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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.

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.

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.

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.

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.

This report describes an automated gel filtration procedure for estimation of molecular weights of glycosaminoglycans using Technicon sugar analyzer. A Sepharose CL-6B column was used for filtration. The method has been applied to determining the distribution of molecular weights of glycosaminoglycans from human aorta. The glycosaminoglycans were fractionated on a Dowex 1 (Cl-) column prior to gel filtration. On gel filtration, hyaluronic acid resolved into two components with estimated molecular weights of 38,000 and 8000. A molecular weight of 22,000 was estimated for heparan sulfate. Chondroitin 6-sulfate and dermatan sulfate eluted from the column in one peak, suggesting these two glycosaminoglycans have a similar molecular weight, 18,000. The procedure was found suitable for studying the gel filtration behavior of proteoglycans from bovine aorta and lung.