The effect of ethane-1-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.

Ultrastructural and elemental analysis of calcification of advanced swine aortic atherosclerosis.

During progression and in the early phase on a regression regimen, calcification of the necrotic portion of the atheroma of swine abdominal aorta occurred primarily in degenerated cells or in membranous, vesicular cellular degradation products which varied in size, shape, and the amount of mineral deposit. Calcium appeared to be deposited in amorphous granular or needle-like crystalline forms. Energy dispersive X-ray and line profile analysis showed that the major elements in the heavily calcified portions of the plaques were calcium and phosphorus. There was a direct relationship between the distribution and concentration of these elements indicating that the mineral deposit was a calcium phosphate. Select area electron diffraction analysis of grossly calcified portions of the plaque gave a diffraction pattern identical to that of calcium hydroxyapatite. Calcification was not observed to occur on elastic tissue or collagen fibers.

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.

Detection of an antigenic cell wall layer in Histoplasma capsulatum. An immunoelectron microscopic study.

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall of Histoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.

Role of macrophages in regression of atherosclerosis.

The exact role of macrophages in regression is still not clear. It appears that some of their functions are beneficial, while others are detrimental. Among their beneficial functions are: (1) their ability to phagocytize cellular and extracellular debris and remove them outside the arterial wall. This function may be enhanced by the macrophage's own secretion of fibronectin; (2) their ability to solubilize necrotic debris by their complement of hydrolytic enzymes, thus, rendering them diffusible through the arterial wall; and, (3) their secretion of SMC mitogen and components of the arterial wall. Our work supports the role of macrophages in the removal of necrotic debris by the mechanisms cited in (1) and (2) above. On the other hand, macrophages may be detrimental to regression if they secrete an excess of the same hydrolytic enzymes, mentioned above as being beneficial, and if directed towards normal arterial wall components. This can result in disorganization and degradation of these components, and in more necrosis, as was seen at the six-week regression period in our sequential study. Cell debris resulting from necrosis of SMC and from death of macrophages themselves may form nidi for calcific bodies to occur. Our work suggests this may be the case during regression. Finally, excess stimulation of SMC, mitogen, and the secretion of the arterial wall components may contribute to the lesion growth and could explain the lack of regression in some species and under certain conditions. In conclusion, our hypothesis that the macrophage is a "friend" during regression appears to be only partially true, and their presence at this phase of the disease may be a "two-edged sword." On one hand, they may help in the removal of necrosis, while on the other hand, they may accelerate calcification.

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells.

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.

Fibronectin inhibits morphological changes in cultures of vascular smooth muscle cells.

In culture, vascular smooth muscle cells proliferate until they form a confluent sheet of cells. At that time the morphology of the culture becomes altered and the cells form multilayered regions that eventually develop into nodular aggregations. We now demonstrate that the transition from monolayer culture to nodular culture is influenced by the presence of components in conditioned media. The development of nodules is enhanced by conditioned medium made from nodular cultures but is either inhibited or unaffected by monolayer culture-conditioned medium. Examination of the two types of conditioned media using NaDodSO4- polyacrylamide gels reveals many similarities and one major difference. Nodular-conditioned medium contains a prominent 42 kilodalton polypeptide which is not present in monolayer-conditioned medium. Further, we demonstrate that although both nodular and monolayer cultures produce fibronectin the transition to nodular culture does not occur in the presence of exogeneously added plasma fibronectin.

Sequential morphologic studies of regression of advanced atherosclerosis.

Atherosclerosis produced in the abdominal aortas of swine by balloon injury and six-month feeding of a high-cholesterol diet resulted in a spectrum of lesions that varied, by gross and/or microscopic examination, from early nonelevated fatty streaks to markedly advanced necrotic and calcified atheromas. On gross examination, the flat lesions had disappeared within five months on the regression regimen, while the advanced lesions persisted after 14 months of withdrawal of the dietary stimulus. Microscopically, the advanced lesions showed changes compatible with a healing process, with a virtual disappearance of foam cells, a marked decrease in necrosis, and replacement of necrotic debris by fibrous tissue and calcification that increased during the regression period. In the early phase of the regression process, the decrease in the number of foam cells was accompanied by an increase in the number of macrophages. The latter cells were found to be closely associated with necrosis, which suggests that macrophages may play a role in the removal of necrotic debris. These results, together with those of our previous experiment on regression of less advanced lesions, suggest that the rate and degree of regression of an atheroma may be a function of its severity.

Sequential study of biochemical changes during regression of swine aortic atherosclerotic lesions.

Severe, complicated atherosclerotic lesions of swine abdominal aortas were induced by combination of balloon injury and atherogenic (HC) diet. After a reference group was killed at six months, the remaining animals were fed a mash diet for six weeks, five months, or 14 months. Extensive biochemical studies of lesions and nonlesion areas of aortas from HC diet-fed swine, and nonlesion aortic tissue from mash-fed animals emphasize the following points. Nonlesion tissues from either mash or HC diet-fed animals were remarkably similar in all features studied, and did not change much over a period of at least 20 months. Lesions differed from the adjacent nonlesion tissue in most aspects; the HC nonlesion tissue does not appear to be an intermediate in lesion development. The lesions were dynamic, changing generally in the direction of normalcy, from relatively active to more quiescent. Lesion changes did not appear immediately after institution of the regression regimen; some were delayed several months. When changes did occur, in general they were not linear with time. Whether a complete return to normalcy would result from a longer regression period needs further investigation.

Non-specific esterase activity during regression of swine aortic atherosclerosis.

Non-specific esterase (NSE) activity has been studied by light and electron microscopic enzyme histochemistry in lesion and non-lesion areas of swine abdominal aortas in a sequential study of the regression of atherosclerosis. On the light microscope level, no activity was demonstrable in normal artery but in atherosclerotic lesions it was seen in round cells, elongate cells and foamy cells. Ultrahistochemistry identified these reactive cells as macrophages, smooth muscle cells and foam cells of undetermined origin. Since NSE has been shown to hydrolyze cholesteryl oleate, it is possible that histochemical demonstration of NSE activity may be, in part, localization of cholesteryl ester hydrolase activity.

Study of esterase-positive cells in swine atherosclerosis.

Non-specific esterase activity was studied by light and electron microscopic histochemistry after 6 mos of progression of injury and diet-induced atherosclerotic lesions in the swine aorta, and after 6 wks, 5 mos and 14 mos on regression regimen. At each time period three cellular patterns of reaction product (RP) were seen, round cells with dense RP, elongate cells with dense "granules" of RP, and large "foamy" cells with very faint RP. Ultrastructurally, RP was found in macrophages, smooth muscle cells and foam cells. The number of esterase-positive cells decreased with time and the disappearance of necrosis.

Morphological and biochemical changes in propylthiouracil-diet induced coronary atherosclerosis under a regression regimen.

Many of the morphologic and biochemical features of porcine coronary atherosclerosis produced by high cholesterol, high fat diet and propoylthiouracil returned to control values after cessation of the atherogenic regimen. These include disappearance of foam cells and a decrease in lipid content, DNA concentration, and DNA and protein synthesis. The morpholigic and biochemical features of te atherosclerotic lesions described herein were similar to those produced in swine and other species by a variety of inciting agents during both the progression and regression phase of the disease. These results indicate that the porcine propylthiouracil-diet model may be useful for the study of coronary lesions. Second, the similarities of response of arterial tissue in several experimental animals suggest the possibility that human coronary lesions by analogy may regress under therapeutic regimens.

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 effects of moderate diet and clofibrate on swine atherosclerosis.

Early proliferative coronary atherosclerosis was produced in swine by feeding them a high-fat, high-cholesterol diet for 17 months, at which time one group of animals was killed (reference group), while the remainder was transferred for 12 months to a moderate diet that resulted in serum cholesterol levels of about 190 mg/100 ml. The moderate diet only did not decrease the size of coronary lesions, but prevented their progression. The addition of clofibrate therapy caused regression that involved a significant decrease in size, gross sudanophilia, and extent of calcification and the disappearance of foam-cell lesions. Resultant serum cholesterol levels appear to be more important than the amount of dietary cholesterol in the progression, prevention, and regression of swine coronary atherosclerosis.

Biochemical effects of moderate diet and clofibrate on swine atherosclerosis.

Aortic atherosclerotic lesions were induced in swine by feeding them an atherogenic diet for 17 months. The effect of a moderate diet (up to 8 gm of cholesterol per day), with or without added clofibrate therapy, for the subsequent 12 months was assessed by biochemical analysis of carefully dissected lesions and adjacent nonlesion areas. The moderate diet alone prevented "progression," except for accumulation of free cholesterol and enhancement of total protein and collagen synthesis, and caused regression of DNA concentration to nonlesion levels. The addition of clofibrate therapy enhanced regression, with significant decreases in DNA and esterified cholesterol concentrations and in the rate of DNA synthesis. Extrapolation of these results to man suggests that a "sensible" level of dietary lipid may be prophylactic against further progression, while addition of an effective hypolipemic drug may be therapeutically useful.

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

(1)Using both an explant system from swine thoracic aorta and an aortic smooth muscle cell culture system, we have investigated the effect of lipoproteins on the synthesis of DNA in the absence of serum or serum derivatives. (2)Chemical and morphological data from both systems confirm that (a) neither VLDL, LDL nor HDL account for any appreciable part of the DNA synthesis activity of whole serum. (b) There is no difference in activity between lipoprotein fractions derived from either normocholesterolemic or hypercholesterolemic serum. (c) The activity resident in whole serum is present largely in the pelleted serum protein fractions and in the soluble resideu. (3) The data suggest that smooth muscle cell proliferation results from the interaction of lipoproteins with other factor(s) in serum.