Harley D. Sybers

Control of 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity in Cultured Human Fibroblasts by Very Low Density Lipoproteins of Subjects with Hypertriglyceridemia

Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from human normolipemic plasma, and the VLDL, the intermediate density lipoprotein (IDL), and LDL from patients with Type III hyperlipoproteinemic plasma were tested for their abilities to suppress the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase in cultured human fibroblasts from normal subjects and a Type III patient. Regulation of cholesterol synthesis in the fibroblasts of a patient with Type III hyperlipoproteinemia appears to be normal. VLDL from normal subjects, isolated by angle head ultracentrifugation (d < 1.006) or by gel filtration on BioGel A-5m, were about 5 times less effective than LDL in suppressing HMG-CoA reductase activity, based on protein content, in agreement with previous reports with normal fibroblasts. Zonal centrifugation of normal VLDL isolated by both methods showed that the VLDL contained IDL. Normal VLDL from the angle head rotor, refractionated by the zonal method, had little, if any, ability to suppress the HMG-CoA reductase activity in either normal or Type III fibroblasts. VLDL, IDL, and LDL fractionated by zonal ultracentrifugation from Type III plasma gave half-maximum inhibition at 0.2-0.5 mug of protein/ml, indistinguishable from the suppression caused by normal LDL. Type III VLDL did not suppress HMG-CoA reductase in mutant LDL receptor-negative fibroblasts. Zonally isolated VLDL obtained from one Type IV and one Type V patient gave half-maximal suppression at 5 and 0.5 mug of protein/ml, respectively. Molecular diameters and apoprotein compositions of the zonally isolated normal and Type III VLDL were similar; the major difference in composition was that Type III VLDL contained more cholesteryl esters and less triglyceride than did normal VLDL. The compositions and diameters of the Type IV and Type V VLDL were similar to normal VLDL. These findings show that the basic defect in Type III hyperlipoproteinemia is qualitatively different from the cellular defect found in familial hypercholesterolemia, since the regulation of HMG-CoA reductase activity is normal in Type III fibroblasts. The metabolic defect in hypertriglyceridemia is related to the triglyceriderich lipoproteins which, free of other lipoproteins, have an enhanced ability to interact with cultured fibroblasts to regulate HMG-CoA reductase activity. These studies suggest that, in hypertriglyceridemia, there is a mechanism for direct cellular catabolism of VLDL which is not functional for normal VLDL.

Pulmonary asbestos body counts and electron probe analysis of asbestos body cores in patients with mesothelioma. A study of 25 cases

Malignant mesotheliomas of the pleura and peritoneum are well‐recognized risks of asbestos exposure. We determined the asbestos body content of the lungs from 24 cases of malignant mesothelioma (19 pleural, five peritoneal) and compared such to the content of lungs from 50 consecutive adult autopsies and four cases of overt asbestosis using a Clorox‐digestion concentration technique. The cores of 90 asbestos bodies were examined by energy dispersive x‐ray analysis and compared with similar data from 120 standard asbestos fibers and 20 fiberglass fibers. The malignant mesothelioma patients had asbestos body counts intermediate between those of the general population and those of patients with asbestosis, although some of the mesothelioma cases overlapped with the general population. These latter cases often lacked an identifiable occupational exposure to asbestos. EDXA studies demonstrated an amphibole core in 88 of the 90 asbestos bodies (amosite or crocidolite in 80 of 88, anthophyllite or tremolite in eight of 88), and chrysotile in two instances.

COMPARISON OF TISSUE-CULTURED BOVINE ENDOTHELIAL CELLS FROM AORTA AND SAPHENOUS VEIN

SummaryEndothelial cells were harvested from bovine aorta and saphenous vein with collagenase and cultured in McCoys 5a medium (modified GIBCO) supplemented with 10% fetal bovine serum. The cells were subcultured through 17 passages over 4 to 5 months. The growth properties in culture of the two cell types were compared. Morphological comparisons included phase microscopy and scanning and transmission electron microscopy. Comparisons with cultured aortic smooth-muscle cells were made using phase and scanning electron microscopy. No differences were found between cultured endothelial cells from aorta and saphenous vein. Differences in growth patterns in culture clearly distinguished both endothelial cell types from smooth-muscle cells. The presence of Weibel-Palade bodies identified the cells from both sources as endothelial.

Abnormal suppression of 3-hydroxy-3-methylglutaryl-CoA reductase activity in cultured human fibroblasts by hypertriglyceridemic very low density lipoprotein subclasses

Our previous studies showed that hypertriglyceridemic very low density lipoproteins (HTG VLDL) are functionally abnormal. HTG VLDL, but not normal VLDL, suppress HMG-CoA reductase in cultured normal human fibroblasts. To determine if the suppression by HTG VLDL resulted from a subpopulation of smaller suppressive particles, more homogeneous subclasses of VLDL-VLDL1 (Sf 100–400), VLDL2 (Sf 60–100), and VLDL3 (Sf 20–60) were obtained from the d<1.006 (g°ml−1) fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient and tested for suppression in normal human fibroblasts. VLDL1 and VLDL2 from each of the 12 normolipemic subjects tested failed to suppress HMG-CoA reductase activity in normal fibroblasts. Eleven out of 12 preparations of normal VLDL3 suppressed HMG-CoA reductase, but only one-third as effectively as LDL. By contrast, the VLDL1, VLDL2 and VLDL3 from 15 out of 17 hypertriglyceridemic patients (hyperlipoproteinemia Types IIb, III, IV and V) were highly effective in suppression, with half-maximal suppression at 0.1–2.0 μg VLDL protein/ml. The VLDL abnormality is apparently associated with hypertriglyceridemia and not hypercholesterolemia, since VLDL from a homozygous familial hypercholesterolemia patient with a Type IIa pattern did not suppress whereas each of the VLDL subclasses from a Type IIb patient suppressed. Suppression by HTG VLDL in normal cells is apparently a consequence of interaction of the protein portion of the VLDL with the specific LDL cell surface receptor since HTG VLDL1 treated with 0.1 M 1,2-cyclohexanedione to block arginyl residues failed to suppress the enzyme. Moreover, hypertriglyceridemic Sf 60–400 VLDL failed to suppress HMG-CoA reductase activity in LDL receptor-negative fibroblasts. There were no consistent major compositional differences between comparable normal and hypertriglyceridemic VLDL subclasses which could account for differences in suppression. All VLDL subclasses from Type III subjects were enriched in cholesteryl esters and depleted in triglyceride, relative to the corresponding normal VLDL subclasses. However, Type IV and Type V VLDL subclasses were normal in this repect. We conclude from these studies that small particle diameter is not required for suppression, since HTG VLDL1 and VLDL2 which contained few, if any, small particles were effective in suppression.

Hypertension and atherosclerosis in cholesterol-fed rabbits: Part 1. Mild, two-kidney, one-clip goldblatt hypertension treated with enalapril

Ten groups of New Zealand white rabbits were used to study the effects of mild, chronic two-kidney, one-clip hypertension (HT) and long-term antihypertensive therapy on atherogenesis. Five groups were fed a normal diet (ND) over the 8-month study period; 2 groups, one of which was given enalapril, remained normotensive (NT) throughout the study. Of the 3 HT groups, one was hypertensive for 7 months; the blood pressures of the other groups were normalized after 2 months with enalapril, or by removal of the clipped kidney. The other 5 groups were similar except that they were fed at 0.1% cholesterol diet (CD). The results showed that: neither mild chronic HT nor abrupt, short-term HT exacerbated atherogenesis in the CD-animals; although fibromuscular vascular lesions were present in the aorta of normal-diet, HT animals no atheroma was observed; enalapril therapy had no effect on atherogenesis; enalapril therapy reduced the total weight and the cholesterol and triglyceride content of the aorta of the ND groups regardless of blood pressure history; the aortic triglyceride content, but not the cholesterol content, of the CD group, was reduced by enalapril; and although heart size was unaffected by either diet or blood pressure levels, the mitochondria volume per unit volume of the left ventricle was reduced in both NT-ND and HT-CD groups treated with enalapril.

Hypertension and atherosclerosis in cholesterol-fed rabbits II. One-kidney, one clip Goldblatt hypertension treated with nifedipine

Eight groups of New Zealand white rabbits were used to study the effects of moderate chronic one-kidney, one clip hypertension (HT) and long-term nifedipine therapy on atherogenesis. Four groups were fed a normal diet (ND) over an 8-month study period; two groups, one of which was given nifedipine, remained normotensive (NT) throughout the study. Of the two HT groups, one remained hypertensive for 7 months; the blood pressure of the other group was normalized after 2 months with nifedipine. The other four groups of animals were similarly constructed except that they were fed a 0.1% cholesterol diet (CD). The results showed that: although scattered fibromuscular vascular lesions were present in the aortas of normal-diet, HT animals no atheroma was observed; neither moderate chronic HT nor abrupt, short-term HT exacerbated atherogenesis in the CD-animals; nifedipine therapy had no suppressive effect on either fibromuscular lesions or atherogenesis; nifedipine therapy reduced the aorta weight of the normotensive ND and CD groups; the aortic triglyceride content of both dietary groups was reduced by nifedipine; cholesterol content was unaffected; left ventricular hypertrophy was evident only in HT-untreated groups; and only the weight of the left ventricle of the ND-NT-treated group was significantly reduced, but the mitochondria volume per unit volume of left ventricle myocardial cells was reduced only in the NT-CD group treated with nifedipine. It is concluded that an antihypertensive dosage of nifedipine administered to animals with atherosclerosis does not suppress subsequent atherogenesis.

Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall

SummarySmooth muscle cells (SMC) were cultured from atherosclerotic plaques and uninvolved arteries to determine if differences exist between growth characteristics or ultrastructure of the cultured cells. Eighteen aortic punch biopsies provided the uninvolved tissue, and 58 carotid plaques provided the atherosclerotic tissue. Eighty percent of the sample yielded viable cultured cells, which reached a maximum population doubling time during log phase growth of 72 h (seeding density=1.0×104 cells/cm2, 2nd passage). Growth characteristics of both normal and plaque-derived cells were the same in vitro. Growth rate declined with time in culture, and cell division ceased by the 5th or 6th passage. In culture, spindle shaped cells formed the “hill and valley” configuration typical of SMC. Plaquederived SMC were ultrastructurally similar to SMC from uninvolved vessel wall. Proliferative potential did not vary with age of sex, with method of culture, or with whether the cells were plaque derived or not.

Tissue Cultured Cells: Potential Blood Compatible Linings for Cardiovascular Prostheses

Polymers currently used for cardiovascular replacement, both clinically and experimentally, fall into two categories; rough surfaced and smooth surfaced materials. The reaction of the blood upon contact with each type material is different. The rough surfaced materials, e.g., Dacron polyester (polyethylene terephthalate), are porous, and upon blood contact encourage the formation of adherent thrombus on their surfaces. The smooth surfaced materials, e.g., polyether urethanes, are generally impervious to blood and resist the formation of thrombus; although none of the smooth surfaced materials approach the thromboresistance of the natural endothelial lining of the vessel wall (1,2).

Renin as a risk factor for atherogenesis. Part 2. Effects of hypercholesterolemia and hyporeninemia in the rabbit.

Four groups of New Zealand rabbits were used to study the effect of suppressed plasma renin activity (PRA) on atherogenesis. Control groups were fed normal rabbit chow (Group I) or normal chow supplemented with 0.25% cholesterol--0.75% corn oil (Group III). Group II animals were fed normal chow and received periodic injections of 11-desoxycorticosterone (DOC)pivalate and 0.5% saline to drink, while Group IV animals were treated similarly except that they were also fed the atherogenic diet. Blood pressure and blood chemistry measurements were performed monthly over a 7-month period. The blood pressure was unaffected by either the diet or the DOC-saline treatment, however, the PRA was greatly reduced in the animals receiving DOC-saline (Groups II and IV). Similarly, plasma aldosterone was significantly (P less than 0.05) reduced in the DOC-saline-treated animals. No atheromata were observed in the animals consuming the regular diet, regardless of DOC-saline treatment. All of the animals fed the atherogenic diet showed extensive aortic atheromata. However, there was no difference in the lesion index between the animals with normal PRA levels (Group III) and those with suppressed PRA levels (Group IV). Likewise, microscopic evaluation of the aorta, coronary arteries, and renal arteries failed to show a consistent difference in the vascular involvement between animals of Groups III and IV. We therefore conclude that the suppression of PRA does not have a protective effect on atherogenesis in the cholesterol-fed normotensive rabbit.

Renin as a risk factor for atherogenesis. Part 3. Effects of hypercholesterolemia, hyporeninemia and one-kidney-one clip hypertension in the rabbit.

Abstract The purpose of this study was to test the hypothesis that plasma renin activity (PRA) is a risk factor for cardiovascular disease. Four groups of New Zealand rabbits were used to study the effects of PRA and hypertension on atherogenesis. Control groups were fed normal rabbit chow (Group I), or normal chow supplemented with 0.25% cholesterol (Group III) Group II animals were fed normal chow, had a clip placed around the left renal artery and were contralaterally nephrectomized (one-kidney-one-clip, 1K-1C). The renal arteries and kidneys of Group IV animals, which were fed a cholesterol diet, were manipulated similarly. Blood pressure and blood chemistry measurements were performed periodically over a 7-month period. The blood pressure was unaffected by either diet; however, PRA and aldosterone levels were greatly reduced in the 1 K-1 C groups (Groups II and IV). No atheroma were observed in any of the animals consuming the normal diet (Groups I and II) despite sharply elevated blood pressure in the Group II animals. Medial hypertrophy of small muscular arteries was observed in several Group II animals. All of the animals fed the atherogenic diet showed extensive aortic atheromata. There was, however, a significantly smaller lesion index for the normotensive cholesterol-fed animals (Group III) than those with increased blood pressure (Group IV). Likewise, microscopic evaluation of the aorta, coronary arteries, renal arteries, and kidneys mirrored this difference in the vascular involvement between the animals of Groups III and IV. We conclude that neither relatively high nor low PRA levels affect the development of atherosclerosis or other cardiovascular lesions. However, hypertension with a concomitantly high cholesterol diet acts synergistically in exacerbating atherogenesis.