Does the addition of active body warming to in-line intravenous fluid warming prevent maternal hypothermia during elective caesarean section? A randomised controlled trial.

INTRODUCTION: Inadvertent perioperative hypothermia occurs frequently during elective caesarean section but perioperative active body warming is not widely used. There is a paucity of evidence of its use in the obstetric population, and no applicable guidelines. We set out to identify a superior active warming method for preventing inadvertent perioperative hypothermia. METHODS: Following ethical approval, 132 women presenting for uncomplicated elective caesarean section under spinal anaesthesia were recruited. All participants received in-line intravenous fluid warming and were randomised to one of three parallel groups: no active body warming; forced air warming; and conduction mattress warming. The primary outcome was the difference in mean core temperature, measured on admission to the recovery room, between study groups. Core temperature and thermal comfort were measured perioperatively at 15-min intervals. Estimated blood loss, haemoglobin change, length of hospital stay and neonatal core temperature were also recorded. RESULTS: One-hundred-and-thirty-one women completed the study. There was no significant difference in mean core temperature on admission to the recovery room (36.6°C vs. 36.6°C vs. 36.6°C, η2=0.005, P=0.74). Maternal hypothermia was prevented in all groups with only 0.3% hypothermic at any of the temperature measurements (3/1016). There was no difference in mean neonatal core temperature (36.3°C vs. 36.3°C vs. 36.3°C, η2=0.003, P=0.82); however, 59.4% (76/128) of all neonates were hypothermic. CONCLUSION: In-line intravenous fluid warming is sufficient to prevent maternal hypothermia and maintain core temperature. The addition of active body warming conferred no added benefit.

The magnitude of decrease in hepatic very low density lipoprotein apolipoprotein B secretion is determined by the extent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition in miniature pigs.

It has been postulated that the rate of hepatic very low density lipoprotein (VLDL) apolipoprotein (apo) B secretion is dependent upon the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. To test this hypothesis in vivo, apoB kinetic studies were carried out in miniature pigs before and after 21 days treatment with high-dose (10 mg/kg/day), atorvastatin (A) or simvastatin (S) (n = 5). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/day; 0.1%). Statin treatment decreased plasma total cholesterol [31 (A) vs. 20% (S)] and low density lipoprotein (LDL) cholesterol concentrations [42 (A) vs. 24% (S)]. Significant reductions in plasma total triglyceride (46%) and VLDL triglyceride (50%) concentrations were only observed with (A). Autologous [131I]VLDL, [125I]LDL, and [3H]leucine were injected simultaneously, and apoB kinetic parameters were determined by triple-isotope multicompartmental analysis using SAAM II. Statin treatment decreased the VLDL apoB pool size [49 (A) vs. 24% (S)] and the hepatic VLDL apoB secretion rate [50 (A) vs. 33% (S)], with no change in the fractional catabolic rate (FCR). LDL apoB pool size decreased [39 (A) vs. 26% (S)], due to reductions in both the total LDL apoB production rate [30 (A) vs. 21% (S)] and LDL direct synthesis [32 (A) vs. 23% (S)]. A significant increase in the LDL apoB FCR (15%) was only seen with (A). Neither plasma VLDL nor LDL lipoprotein compositions were significantly altered. Hepatic HMG-CoA reductase was inhibited to a greater extent with (A), when compared with (S), as evidenced by 1) a greater induction in hepatic mRNA abundances for HMG-CoA reductase (105%) and the LDL receptor (40%) (both P < 0.05); and 2) a greater decrease in hepatic free (9%) and esterified cholesterol (25%) (both P < 0.05). We conclude that both (A) and (S) decrease hepatic VLDL apoB secretion, in vivo, but that the magnitude is determined by the extent of HMG-CoA reductase inhibition.

9-cis retinoic acid induces monocyte chemoattractant protein-1 secretion in human monocytic THP-1 cells.

Monocyte migration and activation are regulated by monocyte chemoattractant protein-1 (MCP-1). Prior studies have shown MCP-1 expression is modulated by a variety of ligands that act through extracellular receptors. In the current study, we show 9-cis retinoic acid (RA), a ligand for the nuclear hormone receptor retinoid X receptor (RXR) and retinoic acid receptor (RAR), markedly induces the expression of MCP-1. In human THP-1 monocytic leukemia cells cultured with RA (0.05 to 500 nmol/L), MCP-1 expression was induced rapidly, significantly, and dose-dependently by as much as 165-fold. MCP-1 RNA level was also increased in RA-treated cells. Expression of PPARgamma, a heterodimer partner of RXR, is also markedly induced by RA in THP-1 cells. However, BRL49653, a PPARgamma ligand, failed to induce MCP-1 secretion either alone or to modify the expression level induced by RA. In contrast, BRL49653 significantly increased MCP-1 (biotinylated MCP-1) binding to THP-1 cells, whereas RA had no effect. Other peroxisome proliferator activated receptor (PPAR) ligands, 15d-PGJ(2) and troglitazone (PPARgamma), Wy14,643 (PPARalpha), and PD195599 (PPARbeta) inhibited the induction of MCP-1 by RA. RA's effect on MCP-1 expression in human elutriated monocytes were similar to that observed in the THP-1 cells. These studies identify RA as a nuclear signal for MCP-1 induction in undifferentiated human monocytic cells. These studies also suggest monocyte MCP-1 expression induced through RA may modulate cell migration.

Hypocholesterolemic effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in the guinea pig: atorvastatin versus simvastatin.

Male Hartley guinea pigs were fed a hypercholesterolemic diet rich in lauric and myristic acids with 0, 10, or 20 mg/kg of simvastatin or atorvastatin for 21 days. Atorvastatin and simvastatin resulted in a lowering of plasma low-density lipoprotein (LDL) cholesterol in a dose-dependent manner by an average of 48 and 61% with 10 and 20 mg/kg, respectively. Both statins were equally effective in lowering plasma LDL cholesterol and apolipoprotein B (apo-B) levels. Atorvastatin and simvastatin treatments yielded LDL particles that differed in composition from the control. Due to the relevance of LDL oxidation and cholesteryl ester transfer in plasma to the progression of atherosclerosis, these parameters were analyzed after statin treatment. Atorvastatin and simvastatin treatment decreased the susceptibility of LDL particles to oxidation by 95% as determined by the formation of thiobarbituric acid reactive substances. An 80% decrease in the transfer of cholesteryl ester between high-density lipoprotein (HDL) and the apo-B-containing lipoproteins was observed after simvastatin and atorvastatin treatment. In addition, statin effects on plasma LDL transport were studied. Simvastatin- and atorvastatin-treated guinea pigs exhibited 125 and 175% faster LDL fractional catabolic rates, respectively, compared with control animals. No change in LDL apo-B flux was induced by either treatment; however, LDL apo-B pool size was reduced after statin treatment. Hepatic microsomal free cholesterol was lower in the atorvastatin and simvastatin groups. However, only atorvastatin treatment resulted in an 80% decrease of acyl-CoA:cholesterol acyltransferase activity (P < 0.001). In summary, atorvastatin and simvastatin had similar LDL cholesterol lowering properties, but these drugs modified LDL transport and hepatic cholesterol metabolism differently.

Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs.

An orally bioavailable acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, avasimibe (CI-1011), was used to test the hypothesis that inhibition of cholesterol esterification, in vivo, would reduce hepatic very low density (VLDL) apolipoprotein (apo) B secretion into plasma. ApoB kinetic studies were carried out in 10 control miniature pigs, and in 10 animals treated with avasimibe (10 mg/kg/d, n = 6; 25 mg/kg/d, n = 4). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/d; 0.1%). Avasimibe decreased the plasma concentrations of total triglyceride, VLDL triglyceride, and VLDL cholesterol by 31;-40% 39-48%, and 31;-35%, respectively. Significant reductions in plasma total cholesterol (35%) and low density lipoprotein (LDL) cholesterol (51%) concentrations were observed only with high dose avasimibe. Autologous 131I-labeled VLDL, 125I-labeled LDL, and [3H]leucine were injected simultaneously into each pig and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). Avasimibe decreased the VLDL apoB pool size by 40;-43% and the hepatic secretion rate of VLDL apoB by 38;-41%, but did not alter its fractional catabolism. Avasimibe decreased the LDL apoB pool size by 13;-57%, largely due to a dose-dependent 25;-63% in the LDL apoB production rate. Hepatic LDL receptor mRNA abundances were unchanged, consistent with a marginal decrease in LDL apoB FCRs. Hepatic ACAT activity was decreased by 51% (P = 0.050) and 68% (P = 0.087) by low and high dose avasimibe, respectively. The decrease in total apoB secretion correlated with the decrease in hepatic ACAT activity (r = 0.495; P = 0.026). We conclude that inhibition of hepatic ACAT by avasimibe reduces both plasma VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion.

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants.

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.

Select 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors vary in their ability to reduce egg yolk cholesterol levels in laying hens through alteration of hepatic cholesterol biosynthesis and plasma VLDL composition.

The inability to markedly attenuate cholesterol levels in chicken eggs has led to speculation that cholesterol is essential for yolk formation and that egg production would cease when yolk cholesterol deposition was inadequate for embryonic survival. However, this critical level hypothesis remains unproven. Here, we determine the relative responsiveness of laying hens to three select inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. A control diet, either alone or supplemented with one of two dietary levels (0.03 or 0.06%) of atorvastatin, lovastatin, or simvastatin, was fed to White Leghorn hens for 5 wk. Liver cholesterol concentrations (mg/g tissue) were decreased (P 0.05), and 22% (P 0.05), and -3% (P > 0.05)], was much less affected. We concluded that cholesterol per se may not be an obligatory component for yolk formation in chickens and, as such, may be amenable to further pharmacological manipulation

ApoB100 secretion from HepG2 cells is decreased by the ACAT inhibitor CI-1011: an effect associated with enhanced intracellular degradation of ApoB.

The concept that hepatic cholesteryl ester (CE) mass and the rate of cholesterol esterification regulate hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to correlate the rate of cholesterol esterification and CE mass with apoB secretion by CI-1011, an acyl CoA:cholesterol acyltransferase (ACAT) inhibitor that is known to decrease apoB secretion, in vivo, in miniature pigs. HepG2 cells were incubated with CI-1011 (10 nmol/L, 1 micromol/L, and 10 micromol/L) for 24 hours. ApoB secretion into media was decreased by 25%, 27%, and 43%, respectively (P<0.0012). CI-1011 (10 micromol/L) inhibited HepG2 cell ACAT activity by 79% (P<0.002) and cellular CE mass by 32% (P<0.05). In contrast, another ACAT inhibitor, DuP 128 (10 micromol/L), decreased cellular ACAT activity and CE mass by 85% (P<0.002) and 42% (P=0.01), respectively, but had no effect on apoB secretion into media. To characterize the reduction in apoB secretion by CI-1011, pulse-chase experiments were performed and analyzed by multicompartmental modelling using SAAM II. CI-1011 did not affect the synthesis of apoB or albumin. However, apoB secretion into the media was decreased by 42% (P=0.019). Intracellular apoB degradation increased proportionately (P=0.019). The secretion of albumin and cellular reuptake of labeled lipoproteins were unchanged. CI-1011 and DuP 128 did not affect apoB mRNA concentrations. These results show that CI-1011 decreases apoB secretion by a mechanism that involves an enhanced intracellular degradation of apoB. This study demonstrates that ACAT inhibitors can exert differential effects on apoB secretion from HepG2 cells that do not reflect their efficacy in inhibiting cholesterol esterification.

Lipoprotein lipase greatly enhances the retention of lipoprotein(a) to endothelial cell-matrix.

The trapping of apolipoprotein (apo)B containing lipoproteins within the arterial subendothelial matrix (ECM) is an early event in atherosclerosis. When lipoprotein lipase, a constituent of the ECM, is prebound to ECM both LDL and oxidized LDL binding is greatly enhanced. In this study we compared the binding of lipoprotein(a) (Lp(a)), a lipoprotein correlated with atherosclerosis and restenosis, to ECM in the presence of varying concentrations of LPL. Without LPL, Lp(a) binding was low and non-saturable. In the presence of LPL, Lp(a) retention increased from 2.7 x 10(-7) to 1.13 x 10(-4) nmoles. Scatchard analysis demonstrated that the affinities of both Lp(a) and LDL to lipase were similar. In competition experiments, LDL, apoE, polymers of lysine and arginine were all capable of preventing the lipase specific [125I]Lp(a) retention. However, neither collagen nor fibronectin were capable of blocking or displacing [125I]Lp(a) from the lipase bound to ECM. In a separate set of experiments, when ECM was not saturated with lipase, both fibronectin and collagen (at 10-fold protein excess) prevented approximately 40% of total [125I]Lp(a) retention to ECM. These data suggest, in the absence of lipase, apo(a) may regulate the binding of Lp(a) to ECM. Whereas, lipase enhanced the binding of Lp(a) to ECM, most probably through the apoB moiety of the Lp(a) particle.

Atorvastatin and simvastatin have distinct effects on hydroxy methylglutaryl-CoA reductase activity and mRNA abundance in the guinea pig.

The effects of atorvastatin and simvastatin on hydroxy methylglutaryl (HMG)-CoA reductase activity and mRNA abundance were studied in guinea pigs randomized to three groups: untreated animals and those treated with 20 mg/kg of atorvastatin or simvastatin. Guinea pigs were fasted for 0, 6, 12, or 18 h in an attempt to remove the drug from their systems. Reductase activity and mRNA levels were analyzed after each time point. Reductase inhibitor treatment resulted in 50-62% lower cholesterol concentrations compared to untreated guinea pigs (P < 0.0001), while plasma triacylglycerol (TAG) concentrations did not differ among groups. Plasma cholesterol and TAG were 50-70% lower after 18 h fasting in the three groups (P < 0.001). In the nonfasting state, simvastatin and atorvastatin treatment did not affect HMG-CoA reductase activity compared with untreated animals. However, after 6 h of fasting, simvastatin-treated guinea pigs had higher HMG-CoA reductase activity than untreated animals (P < 0.01), suggesting that the drug had been removed from the enzyme. In contrast, atorvastatin-treated guinea pigs maintained low enzyme activity even after 18 h of fasting. Further, HMG-CoA reductase mRNA abundance was increased by sevenfold after atorvastatin treatment and by twofold after simvastatin treatment (P < 0.01). These results suggest that simvastatin and atorvastatin have different half-lives, which may affect HMG-CoA reductase mRNA levels. The increase in reductase activity by simvastatin during fasting could be related to an effect of this statin in stabilizing the enzyme. In contrast, atorvastatin, possibly due to its longer half-life, prolonged inhibition of HMG-CoA reductase activity and resulted in a greater increase in mRNA synthesis.

Induction of PPARgamma1 expression in human THP-1 monocytic leukemia cells by 9-cis-retinoic acid is associated with cellular growth suppression.

9-cis-Retinoic acid (RA) and peroxisome proliferator activated receptor gamma (PPARgamma) regulates cellular growth and differentiation. In THP-1 cells, a human monocytic leukemia cell line, RA markedly induced PPARgamma1 RNA, nuclear PPARgamma1 protein and suppressed cell growth. The PPARgamma ligand, BRL49653 enhanced RA's growth suppression ability. With BRL49653 alone, THP-1 cell growth was only marginally suppressed. Cell cycle analysis revealed the G1 phase cell population was significantly increased when cells were treated with both ligands. RA induced growth suppression did not differentiate the THP-1 cells to macrophages. Phorbol ester (PMA) induced differentiation of cells to macrophage also induced PPARgamma1 expression, however when RA is given either simultaneously or sequentially to these cells, no further increase in expression of the nuclear receptor was observed. Overall, these data suggest RA induction of PPARgamma1 may block cell growth and may have application for the treatment of proliferative diseases.

Atorvastatin and gemfibrozil metabolites, but not the parent drugs, are potent antioxidants against lipoprotein oxidation.

Increased atherosclerosis risk in hyperlipidemic patients may be a result of the enhanced oxidizability of their plasma lipoproteins. We have previously shown that hypocholesterolemic drug therapy, including the 3-hydroxy-3-methyl-glutaryl CoenzymeA (HMG-CoA) reductase inhibitors, and the hypotriglyceridemic drug bezafibrate, significantly reduced the enhanced susceptibility to oxidation of low density lipoprotein (LDL) isolated from hyperlipidemic patients. Although this antioxidative effect could not be obtained in vitro with all of these drugs, the active drug metabolites, which are formed in vivo, could affect lipoprotein oxidizability. We thus sought to analyze the effect of atorvastatin and gemfibrozil, as well as specific hydroxylated metabolites, on the susceptibility of LDL, very low density lipoprotein (VLDL), and high density lipoprotein (HDL) to oxidation. LDL oxidation induced by either copper ions (10 microM CuSO4), by the free radical generator system 2'-2'-azobis 2-amidino propane hydrochloride (5 mM AAPH), or by the J-774A.1 macrophage-like cell line, was not inhibited by the parent forms of atorvastatin or gemfibrozil, but was substantially inhibited (57-97%), in a concentration-dependent manner, by pharmacological concentrations of the o-hydroxy and the p-hydroxy metabolites of atorvastatin, as well as by the p-hydroxy metabolite (metabolite I) of gemfibrozil. On using the atorvastatin o-hydroxy metabolite and gemfibrozil metabolite I in combination an additive inhibitory effect on LDL oxidizability was found. Similar inhibitory effects (37-96%) of the above metabolites were obtained for the susceptibility of VLDL and HDL to oxidation in the oxidation systems outlined above. The inhibitory effects of these metabolites on LDL, VLDL, and HDL oxidation could be related to their free radical scavenging activity, as well as (mainly for the gemfibrozil metabolite I) to their metal ion chelation capacities. In addition, inhibition of HDL oxidation was associated with the preservation of HDL-associated paraoxonase activity. We conclude that atorvastatin hydroxy metabolites, and gemfibrozil metabolite I possess potent antioxidative potential, and as a result protect LDL, VLDL, and HDL from oxidation. We hypothesize that in addition to their beneficial lipid regulating activity, specific metabolites of both drugs may also reduce the atherogenic potential of lipoproteins through their antioxidant properties.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.

A novel compound that elevates high density lipoprotein and activates the peroxisome proliferator activated receptor.

In the current studies we describe the effects of PD 72953 and related compounds on lipoprotein levels in chow-fed male rats. After 2 weeks, 10 mg/kg of PD 72953 daily was as effective as 100 mg/kg gemfibrozil for elevating HDL-cholesterol. At 100 mg/kg, PD 72953 further elevated HDL-cholesterol to 232% of control levels, and was associated with increased HDL size and plasma apoE (169% of control), despite no change in hepatic apoE mRNA. ApoA-I rose transiently (at 1 week), but by 2 weeks only apoE remained elevated. PD 72953 dose-dependently reduced plasma apoB, VLDL-cholesterol, LDL-cholesterol, and triglyceride. Hepatic apoC-III mRNA reduction parallelled triglyceride lowering. After 1 week, 30 and 100 mg/kg per day PD 72953 reduced plasma apo-CIII levels by 30 and 34%, and triglycerides by 60 and 83%, respectively. PD 72953 treatment had no effect on triglyceride production rates; however, 125I-labeled VLDL apoB disappearance was enhanced. We compared PD 72953 to a structurally similar diacid, PD 69405, that also reduced VLDL and LDL, but had no effect on HDL elevation. Compared to PD 72953, PD 69405 further accelerated 125I-labeled VLDL apoB disappearance, decreased triglyceride production, and elevated the ratio of post-heparin hepatic to lipoprotein lipase activity. Whole animal studies, transient transfection studies in HepG2 cells, and chimeric receptor studies in kidney 293 cells suggest that PD 72953 is a ligand for the peroxisomal proliferation activated receptor alpha (PPARalpha), and PPARgamma. Overall, PD 72953 may act through a peroxisomal proliferation activated receptor and result in plasma triglycerides and apoB-containing lipoprotein reduction, while also raising HDL cholesterol. Reduced apoC-III may allow triglyceride-rich remnants to more efficiently bind and present substrate to peripheral tissue lipoprotein lipase, and therefore allow enhanced shedding of remnant phospholipid surface for HDL production.

Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoprotein B production in miniature pigs.

In the present studies, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor atorvastatin was used to test the hypothesis that inhibition of cholesterol biosynthesis in vivo with a consequent reduction in the availability of hepatic cholesterol for lipoprotein synthesis, would (1) reduce very low density lipoprotein (VLDL) apolipoprotein B (apoB) secretion into the plasma, (2) reduce the conversion of VLDL apoB to LDL apoB, and (3) reduce LDL apoB direct synthesis. ApoB kinetic studies were carried out in six control miniature pigs and in six animals after 21 days of administration of atorvastatin (3 mg/kg per day). Pigs were fed a fat- (34% of calories; polyunsaturated to monounsaturated to saturated ratio, 1:1:1) and cholesterol- (400 mg/d cholesterol; 0.1%; 0.2 mg/kcal) containing pig chow-based diet. Atorvastatin treatment significantly reduced plasma total cholesterol, LDL cholesterol, total triglyceride, and VLDL triglyceride concentrations by 16%, 31%, 19%, and 28%, respectively (P < .01). Autologous 131I-VLDL, 125I-LDL, and [3H]leucine were injected simultaneously into each pig, and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). The VLDL apoB pool size decreased by 29% (0.46 versus 0.65 mg/kg; P = .002), which was entirely due to a 34% reduction in the VLDL apoB production rate (PR) (1.43 versus 2.19 mg/kg per hour; P = .027). The fractional catabolic rate (FCR) was unchanged. The LDL apoB pool size decreased by 30% (4.74 versus 6.75 mg/kg; P = .0004), which was due to a 22% reduction in the LDL apoB PR (0.236 versus 0.301 mg/kg per hour; P = .004), since the FCR was unchanged. The reduction in LDL apoB PR was primarily due to a 34% decrease in conversion of VLDL apoB to LDL apoB; however, this reduction was not statistically significant (P = .114). Hepatic apoB mRNA abundance quantitated by RNase protection assay was decreased by 13% in the atorvastatin-treated animals (P = .003). Hepatic and intestinal LDL receptor mRNA abundances were not affected. We conclude that inhibition of hepatic HMG-CoA reductase by atorvastatin reduces both VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion into the plasma and not by an increase in hepatic LDL receptor expression. This decrease in apoB secretion may, in part, be due to a reduction in apoB mRNA abundance.

Attenuation of plasma low density lipoprotein cholesterol by select 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in mice devoid of low density lipoprotein receptors.

Low density lipoprotein (LDL) reduction independent of LDL receptor regulation was investigated using HMG-CoA reductase inhibitors in LDL receptor-deficient mice. In males, LDL cholesterol dose-dependently decreased with atorvastatin treatment after 1 week. As untreated mice grew older, their LDL cholesterol progressively rose above basal levels, but was quelled with atorvastatin treatment. In females, atorvastatin treatment time-dependently decreased LDL cholesterol levels and induced hepatic HMG-CoA reductase activity. Unlike males, cholesterol-lowering effects of the drug were sustained in females. Lovastatin, simvastatin, and pravastatin also reduced total and LDL cholesterol; however, additional studies in females demonstrated that atorvastatin caused the greatest dose-dependent and sustained effect after 2 weeks. In females, hepatic HMG-CoA reductase mRNA inversely correlated with LDL cholesterol lowering, with atorvastatin showing the greatest increase in mRNA levels (17.2-fold), followed by lovastatin (10.7-fold), simvastatin (4.1-fold), and pravastatin (2.5-fold). Atorvastatin effects on lipoprotein production were determined after acute (1 day) or chronic (2 week) treatment prior to intraperitoneal injection of Triton WR1339. Acute treatment reduced cholesterol (-29%) and apoB (-16%) secretion, with no change in triglyceride secretion. In contrast, chronic treatment elevated cholesterol (+20%), apoB (+31%), and triglyceride (+57%) secretion. Despite increased cholesterol and apoB secretion, plasma levels were reduced by 51% and 46%, respectively. Overall, under acute or chronic conditions, apoB paralleled cholesterol secretion rates, and triglyceride to cholesterol secretion ratios were elevated by 38% and 32%, respectively. We propose that atorvastatin limits cholesterol for lipoprotein assembly, which is compensated for by triglyceride enrichment. In addition, with either acute or chronic atorvastatin treatment, apoB-100 secretion was blocked, and compensated for by an increased secretion of apoB-48. The apoB-48 particles produced are cleared by LDL receptor-independent mechanisms, with an overall effect of reducing LDL production in these mice. These studies support the idea that HMG-CoA reductase inhibitors modulate lipoprotein levels independent of LDL receptors, and suggest they may have utility in hyperlipidemias caused by LDLreceptor disorders.

Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid. Lack of effect on transcription factor NF-kappa B.

In the present studies, we examined the effect of flavonoids on the endothelial cell expression of adhesion molecules, an early step in inflammation and atherogenesis. Addition of tumor necrosis factor-alpha (TNF) to human aortic endothelial cells (HAECs) led to the induction of vascular cell adhesion molecule-1 (VCAM-1) expression and enhancement in expression of intercellular adhesion molecule-1 (ICAM-1). A flavonoid, 2-(3-amino-phenyl)-8-methoxy-chromene-4-one (PD 098063), markedly inhibited TNF-induced VCAM-1 cell-surface expression in a concentration-dependent fashion with half-maximal inhibition at 19 mumol/L but had no effect on ICAM-1 expression. Another structurally distinct flavonoid, 2-phenyl-chromene-4-one, similarly selectively decreased VCAM-1 expression. The inhibition in cell-surface expression of VCAM-1 by PD 098063 correlated with decreases in steady-state mRNA levels, but there was no effect on ICAM-1 mRNA levels. The decrease in VCAM-1 mRNA levels was not due to changes in mRNA stability but rather resulted from a reduction in the rate of transcription of the gene. However, electrophoretic mobility shift assays using nuclear extracts from TNF-induced HAECs treated with PD 098063 failed to show a decrease in the activation of NF-kappa B, indicating that inhibition of activation of this transcription factor may not be its mode of action. Similarly, PD 098063 did not affect chloramphenicol acetyltransferase reporter gene activity in TNF-inducible minimal VCAM-1 promoter constructs containing two NF-kappa B sites, suggesting that the compound does not affect the transactivation driven by these sites. We conclude that this compound selectively blocks agonist-induced VCAM-1 protein and gene expression in HAECs by NF-kappa B-independent mechanism(s).

Hypocholesterolemic actions of atorvastatin are associated with alterations on hepatic cholesterol metabolism and lipoprotein composition in the guinea pig.

Guinea pigs were fed 15% (w/W) fat, high in lauric and myristic acids, a diet known to produce hypercholesterolemia in these animals. The diet was given alone or in combination with four doses of atorvastatin equivalent to 1, 3, 10, and 20 mg/kg per day. Atorvastatin reduced plasma LDL cholesterol concentrations by 46, 50, 53, and 70%, respectively (P < 0.001). Plasma apoB concentrations were reduced by atorvastatin (P < 0.001) and compositional changes occurred in VLDL and LDL with reductions of the relative proportion of cholesteryl ester and increases in triacylglycerol. A reduction in hepatic cholesteryl ester (66%) was observed only with the highest atorvastatin dose (20 mg/kg per day) while microsomal cholesterol was reduced by 30% with 3-20 mg/kg per day. Hepatic ACAT activity was down-regulated and apoB/E receptor number was increased by atorvastatin. In contrast, HMG-CoA reductase activity and cholesterol 7 alpha-hydroxylase were not affected by the drug. VLDL apoB secretion rates were decreased by atorvastatin treatment 59 and 76% with 3 and 20 mg/kg per day, respectively. Nascent VLDL particles were larger after drug treatment, showing an increased number in triacylglycerol molecules. These results support the hypothesis that the plasma LDL lowering induced by atorvastatin is due to a decreased secretion of apoB in combination with an increase of hepatic apoB/E receptors.

Opposite effects of bezafibrate and gemfibrozil in both normal and hypertriglyceridemic rats.

Chow and sucrose-fed rats were used as animal models to study the dose-responses of bezafibrate and gemfibrozil in normolipidemic and hypertriglyceridemic states, respectively. Although both drugs lowered plasma triglycerides (TG) to about the same extent in chow-fed rats, gemfibrozil lowered liver TG as well as plasma total and LDL-cholesterol (LDL-C), but elevated HDL-cholesterol (HDL-C) and plasma apo E concentrations. Bezafibrate produced opposite effects, namely, decreased HDL-C, apo E and liver TG, and tended to increase LDL-C. TG lowering for both drugs in chow-fed rats was not due to changes in TG secretion (production) in normal rats but was associated with enhanced LPL activity. In hypertriglyceridemic rats both drugs modestly reduced TG secretion rates about 40% at a dose producing maximal TG lowering, but again, gemfibrozil elevated and bezafibrate lowered HDL-C and apo E. Unlike gemfibrozil, bezafibrate induced the appearance of LDL-C in hypertriglyceridemic rats which was not detected in control animals, and also tended to increase rather than decrease plasma apo B levels. Finally, changes in liver TG concentration (mg/g) in hypertriglyceridemic rats were opposite for these drugs, resulting in significant drug-related differences in liver TG content (mg/organ). From these data we postulate that, although similar with regard to TG lowering activity and mechanisms thereof, gemfibrozil and bezafibrate produce fundamentally different effects on LDL, HDL and apolipoprotein metabolism (apo B and apo E) in rats which may relate to potential differential effects on reverse cholesterol transport and atherogenesis.

Lack of correlation between ACAT mRNA expression and cholesterol esterification in primary liver cells.

A partial rabbit cDNA clone (14b) for ACAT has been characterized and used to demonstrate that hepatic and aortic ACAT mRNA14b abundance increased 2-3-fold in rabbits receiving a high fat/high cholesterol-diet compared to chow fed animals (Pape et al. (1995) J. Lipid Res. 36, 823-838). Because of those data we hypothesized that increased hepatic cholesteryl ester mass and synthesis rates in rabbit liver cells are associated with an increase in ACAT mRNA14b levels. To test this hypothesis we altered cellular cholesteryl ester mass and synthesis rates in primary parenchymal and nonparenchymal cells using various extracellular agents and measured the accumulated mass of ACAT mRNA14b. Parenchymal cells incubated with rabbit beta VLDL or mevalonolactone displayed a 6-10-fold increase in cellular cholesteryl ester mass over a three day treatment with no significant changes in cellular free cholesterol, triacylglycerols, or ACAT mRNA14b levels; HMG CoA reductase and LDL receptor mRNA mass decreased initially as a result of cholesteryl ester loading. Treatment of parenchymal cells with CI-976, an ACAT inhibitor, showed a marked reduction in cholesteryl ester synthetic rate compared to beta VLDL controls but displayed no change in ACAT mRNA14b levels. A mixed population of rabbit hepatic nonparenchymal cells was incubated with beta VLDL for 24 h in culture which resulted in a 6-fold increase in cellular cholesteryl ester mass; there was no change in ACAT mRNA14b levels. In an in vivo study, rabbits consuming a high fat/high cholesterol-diet for three weeks showed a 10-fold increase in hepatic cholesteryl ester with no significant changes in ACAT mRNA14b levels. Together these data indicate that rabbit liver cellular cholesteryl ester mass increases of up to 10-fold are not correlated with ACAT mRNA14b changes. Thus, hepatic ACAT mRNA14b expression and cellular cholesterol esterification do not appear to be coordinately regulated at this level of cholesteryl ester loading.