K.E. Fritz

Aortic media explants cell proliferation and production of mucopolysaccharides, collagen, and elastic tissue☆

Abstract Segments of pig aortic medial tissue, cultured in a semisynthetic medium, develop a new growth starting about the fourth day. In the first week most cells in this new growth are primitive cells and fibroblast-like cells with only a few modified smooth muscle cells. At this period, only mucopolysaccharides can be identified in the extracellular substances. By the third week, the great majority of cells are modified smooth muscle cells. The extracellular substance is composed of collagen and elastic tissue in addition to mucopolysaccharides. Since the two phenomena, cell proliferation and the production of these specific extracellular substances, are among the most prominent features of early atherosclerosis, it would seem that this in vitro system could be used advantageously in experiments designed to study the role of individual factors implicated in atherogenesis.

Association of DNA synthesis and apparent dedifferentiation of aortic smooth muscle cells in vitro

Abstract Smooth muscle cells (SMC) of medial explants of swine thoracic aortas appear to undergo in the first few days of culture rapid dedifferentiation toward more primitive forms. The number of cells synthesizing DNA in the explants increases as the number of well differentiated SMC decreases. Electron microscopic autoradiography revealed that more of the cells incorporating 3 H-thymidine were poorly differentiated SMC. Thus it appears that in this in vitro situation dedifferentiation of SMC precedes rapid cell proliferation. In early cholesterol-induced atherosclerosis in swine many partially or completely undifferentiated cells are present. The results of the current in vitro study suggest that the undifferentiated cells in the atherosclerotic lesions could arise from mature SMC through dedifferentiation.

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.

Comparison of pathologic and angiographic findings in a porcine preparation of coronary atherosclerosis.

Coronary atherosclerosis was induced in Yorkshire swine by diet-induced hyperlipidemia and balloon intimal abrasion of a coronary artery. Severe stenoses pathologically similar to the lesions of human atherosclerosis were seen after 8 months of the atherogenic regimen. To examine the relationship between the angiogram and pathology in the assessment of the extent and location of coronary atherosclerosis, antemortem angiographic results were compared with results of pathologic examination. Vernier caliper measurements of the coronary angiogram were compared with results of morphometric evaluation of perfusion-fixed coronary arteries. Isolated focal stenoses were correctly localized and quantified, as were focal lesions within vessels diffusely diseased. Both overestimation and underestimation of lesions occurred at bifurcation sites. Diffuse disease without focal stenoses was not well demonstrated angiographically. Vessels that were angiographically thought to be normal or only minimally diseased demonstrated significantly larger lumens angiographically than pathologically. This is believed to be due to fixation and paraffin-processing artifact, even though fixation was performed by perfusion at physiologic pressure. The demonstration of an excellent correlation between the luminal size as determined angiographically and morphometrically at sites of focal obstruction confirms the value of quantitation of coronary angiograms in vivo as a diagnostic tool in coronary atherosclerosis.

Increased DNA synthesis in aortic explants from swine fed a high-cholesterol diet☆

Abstract Medical explants from the thoracic aorta of swine fed a high-cholesterol (HC) diet, when cultured 4 or 9 days in semisynthetic medium containing serum at 20, 40, 60, or 80% concentration, show a higher rate of DNA synthesis, as measured by incorporation of 3 H-thymidine into DNA, than their paired controls from swine on a normal diet. This increased rate of DNA synthesis was independent of whether the serum supplement was normal or HC. Thus it appears that the HC diet has produced some changes in the medical cells which enhanced the growth potential of these cells. In general, HC serum in the medium resulted in a higher rate of DNA synthesis than normal serum in explants from swine on either a conventional or an HC diet.

The effect of ethane- l-hydroxy-1,1-diphosphonate (EHDP) on necrosis of atherosclerotic lesions

Administration of ethane-1-hydroxy-1,1-diphosphonate (EHDP) to swine with pre-established atherosclerosis resulted in lower lesion calcium concentration, smaller lesions and a decrease in the area of lesions involved in necrosis. Atherosclerosis was developed in Yorkshire swine by balloon catheter-injury to the abdominal aorta, followed by a high cholesterol-high lipid (HL) diet for 4 months. The administration of EHDP (20 mg/kg/day) was begun after these 4 months and continued for 5 additional months along with the atherogenic diet. Other swine were ballooned and fed HL diet for nine months. Morphometric analysis showed that the extent of lesions, expressed as ratio of intima to media was significantly less (P less than 0.05) in the EHDP-treated HL swine, compared to the HL diet-only group. The ratio of lesion areas showing lipid-rich necrotic debris to the area of media was also significantly smaller (P less than 0.05). Biochemical analysis showed that the lesion from the HL drug-treated group contained significantly less (P less than 0.05) calcium compared to that from the HL diet only. Finally, there was significant correlation between average lesion area and average lesion calcium concentration (P less than 0.02) for both groups. While the effect of EHDP on lesion size and calcium concentration has been previously reported for various species such as rabbit and monkey, this study is believed to be the first where a beneficial effect of EHDP on one of the most serious complications of atherogenesis - necrosis - has been documented. The mechanisms by which EHDP may affect necrosis are discussed.

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.