Potent Inhibitors Of Cholesterol Synthesis Research Articles

255-POS

Objectives 27-hydroxycholesterol (27-OHC) is the hydroxylation product of cholesterol by the enzyme sterol 27-hydroxylase (CYP27A1). 27-OHC is a potent inhibitor of cholesterol synthesis and increases cholesterol efflux from extrahepatic cells in vitro. Its transport from peripheral cells to the liver contributes to the high-density lipoprotein HDL-dependent reverse-cholesterol-transport and represents a potentially anti-atherogenic mechanism. Our hypothesis was (1) that the rise in cholesterol concentration during pregnancy is attributed to a lower catabolism and excretion by a relative decrease in 27-OHC concentration during the course of pregnancy and (2) that 27-OHC plays a role in pregnancy related pathologies linked to a disturbed lipid metabolism. Methods 223 serum samples were analyzed. Group A: 33 patients with uncomplicated pregnancies were analyzed at three different time points (first to third trimester). Group B consists of 45 patients (24–42 weeks of gestation) with pregnancy pathologies (IUGR n = 14, preeclampsia n = 14, HELLP n = 10, intrahepatic cholestasis n = 7) and 45 gestational age matched controls. 27-OHC was measured by GC–MS and related to cholesterol concentrations. Statistics: Spearman’s rank coefficient and Kruskal–Wallis test with Dunn’s multiple comparisons. Results Group A: 27-OHC was positively correlated to total cholesterol concentration ( ρ = 0.518). The 27-OHC/cholesterol ratio dropped with the course of pregnancy with higher values in first and second trimester as compared to the non-pregnant state postpartum (mean (SD), 1st trimester: 1.367 (0.38) p p Conclusions Our analysis revealed an increased 27-OHC/cholesterol ratio in the first and second trimester, indicating a lower cholesterol catabolism accounting for increased cholesterol concentrations throughout gestation. However, it does not support a role of 27-OHC in the patho-mechanism of pregnancy pathologies. Disclosures B. Winkler: None. N. Kleine-Eggebrecht: None. N. Maass: None. M. Mohaupt: None. G. Escher: None. U. Pecks: None.

Simvastatin induces osteogenic differentiation of murine embryonic stem cells

Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications.

Identification and characterization of the cholesterol 25‐hydroxylase gene promoter

25‐Hydroxycholesterol (25HC) is the product of the cholesterol 25‐hydroxylase (CH25H) gene, and is one of the most potent inhibitors of cholesterol synthesis, through the suppression of SREBP activation. 25HC has also been implicated in neurodegenerative disease and shown to be toxic to primary cultured neuronal cells. Very little is known about the transcriptional regulation of the CH25H gene. Using in silico analysis of the mouse and human promoters, potential binding sites for several transcription factors were identified including p53, CREB and Sp1. The functionality of the putative promoter elements of the mouse promoter was investigated using an in vitro reporter assay with deletion constructs. The results suggest the proximal CREB binding site may be involved in driving expression from the CH25H promoter. While a high proportion TATA‐less promoters contain CREB binding sites, these promoters appear to be generally unresponsive to cAMP, suggesting activation of these promoters depends on co‐stimulatory signals. In this study we observed co‐activation of the CH25H promoter by Sp1 and CREB in a synergistic manner. A negative feedback mechanism involving 25HC was also observed in the in vitro reporter assays and this effect appeared to be mediated by an element within the proximal 250 bp of the promoter. The region containing the p53 consensus site was demonstrated to repress transcription.

The Discovery of Statins

This year, the Lasker Foundation confers its Clinical Medical Research Award on Akira Endo for his isolation from fungi of statins, potent inhibitors of cholesterol synthesis in the liver. The introduction of statins to clinical practice has markedly reduced morbidity and mortality from atherosclerotic cardiovascular disease.

Hydrophobic but not hydrophilic statins enhance phagocytosis and decrease apoptosis of human peripheral blood cells in vitro

The engulfing ability of phagocyting cells is related to the fluidity of the cell membrane that in turn depends on its chemical composition. Changes in membranal lipid content may increase or decrease membranal fluidity with a subsequent enhanced or impaired phagocytosis, respectively. Statins are recognized as potent inhibitors of cholesterol synthesis and therefore, are successfully administered to patients with hypercholesterolemia. Since it is considered that cholesterol affects cell function via changes in membrane composition, the present study was designed to examine the in vitro effect of three hydrophobic statins – atorvastatin, lovastatin and simvastatin, and a hydrophilic one – pravastatin, on the engulfing capacity, phagocytic index and apoptosis of peripheral blood phagocytes from healthy volunteers. Peripheral white blood cells obtained from 20 healthy normocholesterolemic individuals were incubated for 2 h with 10 and 50 μM of the four statins and phagocytosis of fluorescent latex particles was detected by flow cytometry. Apoptosis was examined using annexin V and propidium iodide staining. An increase in the percentage of phagocyting cells was observed after incubation with 50 μM of lovastatin and simvastatin. On the other hand, all three hydrophobic statins induced a dose-dependent increase in the phagocytic index. The hydrophilic pravastatin did not affect phagocytosis, phagocytic index and apoptosis. All three hydrophobic statins at 50 μM exerted a slight, but significant decrease of apoptosis. The results suggest that the effect of hydrophobic statins on the engulfing capacity of human peripheral blood phagocytes and apoptosis is dependent on their dosage and physiochemical properties. This observation is an additional contribution to the statins' pleiotropic effect.

Simvastatin causes the formation of cholesterol-rich remnants in mice lacking apoE

Mice that lack apolipoprotein E (apoE) display a severe hypercholesterolemia, caused by the accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins in the plasma. Statins are potent inhibitors of cholesterol synthesis that, when administered to mice lacking apoE, cause paradoxical further increases in plasma cholesterol levels. In the present study, we examined the mechanisms responsible for this phenomenon. ApoE-deficient mice fed a chow diet containing simvastatin developed, as anticipated, an enhanced increase in plasma cholesterol and a decrease in plasma triglycerides. Fractionation of the plasma lipoproteins by FPLC revealed that the lipid changes were confined to the lipoprotein remnants. Western blot analysis of the remnants from the untreated and simvastatin-treated mice showed no differences in their apoB-48 content, indicating that both groups of animals accumulated similar numbers of remnant particles in the plasma. Following the injection of Triton WR-1339, the simvastatin-treated mice accumulated in the plasma significantly more cholesterol and significantly less triglycerides than the untreated animals. These results indicate that the enhanced hypercholesterolemia observed in apoE-deficient mice treated with simvastatin is not the result of an increased number of remnant particles in circulation but is caused by synthesis and secretion into the plasma of lipoproteins that are enriched in cholesterol and depleted of triglycerides.

The Role of Rho-Associated Kinase in Differential Regulation by Statins of Interleukin-1β- and Lipopolysaccharide-Mediated Nuclear Factor κB Activation and Inducible Nitric-Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells

An optimal level of NO has protective effects in atherosclerosis, whereas large amounts contribute to septic shock. To study how statins, the potent inhibitors of cholesterol synthesis, regulate NO in the vascular wall, we determined their effects on interleukin-1beta (IL-1beta)- and lipopolysaccharide (LPS)-induced NO production in aortic vascular smooth muscle cells (VSMCs). Compared with the large amounts of NO and inducible NO synthase (iNOS) protein expression induced by LPS, the responses of IL-1beta were modest. Various statins were found to inhibit LPS-induced iNOS expression and NO production, although they potentiated IL-1beta responses. In addition, fluvastatin increased IL-1beta-induced p65 nuclear translocation and nuclear factor kappaB (NF-kappaB) activity, although it inhibited those induced by LPS. To address the role of small G proteins in statin's actions, farnesyl transferase inhibitors [alpha-hydroxyfarne-sylphosphonic acid and (2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester (L-744382)], Rac inhibitor (NSC23766), and Rho-associated kinase (ROCK) inhibitor [N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride (Y-27632)] were used. We found that Y-27632 potentiated IL-1beta-induced iNOS expression, p65 nuclear translocation, IkappaB kinase (IKK), and NF-kappaB activation, whereas it had minimal effects on LPS-induced responses. In contrast, farnesyl transferase inhibitors blocked iNOS protein expression induced by LPS and IL-1beta, whereas NSC23766 had no effect. Further studies showed that LPS down-regulated Rho and ROCK activity, whereas IL-1beta increased them, suggesting a negative role of Rho and ROCK signaling, which is regulated in contrary manners by IL-1beta and LPS, in IKK/NF-kappaB activation. Through abrogating this negative signaling, statins differentially regulate iNOS expression induced by LPS and IL-1beta in VSMCs. These differential actions of statins on iNOS gene regulation might provide an additional explanation for the pleiotropic beneficial effects of statins.

Drug Insight: statins and stroke

Stroke is the third leading cause of death in the US and a common cause of long-term disability worldwide. Ischemic strokes, which are often atherothrombotic, account for more than 80% of all strokes. Current stroke prevention focuses on optimizing the treatment of modifiable risk factors, such as hypertension, diabetes and dyslipidemia. The epidemiologic association between serum cholesterol levels and adjusted stroke rates is not as strong as the link between serum cholesterol levels and coronary heart disease. Clinical trials of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins), which are potent inhibitors of cholesterol synthesis, have demonstrated, however, a marked reduction in stroke risk in hypercholesterolemic and atherosclerotic individuals, with benefits extending to normocholesterolemic individuals. These findings suggest that statins might have additional effects in stroke protection beyond cholesterol reduction. Because statins inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, which are important lipid attachments for intracellular signaling molecules, they might have direct noncholesterol-dependent effects on inflammatory and endothelial cells. Here we discuss data from clinical trials assessing the effects of statins on stroke risk, as well as outline the mechanisms underlying the cholesterol-independent effects of statins and provide evidence-based recommendations for stroke prevention, based on achieved serum cholesterol levels in patients at risk of stroke.

Abstract no.: 4 Effect of statins on macrophage activation after platelet phagocytosis

Statins, through HMGCoA reductase inhibition, are potent inhibitors of cholesterol synthesis and are widely used in cardiovascular disease. Recent evidence suggests that the beneficial effects of statins extend beyond their action on serum cholesterol levels. The so called pleiotropic effects can be attributed to reduced production of isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). We investigated the effects of statins on macrophage activation after platelet phagocytosis. Platelet phagocytosis is known as an alternative mechanism for foam cell formation and macrophage activation in human atherosclerotic plaques. Processing of amyloid precursor protein (APP), present in platelets, to amyloid‐β (Aβ) might be responsible for induction of the inducible nitric oxide synthase (iNOS) in macrophages. It has been reported that statins can affect APP processing not only through reduction of cholesterol levels, but also via other mechanisms such as reduced isoprenylation of small GTP binding proteins. Interferon‐γ primed murine J774 macrophages were incubated with washed human blood platelets for 18 h with or without different statins. Nitrite was quantified in the supernatant as a measure for iNOS activity. Macrophage activation following platelet phagocytosis was reduced in the presence of fluvastatin and atorvastatin, but not with simvastatin and rosuvastatin. Besides 30 μM of simvastatin, there was no effect of statins on LPS‐stimulated macrophages, indicating that LPS activates macrophages via a different pathway than platelets. The effect of fluvastatin could be reversed by mevalonic acid, FPP and GGPP. Our results show that some statins decrease macrophage activation after platelet phagocytosis. This effect may be explained by interference with cholesterol synthesis or through reduced isoprenylation of small GTP binding proteins. Diminished macrophage activation in an atherosclerotic plaque can contribute to plaque stabilisation.

Diverse effects of statins on endothelial cells?

T he vascular endothelium is a dynamic interface, normally providing a non-adhesive, non-thrombogenic surface for blood constituents. However, in response to proinflammatory stimuli from cytokines and bacterial products, or in response to vascular wall shear stress, induction of adhesion molecules and the expression of endothelial cell (EC) surface procoagulant proteins such as tissue factor (TF) may occur. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins, are potent inhibitors of cholesterol synthesis in the mevalonate pathway. Clinical trials of statin therapy have demonstrated beneficial effects in primary and secondary prevention of coronary heart disease as well as ischemic stroke (1–6). Because mevalonate is a precursor of not only cholesterol, but also a variety of nonsteroidal isoprenoid compounds essential for normal cellular activity, the inhibition of HMGCoA reductase may have potential pleiotropic effects. Prenylation . . .

HMG-CoA reductase inhibitors inhibit inducible nitric oxide synthase gene expression in macrophages.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use in cardiovascular diseases. Recent evidence, however, suggests that the beneficial effects of statins may extend beyond their action on serum cholesterol levels. In this study, we investigated the effects of lovastatin, pravastatin, atorvastatin and fluvastatin on macrophage formation of nitric oxide (NO) in murine RAW 264.7 cells. Stimulation of macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in inducible NO synthase (iNOS) expression, which was accompanied by a large amount of NO formation. At concentrations of 0.1-30 microM, statins can inhibit stimuli-induced NO formation and iNOS induction to different extents. This inhibition occurs at the transcriptional level, and displays potency in the order of lovastatin > atorvastatin > fluvastatin >> pravastatin. We found that LPS-induced I kappa B kinase and nuclear factor-kappa B (NF-kappa B) activation, as well as IFN-gamma-induced signal transducer and activator of transcription 1 (STAT1) phosphorylation, were reduced by lovastatin. Moreover, inhibition by lovastatin of NO production and kappa B activation was reversed by mevalonate, geranylgeranyl pyrophosphate and farnesyl pyrophosphate. All these results suggest that inhibition of iNOS gene expression by statins can be attributed to interference with protein isoprenylation, which mediates both NF-kappa B and STAT1 activation in the upstream signaling pathways for iNOS gene transcription.

Statins and ischemic stroke.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors or statins are potent inhibitors of cholesterol synthesis. Several large clinical trials have demonstrated that these agents reduce serum cholesterol levels and the incidence of cardiovascular diseases. However, overlap and meta-analyses of these clinical trials suggest that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Because statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, they may have pleiotropic effects on the vascular wall. In particular, the ability of statins to decrease the incidence of ischemic stroke highlights some of their non-cholesterol effects since serum cholesterol levels are poorly correlated with the risk for ischemic stroke.

Dietary cholesterol absorption

In the early 1970s, Bhattacharyya and Connor studied two sisters who complained of arthralgias, and had tendon xanthomas and haemolytic anaemia. The doctors recognised the symptoms as those of autosomal dominant familial hypercholesterolaemia. However, neither girl had raised concentrations of blood cholesterol—a classic sign of the disease. Furthermore, neither of the sisters’ parents had the disorder, suggesting autosomal recessive inheritance. After assessing the girls, Bhattacharyya and Connor wrote a seminal paper, published in 1974, in which they identified the cause of the newly identified disorder as raised concentrations of plant sterols. And since sitosterol was the major plant sterol present in the sisters’ blood, they named the disease sitosterolaemia. The disorder is not well recognised by the general medical community, or by many lipid specialists. The true prevalence is thus unknown, although more than 50 families worldwide have been reported in medical publications. A standard cholesterol test cannot distinguish between cholesterol and non-cholesterol sterols, and a specialised test (gas-liquid or high performance liquid chromatography analyses) therefore has to be done to confirm the diagnosis. The disease is associated with an increased cardiovascular risk, as highlighted by the presentation some years ago of two men, not yet 20 years old, who had fatal heart attacks. Subsequent investigation of their families led to a diagnosis of sitosterolaemia among their relatives. Although a formal study of the prevalence of premature atherosclerotic disease has not been published, we have followed up more than 34 families with a history of sitosterolaemia, and more than half have documented atherosclerotic disease. The identification of sitosterolaemia has focused attention on the basic processes that govern how we normally absorb dietary cholesterol and keep all other sterols out. Our diets generally contain about equal amounts of cholesterol and plant sterols. Plants do not make cholesterol, but use molecules that are similar, such as sitosterol. Under normal circumstances, our bodies do not retain any of these non-cholesterol sterols and, on average, we absorb about 55% of dietary cholesterol. Thus, in healthy individuals, there exists an exquisite mechanism that allows the body to distinguish between sterols. The genetic defect in sitosterolaemia has been identified. Human beings have two genes located in a head-to-head configuration, a mutation in either of which results in sitosterolaemia. ABCG5 and ABCG8, and the proteins that they encode (sterolin-1 and sterolin-2, respectively) are highly homologous to each other and might have arisen through gene duplication during evolution. These genes are part of a larger ABC family of proteins, characterised by the presence of an ATP-binding cassette motif. Better known members of this family are the cystic fibrosis transconductance regulator, the sulphonylurea receptor, and the multidrug resistance proteins. Sterolins are expressed in the intestine and the liver only. Sterolin-1 and sterolin-2 work together to exclude non-cholesterol sterols from our bodies, probably by preventing initial entry at the intestinal level, and by rapidly excreting any residual amounts that enter the bloodstream, by rapid uptake by the liver and excretion into bile (figure). Findings of genetic studies also suggest that some of the mutations that cause sitosterolaemia might be old; analyses of the Amish-Mennonite community indicate that their mutation might have been brought from Europe to the Americas around the late 1700s. Genetic analyses of other families suggest that some mutations occurred more than 1800 years ago. Cholesterol, once synthesised or absorbed, can only be disposed of by the body via the bile system, either by direct excretion or by breakdown into bile acids. Enterohepatic circulation is therefore involved in the removal of cholesterol. Statin drugs (simvastatin, atorvastatin, &c) are potent inhibitors of cholesterol synthesis. A more effective therapy for reducing raised concentrations of cholesterol might also affect loss of cholesterol. Although not proven, sterolins in the liver could be responsible for the direct excretion of cholesterol into bile. Cholesterol is an important molecule for normal embryonic development, but can also be a potent risk factor for heart disease in later life. A greater understanding of the mechanisms involved in its metabolism would not only result in treatment options for sitosterolaemia, but would also lead to a greater understanding of the processes of cholesterol absorption and excretion in the general population. Dietary sterol absorption and the sites of defects in sitosterolaemia

Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymes.

Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered.

7 alpha-hydroxylation of 27-hydroxycholesterol: biologic role in the regulation of cholesterol synthesis

The report of a novel cytochrome P450 enzyme in mouse hippocampus (cyp7b) with close homology to cholesterol 7 alpha-hydroxylase led us to determine the substrate specificity with respect to 27-hydroxycholesterol, known to be a potent inhibitor of cholesterol synthesis. Transfection of 293/T-cells with PcDNA3.1(+)-mcyp7b was followed by metabolism of 2.5 microM 27-hydroxycholesterol to the 7 alpha-hydroxy intermediate, cholest-5-ene,3 beta,7 alpha,27-triol, with complete loss of down-regulation of cholesterol synthesis. Addition of 5 microM and 10 microM concentrations of the triol to HepG2 and CHO cells, respectively, also did not reduce cholesterol synthesis. The contrast between the biologic effect on cholesterol synthesis by these two C27 hydroxysterols and the wide tissue distribution of both cholesterol 27-hydroxylase and hydroxysterol 7 alpha-hydroxylase implies local regulatory effects prior to their further catabolism in the liver to chenodeoxycholic and cholic acids.

EFFECT OF THE NEW HMG-CoA REDUCTASE INHIBITOR CERIVASTATIN (BAY W 6228) ON MIGRATION, PROLIFERATION AND CHOLESTEROL SYNTHESIS IN ARTERIAL MYOCYTES

The major relation existing between cell growth, migration and cholesterol homeostasis prompted us to investigate the effect of the new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin (BAY W 6228) on these cellular events. The molecule inhibited in a dose-dependent manner the migration and the replication (evaluated as cell number and nuclear incorporation of 3H-thymidine) of rat arterial SMC with IC 50values of 2.7μ Mand 0.5μ M, respectively. Among the tested statins BAY W 6228 resulted to be the most potent inhibitor of cholesterol synthesis and cell proliferation. Conditions producing 80–90% inhibition of cholesterol synthesis correlate with approximately 50% inhibition of cell growth. Similar results were obtained in SMC from human femoral artery. The in vitroinhibition of cell migration and proliferation induced by BAY W 6228 (80% decrease) was completely prevented by the addition of mevalonate and partially prevented (60–80%) by farnesol and geranylgeraniol, confirming the specific role of isoprenoid metabolites— probably through a prenylated protein(s)—in regulating these cellular events. The present results provide evidence that BAY W 6228 interferes, at least in vivo, with smooth muscle cells migration and proliferation, major processes involved in atherogenesis.

The Lovastatin-Treated Rodent: A New Model of Barrier Disruption and Epidermal Hyperplasia

Recent studies have linked epidermal cholesterol synthesis with maintenance of the permeability barrier. To assess directly the importance of cholesterol synthesis, we applied lovastatin, a potent inhibitor of cholesterol synthesis, to hairless mouse skin. Transepidermal water loss (TEWL) began to increase after four to six daily applications. Co-application of cholesterol blocked the expected increase in TEWL, demonstrating the importance of cholesterol for development of the lesion. The histology of lovastatin-treated skin revealed epidermal hyperplasia, accompanied by accelerated DNA synthesis. Whereas cholesterol synthesis initially was reduced in lovastatin-treated epidermis, with further treatment cholesterol synthesis normalized, while fatty acid synthesis accelerated greatly. Although the total free sterol content of lovastatin-treated epidermis remained normal, the fatty acid content increased coincident with barrier disruption. Finally, morphologic abnormalities of both lamellar body structure and their deposited, intercellular contents occurred coincident with the emerging biochemical abnormalities. Thus, the abnormal barrier function in this model can be ascribed to an initial inhibition of epidermal sterol synthesis followed by an alteration in cholesterol and fatty acid synthesis, leading to an imbalance in stratum corneum lipid composition and abnormal membrane bilayer structure.

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.

Effects of a competitive inhibitor (mevinolin) of 3-hydroxy-3-methylglutaryl coenzyme A reductase on human and bovine endothelial cells, fibroblasts and smooth muscle cells in vitro.

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, is a potent inhibitor of cholesterol synthesis. We have tested the effects of mevinolin on cell replication (3H-thymidine incorporation), prostacyclin production (6-keto-PGF1 alpha) and cell death (51Cr release) in cell cultures (human umbilical vein endothelial cells, bovine endothelial cells, human fibroblasts and bovine smooth muscle cells). Mevinolin concentrations ranging from 0.05 mumol/l (reported therapeutic concentration) to 20 mumol/l were used. In human endothelial cells the replication was reduced by 11% at a concentration of 2.0 mumol/l (P less than 0.01). In fibroblasts and smooth muscle cells the reduction was significant already at 0.1 mumol/l (10%, P less than 0.01). The prostacyclin production was reduced in endothelial cells at 1.0 mumol/l (19%, P less than 0.01) and in smooth muscle cells at 2.0 mumol/l (15%, P less than 0.05). At 20 mumol/l both cell replication and prostacyclin production was markedly reduced by about 40% in all cell types. No effects on 51Cr release or trypan blue staining was seen at any concentration. It is concluded that mevinolin has an effect on DNA synthesis and prostacyclin production on the tested cell types in vitro. These effects were, however, observed only at concentrations higher than those recommended for therapeutical use.

Inhibitors of sterol synthesis. Metabolism of [2,4-3H]5 alpha-cholest-8(14-)-en-3 beta-ol-15-one after intravenous administration to a nonhuman primate.

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent inhibitor of cholesterol synthesis with marked hypocholesterolemic activity, has been studied after the intravenous administration of a mixture of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one and [4-14C] cholesterol to a baboon. The levels of 3H in plasma which was associated with the free 15-ketosterol decreased very rapidly (T1/2 approximately 9 min) after injection of the labeled sterol. By 4 h, the level of the [3H]15-ketosterol in plasma was negligible. The rapid decrease in the levels of the free 15-ketosterol was associated with rapid formation of fatty acid esters of the 15-ketosterol. The maximum level of 3H-labeled 15-ketosteryl esters was observed at 20 min after the injection of the 15-ketosterol. Thereafter, the levels of the 15-ketosteryl esters decreased rapidly with an apparent T1/2 of approximately 3.5-4.0 h. The results also indicated rapid formation of 3H-labeled cholesterol and cholesteryl esters. Substantial formation of [3H]cholesterol was observed at 20 min after the injection of the 15-ketosterol and reached a maximum level in plasma at 2 h. The maximum levels of [3H]cholesteryl esters in plasma were observed much later. These and other findings indicated that the observed slow clearance of total 3H from plasma is a consequence of metabolism of the 15-ketosterol to cholesterol and cholesteryl esters, normal constituents of plasma whose turnover in the whole animal is known to be relatively slow.