Atherosclerosis-related remodeling of aortic relaxation to purines in the Watanabe heritable hyperlipidemic rabbit

The mechanism of the unimpaired relaxant effect of ATP in the Watanabe heritable hyperlipidemic rabbit aorta was investigated to elucidate the involvement of P2y purinoceptor at the endothelial level during atherosclerosis. Experiments were carried out on isolated thoracic aorta from such rabbits that were 12 months of age. The potent P2y purinoceptor agonist, 2-methylthio-ATP, did not induce any endothelium- or smooth muscle-dependent relaxation, thus excluding any involvement by the P2y purinoceptor. ADP, but not AMP, produced relaxation of the aorta by acting at both endothelial and smooth muscle levels. Adenosine relaxed the vessel by acting only in smooth muscle. The maintained endothelial relaxant effect of ATP and ADP is therefore not due to activation of P1 or P2y purinoceptors but may involve activation of a remodeled purinergic receptor site that emerges with the progression of atherosclerosis. This site is antagonized by methylene blue. The disorganization of the endothelial monolayer observed in the morphological analysis may be related to functional remodeling of the endothelial purinergic activity in atherosclerosis.     

The effect of new synthetic cholesterol derivatives on cholesterol metabolism in cultured rabbit hepatocytes

The effects of three novel synthetic derivatives of cholesterol with ethoxy (I), aminoethoxy (II), azidoethoxy (III) and toluenesulfonyloxyethoxy (IV) groups in the 3 beta-hydroxy position of cholesterol on cholesterol synthesis as well as on apolipoprotein B and bile acid secretion in cultured rabbit hepatocytes have been studied. 3 beta-(2-hydroxyethoxy)-cholest-5-en (I) was used as a standard. It was found that the inhibiting effect of these compounds on cholesterol synthesis depends on their structure. Compound II (1 microgram/ml), which inhibited acetate incorporation into cholesterol by 30-50%, appeared to be the most effective among the other compounds tested. This derivative had no effect on the production of bile acids. Compound III was less effective, while compound IV had no effect on cholesterol synthesis. All the compounds under study reduced by 20-36% the secretion of the total apolipoprotein B as measured by the enzyme-linked immunosorbent assay (ELISA). None of the synthetic cholesterol derivatives influenced the leucine incorporation into the total protein fraction. The results obtained indicate that 3 beta-(2-aminoethoxy)cholest-5-en, the most effective compound among other cholesterol derivatives tested in the study, can serve as a basis for synthesizing novel cholesterol derivatives able to inhibit cholesterol biosynthesis in liver cells and to decrease the secretion of very low density lipoproteins in cultured rabbit hepatocytes.     

Growth of rabbit aortic smooth muscle cells in serum from patients with juvenile diabetes

The effect of human diabetic serum on the growth of rabbit arterial smooth muscle cell cultures was studied in the stationary phase of growth. The serum was obtained from young, male, non-obese, juvenile diabetics and non-diabetics. The experiments were carried out using dialysed as well as non-dialysed serum. The concentration of cholesterol and triglycerides were equal in normal and diabetic serum. Media supplemented with diabetic serum from both short term and long term diabetics stimulated the outgrowth of the smooth muscle cells significantly (2p less than 0.01). A statistically significantly stimulation of growth was also observed using dialysed human diabetic serum (2p less than 0.05). Autoradiographic studies showed that the number of 3H-thymidine labelled cells and of cells in mitosis increased appreciably after incubation in diabetic human serum (2p less than 0.005). The present data show that human serum from juvenile diabetics contains a factor or factors which promote an excessive growth of arterial medial cells. The factor(s) is not lipids as hyperlipemia was not present nor is it glucose, aminoacids, fructose or ketones, as the growth effect remained after dialysis.     

Effect of pectin on serum cholesterol, fecal bile acids and biliary lipids in normolipidemic and hyperlipidemic individuals

Pectin, 40-50 g/day for two weeks administered to nine normolipidemic and hyperlipidemic patients, had no effect on serum triglycerides but did cause a significant decrease in the serum total and unesterified cholesterol of hypercholesterolemic subjects in particular. This was associated with increased excretion of fecal bile acids and total steroids and increased concentration of plasma methyl sterols. Thus, the serum cholesterol reduction by pectin appears to be caused by increased cholesterol elimination into stools as bile acids which is then balanced by enhanced cholesterol synthesis. The composition of biliary bile acids and lipids was not changed and secondary bile acids and sterols decreased inconsistently in feces. The measurement of fecal dry weight suggested that the bulk of the pectin was degraded by bacteria during passage through the intestine. Consequently fecal mass and dry weight were not consistently increased, suggesting that pectin may not be an ideal fibre for increasing fecal bulk in functional colonic disorders.     

Coenzyme-A-dependent esterification of cholesterol in rat intestinal mucosa

Rat intestinal mucosa contains an enzyme which catalyses the esterification of cholesterol. The enzyme is CoA-dependent and probably an acyl-CoA: cholesterol acyltransferase (ACAT) (E.C.2.3.1.26). Maximal activity in vitro is obtained when long chain acylcarnitines, CoA and carnitine palmityltransferase are used as an acyl-CoA generating system. The enzyme is localized in the microsomal fraction, has a pH optimum between 6.4-7.2, and oleate is the preferred acyl group. The activity of the enzyme is highest in the proximal jejunum, and has a capacity that can account for all cholesteryl esters found in intestinal rat lymph.     

Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes

The objective of this study was to investigate the mechanisms by which tamoxifen modifies cholesterol metabolism in cellular models of liver metabolism, HepG2 cells and rat hepatocytes. The effect of tamoxifen on cholesterol and triglyceride-palmitate synthesis was measured using isotopomer spectral analysis (ISA) and gas chromatography-mass spectrometry (GC-MS) and compared with the effects of progesterone, estradiol, the antiestrogen ICI 182,780, and an oxysterol, 25-hydroxycholesterol (25OHC). Cholesterol synthesis in cells incubated in the presence of either [1-(13)C]acetate, [U-13C]glucose, or [4,5-(13)C]mevalonate for 48 hours was reduced in the presence of 10 micromol/L tamoxifen and 12.4 micromol/L 25OHC in both HepG2 cells and rat hepatocytes. The ISA methodology allowed a clear distinction between effects on synthesis and effects on precursor enrichment, and indicated that these compounds did not affect enrichment of the precursors of squalene. Progesterone was effective in both cell types at 30 micromol/L and only in HepG2 cells at 10 micromol/L. Estradiol and ICI 182,780 at 10 micromol/L did not inhibit cholesterol synthesis. None of the compounds altered the synthesis of triglyceride-palmitate in either cell type. Treatment of cells with tamoxifen produced accumulation of three sterol precursors of cholesterol, zymosterol, desmosterol, and delta8 cholesterol. This pattern of precursors indicates inhibition of delta24,25 reduction in addition to the previously described inhibition of delta8 isomerase. We conclude that tamoxifen is an effective inhibitor of the conversion of lanosterol to cholesterol in cellular models at concentrations comparable to those present in the plasma of tamoxifen-treated individuals. Our findings indicate that this mechanism may contribute to the effect of tamoxifen in reducing plasma cholesterol in humans.     

Synthesis and removal of different cholesterol esters by aortic smooth muscle cells in culture

The incorporation of 3H-labelled oleic acid and of 14C-labelled linoleic acid into phospholipid, triglyceride and cholesterol ester in smooth muscle cells grown in incubation medium supplemented with either 5% normal or 5% hyperlipemic serum has been studied. Both fatty acids were incorporated into cholesterol esters to a greater extent when cells grown in incubation medium containing hyperlipemic serum. Oleic acid was incorporated into cholesterol esters in preference to linoleic acid. The addition of hyperlipemic serum to the incubation medium did not increase the incorporation fo either 3H-labelled oleic acid or of 14C-labelled linoleic acid into phospholipid or triglyceride. The removal of labelled lipid fractions has also been followed for four days in cells pulse labelled for 24 hours with 3H-labelled oleic acid and 14C-labelled linoleic acid. Both 3H- and 14C-labelled cholesterol esters were removed more rapidly when the smooth muscle cells were grown in medium containing normal serum than in medium containing hyperlipemic serum. The removal of both phospholipid and triglyceride was similar in normal and hyperlipemic serum. Comparison of the 3H/14C ratio indicated that the cholesterol oleate and cholesterol linoleate were removed at similar rates.     

OxLDL-induced cytotoxicity and its inhibition by API0134 in cultured porcine aortic endothelial cells

Protective effects of API0134 on endothelial cells (EC) damaged by oxidatively modified low density lipoprotein (oxLDL) were studied. The results showed that the content of endothelia (ET) and malondialdehyde (MDA) in the media of porcine aortic EC incubated with oxLDL were increased and the cGMP was decreased significantly, and the activity of superoxide dismutase (SOD) was inhibited. The effect of cytotoxicity of oxLDL can be eliminated by API0134. These results suggest that API0134 may protect EC against damages elicited by oxLDL.     

Increased resistance to streptolysin O in mammalian cells treated with oxygenated derivatives of cholesterol

L-cell fibroblast cultures were treated with certain oxygenated derivatives of cholesterol which are known to inhibit cholesterol biosynthesis in mammalian cells. After incubation in the presence of 20-alpha-hydroxycholesterol or 25-hydroxycholesterol for 18 h, the cells became increasingly resistant to streptolysin O. Maximum resistance to toxin was obtained by incubation for 48 h in 0.5 microgram of 20-alpha-hydroxycholesterol or 0.25 microgram of 25-hydroxycholesterol per ml; under these conditions, the cells were 10 to 50 times more resistant than were untreated controls. The ability of the hydroxycholesterol compounds to render the cells resistant was related to the age of the cultures. Maximum protection was found when more sparsely populated cultures were treated with 25-hydroxycholesterol. Older, heavily populated cultures could not be protected even with the high concentrations of 25-hydroxycholesterol. In contrast to control cultures, most of the toxin activity remained in the medium after being incubated with hydroxycholesterol-treated cultures. The results indicate that less toxin binds to the resistant cells and suggest that a reduction in membrane cholesterol content may account for resistance to streptolysin O.     

Mucosal coenzyme A-dependent cholesterol esterification after intestinal perfusion of lipids in rats

Coenzyme A-dependent esterification of cholesterol was determined in intestinal mucosal homogenates prepared after duodenal perfusion of cholesterol-free lipid emulsions for 5 h in unanesthetized rats. Cholesterol esterification rates were lowest and the mucosal cholesterol pool was greatly reduced after the same lipid infusions that, in lymph fistula rats, had produced chylomicrons deficient in cholesterol esters. CoA-dependent esterification rates were sufficient to account for all the cholesterol esters secreted in mesenteric lymph chylomicrons. During triglyceride secretion by the gut, unesterified cholesterol for chylomicron membranes may be maintained both by suppressing mucosal CoA-dependent cholesterol ester formation and from a mobilizable unesterified cholesterol pool within the mucosa.     

Hypercholesterolemia and aortic collagen synthesis in rabbit aortas

Male adult New Zealand rabbits were fed a 2% cholesterol diet for 30 of 60 days in order to determine the effect of hypercholesterolemia on aortic collagen synthesis. Collagen synthetic activity was estimated by measuring tissue prolyl hydroxylase activity and the amount of tissue collagen was estimated by measuring tissue hydroxyproline levels. Following 30 or 60 days of feeding there was a significant increase in both tissue and serum cholesterol indicating the onset of hypercholesterolemia. Measurement of collagen synthetic activity and tissue collagen levels demonstrated no increase over control tissues. These data therefore indicate that hypercholesterolemia is not a direct stimulus of tissue collagen synthetic activity.     

Evaluation and clinical application of a direct low-density lipoprotein cholesterol assay in normolipidemic and hyperlipidemic adults

This study examines the performance and clinical use of a commercial immunoseparation assay for low-density lipoprotein (LDL) cholesterol in a sample population of normolipidemic and hyperlipidemic adult volunteers. Using paired fasting and nonfasting samples, we compared the direct LDL assay with the beta quantification method and the Friedewald calculation. Overall, the direct LDL assay correctly classified 82% and 60% of fasting and nonfasting subjects, respectively, into National Cholesterol Education Program risk groups. The Friedewald method correctly classified 84% of subjects. The fasting direct LDL assay has comparable positive and negative predictive values to the Friedewald method, except at an LDL cholesterol of 100 mg/dl. The nonfasting direct LDL assay demonstrates unacceptable positive predictive values when LDL cholesterol decreases to the 130 to 159 and > or = 160 mg/dl categories. Overall, direct LDL assay demonstrates limitations in the nonfasting state and at the LDL cholesterol level of 100 mg/dl used for patients with established coronary heart disease.     

Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals

The microsomal enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) catalyzes the esterification of cellular cholesterol with fatty acids to form cholesterol esters. ACAT activity is found in many tissues, including macrophages, the adrenal glands, and the liver. In macrophages, ACAT is thought to participate in foam cell formation and thereby to contribute to atherosclerotic lesion development. Disruption of the gene for ACAT (Acact) in mice resulted in decreased cholesterol esterification in ACAT-deficient fibroblasts and adrenal membranes, and markedly reduced cholesterol ester levels in adrenal glands and peritoneal macrophages; the latter finding will be useful in testing the role of ACAT and macrophage foam cell formation in atherosclerosis. In contrast, the livers of ACAT-deficient mice contained substantial amounts of cholesterol esters and exhibited no reduction in cholesterol esterification activity. These tissue-specific reductions in cholesterol esterification provide evidence that in mammals this process involves more than one form of esterification enzyme.     

Antiproliferative gene BTG1 is highly expressed in apoptotic cells in macrophage-rich areas of advanced lesions in Watanabe heritable hyperlipidemic rabbit and human

In the present study, the expression and potential role of the highly conserved antiproliferative gene BTG1 in the process of apoptosis as it occurs in atherosclerotic lesions was examined. In situ hybridization and immunodetection studies demonstrated that BTG1 localized to specific macrophage-rich regions of lesions in both Watanabe heritable hyperlipidemic rabbits and humans. In addition, the spatial distribution of BTG1 mRNA was shown to colocalize not only with macrophage-rich regions but also to cells that exhibited changes consistent with apoptosis, such as DNA fragmentation and nuclear condensation. In vitro studies demonstrated that forced overexpression of BTG1 induced a 3-fold increase in apoptosis in NIH/3T3 cells. Furthermore, significantly increased expression of BTG1 mRNA resulted from lipid loading of human monocyte-derived macrophages in vitro. The process of increasing lipid content in macrophages is frequently associated with decreased macrophage viability. Taken together, these data underscore a potentially important role for BTG1 in regulating the cellularity of advanced atherosclerotic lesions.     

Effect of eicosapentaenoic acid on glucose-induced diacylglycerol synthesis in cultured bovine aortic endothelial cells

Endoscopic harvest of the jejunum is indicated for the reconstruction of complex defects of the head and neck and specifically the reconstruction of circumferential defects of the esophagus. However, open laparotomies are associated with systemic and local morbidity and are often seen as prohibitively invasive procedures. Laparoscopy for intra-abdominal procedures is a proven technique that yields less morbidity and provides an anatomical exposure similar to that of the open approach. We report on 11 consecutive clinical cases of free jejunal flap harvesting with the laparoscopic technique and show that the procedure is feasible.     

Localization of 17-kDa myosin light chain isoforms in cultured aortic smooth muscle cells

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.     

Plasma protein-facilitated coupled exchange of phosphatidylcholine and cholesteryl ester in the absence of cholesterol esterification

A protein(s) which catalyzes the exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins has been purified 10,000-fold from lipoprotein-free human plasma. The apparent molecular weight of the protein of the active fraction, designated lipid transfer complex (LTC), is approximately 61,000; when electrophoresed in 6 M urea, 0.1% sodium dodecyl sulfate on a 3-20% polyacrylamide gradient, the protein appears as a doublet of molecular weights 58,000 and 63,000. The active material is a glycoprotein which binds to concanavalin A. Human LTC is a lipid-protein complex with phospholipid, cholesterol, cholesteryl ester, and glyceride comprising 7% of the total mass. A similar glycoprotein (or glycoproteins) exists in rat plasma, although the fold-purification thus far achieved is low: about 500-fold. Moreover, the rat preparation enhances exchange of phosphatidylcholine, but does not appreciably enhance exchange of cholesteryl ester. Partially purified LTC (less than or equal to 3500-fold) exists in a complex with lecithin: cholesterol acyltransferase. Active lecithin: cholesterol acyltransferase is not, however, required for exchange of phosphatidylcholine or cholesteryl ester facilitated by human LTC. The rates of exchange of phosphatidylcholine and cholesteryl ester facilitated by human LTC are equal. Coupled lipid exchange occurs at all stages of LTC purification, at values of pH between 5 and 10, and at ionic strengths as great as 0.9. Moreover, phosphatidylcholine and cholesteryl ester are exchanged with 1:1 stoichiometry in the presence of thiol group reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid). Both lipid exchange activities are relatively resistant to elevated temperatures. Coupled exchange of phospholipid and neutral lipid is not dictated by the nature of the lipoprotein donor and acceptor substrates: bovine liver phospholipid exchange protein catalyzes exchange of phosphatidylcholine but not cholesteryl ester between low and high density lipoproteins under conditions identical with those in which human LTC facilitates exchange of both lipids.