Lovastatin reversed the enhanced sphingomyelin caused by 27-hydroxycholesterol in cultured vascular endothelial cells.

Statins have pleiotropic properties which are involved in inhibiting the thrombogenic response. In this study, the effects of lovastatin on two phospholipids, phosphatidylcholine and sphingomyelin, were studied in cultured endothelial cells in the presence of an oxysterol, 27-hydroxycholesterol. After the cells were cultured with 50 nM of lovastatin for 60 h, lovastatin was found to decrease the incorporation of [3H]choline into phosphatidylcholine and sphingomyelin, inhibited CTP: phosphocholine cytidylyltransferase (CT) activity without altering the activity of sphingomyelin synthase and neutral sphingomyelinase. And lovastatin was not found to have a direct inhibitive effect on activity of CT. Exogenous mevalonic acid or cholesterol reversed the reduction of cholesterol concentration that was caused by lovastatin, but had no significant effect on the diminished [3H]sphingomyelin by lovastatin. The increase of [3H]sphingomyelin by 27-hydroxycholesterol was not detected in the presence of lovastatin. These findings suggest that (1) lovastatin can reduce sphingomyelin content by means of inhibiting phosphatidylcholine synthesis; and (2) The decrease in sphingomyelin is not related to the diminished cholesterol concentration or mevalonate-derived intermediates. This inhibitive effect of lovastatin on sphingomyelin may benefit cellular calcification caused by sphingomyelin.

Effects of trans fats on prostacyclin production.

OBJECTIVES: Prostacyclin is a prostanoid derived from arachidonic acid that prevents thrombosis and is thereby expected to protect against heart disease, while trans fats present in partially hydrogenated oils interfere with arachidonic acid metabolism. Therefore, the objective of the present study was to investigate how fats with different proportions of linoleic acid and trans-18:1 affect prostacyclin released by cultured endothelial cells, and to compare these proportions with those found in commercially available foods. DESIGN: Soybean oil and hydrogenated soybean oil (coating fat) were mixed in different proportions to yield seven fat mixtures with proportions of linoleic acid ranging from 54.1% to 5.7% and trans-18:1 acid ranging from 0.4% to 43.9%. Human endothelial cells were cultured in each of the mixtures, and their phospholipid fractions were then separated and their fatty acids were analyzed by gas chromatography. The prostacyclin released by the cells was measured using RIA kits. Margarines and processed foods were purchased from the supermarket for comparison. RESULTS: Our work revealed that as the percentage of trans fat was increased, the amount of prostacyclin released dose-dependently and significantly (P < 0.0001) decreased, the concentration of linoleic and arachidonic acid decreased in the membrane phospholipids while the concentration of trans 18:1 acids increased, the prostacyclin decreased by 35-98%. Supermarket margarines had levels of trans fats similar to those that suppressed prostacyclin by 35-54%. Most processed foods labeled as trans-free contained trans fats. CONCLUSIONS: Trans fatty acids suppress prostacyclin production at levels found in commercial margarines, and processed foods labeled as trans-free could contribute to this effect if consumed in multiple servings or in addition to foods containing larger amounts of trans fats.

Interaction between sphingomyelin and oxysterols contributes to atherosclerosis and sudden death.

Despite major public health efforts, coronary heart disease continues to be the leading cause of death in the United States. Oxidized lipids contribute to heart disease both by increasing deposition of calcium on the arterial wall, a major hallmark of atherosclerosis, and by interrupting blood flow, a major contributor to heart attack and sudden death. Oxidized cholesterol (oxysterols) enhances the production of sphingomyelin, a phospholipid found in the cellular membranes of the coronary artery. This increases the sphingomyelin content in the cell membrane, which in turn enhances the interaction between the membrane and ionic calcium (Ca(2+)), thereby increasing the risk of arterial calcification. Patients undergoing bypass surgery had greater concentrations of oxysterols in their plasma than cardiac catheterized controls with no stenosis, and had five times more sphingomyelin in their arteries than in the artery of the placenta of a newborn. The oxysterols found in the plasma of these patients were also found in the plasma of rabbits that had been fed oxidized cholesterol and in frying fats and powdered egg yolk intended for human consumption. Together these findings suggest that oxysterols found in the diet are absorbed and contribute to arterial calcification. Oxidized low-density lipoprotein (OxLDL) further contributes to heart disease by increasing the synthesis of thromboxane in platelets, which increases blood clotting. Cigarette smoke and trans fatty acids, found in partially hydrogenated soybean oil, both inhibit the synthesis of prostacyclin, which inhibits blood clotting. By increasing the ratio of thromboxane to prostacyclin, these factors interact to interrupt blood flow, thereby contributing to heart attack and sudden death. Levels of oxysterols and OxLDL increase primarily as a result of three diet or lifestyle factors: the consumption of oxysterols from commercially fried foods such as fried chicken, fish, and french fries; oxidation of cholesterol in vivo driven by consumption of excess polyunsaturated fatty acids from vegetable oils; and cigarette smoking. Along with the consumption of trans fatty acids from partially hydrogenated vegetable oil, these diet and lifestyle factors likely underlie the persistent national burden of heart disease.

Intracellular free calcium content plays a role in low magnesium-induced increases in prostacyclin production.

In a previous study, we found that magnesium deficiency stimulated prostacyclin production and suggested that this stimulation resulted from an enhanced Ca2+ influx induced by magnesium deficiency. In this study, we further examined prostacyclin generation in cultured human umbilical vein endothelial cells after intracellular free calcium content ([Ca2+]i) was altered by addition of diltiazem, nifedipine, verapamil, sodium cyanide (NaCN), ruthenium red or quinidine to a low magnesium medium. The results showed that diltiazem, nifedipine, verapamil and ruthenium red inhibited 45Ca2+ influx, and NaCN and quinidine had no effect on 45Ca2+ influx. However, all of these compounds decreased [Ca2+]i, [3H]arachidonic acid release and prostacyclin production. The reduced [3H]arachidonic acid content in cellular phospholipids caused by low magnesium treatment was not altered by the added compounds. We suggested that arachidonic acid release and prostacyclin production was calcium-dependent in cultured endothelial cells.

Effect of curcumin on LDL oxidation in vitro, and lipid peroxidation and antioxidant enzymes in cholesterol fed rabbits.

In this study we examined the antioxidant effect of curcumin on lipid oxidation in vitro and in vivo. In vitro, curcumin at 5 microgM concentration completely prevented low-density lipoprotein (LDL) oxidation by CuS0(4), indicating that curcumin is an effective antioxidant in vitro. In vivo, feeding a pure cholesterol (PC)-rich diet to rabbits significantly increased the plasma and liver lipids as well as thiobarbituric acid reactive substances (TBARS) levels. Addition of curcumin to the PC diet did not show any effect on either plasma lipid and TBARS or liver lipids. Liver TBARS tended to decrease but that decrease was not significant. Erythrocyte glutathione peroxidase (GSH-Px) activity was significantly decreased while catalase activity was significantly increased in rabbits fed a PC diet. The addition of curcumin to a PC diet did not show any significant effect on erythrocyte enzyme activities compared to the rabbits fed a PC diet. The liver GSH-Px and catalase activities were significantly decreased in rabbits fed a PC diet, but the addition of curcumin to the PC diet enhanced the liver GSH-Px activity, which became nonsignificantly different from the control group. These results were discussed considering that curcumin may not be well absorbed and it did not reach a level high enough in vivo to overcome the severe hypercholesterolemia and oxidative stress produced by the PC-rich diet.

Curcumin prevents the oxidation and lipid modification of LDL and its inhibition of prostacyclin generation by endothelial cells in culture.

Low-density lipoprotein (LDL) was isolated from human plasma and oxidized by 5microM copper sulfate for 4h at 37 degrees C in the absence and presence of 1, 3, 5, 10, or 20microM of curcumin. LDL oxidized in the absence of curcumin (oxLDL) showed an increased levels of conjugated dienes, lipid peroxides (TBARS) and lysolecithin (lysoPC) and a significant loss of polyunsaturated fatty acids (PUFA). LDL oxidized with 5microM copper sulfate in the presence of curcumin caused a significant decrease of conjugated diene, lipid peroxides, lysoPC and significant increase of PUFA compared to oxLDL. These changes were dose dependent and reached a maximum at 5microM curcumin. Incubation of human endothelial cells (EC) with 200microg protein/ml of oxLDL caused a significant decrease of prostacyclin (PGI(2)) generation. LDL oxidized in presence of 5microM curcumin did not show any inhibition of PGI(2) generation compared to the control cells. These results indicate that curcumin is an effective chain-breaking antioxidant which prevents oxidation and lipid modification of LDL. The inhibition of oxLDL on PGI(2) is considered a contributing factor in the pathogenesis of thrombosis and atherosclerosis. Curcumin supplementation could be an effective strategy in preventing LDL oxidation and its impact on atherosclerosis and lesion formation.

The negative effects of hydrogenated trans fats and what to do about them.

Partially hydrogenated vegetable oils have been in the American diet since 1900. More than 50 years ago they were found to contain trans fatty acids that were different from natural fatty acids in plant oils and in animal fat. There was growing evidence that the consumption of trans fats have negative health effects, including increasing plasma lipid levels. In 2003, the Food and Drug Administration (FDA) ruled that the amount of trans fat in a food item must be stated on the label after January 1, 2006; food items could be labeled 0% trans if they contain less than 0.5g/serving. Since the initial ruling, it is now known that the fatty acids in partially hydrogenated vegetable oil are 14 cis and trans isomers of octadecenoic and octadecadienoic acids that are formed during hydrogenation. They cause inflammation and calcification of arterial cells: known risk factors for coronary heart disease (CHD). They inhibit cyclooxygenase, an enzyme required for the conversion of arachidonic acid to prostacyclin, necessary for the regulation of blood flow. There have been several reformulations of hydrogenated fat containing varying amounts of trans fatty acids and linoleic acid, an essential fatty acid that is converted to arachidonic acid. Epidemiological data suggest that when trans fat percentages go up and linoleic acid percentages go down, death rates rise; when trans goes down, death rates go down. In spite of the harmful effects of trans fats, the FDA allows it in the food supply as long as the amount in a food item is declared on the label. Trans fat should be banned from the food supply.

High-dose lovastatin decreased basal prostacyclin production in cultured endothelial cells.

The effect of lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on prostacyclin production was studied in cultured human umbilical vein endothelial cells. The results indicated that lovastatin induced a significant dose- and time-dependent decrease of arachidonic acid release from the cells, an inhibition of phospholipase A(2) (PLA(2)) activity, a reduction of [(3)H]choline in lysophosphatidylcholine (lysoPC), and a diminishment of (45)Ca(2+) influx into the cells. The decreased arachidonic acid release was not reversed by addition of either intermediate products during cholesterol synthesis, such as mevalonate, geranylgeraniol, farnesol, or cholesterol and lipoprotein although a reduced concentration of cholesterol in the cells, caused by lovastatin, was reversed by added cholesterol. Lovastatin, furthermore, diminished prostacyclin production and inhibited activity of cyclooxygenase-1. 27-Hydroxycholesterol (27OHC), an oxidized cholesterol, had the same effect on HMG-CoA reductase as lovastatin, but 27OHC increased arachidonic acid release and (45)Ca(2+) influx. Our results indicated that lovastatin inhibited significantly activities of PLA(2) and cyclooxygenase-1, resulting in a marked reduction in arachidonic acid release, lysoPC content and prostacyclin production in the cultured vascular endothelial cells.

Vitamin B6 compounds are capable of reducing the superoxide radical and lipid peroxide levels induced by H2O2 in vascular endothelial cells in culture.

Pyridoxamine, pyridoxine, and pyridoxal phosphate were tested to examine if they have antioxidant properties. Endothelial cells exposed to 0.5 mM H(2)O(2) for 2 hours increased the superoxide anion and lipid peroxide levels as biomarkers of oxidative stress. The increase of superoxide was mainly due to the activation of NADPH-oxidase by H(2)O(2). Preincubation of the endothelial cells with 0.1 or 1.0 mM of pyridoxamine or pyridoxal phosphate for one-half hour before H(2)O(2) exposure significantly reduced the superoxide and lipid peroxide compared to the cells exposed to H(2)O(2) only. Preincubation of the cells with 0.1 or 1.0 mM of pyridoxine also significantly reduced the lipid peroxide but did not significantly affect the superoxide level unless the preincubation time was extended to 24 hours. The prostacyclin release by endothelial cells was also significantly inhibited by H(2)O(2). However, the preincubation of endothelial cells with 1.0 mM of pyridoxamine, pyridoxine, or pyridoxal phosphate did not prevent that inhibition. These results indicate that pyridoxamine, pyridoxine, and pyridoxal phosphate acted as antioxidants and reduced the superoxide and lipid peroxides induced by H(2)O(2), but did not protect the cells from the effects directly related to H(2)O(2) itself.

Low magnesium stimulated prostacyclin generation in cultured human endothelial cells.

Prostacyclin, synthesized from arachidonic acid, is a strong vasodilator and the most powerful inhibitor known for platelet aggregation. Magnesium deficiency as a risk factor for cardiovascular diseases was related to imbalance of thromboxane and prostacyclin in the vasculature. In this study, we examined the effect of a low level of magnesium on prostacyclin generation in cultured human umbilical vein endothelial cells by measuring arachidonic acid release, 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) production, calcium ((45)Ca2+) influx, and activity of phospholipase A2 (PLA2) and cyclooxygenases (COX), which are the two main enzymes that control the synthesis of prostacyclin. We found that lower levels of magnesium in the culture medium induced a time- and dose-dependent increase in arachidonic acid release. Low magnesium also enhanced 6-keto-PGF1alpha production, activated PLA2 and COX, enhanced (45)Ca2+ influx and decreased the remaining arachidonic acid in phospholipids. Our data indicate that the enhanced 6-keto-PGF1alpha production could be due to (1) the stimulated (45)Ca2+ influX resulting in an activation of PLA2, (2) the increased arachidonic acid liberation from the cell phospholipid, and (3) the activated COX activity. The increased prostacyclin production could provide protection against the cardiovascular effect of thromboxane which was increased by magnesium deficiency.

Trans fatty acids in partially hydrogenated soybean oil inhibit prostacyclin release by endothelial cells in presence of high level of linoleic acid.

Partially hydrogenated soybean oil (PHSBO) and natural soybean oil (SBO) were obtained from a commercial source and their fatty acids were fractionated into saturates, monoenes and diene fractions. The present study compared the effect of the total, monoene and diene fatty acid fractions of PHSBO with those of the SBO on the fatty acid composition of the cell phospholipids (PL) and the prostacyclin (PGI(2)) release by endothelial cells (EC) in culture. Results showed that arachidonic acid (AA) level decreased significantly and linoleic acid (LA) significantly increased in the cells incubated with the diene fraction or the monoene fraction of PHSBO plus 18:2 at 3:1 ratio compared to the cell incubated with those fractions of SBO. These changes were attributed to the inhibition of LA conversion to AA by trans 18:1 and 18:2 isomers present in the monoene or diene fractions of PHSBO leading to a significant decrease of PGI(2) released by the cells incubated with monoene or diene fractions of PHSBO. The cells incubated with the monoene of PHSBO or SBO plus 18:2 at a 1:1 ratio showed no inhibition of LA conversion to AA and the level of AA was almost equal in their PL, but the PGI(2) released by the cells incubated with the monoene of PHSBO was significantly less than the cells incubated with the monoene of SBO. This decrease was not related to the inhibition of PGI(2) synthesizing enzymes or phospholipase (PLA(2)) activities. Our data show that trans acids in PHSBO inhibited the PGI(2) release by the cells through controlling the level of AA as substrate, either by (a) inhibiting the conversion of LA to AA or (b) by shunting the free AA released by the PLA(2) action to metabolism by another pathway leaving less AA available for PGI(2) synthesis.

27-Hydroxycholesterol causes remodeling in endothelial cell membrane lipid composition comparable to remodeling in the failed vein grafts of CABG patients.

Our objective is to determine if vascular remodeling in CABG patients is related to oxysterols, therefore, we compared failed vein grafts from 18 patients, available after a second coronary artery bypass grafting (CABG), with human endothelial cells (ECs). The ECs were cultured in minimum essential medium (MEM) with or without 27-hydroxycholesterol (27OHC), one of the oxysterol products of oxidatively modified low density lipoproteins (ox-LDL), as an agent to alter molecular mechanisms in vascular cells. Significant changes in phospholipid composition, in fatty acid profile and in calcium concentration were found in the failed vein compared to the native saphenous vein from the same (CABG) patient. The failed vein contained significantly less phosphatidylethanolamine, more sphingomyelin, less arachidonic acid, more linoleic acid and more calcium than the native saphenous vein. Comparable changes in phospholipid composition, in fatty acid profile and increased calcium influx were reproduced in ECs cultured in medium containing 27OHC indicating that an oxysterol is an agent that can alter the lipid composition of vascular cell membranes. Our study indicates that a lipid agent, as well as protein agents that have previously been linked to the process of vascular remodeling, may be fundamental to many vascular diseases.

27-Hydroxycholesterol inhibits neutral sphingomyelinase in cultured human endothelial cells.

To study the effect of 27-hydroxycholesterol (27OHC) on the catabolism of sphingomyelin, we cultured endothelial cells (ECs) from human umbilical veins with 27OHC, then measured activities of acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) and sphingomyelin consumption by using [14C]sphingomyelin, and determined NSMase mRNA expressions by RT-PCR method. The results indicated that [14C]sphingomyelin accumulated in cells treated with 27OHC, and that the activities of both NSMase and ASMase were inhibited in ECs cultured with 27OHC. To further study the effect of 27OHC on NSMase, we used desipramine, an inhibitor of ASMase, to exclude the possible interference of ASMase's residual activity at neutral condition. Also, we observed the significant inhibition of NSMase activity by using glutathione, an inhibitor of NSMase, but found no further impact when 27OHC was added later. To determine whether the inhibition of NSMase activity was directly due to the effect of 27OHC, we exposed cell homogenate to 27OHC, and found no inhibitive effect of 27OHC on the activity of NSMase. All of our data confirmed that 27OHC had only an indirect inhibitive effect on NSMase. Our finding that no change of the NSMase mRNA expression by 27OHC indicated that the inhibitive effect of 27OHC on NSMase activity occurred at a post-transcriptional level. We suggest that an altered membrane fluidity caused by 27OHC could be involved in the inhibited activity of NSMase.

Trans fatty acids in hydrogenated fat inhibited the synthesis of the polyunsaturated fatty acids in the phospholipid of arterial cells.

Our hypothesis that the trans fatty acids in hydrogenated fat inhibited the synthesis of polyunsaturated fatty acids in the phospholipid of arterial cells was tested with five groups each with six pregnant porcine fed from d 35 of gestation and during lactation. The basal diet contained 2% corn oil (control). The other four diets included the control + 10% butter or 10% hydrogenated fat plus two levels of Mg. Plasma, milk and aortic phospholipid fatty acids, phospholipid composition and calcium content of the aorta from the piglets were determined. At 48 +/- 2 d of age, the aorta phospholipid of piglets from porcine fed hydrogenated fat contained a significantly higher concentration of linoleic acid, less arachidonic acid, and less long chain polyunsaturated fatty acid (PUFA) than did piglets from porcine fed either butterfat or the control diet. Mg had no effect. These changes in composition in piglets from porcine fed hydrogenated fat indicate that trans fat inhibits the metabolic conversion of linoleic acid to arachidonic acid and to other n-6 PUFA. The aortic calcium content data showed a significant interaction of calcium concentration with age. We concluded: 1) that dietary trans fat perturbed essential fatty acid (EFA) metabolism which led to changes in the phospholipid fatty acid composition in the aorta, the target tissue of atherogenesis, 2) this inhibition of EFA to PUFA by the isomeric fatty acids in hydrogenated fat is a risk factor in the development of coronary heart disease.

High density lipoprotein can modulate the inhibitory effect of oxLDL on prostacyclin generation by rat aorta in vitro.

To examine the effect of oxidized low density lipoprotein (oxLDL) on prostacyclin (PGI2) generation by rat aorta in vitro and whether high density lipoprotein (HDL) has any protective effect against the inhibition of PGI2 generation induced by oxLDL is the objective of this study. Preincubation of aortas with oxLDL resulted in significant inhibition of PGI2 generation compared to preincubation with normal low density lipoprotein (nLDL) or buffer only. The inhibitory effect of oxLDL resided in its lipid moiety while the lipid fraction of nLDL showed no effect. Aortas preincubated with 10 microg/ml of lyso phosphatidycholine (lyso PC) also showed 30% inhibition of PGI2 generation, indicating that lyso PC was among the lipid components of oxLDL which inhibited PGI2 generation. Preincubation of aortas with a mixture of HDL and oxLDL at a ratio of 10:1 showed a significant recovery of PGI2 generation compared to aortas preincubated with only oxLDL, indicating a protective role for HDL. When HDL was incubated with oxLDL the transfer of lyso PC from oxLDL to HDL suggested that HDL trapped lyso PC from oxLDL thus preventing it from acting on the aorta. However, when a mixture of HDL and oxLDL at a ratio of 3:1 was preincubated with aortas, no protective effect of HDL was observed. Preincubation of aortas with a mixture of HDL plus oxLDL at a ratio of 8:1, which was incubated for 1 h at 37 degrees C, produced significantly less PGI2 than aortas preincubated only with oxLDL, indicating that HDL under these conditions was not protective but even enhanced the inhibitory effect of oxLDL. Similarly, aortas preincubated with HDL plus whole oxLDL (at a ratio of 10:1); containing all the small molecular weight oxidation products and characterized by high levels of thiobarbituric acid reactive substance (TBARS) and lipid hydroperoxides; produced significantly less PGI2 than aortas preincubated with whole oxLDL. These results were evaluated in light of possible modification of HDL by oxLDL and its lipid oxidation products such as aldehydes and lipid peroxides. The modified HDL can add more lipid peroxides and increase the effectiveness of lipid peroxides originally present in oxLDL.

Antioxidative effects of lovastatin in cultured human endothelial cells.

The effects of lovastatin on glutathione peroxidase activity, hydrogen peroxide consumption, [3H]cholesterol uptake and [14C]acetate incorporation were investigated in cultured human endothelial cells. Treatment of endothelial cells with lovastatin in a medium without serum for 4 hr significantly increased both glutathione peroxidase activity and hydrogen peroxide consumption. This treatment also significantly inhibited cholesterol synthesis and cholesterol esterification. However, lovastatin stimulated cholesterol uptake by the cells. These alterations produced by lovastatin continued up to 24 hr. When serum was present in the culture medium, only decreased cholesterol synthesis and esterification were detected. We suggest that the in vitro antioxidative ability of lovastatin resulted, in part at least, from its activating effect on glutathione peroxidase, its stimulative effect on the ability of endothelial cell to scavenge H(2)O(2), and its hypolipidemic effect.

Effect of low magnesium concentration and cholestane-3beta, 5alpha, 6beta-triol on levels of LDL receptor in cultured fibroblasts.

We investigated the level of low density lipoprotein receptors (LDLR) in fibroblasts upon treatment with low magnesium and/or cholestane-3beta, 5alpha, 6beta-triol (TriolC). After 72 h of incubation with low magnesium at a level of 188 micromol or lower, the LDLR levels, determined with immunoblotting analysis, were significantly reduced in the cultured cells. TriolC at a level of 1 microg/ml or higher also decreased LDLR level. However, no further decrease of LDLR level was detected in the cells treated with low magnesium plus TriolC at a level of 0.1 microg/ml, compared with the LDLR level from the cells treated with low magnesium only. Our results showed that both low magnesium concentration and TriolC reduced the LDLR level, but the reducing effect was not accentuated when both were present in the culture medium.