Effect of sirolimus on the cholesterol content of aortic arch in ApoE knockout mice.

Chronic inflammatory responses involving the immune system have been implicated in the process of atherosclerosis. Sirolimus (Rapamycin, Rapamune), a potent immunosuppressant used to prevent rejection of transplanted kidneys, has also proven effective at inhibiting restenosis in humans when eluted from implanted stents. The aim of this study was to evaluate the effect of sirolimus treatment on the development of atherosclerosis in the aortic arch of apo E-/- mice fed a high-fat (Western) diet. Following 12 weeks of treatment with sirolimus (4 mg/kg/d), the cholesterol content of the arch was reduced by 36% compared to untreated control mice fed the Western diet only. Although the murine model is not comparable to the human situation, the results of this study suggest that sirolimus may exert beneficial effects on atherosclerosis in transplant patients.

Reciprocal antagonism between estrogen receptor and NF-kappaB activity in vivo.

The functional interaction, or "cross-talk," between estrogen receptor (ER) and the proinflammatory transcription factor nuclear factor (NF)-kappaB demonstrated in vitro has been suggested to play a role in estrogen prevention of cardiovascular disease. Here, we demonstrate that this reciprocal cross-talk occurs in vivo. Ovariectomized C57BL/6 mice fed an atherogenic diet had increased hepatic levels of active NF-kappaB and numerous inflammatory genes, including MHC invariant chain (Ii), vascular cell adhesion molecule-1, tumor necrosis factor-alpha, and RANTES. Treatment with 17alpha-ethinylestradiol (EE) strongly blocked induction of these genes but had no effect on their basal expression levels. ER was required for this activity, because the antagonist ICI 182,780 completely blocked the inhibitory activity of EE. Gene activation by EE was not required for inhibition of inflammatory gene expression, because both the phytoestrogen genistein and low doses of EE were effective in blocking inflammatory gene induction without inducing marker genes such as intestinal trefoil factor (ITF) or myo-inositol-1-phosphate synthase (IPS). The in vivo transcriptional interference was reciprocal, with EE induction of ITF and IPS greatly reduced in animals fed the atherogenic diet versus chow-fed controls. This interference was specific to the liver, because diet had no effect on uterine weight increases produced by EE. Transfection experiments confirmed that the extent of inhibition of ER-mediated transcription by inflammatory stimuli correlated with the extent of NF-kappaB activation. These results suggest that the cross-talk between ER and NF-kappaB does occur in vivo and may indeed contribute significantly to the cardioprotective effects of estrogen.

The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in HepG2 cells.

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-kappaB function. In the current study 17beta-estradiol-bound ERalpha interfered with cytokine-induced activation of a NF-kappaB reporter in HepG2 cells. The estrogen metabolite, 17alpha-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-kappaB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-kappaB interfered with the trans-activation properties of ERalpha. Ligand-bound ERalpha did not inhibit NF-kappaB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-kappaB transcriptional activity. Coexpression of the transcription coactivator CREB binding protein (CBP), but not steroid receptor coactivator 1a, reversed the ERalpha-mediated inhibition of NF-kappaB activity. Mammalian two-hybrid experiments also revealed that ligand-bound ERalpha can interact functionally with CBP-NF-kappaB complexes. We suggest that CBP targeting by ERalpha results in the inhibition of NF-kappaB and may occur through formation of transcriptionally inert multimeric complexes that are dependent upon the nature of the ERalpha ligand.

Future therapies for the prevention and treatment of venous and arterial thrombosis.

The incidence of thrombosis as a complication of invasive surgery, in cancer patients, as a cause or complication of stroke, acute myocardial infarction (AMI), thrombolysis, unstable angina (UA) or angioplasty is substantial. To better serve this patient population in the prevention and prophylaxis of thrombosis, new types of anticoagulant drugs are under development by the pharmaceutical industry. The goal of these efforts are orally-active anticoagulants with specificity and pharmacokinetic properties that could translate into better control of anticoagulation and thrombosis and less bleading liability compared to the currently used anticoagulants: heparin, the low molecular weight heparins and warfarin. Various approaches for which there is a great deal of activity include: tissue factor/Factor VIIa inhibitors, Factor Xa inhibitors, thrombin inhibitors, glycoprotein IIb/IIIa antagonists. There is also interest in Factor IXa inhibitors, thrombin receptor antagonists and inhibitors of plasminogen activator inhibitor-1.

Effects of 17 alpha-dihydroequilenin sulfate on atherosclerotic male and female rhesus monkeys.

OBJECTIVES: Our purpose was to determine the effects of 17 alpha-dihydroequilenin on plasma lipid and lipoprotein, glucose, and insulin concentrations; coronary artery vasomotor function; and reproductive organ and mammary gland proliferation in atherosclerotic male and female rhesus macaques. STUDY DESIGN: Fifty adult female and 33 adult male rhesus macaques were randomized to treatment by lifetime dietary cholesterol exposure and ratio of total plasma cholesterol to high-density lipoprotein cholesterol. The female treatment groups were intact female controls (n = 9), ovariectomized controls (n = 16), ovariectomized plus 0.3 mg/kg/day 17 alpha-dihydroequilenin (n = 17) and ovariectomized plus subcutaneous estradiol (n = 7). The male treatment groups were control (n = 16) and 1.25 mg/kg/day 17 alpha-dihydroequilenin (n = 17). Treatment lasted 5 weeks. Longitudinal assessments of plasma lipid and lipoprotein and glucose and insulin concentrations were performed. Coronary artery vasomotor function was assessed by quantitative coronary angiography 1 week after initiation of treatment. Morphologic and immunohistochemical assessments of proliferation index values of reproductive organs and mammary glands were done at necropsy. RESULTS: 17 alpha-Dihydroequilenin prevented endothelium-dependent vasoconstriction in males (p < 0.05) and ovariectomized females (p < 0.08). 17 alpha-Dihydroequilenin treatment increased plasma apolipoprotein A-1 concentrations (p < 0.05) and lowered fasting insulin concentrations (p < 0.05) without changing fasting plasma glucose concentrations in males. 17 alpha-Dihydroequilenin had no other effects on plasma lipid and lipoprotein concentrations in either males or females. It had no trophic effects on uterus, endometrium, or breast. There was no effect on either prostatic or testicular weight. CONCLUSION: 17 alpha-Dihydroequilenin may represent a single-agent hormone therapy for reduction of ischemic hear disease risk for both menopausal women and men. It has no apparent trophic effects on reproductive organs or mammary glands of female and male rhesus macaques.

In vitro antioxidant effects of estrogens with a hindered 3-OH function on the copper-induced oxidation of low density lipoprotein.

Estrogens with some bulky alkyl substituents in both the 2- and 4-positions have been synthesized and evaluated for the ability to inhibit the in vitro oxidation of low density lipoprotein as determined by the thiobarbituric reactive substances method. The present compounds with bulky groups in either the 2- or the 4-position (but not both the 2- and 4-) were especially effective as antioxidants, having IC50 values lower than either estradiol or probucol; however, they do not bind to the estrogen receptor with any great affinities (RBA < 0.1 versus estradiol). This separation of antioxidant efficacy from estrogenicity may allow these compounds to serve as useful probes for ascertaining the relative importance of these effects in the cardioprotective role played by estrogens.

Determination of 17 alpha-dihydroequilenin in rat, rabbit and monkey plasma by high-performance liquid chromatography with fluorimetric detection.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 17 alpha-dihydroequilenin in male and female rat, female rabbit and male and female rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14 beta-equilenin) and solvent extraction. The extracts were chromatographed on a C6, 5-microns reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences < 14.5; coefficients of variation < 9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17 alpha-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17 alpha-dihydroequilenin sulfate intragastrically.

Effect of 17 alpha-dihydroequilin sulfate, a conjugated equine estrogen, and ethynylestradiol on atherosclerosis in cholesterol-fed rabbits.

The effect of 17 alpha-dihydroequilin sulfate (DHES), a water-soluble estrogen of conjugated estrogens (Premarin), and ethynylestradiol (EE), a commonly used estrogen found in many oral contraceptives, on the development of atherosclerosis was studied in rabbits fed an atherogenic diet (0.2% cholesterol) for 24 weeks. Ten animals were given 15 micrograms. kg-1.d-1 EE, 10 received 3.8 mg.kg-1.d-1 of DHES, and the remaining 10 sham-ovariectomized and 10 ovariectomized animals served as cholesterol-fed controls. These doses were chosen to have similar estrogenic potency. Plasma cholesterol concentrations increased to about 900 mg/dL and did not differ among the experimental groups. After 24 weeks, plasma beta-VLDL and HDL cholesterol concentrations were the same for all cholesterol-fed groups, while LDL cholesterol was significantly higher in the two estrogen-treated groups. In spite of this, both EE and DHES significantly reduced atherosclerosis by 35% in the aortic arch and 75% to 80% in the thoracic and abdominal aorta. The reduction in atherosclerosis was seen in animals with a wide range (400 to 1400 mg/dL) of plasma cholesterol concentrations and was independent of lipoprotein profile. beta-VLDL isolated from estrogen-treated animals was not significantly different from control beta-VLDL in its ability to stimulate cholesterol accumulation in THP-1 macrophages in culture. This suggests that the protective effect of estrogens on the development of atherosclerosis is not mediated by qualitative differences in beta-VLDL that affect uptake by macrophages. The results of this study extend our knowledge of the range of estrogens that reduce atherosclerosis. Given the lack of effect on plasma lipid and lipoprotein concentrations, these data are consistent with the conclusion that estrogens exert some of this beneficial effect directly at the level of the arterial wall by influencing certain key components in the pathogenesis of atherosclerosis.

A conjugated equine estrogen with differential effects on uterine weight and plasma cholesterol in the rat.

OBJECTIVE: Our purpose was to determine the effect of Premarin (conjugated estrogens) and three of its component estrogens on uterine weight and plasma cholesterol concentrations in surgically menopausal female rats. STUDY DESIGN: A randomized trial of Premarin and three component estrogens--estrone sulfate, 17 alpha-estradiol sulfate, and 17 alpha-dihydroequilenin sulfate--in female rats after oophorectomy. RESULTS: High-dose Premarin and high- and middle-dose estrone sulfate significantly increased uterine weight relative to untreated controls (high-dose Premarin, 243.34 +/- 0.15 mg; high-dose estrone sulfate, 376.1 +/- 9.36 mg; middle-dose estrone sulfate, 249.0 +/- 6.34 mg; untreated controls, 124.63 +/- 3.17 mg; for all, p < 0.05). 17 alpha-Dihydroequilenin sulfate had no effect on uterine weight relative to controls. All 17 alpha-dihydroequilenin sulfate doses markedly reduced total plasma cholesterol concentrations versus controls (34.02 +/- 3.44 mg/dl, 32.49 +/- 1.08 mg/dl, and 71.55 +/- 5.16 mg/dl vs 90.44 +/- 1.06 mg/dl; for all, p < 0.02). 17 alpha-Dihydroequilenin sulfate had a more pronounced effect on low- or very-low-density lipoprotein cholesterol than total plasma cholesterol or high-density lipoprotein cholesterol concentrations. CONCLUSIONS: 17 alpha-Dihydroequilenin sulfate reduced total plasma cholesterol concentrations without inducing uterine growth in rats after oophorectomy.

Beta-VLDL metabolism by pigeon macrophages. Evidence for two binding sites with different potentials promoting cholesterol accumulation.

Previous studies from our laboratory (J Lipid Res 1988;29:643-656) have shown that thioglycolate-elicited peritoneal macrophages from White Carneau and Show Racer pigeons, like mammalian macrophages, have on their surfaces specific receptors for acetylated low density lipoprotein (acLDL) and beta-migrating very low density lipoproteins (beta-VLDL). The binding kinetics of beta-VLDL were complex, however, suggesting more than one binding site. The purpose of the present study was to further characterize these beta-VLDL binding sites. Scatchard analysis of 125I-beta-VLDL binding curves indicated at least two classes of binding sites. The first binds pigeon beta-VLDL and LDL with high affinity (Kd approximately 7 micrograms/ml), is down-regulated by cholesterol loading, requires calcium, and is destroyed by the proteolytic enzyme, pronase. This pigeon beta-VLDL receptor is specific for pigeon beta-VLDL and LDL and does not recognize HDL, acLDL, methyl LDL, cynomolgus monkey LDL, or rabbit beta-VLDL. Like the mammalian macrophage beta-VLDL receptor, the "pigeon beta-VLDL receptor" has many of the characteristics of an LDL receptor. The second class of binding sites is relatively nonspecific, recognizing both pigeon and rabbit beta-VLDL, LDL, acLDL, methyl LDL, and HDL. Binding to this site is not altered by incubation of macrophages with pronase or by cholesterol loading. This binding site has low affinity for beta-VLDL (Kd approximately 100 micrograms/ml), but high capacity. We have called this the "lipoprotein binding site," a term used by others to describe similar lipoprotein binding characteristics on a variety of cells. Not only does binding to this site promote the internalization and degradation of lipoproteins, but it may also facilitate the independent uptake of cholesterol. This conclusion is based on the observation that more cholesterol accumulates in cells incubated with rabbit beta-VLDL, which binds only to the lipoprotein binding site, than can be accounted for by beta-VLDL uptake and degradation. Since the lipoprotein binding site recognizes a variety of normal, as well as abnormal, lipoproteins, it would not require the generation of abnormal lipoprotein products, as must occur with the scavenger receptor, to promote the accumulation of cholesteryl esters in macrophages of atherosclerotic lesions. This, coupled with the fact that the lipoprotein binding site is not down-regulated by cholesterol loading, suggests that it could provide an alternative mechanism to the scavenger receptor pathway for the formation of foam cells.

Lipoprotein metabolism by macrophages from atherosclerosis-susceptible White Carneau and resistant Show Racer pigeons.

The presence of specific receptors for the metabolism of acetylated low density lipoprotein (AcLDL) and beta-migrating very low density lipoprotein (beta-VLDL) was demonstrated in thioglycolate-elicited peritoneal macrophages from both atherosclerosis-susceptible White Carneau (WC) and resistant Show Racer (SR) pigeons. Macrophages from both breeds metabolized AcLDL through a single class of receptors that were similar, but not identical, to the scavenger receptors described in mammalian macrophages. Both pigeon and mammalian AcLDL bound to this receptor. At 37 degrees C, AcLDL was internalized and degraded in the lysosomes, and cholesterol esterification and cholesteryl ester accumulation were stimulated. As in mammalian macrophages, AcLDL receptor activity was not down-regulated by cholesterol loading. In contrast, AcLDL binding was poorly competed for by fucoidin or polyinosinic acid, and the magnitude of cholesteryl ester accumulation was only about one-half of that seen with mouse peritoneal macrophages. Pigeon beta-VLDL bound to both a high and a low affinity site on pigeon macrophages. Binding to the high affinity site was calcium-dependent, pronase-sensitive, and down-regulated by cholesterol loading. Cholesterol esterification and cholesteryl ester accumulation with beta-VLDL were stimulated to an equal or greater extent than with AcLDL. Unlike mammalian macrophages, the pigeon beta-VLDL receptor did not require apolipoprotein E, as evidenced by the lack of apoE in pigeon lipoproteins and by the failure of rabbit beta-VLDL, containing apoE, to compete for binding. Pigeon LDL, but not mammalian LDL, was recognized by the pigeon beta-VLDL receptor, suggesting that like the mammalian beta-VLDL receptor, the pigeon beta-VLDL receptor may be a form of an LDL receptor. This was an unexpected finding since pigeon fibroblasts and smooth muscle cells in culture do not express LDL receptors. Thus, pigeon macrophages have receptors for the uptake of abnormal lipoproteins that could play a role in the development of macrophage-derived foam cells that are prevalent in the early stages of atherosclerosis in this species. No quantitative or qualitative differences in these receptors, however, were identified that could account for the differences in atherosclerosis susceptibility between the WC and SR breeds.

Cholesteryl ester cycle in cultured hepatoma cells.

The existence of a cholesteryl ester cycle in cultured Fu5AH hepatoma cells was documented and factors affecting the rate of turnover of the cholesteryl ester cycle in this cell line were explored. The influence of the physical state of the lipid inclusion in which the cholesteryl esters are stored could be addressed in this cell line because these cells can be induced to store cholesteryl esters in anisotropic (liquid-crystalline) cytoplasmic inclusions by exposure to free cholesterol-rich phospholipid dispersions or in isotropic (liquid) inclusions by addition of oleic acid to the phospholipid dispersions. To examine the relative rates of turnover of the cholesteryl ester cycle in the cells with the two types of inclusions, the fraction of cholesteryl linolenate, a cholesteryl ester present in low amounts in these inclusions, was examined after cells were exposed to medium containing linolenate. After 12 h, cells with anisotropic inclusions contained 17.5% cholesteryl linolenate and cells with isotropic inclusions contained 29.8% cholesteryl linolenate, suggesting an approximately 2-fold difference in turnover of the cholesteryl ester pool. To determine whether this difference was due to a differential rate of cholesteryl ester hydrolysis, the acyl CoA: cholesterol acyl transferase arm of the cholesteryl ester cycle was blocked using a specific inhibitor, Sandoz 58-035. In the presence of this compound, cholesteryl ester was hydrolysed twice as fast in cells with isotropic inclusions as compared to that in cells with anisotropic inclusions. The difference in rate of turnover of the cholesteryl ester cycle was shown to be related to the rate of hydrolysis of cholesteryl ester which, in turn, is related to the physical state of the stored cholesteryl ester.

Lipid composition and physical state effects on cellular cholesteryl ester clearance.

The influence of lipid composition and physical state on the rate of cholesteryl ester clearance from cytoplasmic inclusions has been investigated. Our findings demonstrate that the increased rate of clearance correlates with an increased cellular triglyceride content and a more fluid cholesteryl ester physical state. Cultured rat hepatoma cells were induced to accumulate esterified cholesterol in a smectic liquid-crystalline state by exposure to free cholesterol-rich phospholipid dispersions. Addition of cis-unsaturated fatty acids to this loading medium (either oleate, linoleate, linolenate, or eicosadienoate) resulted in a substantial increase in cellular triglyceride content (greater than 7 times non-fatty acid-treated), cellular cholesteryl esters in a liquid state, and a rate of cholesteryl ester clearance twice that of control (approximately 34% versus 17% in 12 h). In studies with oleic acid, storage of cellular cholesteryl esters in a liquid state was found to be dependent on the presence of triglycerides, and the rate at which these cells hydrolyzed cholesteryl esters was proportional to triglyceride levels. Cells exposed to either linoleic or linolenic acid hydrolyzed cholesteryl esters at the faster rate, but in contrast to findings with oleate and eicosadienoate, the storage of cholesteryl esters in a liquid state may also be a consequence of the modified fatty acyl composition of the cholesteryl esters themselves. Addition of a saturated fatty acid (palmitate) or a fatty acid with a trans-double bond (elaidate) to the cholesterol loading media had little effect on cellular triglyceride content, cholesteryl ester physical state, or the rate of cholesteryl ester clearance.

Cellular cholesteryl ester clearance. Relationship to the physical state of cholesteryl ester inclusions.

The hypothesis that clearance of cellular cholesteryl ester deposits may be a function of the physical state of the stored lipid has been investigated. Cultured rat hepatoma cells were induced to store cholesteryl ester in either anisotropic inclusions by exposure to free cholesterol-rich phospholipid dispersions or isotropic inclusions by exposure to identical dispersions supplemented with oleic acid. Differential scanning calorimetry demonstrated an order/disorder transition at 43 degrees C for cholesteryl esters stored in anisotropic inclusions; the enthalpy of this transition was consistent with a smectic liquid crystalline to liquid transition. Lipids in cells with isotropic inclusions displayed no order/disorder transitions over the range 20-80 degrees C, indicating that the lipids are in a liquid state. The presence of oleic acid did not influence the mass of cholesteryl ester stored but increased the amount of stored triglyceride. Fatty acyl compositions of the cholesteryl esters were different under the two loading conditions; in particular, there was 38% cholesteryl oleate in anisotropic inclusions and 65% cholesteryl oleate in isotropic inclusions. Kinetics of cholesteryl ester clearance from cells with either anisotropic or isotropic inclusions were studied during a 12-h exposure to acceptors of free cholesterol. In both cases, cholesteryl ester clearance is essentially linear over 12 h and is directly proportional to the initial content of cholesteryl ester. However, the fraction of initial content of cholesteryl ester cleared in 12 h is 0.17 +/- 0.05 for cells with anisotropic inclusions and 0.34 +/- 0.09 for cells with isotropic inclusions. Our data demonstrate that the more rapid clearance of cholesteryl ester by cells with isotropic inclusions can be correlated with the physical state of the cholesteryl ester.

Established cell lines from rat adipose tissue that secrete lipoprotein lipase.

A number of cell lines derived from the stromal-vascular fraction of rat adipose tissue have been established that represent a variety of morphologic types. Despite their differing morphology, all of these cell lines secrete lipoprotein lipase in response to heparin. Because lipoprotein lipase secretion has been attributed to the presence of preadipocytes in the stromal-vascular fraction, we examined these cell lines for adipocyte conversion. None of the cell lines converted to adipocyte morphology when held at confluency or when exposed to media supplemented with high concentrations of fatty acid or very low density lipoproteins. These cell lines therefore do not seem to be preadipocytes, despite the presence of lipoprotein lipase. Among these cell lines are several that display the "cobblestone" morphology of endothelial cells, although they lack angiotensin-converting enzyme activity, reactivity with Factor VIII antibodies, and Weibel-Palade bodies. A number of authentic endothelial cells were found to be negative for lipoprotein lipase secretion. These data suggest that the "endothelial-like" cell lines established from adipose tissue are not endothelial cells.