Atherogenic effect of beta-blocker propranolol revealed in the rabbit denudated aorta

The influence of beta-blocker propranolol onto atherogenic properties of blood serum and to formation of myointimal thickening in rabbit aorta, which was caused by denudation, were investigated. The preparation was introduced per os in the dose 6 mg/kg. Culture of mouse peritoneal macrophages was used to estimate the atherogenic properties of the serum. Serum of propranolol-treated rabbits induced accumulation of cholesterol in cultivated cells. Propranolol also induced an increase of the thickness of aortic intima, lipid accumulation and increasing of cell's number in myointimal thickening. Thus, atherogenic effect of beta-blocker propranolol was found both in vitro and in vivo.     

Effects of blocking plasma lipid transfer protein activity in the rabbit

Plasma lipid transfer protein activity was completely blocked in rabbits for up to 48 h by infusion with goat antibody to rabbit lipid transfer protein. Lipid transfer protein activity in plasma of control animals, infused with antibody from a non-immune goat, decreased during the experiment but was never less than 50% of pre-infusion levels. During the period that lipid transfer protein activity was completely blocked, there were changes in high-density lipoprotein composition (expressed as % by weight) with a reduction in triacylglycerol from 8.4 +/- 2.4% to 1.0 +/- 0.2% (P less than 0.05) and an increase in esterified cholesterol from 10.7 +/- 1.7% to 14.5 +/- 0.3% (P less than 0.1). In conjunction with the observed changes in high-density lipoprotein composition, there was an increase in high-density lipoprotein particle size from a mean radius of 4.7 to 5.4 nm. The change in composition and particle size was not observed in high-density lipoproteins from control animals. There was a change in the distribution of plasma cholesterol in control animals, with a fall in the proportion of cholesterol in high-density lipoproteins (P less than 0.02) and consequently an increase in the proportion of cholesterol in low-density lipoproteins (P less than 0.02). However, the distribution of plasma cholesterol in animals in which lipid transfer protein activity was inhibited was maintained at original levels during the period of inhibition. Consequently, in these animals, there was a less atherogenic distribution of cholesterol during the period of lipid transfer protein inhibition when compared with control animals. The changes observed in lipoproteins, in the absence of lipid transfer protein activity, demonstrate that lipid transfer protein modifies lipoproteins in vivo and appears to contribute to a more atherogenic lipid profile.     

Intracellular cholesterol accumulation is accompanied by enhanced proliferative activity of human aortic intimal cells

Cholesterol accumulation in smooth muscle cells of unaffected human aortic intimal tissue occurred in the following conditions: (1) incubation of cells with atherogenic blood serum from patients with coronary heart disease (CHD), (2) cultivation of cells in the presence of insoluble associates containing low density lipoprotein (LDL). Preincubation of cells with blood serum from CHD patients resulted in a 2-5-fold increase in intracellular cholesterol and in 1.5-3-fold increase in cellular [3H]thymidine uptake. Blood serum collected from healthy donors had no significant effect on cultivated smooth muscle cells. When intimal cells were preincubated with insoluble associates containing LDL and components of fibrous extracellular matrix, the level of intracellular cholesterol increased from 2-4 times and uptake of [3H]thymidine increased 1.5-2.5-fold. Thus, a strong correlation was found between [3H]thymidine incorporation and intracellular cholesterol accumulation. The current study suggests that intracellular lipid accumulation may stimulate the proliferative activity of human aortic intimal cells from uninvolved tissue.     

Effect of papaverine on monosaccharide and glycine transport in the small intestine in vitro

Papaverin is shown to have a significant inhibitory effect on the intestinal transport systems for glucose, galactose and glycine but not for fructose. In vitro experiments have revealed that the inhibitory effect of papaverin on the glucose transport take place under mucosal application, nevertheless the serosal one is of a stimulatory character. Papaverin inhibits only the active component of the glucose transport.     

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.     

The effect of calcium antagonists on cholesterol metabolism in human aortal intima cells and macrophage lines]

Effects of two Ca-antagonists, verapamil and nifedipine, on the total cellular cholesterol content and accumulation, as well as on the synthesis and hydrolysis of cholesteryl esters in human aortic intimal smooth muscle cells and P388D1 cell line have been studied. Verapamil and nifedipine used at 10(-6) M and higher concentrations decreased the total cellular cholesterol content (by 25-40%) in intimal cells isolated from atherosclerotic lesions without any effect on the cholesterol content in normal intimal cells or P388D1 cells. At 2 x 10(-5) M verapamil and nifedipine prevented the accumulation of cholesterol induced by atherogenic blood serum or atherogenic low density lipoproteins in both types of cells. At 10(-5) M and higher concentrations verapamil and nifedipine inhibited (2-3-fold) cholesteryl ester synthesis in intimal cells and, used at 10(-6) M and higher doses, in P388D1 cells as well. Verapamil and nifedipine (2 x 10(-5) M) enhanced the hydrolysis of cholesteryl esters in both types of cells. The Ca-channel agonist Bay K8644 had no effect on cholesteryl ester synthesis, nor did it suppress its inhibition by Ca-antagonist. The beta-receptor blocker propranolol induced the accumulation of cholesterol in intimal cells and inhibited the synthesis and hydrolysis of cholesterol esters in these cells. The data obtained suggest that the antiatherosclerotic action of Ca-blockers is determined by their ability to reduce the cellular cholesterol content which is suggested to be the result of enhanced hydrolysis of cellular cholesteryl esters.     

Improvement in cholesterol metabolism in mice given chronic treatment of taurine and fed a high-fat diet

The effects of chronic treatment of taurine on hypercholesterolemia and atherosclerosis were examined in C57BL/6J mice fed a high-fat diet containing 15% fat and 1.25% cholesterol. Taurine was dissolved in drinking water at 1% (w/v) and was given to mice ad libitum during 6 months-feeding of a high-fat diet. Hypercholesterolemia occurred and lipid accumulation on the aortic valve was evident. Taurine treatment lowered serum LDL + VLDL cholesterol by 44% in mice fed a high-fat diet, while it elevated serum HDL cholesterol by 25%. As a result, the atherogenic index, the ratio of HDL to LDL + VLDL was markedly improved. Cholesterol content in the liver also decreased by 19% with taurine. Similar tendencies were seen in mice fed regular chow, but the changes were not significant. The area of aortic lipid accumulation, which served as an index of atherosclerosis, was reduced by 20% with taurine. In the liver, taurine doubled the activity of cholesterol 7alpha-hydroxylase. These observations, together with prior findings, suggest that the cholesterol-lowering action of taurine may relate to the increased conversion of cholesterol to bile acids via stimulation of cholesterol 7a-hydroxylase activity. Thus, chronic treatment of high-fat mice with taurine improves the abnormal profile of the serum lipoproteins, and thereby retards the progression of atherosclerosis.     

Effects of conjugated linoleic acid on body fat accumulation and serum lipids in hamsters fed an atherogenic diet

Conjugated linoleic acid (CLA) refers to a mixture of naturally occurring positional and geometric isomers of linoleic acid that exist in dairy products and meat. The aim of the present work was to study the effects of c-9,t-11 and t-10,c-12 CLA isomers on body fat accumulation and serum lipids in hamsters fed an atherogenic diet. Hamsters were divided in four groups: one group was fed a chow diet (control) and the other three groups were given semi-purified atherogenic diets with 0.5% linoleic acid (LA), c-9,t-11 or t-10,c-12 CLA. Body weight and food intake were measured daily. After 6 weeks, adipose tissues from different anatomical locations and liver were dissected and weighed. Serum glucose, total cholesterol, HDL-c, LDL-c and triacylglycerol levels, as well as total and free cholesterol, triacylglycerol and phospholipid content in liver were determined by enzymatic methods. No differences in either energy intake or final body weight were found. The addition of t-10,c-12 CLA reduced fat accumulation and led to lower serum cholesterol, as compared with LA group. Nevertheless the level remained higher than in the control animals. The reduction in serum cholesterol was limited to LDL-c. This isomer also reduced triacylglycerol content in liver but did not modify serum triacylglycerol level. In summary, the present study demonstrates that t-10,c-12 CLA is the biologically active agent when anti-obesity and hypocholesterolaemic properties of CLA are considered. In contrast, the isomer c-9,t-11 has no effect on lipid metabolism in hamsters.     

Lipoproteins and diet in coronary heart disease; a five-year study

In a follow-up study for a five-year period of 351 patients with myocardial infarction and 119 patients with angina pectoris, the following observations were made:(a) The previously reported lipoprotein atherogenic index elevation in coronary heart disease was confirmed.(b) The prognosis in angina pectoris is strikingly and significantly worse when the lipoprotein atherogenic index is high.(c) Patients who died in the follow-up period showed significantly higher atherogenic index values than those who survived.(d) The lipoprotein atherogenic index measure is much superior to the serum cholesterol measurement as an indicator of the lipid disorder in coronary disease.(e) The low fat, low cholesterol diet is effective in maintaining chronically lowered lipoprotein atherogenic index values.(f) In patients who said they did not adhere to a low fat, low cholesterol diet, the recurrence and death rate was four times as high as in patients who stated they adhered to the diet.     

LIPOPROTEINS AND DIET IN CORONARY HEART DISEASE—A Five-Year Study

In a follow-up study for a five-year period of 351 patients with myocardial infarction and 119 patients with angina pectoris, the following observations were made:(a) The previously reported lipoprotein atherogenic index elevation in coronary heart disease was confirmed.(b) The prognosis in angina pectoris is strikingly and significantly worse when the lipoprotein atherogenic index is high.(c) Patients who died in the follow-up period showed significantly higher atherogenic index values than those who survived.(d) The lipoprotein atherogenic index measure is much superior to the serum cholesterol measurement as an indicator of the lipid disorder in coronary disease.(e) The low fat, low cholesterol diet is effective in maintaining chronically lowered lipoprotein atherogenic index values.(f) In patients who said they did not adhere to a low fat, low cholesterol diet, the recurrence and death rate was four times as high as in patients who stated they adhered to the diet.     

Biochemical reactivity of aortic connective tissue in chickens treated with cholesterol diet in combination with a nonspecific immunization or with epinephrine and thyroxine

Our previous work showed that only changes in aortic lipids and no change in connective tissue components occurred in hatched chickens, fed for four months a cholesterol atherogenic diet. The aim of this study was to test if combination of atherogenic stimuli will activate the connective tissue of aorta.Newly hatched chickens were fed a diet containing 2% cholesterol and in addition immunized with bovine serum albumen. Another group was administered epinephrine and thyroxine in excessive doses. The treatment lasted 6 to 7 weeks. In isolated aortas the amount and rate of synthesis of collagen, activity of prolyl hydroxylase and collagenase and the amount of total cholesterol were measured. With the exception of significantly increased lipid content, neither parameter reflecting the connective tissue and collagen metabolism was affected by the various atherogenic stimuli. It is concluded that in young chickens the changes in cholesterol content in aortic tissue preceded eventual activation of connective tissue components.     

A novel anti-atherogenic role for COX-2--potential mechanism for the cardiovascular side effects of COX-2 inhibitors

Atherosclerosis, the underlying cause of cardiovascular disease, is characterized by lipid accumulation, lipoprotein oxidation, and inflammation. Products of the cyclooxygenase (COX) pathway participate in acute and chronic inflammation. The inducible form of COX, COX-2, generates lipid mediators of inflammation that are pro-inflammatory and COX-2-selective inhibitors are potent anti-inflammatory agents. However, clinical data suggest an increased risk of cardiovascular side effects in patients using COX-2-selective inhibitors. In this paper, we sought to determine the effect of COX-2 deficiency on atherosclerosis-related lipoprotein metabolism in mice. We demonstrate that COX-2 deficiency resulted in (i) accumulation of lipids in circulation and liver, (ii) pro-inflammatory properties of HDL as measured by HDL's increased reactive oxygen species (ROS) content, decreased paraoxonase 1 (PON1) activity, decreased serum apoA-1, reduced ability to efflux cholesterol and to prevent LDL oxidizability, and (iii) increased TXB(2) in circulation. Moreover, when placed on an atherogenic diet, COX-2 deficiency resulted in (i) increased lipid deposition in the aorta, (ii) a further dramatic imbalance in circulating eicosanoids, i.e. decreased serum PGI(2) coupled with increased PGE(2) and TXB(2), and (iii) a marked elevation of pro-inflammatory cytokines, TNF and IL-6. Our results suggest, for the first time, that COX-2 deficiency contributes to the pro-atherogenic properties of HDL in mice.     

Effect of phenobarbital upon serum cholesterol lipoprotein fractions of three rodent species

Phenobarbital (PB) in doses previously shown to be optimal for induction of hepatic microsomal enzymes had no effect on the cholesterol content of whole serum or of serum lipoprotein fractions in the guinea pig, but reduced high density lipoprotein cholesterol (with increase in very low density lipoprotein cholesterol) in the rabbit, and increased high density lipoprotein cholesterol (as well as total and low density lipoprotein cholesterol) in the rat. These results, when taken together with previous data demonstrating that PB increases serum triglyceride in the rabbit but lowers it in the rat, suggest that the drug may be atherogenic in the rabbit but not in the rat which, in any case, is highly resistant to atheroma.     

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.     

Interferon-gamma requires serum retinoids to promote the expression of tissue transglutaminase in cultured human blood monocytes

The culture of HPBM in serum-containing medium induced a large accumulation of the protein cross-linking enzyme, tissue TGase. Immune IFN enhanced the expression of tissue TGase in cultured monocytes. Enzyme-inducing activity, both in normal and IFN-treated cells, was completely blocked by depleting the serum of the lipid fraction. The readdition of retinol at a physiologic concentration (1 micron) to delipidized serum completely restored the enzyme-inducing activity in cultured monocytes. Thus, serum retinoids seem to play an important regulatory role in the expression of tissue TGase gene in differentiating human monocytes.     

Effects of trans-10, cis-12 conjugated linoleic acid on body fat and serum lipids in young and adult hamsters

The aim of the present work was to determine whether t-10, c-12 conjugated linoleic acid (CLA) feeding was able to reduce body fat accumulation and improve the serum lipid profile in adult hamsters fed an atherogenic diet, in order to compare these effects with those observed in young growing hamsters. Young and adult hamsters were fed semi-purified atherogenic diets supplemented with 0.5 % linoleic acid or 0.5% t-10, c-12 CLA for 6 weeks. Body weight and food intake were measured every two days. Adipose tissue from different anatomical locations, liver and gastrocnemious muscle were dissected and weighed. Cholesterol, triacylglycerols, non-esterified fatty acids and proteins were determined spectrophotometrically and water content by gravimetry. In young hamsters, no significant differences were found in food intake, final body weight and gastrocnemious muscle weight. White adipose tissue weights were reduced, liver weight was increased and cholesterol and triacyl-glycerols in both serum and liver were reduced. In adult hamsters, CLA feeding decreased food intake and adipose tissue weights. No changes were observed in other parameters. The present study demonstrates that age has an influence in hamster responsiveness to t-10, c-12 CLA because, although when this isomer is added to an atherogenic diet it reduces body fat accumulation in both young and adults hamsters, the lessening of the effects on serum lipids brought about by atherogenic feeding is only observed in young animals. Moreover, it is clear that liver is a target for CLA in young but not in adult hamsters.     

Paraoxonase 1 (PON1) and its relationship to lipid variables, age and gender in healthy volunteers

Recent studies implied that low-density lipoprotein (LDL) modified predominantly by oxidation or glycation, significantly contributes to the formation of atherosclerotic lesions. In contrast to oxidized LDL (ox-LDL), high-density lipoprotein (HDL) is able to prevent accumulation of ox-LDL in arterial walls. This antiatherogenic property of HDL is attributed in part to several enzymes associated with the lipoprotein, including HDL-associated paraoxonase 1 (PON1). In this study we analyzed PON1 arylesterase/paraoxonase activities in relation to serum lipid profile, gender and age in thirty clinically healthy Slovak volunteers. Our results showed that PON1 arylesterase and paraoxonase activities were lower in citrated plasma than in serum by 16.6% and 27.3%, respectively. Among serum lipoproteins, only HDL-cholesterol level showed significant positive correlation with PON1 arylesterase activity (p = 0.042). Likewise, we found a significant relationship between atherogenic index (AI = total cholesterol/HDL-cholesterol) and PON1 arylesterase activity (p = 0.023). No significant correlation could be demonstrated between PON1 paraoxonase activity and serum lipid profile, age or gender. Furthermore, it was found that PON1 paraoxonase/arylesterase activities were higher in women compared with both investigated activities in men, but these differences were not statistically significant. These results confirmed a positive correlation between HDL-cholesterol and PON1 arylesterase activity. Moreover, it was found out that PON1 paraoxonase activity is not influenced either by gender or by age. PON1 arylesterase activity was however affected by gender to a limited extent.     

Low density lipoproteins transactivate EGF receptor: role in mesangial cell proliferation

Hyperlipidemia and the glomerular accumulation of atherogenic lipoproteins (low density lipoprotein, LDL; and its oxidatively-modified variants, ox-LDL) are commonly associated with the development of glomerular mesangial proliferative diseases. However, cellular signaling mechanisms by which atherogenic lipoproteins stimulate mesangial cell proliferation are poorly defined. In this study, we examined the effect of atherogenic lipoproteins on the activation of mesangial cell epidermal growth factor (EGF) receptor, mitogen activated protein kinase (MAP kinase), Ras, and mesangial cell proliferation. Stimulation of mesangial cells with LDL, and with greater activity, ox-LDL, markedly induced the transactivation of EGF receptor within 5 min of stimulation; the effect persisted up to at least 60 min LDL, and with a greater degree, ox-LDL, increased the activation of Ras, MAP kinase, and mesangial cell proliferation. Inhibition of EGF receptor kinase activity and/or MAP kinase activation blocked both LDL- and ox-LDL-induced mesangial cell proliferation. We suggest that the accumulation of LDL and more potently its oxidized forms within the glomerulus, through the transactivation of EGF receptor, stimulate down-stream Ras-MAP kinase signaling cascade leading to mesangial cell proliferation. Regulation of glomerular accumulation of atherogenic lipoproteins and/or EGF receptor signaling may provide protective environment against mesangial hypercellularity seen in glomerular diseases.     

Reversible inhibition of rabbit sperm-fertilizing ability by cholesterol sulfate

Experiments were carried out to examine the influences of lipid treatments on the fertilizing ability of rabbit spermatozoa. In vitro insemination of tubal oocytes with in vivo-capacitated sperm resulted in fertilization (IVF) of 81% of the oocytes (38/47) and in vitro development to the morula or blastocyst stage of 92% (35/38) of the embryos within 72 to 96 h. Treatment of capacitated sperm with cholesterol (Ch, up to 100 micrograms/ml) did not reduce the proportion of oocytes fertilized (fertilization rate, 100%, 8/8). Cholesterol-3-sulfate (Chs) at concentrations of 100 and 1,000 micrograms/ml significantly (p less than 0.001) decreased fertilization rates to 13.6% (8/59), and 3.5% (1/29), respectively. Hypercholesterolemic serum (HChS, 1295 mg cholesterol/dl vs. 45 +/- 18 mg/dl in normal serum), incubated for 2 h with in vivo-capacitated sperm, did not inhibit fertilization. However, a decreasing trend in fertilization was associated with increasing levels of HChS cholesterol. ChS effectively inhibited the fertilizing ability of capacitated sperm (p less than 0.05) compared to control, Ch, and HChS. In another experiment the use of ChS at 100 micrograms/ml significantly (p less than 0.05) reduced the fertilization rate from 56.6% (30/53) to 14.3% (7/49). When a phospholipid-enriched serum medium was added to sperm treated with 100 micrograms ChS/ml, the fertilization rate was 57.7% (23/40), which was not significantly (p less than 0.05) different from the fertilization rate of sperm not treated with ChS (56.6%, 30/53). These data suggest that rabbit sperm fertilizing ability can be reversibly inhibited by cholesterol sulfate.     

Regulation by high density lipoproteins of muscarinic acetylcholine receptor function in chick heart cells cultured in defined medium

Activation of cardiac muscarinic acetylcholine receptors (mAChR) on cultured chick heart cells results in a decrease in cellular cAMP levels and a stimulation of phosphoinositide breakdown. A serum-free culture system has been used to investigate the regulation of mAChR number and function by purified serum high density lipoprotein (HDL). Administration of HDL purified from rooster serum to chick heart cells cultured in defined medium results in an attenuation of the ability of muscarinic agonist to inhibit forskolin-stimulated cAMP accumulation, with no change in its ability to stimulate phosphoinositide hydrolysis or to mediate down-regulation of receptor number. The inclusion of HDL in the culture medium did not result in appreciable changes in mAChR number or affinity, nor were the levels of the inhibitory guanine nucleotide-binding regulatory proteins (G-proteins) altered. However, the ability of guanine nucleotides to inhibit forskolin-stimulated adenylate cyclase activity was reduced by HDL treatment, suggesting that HDL interferes with the capacity of G-proteins to interact with adenylate cyclase. In order to determine which component of native HDL mediates the decreased effectiveness of carbachol, the ability of lipid and apoprotein fractions to mimic the effect of HDL was tested. HDL lipid fractions were able to mimic the effect of native HDL, while protein fractions were not. This result suggests that the ability of HDL to attenuate muscarinic receptor function is mediated by its lipid constituents. The effect of HDL and HDL lipid fractions were not correlated with changes in membrane cholesterol content.