Augustyn Jm

Regression of Advanced Atherosclerosis in Swine

Advanced complicated atherosclerosis was produced in the abdominal aorta of swine by a combination of mechanical injury and high-cholesterol, high-fat diet for four months. After removal of the high-cholesterol diet and placing the animal on swine mash for 14 months, there was a significant (P less than .005) decrease in size of lesions with remodeling of the intima toward a smooth surface. Sudanophilia had virtually disappeared and atheromas were almost absent in the regression group, as were thrombosis and hemorrhage in plaques. Cell proliferation, as judged by the number of labeled cells in autoradiography, was less pronounced in this group. There was no decrease in the numbers of segments showing calcification; however, the size of the calcified areas was smaller in the regression group than in the base line. The data suggest that advanced atherosclerosis is susceptible to regression on removal of the atherosclerotic stimulus.

Glycosaminoglycan-lipoprotein complexes from aortas of hypercholesterolemic rabbits. Part 1. Isolation and characterization.

Glycosaminoglycan-lipoprotein complexes were isolated from rabbit aortas exhibiting nearly confluent cholesterol-induced foam cell lesions by extraction with 0.15 M NaCl. Purification and characterization was achieved by gel chromatography, non-ionic differential flotation and by cellulose polyacetate electrophoresis. Analysis showed that these complexes consisted of very low density lipoproteins, heparan sulfate, chondroitin sulfate-C and hyaluronic acid. The demonstration that rabbit intimal foam cell lesions contain extractable glycosaminoglycan-lipoprotein complexes makes this animal model an excellent tool for further studies on the role of these complexes in the atherogenic process.

Morphological and biochemical differences among grossly-defined types of swine aortic atherosclerotic lesions induced by a combination of injury and atherogenic diet.

Abstract In swine, three grossly-defined atherosclerotic lesions can be produced simultaneously by balloon catheter injury combined with atherogenic diet. They are: (1) a flat yellow lesion not raised above the surrounding intimal surface; (2) a raised yellowish gray lesion; and, (3) a raised lesion with grossly detectable complications such as calcification, hemorrhage or thrombosis. All three types of lesions were present in animals fed the atherogenic diet for 6 months, and in those subjected to the same dietary regimen followed by 6 weeks of mash feeding. Some morphologic differences were noted between lesions from animals at the two time periods. However, biochemical analysis of lipid composition, DNA concentration and DNA and protein synthesis at these same time periods showed marked similarities. Also, raised and complicated lesions, at any time period studied, were indistinguishable in the above biochemical parameters. The flat lesions were greatly different from the other types of lesions. Their lipid content was intermediate between the normal intima and the other lesion types and their DNA and protein synthesis was similar to the non-lesion areas and significantly less than that of the raised and complicated lesions. The low rate of DNA and protein synthesis of the flat lesion suggests that these lesions are dormant awaiting some stimulus to transform them into a progressive lesion or that they are relatively inocuous end-stage lesions.

Effect of lipoprotein on in vitro synthesis of DNA in aortic tissue

Several factors such as lipoproteins,1,2 platelets3 and insulin4 have been shown to stimulate the proliferation of aortic smooth muscle cells in culture in the presence of serum or serum derivatives. Using an explant system from swine thoracic aorta we have studied the effect of lipoproteins on the synthesis of DNA. Our system, on which we have previously reported in detail,5 exhibits cell proliferation, cell death and a peripheral growth of smooth muscle cells which secrete collagen, elastic tissue and glycosaminoglycans and is well-suited as a model system for use in the study of the phenomenon characteristic of atherosclerosis. In addition, unlike cultures of smooth muscle cells which are both very time-consuming to obtain and which cannot be easily cultured in large numbers, our system has the advantage of having only a 9-day culture period and of allowing for the assay of large numbers of samples in quadruplicate.

Production of Mucopolysaccharides, Collagen and Elastic Tissue by Aortic Medial Explants

Early atherosclerotic lesions consist mainly of smooth muscle cells and extracellular substances, the principal constituents of which are mucopolysaccharides, collagen, and elastic tissue. Varying amounts of intracellular and extracellular lipids are present. In the late phase of the disease the lesion is complicated by necrosis, hemorrhage, and calcification (Daoud et al., 1964; Haust et al., 1960; Geer et al., 1961). The role of the extracellular substances in the progression of the lesion and the occurrence of complications is not clear. One can postulate that the presence of large amounts of these substances in the lesion leads to an increase in its size and encroachment upon the lumen. It is also possible that these substances may influence the accumulation of lipids in the atheroma, thus aggravating the atherosclerotic process. On the other hand, the presence of collagen and elastic tissue may be beneficial in preventing extensive necrosis or in causing healing of necrotic lesions.

Use of aortic medial explants in the study of atherosclerosis

Abstract An in vitro culture system having many of the features of early atherosclerosis has been developed. Explants of swine aortic media develop a peripheral growth with cellular and extracellular components similar to the early lesion, and show both cell proliferation and necrosis. Using this system it has been shown that aortic tissues themselves have differences in potential for both cell proliferation and cell death, and that swine sera differ in capability to cause cell death, cell proliferation, and the development of a peripheral growth. Either serum or aortic tissue derived from swine fed hypercholesterolemic diet results in more cell proliferation, but no differences in cell death. All swine sera contain, in varying amounts, factors, separable from such macromolecules as lipoproteins, which result in DNA synthesis, and DNA breakdown, and inhibit DNA synthesis. Human sera, when used in the swine explant assay, cause the same effects as normal swine sera, with generally greatly increased capacity for stimulating DNA synthesis. Studies of possible correlation between results of human serum assay in the explant system and angiographic evidence of coronary artery disease are in progress. In any studies of the effects of lipoprotein classes in any in vitro system, careful processing of lipoproteins to insure separation from the other serum DNA synthesis-stimulating and synthesis-inhibiting factors is imperative.

Production of glycosaminoglycans, collagen and elastic tissue by aortic medial explants.

Collagen, glycosaminoglycans and elastic tissue are the principle, extracellular substances of the early atherosclerotic plaque. It is well established by in vivo 1–7 and in vitro 8–10 studies that these elements can be synthesized and secreted by arterial

Protein synthesis and its relation to DNA synthesis in aortic medial explants

mooth muscle cells (SMC). In this laboratory we have devised and characterized an in vitro system which, under our standard culture conditions, develops a peripheral growth having many features resembling those found in the early atherosclerotic plaque.11 Both cell proliferation and cell death, which are two important components of atherosclerotic lesions, are observed early in the culture period and continue throughout the life of the explants. The cells in the peripheral growth are SMC and are indistinguishable from those described in early atherosclerotic lesions. The cells secrete collagen, elastic tissue and glycosaminoglycans.

Regression of Complicated Atherosclerotic Lesions in the Abdominal Aortas of Swine

Abstract Total protein synthesis has been investigated in an in vitro system (swine aortic explants) which has many of the features of the early atherosclerotic lesion. The synthetic activity is measured by the incorporation of [ 14 C]leucine into protein the last 2 h of a 9-day culture period. Human sera differ (up to 10-fold) in their capacity to stimulate protein synthesis, and, in addition, cause significantly greater synthesis than normal swine sera when included in the culture medium. The synthetic activity resulting from an ultrafiltrate of serum (mol. wt. 20 000) is less than that resulting from the whole serum of origin, while the recombination of dialyzed serum and dialysate results in enhanced synthesis. There is a high degree of correlation between protein and DNA synthesis measured during this 2-h interval in every experimental situation. Use of metabolic inhibitors shows that DNA synthesis can be almost completely inhibited with no such effect on protein synthesis, but inhibition of protein synthesis causes concomitant depression of DNA synthesis. The explant system may be useful in assessing the importance of the contribution of protein synthesis to the human disease picture.

Characterization of Nucleating Proteolipids from Calcified and Non-Calcified Atherosclerotic Lesions

A number of studies have shown that diet-induced proliferative non-complicated atherosclerotic lesions can regress after removal of the dietary stimulus. In contrast there is no clear evidence of regression of advanced necrotic lesions with their complications such as calcification, hemorrhage and thrombosis. The reason for this lack of information is the difficulty in producing advanced complicated lesions in experimental animals in a reasonable time.