plasma/serum variables were explored after controlling for race, sex, and age effects and after combining the four selection groups. Less membrane cholesterol was present in the lowest (v highest) quintile of serum LDL-C/high density lipoprotein cholesterol (HDL-C) ratio (P less than 0.01). Membrane phospholipid levels were lower in the highest (v lowest) quintiles of the apoB/apoA-I ratio (P less than 0.01). With respect to measures of carbohydrate tolerance, membrane hexosamine showed low values in the highest (v lowest) quintile of hemoglobin A1c, while the trend was opposite for plasma insulin response (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Two proteoglycan-hyaluronate complexes were extracted from bovine aorta with 4.0 M guanidinium chloride in the presence of protease inhibitors and purified by cesium chloride density gradient centrifugation under associative conditions. Complex I with higher buoyant density had 22% protein and 18% uronate, whereas Complex II had 11% protein and 23.6% uronate. Glycosaminoglycan (GAG) analysis of the complexes indicated that both complexes contained large amounts of hyaluronic acid, 13.6% and 24.0% of the total GAG in Complexes I and II, respectively. Chondroitin sulfates and dermatan sulfate constituted the remainder of the GAG. Chromatography on Sepharose CL 6B suggested that Complex I had an estimated molecular weight of 800,000 and Complex II, 250,000. The complexes were dissociated into proteoglycan and hyaluronate by cesium chloride density gradient centrifugation under dissociative conditions. The proteoglycans free of hyaluronic obtained from the complexes had similar protein and GAG composition. The molecular weight of the proteoglycan was estimated to be 80,000 and that of the hyaluronate of the complex, 90,000.

Extracellular glycoproteins play an important role in wound healing; yet little is known about glycoprotein biosynthesis and its regulation by insulin in inflammation. Using [1-14C] fucose as a marker, glycoprotein biosynthesis was studied in carrageenan-induced granuloma from diabetic and control rats. Fucose incorporation into glycoproteins was followed for 24 h after an intraperitoneal injection of the label. Radioactivity in trichloroacetic acid precipitable serum glycoproteins and saline-soluble and insoluble glycoproteins was assessed in five-, seven-, and ten-day-old granuloma tissues. Fucose incorporation was higher in soluble glycoproteins (P less than 0.01) at all points in controls than in diabetic granulomas, and peak incorporation was reached in both groups on the seventh day. Incorporation of fucose into insoluble glycoproteins was higher in normals on the seventh day than in diabetics. Liver-, kidney-, and intestine-soluble glycoproteins showed a maximum incorporation on the seventh day, but no difference was noted between diabetic and normal rats. Incorporation of fucose in insoluble glycoproteins showed a gradual decline with the age of granuloma in all tissues from both groups, with the exception of the kidney. In the kidney, fucosylation of insoluble glycoproteins was decreased (P less than 0.01) in diabetics compared to controls. These results indicate an active phase of biosynthesis, with an increase in glycosylation during inflammation that is probably insulin dependent.

Proteoglycans were extracted from bovine lung gas exchange tissue, pleura, and bronchioles with 4.0 M guanidinium chloride at 5 degrees C in the presence of protease inhibitors. Preliminary purification of the proteoglycans was achieved by an initial CsCl isopycnic centrifugation (rho 0 = 1.33) and through precipitation with cetylpyridinium chloride in 0.5 M KCl. Further purification and fractionation of proteoglycans was achieved by a second CsCl isopycnic centrifugation (rho 0 = 1.45) in 4.0 M guanidinium chloride. Based on the ultracentrifuge profiles and electrophoretic behavior, the major fractions were pooled. They were purified further by gel filtration on Sepharose CL-2B and characterized by extensive analyses. A heparan sulfate proteoglycan was the major proteoglycan identified in the gas exchange tissue and in the pleura. The major proteoglycan component from the bronchioles was a chondroitin sulfate proteoglycan. Approximate molecular weight of 2 x 10(6) for the heparan sulfate proteoglycan from the pleura and chondroitin sulfate proteoglycan from the bronchioles and 1 x 10(6) for the heparan sulfate proteoglycan from the gas exchange tissue were estimated from gel filtration analyses. After incubation with hyaluronic acid, the chondroitin sulfate proteoglycans from the bronchioles showed an increase in specific viscosity and a higher molecular weight compound eluting near the void volume in Sepharose CL-2B column chromatography. The proteoglycans exhibited varied anticoagulant activities in Stypven, partial thromboplastin and thrombin clotting times and inhibited thrombin-induced platelet aggregation.

Serum proteins and hemoglobins show increased nonenzymatic glycosylation in diabetes mellitus. The measure of glycosylated proteins, particularly hemoglobin, is considered to be a preferred indicator in the control of diabetes. In a study of diabetes and inflammation, we assessed the extent of nonenzymatic glycosylation of proteins of granulation tissue from diabetic rats. Five, seven, and ten days after carrageenan injection, the granuloma proteins were extracted. Nonenzymatic glycosylation was measured in soluble and insoluble granuloma proteins by thiobarbituric acid assay. Protease activities and free amino groups were assayed in soluble extracts. Nonenzymatic glycosylation in soluble proteins of both groups reached a maximum on the seventh day. However, nonenzymatic glycosylation in soluble proteins of the diabetic granulomas was significantly greater than the controls on days five and seven. During the days after granuloma induction, nonenzymatic glycosylation in the insoluble granuloma tissue proteins gradually decreased without any significant differences between controls and diabetics. Significant decreases in the free amino groups in soluble proteins of the diabetic tissues were noted. Greater activities of cathepsins B and D were noted in diabetic tissues over controls. These observations suggest that, in addition to increased proteolysis, increased nonenzymatic glycosylation of tissue proteins could be associated with the impaired process of wound healing in diabetics.

We describe a procedure for determining thiocyanate in plasma with a Technicon AutoAnalyzer I, modified with a 50-mm tubular flow cell to increase the sensitivity of thiocyanate detection. The procedure is suitable for distinguishing cigarette smokers from non-smokers, the mean for smokers being 151 (SD 43) micromol/L and for non-smokers 62 (SD 19) micromol/L.

Since salt-sensitivity to blood pressure (BP) response is a species-related phenomenon, the response of BP to dietary sodium was examined in 14 adult male African green monkeys (Cercopithecus aethiops). The animals were exposed to graded increase of dietary NaCl at 0 (control), 3.0, 4.5, and 6.0%, respectively for 45, 139, 37, and 90 days. Systolic and diastolic BP and body weights were measured twice weekly. Body weight did not differ significantly between successive diets. The mean (+/- SD) systolic/diastolic BP for control, 3.0, 4.5, and 6.0% NaCl diets were 88.8 +/- 15.4/65.9 +/- 13.7, 101.9 +/- 17.6/72.1 +/- 13.0, 90.5 +/- 20.9/62.2 +/- 16.2, and 115.3 +/- 22.6/81.0 +/- 16.4, respectively. BP changes between successive diets were significant (p less than 0.05). Monkeys with high or low initial mean arterial BP levels on control diet tended to maintain the same order with significantly high correlations between BP levels for successive diet periods (r = 0.88 to 0.95, p less than 0.0001). Thus as a group, this nonhuman primate species responds to increased NaCl intake with elevated mean systolic and diastolic BP. Individual variations in salt sensitivity and consistency in persistence of BP levels in this species has bearing on human hypertension.

The effect of obesity on the connective tissue composition of skin was investigated in mice with goldthioglucose (GTG)-induced obesity. Four months after GTG treatment, the obese animals were sacrificed. Acid mucopolysaccharides, glycoproteins, collagen, and elastin were analyzed in the skin and compared to the controls. Total MPS in the skin from obese animals decreased, reflected mostly in hyaluronic acid. Chondroitin showed an increase over controls. The content of soluble glycoproteins varied; total carbohydrate and sialic acid of the glycoprotein tended to increase with obesity. Collagen and elastin both tended to decrease with obesity.

Connective tissue composition of aortas from several non-human primate species has been studied in an effort to relate collagen, elastin, ang glycosaminoglycan (GAG) content to species susceptibility to atherosclerosis. Among the species studied the baboon contained the highest content of GAG in the aorta. While the distribution of individual GAG varied from species to species, heparan sulfate (HS) was the highest GAG in aortas from most of the species. The ratio of HS to chondroitin sulfates (CS) plus dermatan sulfate (DS) was lowest in the baboon, a species relatively less susceptible to atherosclerosis, and highest in the squirrel monkey, a very susceptible primate. If a relationship exists between HS to CS + DS ratio in the aorta and atherosclerosis, the primates can be arranged in the following decreasing order of susceptibility: squirrel, chimpanzee, stump-tailed, rhesus, African green, patas, baboon. In studies of other connective tissue components, the proportion of total collagen to elastin was found lowest in the baboon. Such observations emphasize the importance of connective tissue in the pathogenesis of atherosclerosis.

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.

A special in-depth substudy was conducted on 388 children from a total biracial (black-white) population, who were stratified on levels of serum beta- and pre-beta-lipoprotein cholesterol to explore factors associated with lipoprotein levels in childhood. Biochemical parameters on venous blood samples were obtained both on fasting subjects and after an abbreviated glucose tolerance test, along with selected anthropometric measures like height, weight, and skinfolds. Biochemical and anthropometric relationships were minimal for children with elevated beta-lipoprotein cholesterol and low pre-beta-lipoprotein cholesterol. On the other hand, children with higher levels of pre-beta-lipoprotein cholesterol, with or without elevated beta-lipoprotein cholesterol, showed associations with fatness and slightly higher levels of glucose and insulin, with other biochemical parameters considered within normal levels. These differences noted among free-living children with different levels of serum lipoproteins provide clues to mechanisms involved in the early natural history of coronary artery disease.

Copper deficiency adversely affects the extracellular matrix of the arterial wall, leading to cardiovascular lesions. To study the lesions resulting from copper deficiency, the composition of proteoglycans from aortas of copper-deficient rats was compared with proteoglycans of aortas from copper-supplemented rats. Copper deficiency in rats was verified by copper levels in adrenal glands (mean +/- SE, 0.37 +/- 0.07 vs 1.03 +/- 0.17 micrograms/g wet wt in supplemented rats). The proteoglycans were isolated from the aorta by extraction with 4 M guanidine-HCl and by digestion of the tissue with elastase. The proteoglycans were purified by CsCl isopycnic centrifugation and fractionated by gel filtration. The fractions were characterized for molecular size and glycosaminoglycan composition. Total uronate in the aortas from copper-deficient rats was 25% greater than in aortas from copper-supplemented rats, and the proteoglycans from copper-deficient rat aortas were of greater molecular size. Among the glycosaminoglycans the concentration (microgram/mg tissue) of isomeric chondroitin sulfates, particularly dermatan sulfate, was greater in copper-deficient animals than in copper-supplemented animals. These observations are similar to earlier findings in experimental atherosclerosis and to a response of cardiovascular connective tissue to injury.

Glycoproteins were extracted from human atherosclerotic lesions by a phosphate buffered heparin solution and by 0.15 M NaCl, followed by sequential digestion of the tissue by collagenase and elastase. Proteins obtained from the extracts by fractional precipitation by (NH4)2SO4 at 40%, 60% and 100% saturation of the salt at pH 4.0 were analyzed for total protein and for fibronectin and laminin by immunodiffusion. Greater amounts of proteins were extracted from atherosclerotic lesions than from uninvolved tissue. The buffered heparin solution extracted several-fold more protein than 0.15 M NaCl. Fibronectin was found in most protein fractions from every extract, but the presence of laminin was noted only in the protein fractions precipitated at 40% saturation of (NH4)2SO4 in the extracts of the tissue by heparin.

The effect of various diets on the aortic glycosaminoglycan (GAG) composition was studied in rhesus monkeys. Aortas were obtained from monkeys fed diets containing cholesterol and comparative fats including coconut oil--butter and peanut oil and with and without cholestyramine. Additional groups in each experiment were placed on regression diets of low-fat, low-cholesterol with and without cholestyramine. Further, an atherogenic diet of coconut oil--butter was alternated every 2 months with a diet enriched with corn oil. GAG isolated from intima and media--adventitia indicated slight variations in the concentration of total GAG among different dietary groups but major differences in the concentration of individual GAG. The concentrations of hyaluronic acid and heparan sulfate were generally greater in aortas of monkeys fed corn oil diets than in those fed coconut oil--butter or peanut oil diets. The concentration of dermatan suulfate generally decreased during regression of lesions induced by the saturated fat--cholesterol diet. Furthermore, the aortas of monkeys with lesions from feeding peanut oil showed higher levels of dermatan sulfate and lower levels of chondroitin 4-sulfate than the saturated fat-fed groups. The addition of cholestyramine enhanced the effects of regression. These observations show that the composition of GAG of the arterial wall can be influenced by various dietary programs and that GAG play a role in induction and regression of experimental atherosclerotic lesions.

Heparin-like glycosaminoglycans (GAG) were isolated from commerical Vessel and their biologic properties studied. Vessel was found to be a mixture of chondroitin sulfates, dermatan sulfate and heparin-like GAG. Chondroitin sulfates and dermatan sulfate in Vessel were hydrolyzed by chondroitinase ABC and the residual Vessel was fractionated on a Dowex-1 Cl- column eluting with a stepwise-increasing concentration of NaCl (1.2--4.0 M). The major fractions eluted at 1.6 M and 1.8 M NaCl were tentatively identified by chemical analysis as heparin-like GAG with somewhat lower sulfate content than standard heparin. Both fractions had lipoprotein lipase-releasing activity and anticoagulant activity similar to heparin, but 1.6 M NaCl fraction had a third of the anticoagulant activity of standard heparin. The 1.8 M NaCl fraction complexed with serum lipoproteins similarly to heparin. In preliminary studies cholesterol-fed rabbits treated with Vessel exhibited somewhat less atherosclerosis than controls.

The effect of various antiatherogenic regimens on glycosaminoglycan and collagen concentrations in aortas from cynomolgus monkeys with diet-induced atherosclerosis was studied. The drugs and materials that were studied included d-thyroxine, [4-chloro-6-(2,3-xylidino)-2-pyrimidinyl]thioacetic acid, cholestyramine, alfalfa, and glucophage. The treatments resulted in varied degrees of regression of lesions. The mean hydroxyproline concentration in aortas among groups of animals treated with different regimens was significantly different within the groups (p less than 0.001) and correlated with the severity of the lesions (p less than 0.01). The mean total glycosaminoglycan concentration among different groups did not differ significantly but correlated positively (p less than 0.05) with the severity of lesions. Heparan sulfate and hyaluronic acid increased with regression and decreased with increasing severity of lesions, whereas chondroitin sulfates followed an opposite trend. These observations show connective tissue components are intimately involved in remodeling the aorta during regression of diet-induced atherosclerosis.

Cardiovascular risk factors in childhood are related to arterial wall changes that lead to atherosclerotic coronary artery disease in later life. Atherosclerosis begins early in life. The observations of early arterial wall connective tissue changes and accompanying early lipid deposition show the importance of understanding cardiovascular risk factors in children. Since risk factors found in childhood are potentially predictive of adult coronary heart disease, methods for prevention of atherosclerosis should begin in children. Rational strategies should be directed to removing atherogenic forces that work in a child at high risk. Primary prevention of atherosclerosis has its maximal potential when begun before advanced irreversible lesions can occur. Consideration needs to be directed to how cardiovascular connective tissue changes and lipid and calcium deposition can be modulated in the injury and healing processes. It is important to recognize that adult coronary artery disease is really a major pediatric problem.

We studied the effect of low-density lipoproteins (LDL) on the synthesis and secretion of proteoglycans by cultured human umbilical-vein endothelial cells. Confluent cultures were incubated with [35S]sulphate or [3H]glucosamine in lipoprotein-deficient serum in the presence and in the absence (control) of LDL (100-400 micrograms/ml), and metabolically labelled proteoglycans in culture medium and cell layer were analysed. LDL increased accumulation of labelled proteoglycans in medium and cell fractions up to a concentration of 200 micrograms/ml. At this concentration of LDL the accumulations of proteoglycans in medium and cell layer were 65% and 32% respectively above control for 35S-labelled proteoglycans, and 55% and 28% respectively above control for 3H-labelled proteoglycans. At concentrations above this LDL was found to depress the accumulation of proteoglycans in medium and cell layer. Gel filtration on Sepharose CL-4B showed that in both control and LDL-treated cultures the cell layer contained a large (Kav. = 0) and a small (Kav. = 0.35) heparan sulphate proteoglycan, whereas the culture medium contained a large heparan sulphate proteoglycan (Kav. = 0) and a smaller isomeric chondroitin sulphate proteoglycan (control, Kav. = 0.35; LDL-treated, Kav. = 0.17). The relative increase in hydrodynamic size of the isomeric chondroitin sulphate proteoglycan (Mr 150,000 compared with 90,000) in the medium of cultures exposed to LDL was partly attributable to the larger size of the glycosaminoglycan side chains (Mr 39,000 compared with 21,000). The isomeric chondroitin sulphate proteoglycan in LDL-treated culture was relatively enriched in chondroitin 6-sulphate compared with that in control cultures (39% compared with 29%). Pulse-chase studies showed that LDL treatment did not alter the turnover rate of proteoglycans as compared with controls, implying that the elevation in proteoglycan accumulation in LDL-treated cultures was due to enhanced synthesis. These results demonstrate that LDL can modulate proteoglycan synthesis by cultured vascular endothelial cells, resulting in the secretion of a larger isomeric chondroitin sulphate proteoglycan enriched in chondroitin 6-sulphate.

Glycoproteins from bovine aorta intima were isolated by a sequential digestion of the tissue with collagenase and elastase after extration of the tissue with saline. The proteins in the extracts were precipitated by (NH4)2SO4 and fractionated by Con-A sepharose affinity chromatography. The fractions were analyzed for their carbohydrate composition and by polyacrylamide disc gel electrophoresis. These studies show that considerable heterogeneity of aorta glycoproteins exists. Some of the glycoprotein materials are likely intimately associated with fibrous structures, collagen and elastin, of the aorta intima.

The interaction of lipoproteins and arterial connective tissue macromolecules was studied using human atherosclerotic plaque tissues. After extraction with 0.15 M NaCl, the tissues were repeatedly digested with collagenase followed by elastase. The collagenase-solubilized lipoprotein--GAG complexes were isolated by gel-filtration and ultracentrifugation and analyzed for lipids, GAG and protein. While extraction by 0.15 M NaCl released only about 13% of the total cholesterol from the tissues, subsequent digestions by collagenase and elastase yielded 60% and 17% cholesterol, respectively. Both 0.15 M NaCl and collagenase treatment released equal amounts of GAG and accounted for 84% of the total GAG. Immunologically, lipoproteins resembled serum apoB-containing lipoproteins. Bio-Gel A-50m column chromatography of collagenase-extracted materials gave a single peak which contained lipoproteins of 1.006 and 1.063 floating densities, GAG and hydroxyproline. Hyaluronic acid (HA) and chondroitin 6-sulfate were identified; HA was the major GAG. Although the precise nature of the interaction of arterial connective tissue components with lipoproteins is not completely understood, isolation of such complexes indicates the importance of these macromolecules in sequestration of lipoproteins.

We compared in vitro heparin binding activity and in vivo intravascular clearance and aortic uptake in rabbits of native, reductively methylated and heparin-complexed low density lipoprotein (LDL) in order to explore the extracellular matrix binding vs cellular metabolism of LDL. Reductively methylated LDL formed soluble and insoluble complexes with heparin which was comparable to native LDL. Reductive methylation of LDL produced only 30% reduction in aortic uptake vs 60% reduction in plasma clearance, reflecting the relatively smaller contribution of receptor-mediated pathway in aortic tissue vs whole animal. The intravascular clearance of native and heparin-complexed LDL remained essentially the same, indicating similarities in cellular metabolism of LDL in both cases. But the aortic uptake of the heparin bound LDL was 30% less than the native LDL, suggesting an inhibition in binding of heparin-complexed LDL to tissue proteoglycans. Saline extraction accounted for only part (53-66%) of the LDL preparations that were retained by the tissue while subsequent collagenase and elastase treatments extracted 3-5% and 17-22% of the materials respectively. These results favor the contribution of arterial extracellular matrix components to the retention of LDL.