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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 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.