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Integrins exhibit reversible changes in their ability to bind ligands and these changes enable transient cell adhesion. We recently showed that leukocyte integrin CR3 (complement receptor type three, CD11b/CD18, alpha m beta 2) may be purified in a form that is either capable or incapable of binding soluble, monomeric ligand and that "inactive" CR3 may be rendered capable of binding ligand by addition of an anti-CR3 mAb known as KIM-127 (Cai and Wright, JBC. 270: 14358, 1995). Here, we demonstrate that active CR3 may be rendered inactive by treatment of immobilized receptor with EDTA. EDTA-treated CR3 failed to bind ligand even in the presence of mM Ca2+ and Mg2+, suggesting that EDTA-treatment caused a change in the receptor that is not readily reversed. EDTA-treated receptor did, however, bind ligand upon addition of KIM-127 plus Mg2+ with an affinity (17.8 +/- 4.5 nM) similar to untreated, active receptor (12.5 +/- 4.7 nM). EDTA-treated CR3 thus exhibits the properties of inactive CR3, in which the ligand binding site is cryptic but subject to exposure by KIM-127. A candidate for the cryptic ligand binding site is the I-domain, a Mg2+-binding region in the alpha chain of CR3. We found that monomeric C3bi binds directly to recombinant I-domain in a Mg(2+)-dependent fashion with an affinity of 300 +/- 113 nM. These results thus suggest that CR3 may be inactivated by removing tightly bound divalent cation from a cryptic site in CR3.

Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane G(i)-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D₂, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this "FFA hypothesis" and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin's lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin's lipid efficacy, challenging the long-standing FFA hypothesis.

The ability of HDL to remove cholesterol from macrophages in atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Indeed, cholesterol efflux from macrophages is a functional serum marker that has an even stronger inverse association with coronary heart disease than HDL cholesterol (Khera et al. 2011, NEJM 364;2;127). We recently described CSL112, a novel formulation of full length human apoA-I and phospholipids designed to resemble nascent HDL and to maximize cholesterol efflux from macrophages. Here we studied the effects of CSL112 vs HDL3 on ex vivo cholesterol efflux in human plasma and serum. Addition of HDL3 to pooled plasma significantly increased ABCA1-independent efflux but had little or no effect on ABCA1-dependent efflux. In contrast, the addition of CSL112 increased not only ABCA1-independent efflux but also strongly increased ABCA1-dependent efflux in a concentration dependent fashion. Through a range of concentrations, CSL112 was significantly more efficacious in global cholesterol efflux than was HDL3. For example, at 5 μg/mL, CSL112 increased global efflux by 152% ± 5.6% vs 66% ± 7.4% for HDL3. Additional studies examined serum samples from five subjects with a range of lipid phenotypes including low, normal and high levels of HDL. When spiked into these sera, CSL112 increased both ABCA1-dependent and independent efflux in all subjects, regardless of lipid profile. In all subjects, the relative increase in ABCA1-dependent efflux was larger than that observed with ABCA1-independent efflux indicating selectivity of the interaction of CSL112 with the ABCA1 transporter. We speculate that the disproportionate rise in ABCA1-dependent efflux with CSL112 may be explained by the observation that PreBeta1-HDL levels measured by ELISA increase dramatically upon incubation of CSL112 with human plasma. We conclude that CSL112 robustly enhances the capacity of human serum to promote cholesterol efflux; the enhancement appears independent of lipid phenotype, is greater than that observed with HDL3 and that the efflux derives disproportionately from the ABCA1 pathway.

The greatest risk for recurrent cardiovascular events lies in the first several weeks following ACS, and current therapies (e.g. statins) do not target cholesterol efflux from plaque in this time frame. ApoA-I is able to rapidly remove cholesterol from plaque, and cholesterol efflux from macrophages

Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. Recent studies have shown that experimentally elevating the levels of circulating high-density lipoproteins (HDL) provides protection against death in animal models of endotoxic shock. We sought to define the components of HDL that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. We report here that reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, are not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. In contrast with R-HDL, apoA-I containing lipoproteins (LpA-I) isolated from plasma by selected affinity immunosorption (SAIS) on an anti-apoA-I column, neutralized LPS without addition of exogenous LBP. Several lines of evidence demonstrated that LBP is a constituent of LpA-I in plasma. Passage of plasma over an anti-apoA-I column removed more than 99% of the LBP detectable by ELISA, whereas 31% of the LBP was recovered by elution of the column. Similarly, the ability of plasma to enable activation of neutrophils by LPS (LBP/Septin activity) was depleted and recovered by the same process. Furthermore, an immobilized anti-LBP monoclonal antibody coprecipitated apoA-I. The results described here suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.

The avidity of leukocyte integrin CR3 (Mac-1, CD11b/CD18, α m β 2 ) on neutrophils (PMN) may be rapidly modulated by several agonists. We describe a method for determining the avidity of these receptors by measuring the adhesion of PMN to fibrinogen-coated surfaces. Cells are loaded with a succinimidyl ester of carboxyfluorescein diacetate, which is deacetylated by intracellular esterases yielding a highly fluorescent carboxyfluorescein that remains trapped within the PMN. The number of cells adhering to fibrinogen-coated wells of Terasaki microplates is quantitated with a fluorescence plate reader. Stimulation of PMN with a number of agonists, including PMA, fNLLP, Ca-ionophore A23187, interleukin-8, tumor necrosis factor and lipopolysaccharide strongly increased adhesion to fibrinogen, which was CD11b/CD18 dependent. The extent of cell adhesion depended on stimulus concentration and incubation time. This assay requires little time, utilizes small numbers of cells and does not require hazardous reagents.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

Recently, there has been a strong interest in the ability to increase levels of high density lipoprotein-cholesterol (HDL-C). This interest stems from the hypothesis that such an elevation in HDL-C will decrease the likelihood of cardiovascular disease. Inhibition of cholesteryl ester transfer protein (CETP) has been shown to elevate HDL-C levels in human subjects. This letter describes the discovery of a novel and potent (

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

We have previously reported that lipopolysaccharide (LPS) binding protein (LBP) opsonizes endotoxin (LPS) for recognition by CD14 on phagocytes. Here we show that normal human plasma contains high titers of an activity that also binds LPS (Re, 595) and mediates recognition by CD14. Opsonization of LPS-coated particles with plasma enables the particles to be bound by phagocytes. Further, opsonization with plasma also enables subnanogram-per-milliliter concentrations of LPS to induce dramatic alterations in the function of leukocyte integrins on polymorphonuclear leukocytes and to induce secretion of tumor necrosis factor by monocytes, suggesting that opsonization by factors in plasma may be important in responses of cells to endotoxin. The opsonic activity in plasma appears distinct from LBP since it is not blocked by neutralizing antibodies against LBP. Surprisingly, the opsonic activity of plasma is not present in a single protein species, but at least two species must be combined to observe activity. Further, the opsonic activity of plasma for LPS is blocked by addition of protease inhibitors, suggesting that proteolytic activity or activities are required for opsonization. These properties are suggestive of the action of a protease cascade, but opsonic activity of plasma is not affected by blockade or depletion of either the complement or clotting cascades. We propose the name "septin" to describe this novel LPS-opsonizing activity in plasma.

The avidity of integrin CR3 (also known as α M β 2 , Mac-1, Mo-1, and CD11bCD18) may be reversibly altered without changes in the number of cell surface receptors. Here we describe a molecule termed integrin modulating factor (IMF-1), which controls CR3 avidity. Addition of IMF-1 to polymorphonuclear leukocytes (PMNs) or to purified CR3 causes enhanced binding of ligand. IMF-1 is not present in resting PMNs, but stimulation of cells results in a transient rise in IMF-1 content that parallels a transient rise in CR3 activity. We suggest that PMNs control adhesivity by controlling synthesis of IMF-1, which then acts as an allosteric activator of leukocyte integrins. IMF-1 is an acidic, amphiphilic molecule of M r 340 ± 16 that does not contain ester, phosphate, amide, sialic acid, or glycosidic or vicinal hydroxyl functionalities, but does contain a carbon-carbon double bond. These results suggest that IMF-1 is an unsaturated fatty acid or an isoprenoid acid.

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure-activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC(50) of 28nM.

The lipid A domain of lipopolysaccharide (LPS) is a unique, glucosamine-based phospholipid that makes up the outer monolayer of the outer membrane of most gram-negative bacteria. Because of its profound pharmacological effects on animal cells, especially those of the immune system, lipid A is also known as endotoxin. Despite decades of earlier work, the precise chemistry of endotoxins and the biochemical pathways for their enzymatic synthesis have been elucidated only within the past 5 years. In this review, we summarize the essentials of endotoxin biochemistry and also present recent experiments aimed at identifying surface receptors, signal-transducing elements, transcriptional factors, and key intracellular targets involved in the response of animal cells to endotoxins.

Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.

Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.

During the course of a previous investigation, we noticed that the uptake of liposomes by human polymorphonuclear neutrophils (PMNs) was significantly lower in the presence of heat-inactivated serum compared to that in intact whole serum (Scieszka et al., Pharm. Res. 5:352, 1988). This observation suggested the participation of heat-labile complement components in the phagocytic process. In this report we conclude that complement C3bi is the component responsible for opsonization of the liposome surface. Phagocytosis was not supported by C3-deficient serum, and phagocytosis in whole serum was blocked by the antibody to the receptor for C3bi (CR3) but not by the antibody to the receptor for C3b (CR1). We also found that with C5-deflcient serum the level of uptake was minimal but slightly higher than without any serum. When exogenous C5a was added along with C5-deficient serum, uptake levels similar in magnitude to those observed with intact serum were obtained. We conclude that C5a enhances phagocytosis of opsonized liposomes by activating the phagocytic capacity of CR3 on the PMN.

Improved methods for non-invasive in vivo assessment are needed to guide development of animal models of atherosclerosis and to evaluate target engagement and in vivo efficacy of new drugs. Using novel 3D-micro-ultrasound technology, we developed and validated a novel protocol for 3D acquisition and analysis of imaging to follow lesion progression in atherosclerotic mice. The carotid arteries of ApoE receptor knockout mice and normal control mice were imaged within the proximal 2 mm from the aortic branch point. Plaque volume along that length was quantified using a semi-automated 3D segmentation algorithm. Volumes derived by this method were compared to those calculated using 3-D histology post-mortem. Bland-Altman comparison revealed close correlation between these two measures of plaque volume. Furthermore, using a segmentation technique that captures early positive and 33 week negative remodeling, we found evidence that plaque volume increases linearly over time. Each animal and each plaque served as its own control, allowing accurate comparison. The high fidelity anatomical registration of this protocol provides increased spatial resolution and therefore greater sensitivity for measurement of plaque wall size, an advance over 2-dimensional measures of intimal-medial-thickening. Further, 3-dimensional analysis ensures a point of registration that captures functional markers in addition to the standard structural markers that characterize experimental atherosclerosis. In conclusion, this novel imaging protocol provides a non-invasive, accurate surrogate marker for experimental atherosclerosis over the life of the entire lesion.

4-Methyl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They were active in vitro and in an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). These were active both in vitro and in an in vivo mouse pharmacodynamic (PD) model. Fluorine substitution of the cyclobutane ring improved the pharmacokinetic profile significantly. The synthesis and structure–activity relationships are presented.

Bacterial lipopolysaccharide (LPS) is a potent stimulus of cells, yet a target protein for LPS has not been defined. We used two approaches to define LPS-binding sites on cell surfaces: one assay measured binding of LPS-coated erythrocytes (ELPS) to cultured human cells, and a second measured binding of a radiolabeled probe, [32P]lipid IVA, to intact leukocytes. The first approach identified the CD11-CD18 family of integrins as lipid A-binding sites in human phagocytes, and the latter approach demonstrated saturable lipid A binding to intact murine macrophages, as well as to an approximately 95-kDa binding protein in purified membrane preparations. Because CD18 has a known molecular mass of 95 kDa, we sought to determine whether the [32P]lipid IVA-binding site was CD18. Binding of ELPS and [32P]lipid IVA to human macrophages was found to differ with respect to temperature, divalent cation dependence, cellular specificity, and susceptibility to competition by polyanions. To determine whether the previously described 95-kDa lipid A-binding protein was CD18, nitrocellulose-immobilized RAW264.7 membrane proteins were probed with [32P]lipid IVA and subsequently immunoblotted with a monoclonal antibody to murine CD18. The lipid A-binding protein has an electrophoretic mobility slightly different from that of CD18. Moreover, monoclonal antibodies and polyclonal antiserum to the CD11-CD18 family of proteins did not inhibit lipid IVA binding to intact human macrophages. Finally, mononuclear cells from two patients with CD18 deficiency failed to form rosettes with ELPS but bound [32P]lipid IVA normally. Thus, different LPS preparations may bind to cells in a CD18-dependent or -independent manner. Since ELPS is particulate and lipid IVA is a fine dispersion, the identity of the binding site may depend on the physical state of the LPS.

Adherence of leukocytes to the endothelium is a prerequisite for infiltration and accumulation of cells at an inflammatory site. Recent studies suggest that the CD11/CD18 family of adhesion-promoting receptors plays a crucial role in the initial adherence of polymorphonuclear leukocytes (PMNs) to endothelium. We have studied the effect of the anti-CD18 monoclonal antibody (MoAb) IB4, on movement of PMN in rabbits. Accumulation of PMNs in the skin induced by a local injection of the chemoattractant, zymosan-activated serum (ZAS), was strongly inhibited, in a dose-dependent fashion, by intravenous injection of IB4. A greater than 95% reduction in PMN accumulation was seen with 1 mg IB4/kg body weight, the highest dose used. PMN-dependent plasma leakage in the ZAS-injected skin sites was also inhibited by pretreatment with MoAb IB4, with a similar dose dependence. Histamine- induced plasma leakage, which is PMN independent, was not affected by this treatment. F(ab)2 fragments of IB4 were as effective as the whole immunoglobulin G molecule in reducing PMN accumulation. The half-life of circulating IB4 in rabbits was found to be 11.5 hours. These results are consistent with in vitro studies that show that binding of PMNs to endothelium requires both expression of CD11/CD18 molecules and activation of the PMNs by agonists, and confirm that sites on CD11/CD18 that recognize endothelial cells are blocked by IB4. Other investigators have shown that injection of chemoattractants into the blood stream causes a rapid neutropenia associated with accumulation of PMNs in the lung. We find that intravenous treatment of animals with IB4 did not block the transient accumulation of PMNs in the lung induced by formyl-methionyl-leucyl-phenylalanine, suggesting that this accumulation occurs by a mechanism that does not require CD11/CD18 molecules.

During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 (alpha M beta 2, CD11b/CD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097-1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. Recent studies have shown that PCSK9 directly binds the LDL receptor (LDLR) and that addition of PCSK9 to cells promotes degradation of LDLR. Mutations associated with hypercholesterolemia (S127R and D374Y) bind the LDLR stronger and are more potent in decreasing LDL-uptake. Based on these data, we sought to better understand the mechanism by which mutations at both the S127 and D374 residues of PCSK9 affect PCSK9 function. A limited vertical scanning mutagenesis was done, resulting in the following mutants: S127(R, A, D, K, L, T) and D374(Y, A, E, F, K, L). Expression of several S127 alleles in HEK293 cells resulted in less efficient processing of PCSK9, with S127L severely defective in processing. In contrast, all D374 alleles tested had no effect on PCSK9 processing. In a cell-based functional assay of PCSK9 activity, both S127R and S127K proteins were significantly more potent (2– 4 fold) in decreasing LDL-uptake than wild-type PCSK9, while all other S127 mutants were similar to wild-type. Each D374 mutant tested was more potent than wild-type PCSK9 in reducing LDL-uptake (D374Y, F > A, L > E, K > wild-type). To determine if the potencies of S127 and D374 mutations in lowering LDL-uptake correlated with their ability to interact with the LDLR, each mutant was tested in a PCSK9-LDLR time-resolved FRET assay. Results indicate that potency in the cell-based LDL-uptake assay correlates with PCSK9’s binding affinity for the LDLR. Combination of S127R and D374Y was also found to have an additive effect in enhancing PCSK9’s ability to reduce LDL-uptake. Molecular modeling indicates that mutations at S127 and D374 which enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at S127 and D374 residues modulate PCSK9’s ability to regulate LDLR function through distinct mechanisms.

Nicotinic acid remains the most effective therapeutic agent for the treatment and prevention of atherosclerosis resulting from low high density lipoprotein cholesterol. The therapeutic actions of nicotinic acid are mediated by GPR109A, a Gi protein-coupled receptor, expressed primarily on adipocytes, Langerhans cells, and macrophage. Unfortunately, a severe, cutaneous flushing side effect limits its use and patient compliance. The mechanism of high density lipoprotein elevation is not clearly established but assumed to be influenced by an inhibition of lipolysis in the adipose. The flushing side effect appears to be mediated by the release of prostaglandin D2 from Langerhans cells in the skin. We hypothesized that the signal transduction pathways mediating the anti-lipolytic and prostaglandin D2/flushing pathways are distinct and that agonists may be identified that are capable of selectively eliciting the therapeutic, anti-lipolytic pathway while avoiding the activation of the parallel flush-inducing pathway. We have identified a number of GPR109A pyrazole agonists that are capable of fully inhibiting lipolysis in vitro and in vivo and not only fail to elicit a flushing response but can antagonize the ability of nicotinic acid to elicit a flush response in vivo. In contrast to flushing agonists, exposure of cells expressing GPR109A to the non-flushing agonists fails to induce internalization of the receptor or to activate ERK 1/2 mitogen-activated protein kinase phosphorylation.

Beginning with a moderately potent PPARγ agonist 9, a series of potent and highly subtype-selective PPARα agonists was identified through a systematic SAR study. Based on the results of the efficacy studies in the hamster and dog models of dyslipidemia and the desired pharmacokinetic data, the optimized compound 39 was selected for further profiling.

Adamantyl triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They are active both in in vitro and in in vivo pharmacodynamic models. The synthesis and structure-activity relationships of these inhibitors are presented.

Diabetes mellitus is one of the major risk factors associated with atherosclerosis and coronary heart disease but the mechanistic links between the disease and atherosclerosis are not well understood. In this study, we investigated the effect of the deletion of the long-form leptin receptor on the progression of atherosclerosis in ApoE-/- mouse. ApoE-/-;db/db double knockout mice as well as ApoE-/-;db/+ and ApoE-/- littermates were generated by crossing ApoE-/- and db/+ mice. On a regular chow diet, ApoE-/-;db/db mice at 20 weeks of age exhibited features typical of type 2 diabetes: obesity, hyperglycemia, hyperinsulinemia and dyslipidemia and had significantly accelerated atherosclerosis compared with their age-matched ApoE-/- littermates as assessed by either the percentage of the aorta bearing lesion (5.3+/-0.9% for ApoE-/-;db/db versus 1.5+/-0.5% for ApoE-/-) or by aortic lipid content ( approximately 1.5-2-fold increase in free cholesterol and approximately 3-4-fold increase in cholesteryl ester). The atherosclerosis in these ApoE-/-;db/db mice was further accelerated by feeding mice with a Western diet and markedly inhibited by fenofibrate with a 2.5- and 5.3-fold reduction of the lesion in male and female mice, respectively. The results from this study demonstrate that type 2 diabetes can accelerate atherogenesis in mice. This mouse model may provide insight into the mechanistic link between type 2 diabetes and atherosclerosis as well as serve as a valuable tool for evaluating therapeutics.

Polyunsaturated fatty acids (PUFAs) have been previously reported as agonists of peroxisome proliferatoractivated receptor and antagonists of the liver X receptor. The activities on these two nuclear receptors have been attributed to their beneficial effects such as improvement of dyslipidemia and insulin sensitivity and decrease of hepatic lipogenesis. Here we report that PUFAs are ligands of farnesoid X receptor (FXR), a nuclear receptor for bile acids. In a conventional FXR binding assay, arachidonic acid (AA, 20:4), docosahexaenoic acid (DA, 22:6), and linolenic acid (LA, 18:3) had an affinity of 2.6, 1.5, and 3.5 microM, respectively. In a cell-free coactivator association assay, AA, DA, and LA decreased FXR agonist-induced FXR activation with IC(50)s ranging from 0.9 to 4.7 microM. In HepG2 cells, PUFAs regulated the expression of two FXR targets, BSEP and kininogen, in an opposite fashion, although both genes were transactivated by FXR. All three PUFAs dose-dependently enhanced FXR agonist-induced BSEP expression but decreased FXR agonist-induced human kininogen mRNA. Saturated fatty acids such as stearic acid (SA, 18:0) and palmitic acid (PA, 16:0) did not bind to FXR and did not change BSEP or kininogen expression. The pattern of BSEP and kininogen regulation by PUFAs is closely similar to that of the guggulsterone, previously reported as a selective bile acid receptor modulator. Our results suggest that PUFAs may belong to the same class of FXR ligands as guggulsterone, and that the selective regulation of FXR targets may contribute to the beneficial effects of PUFAs in lipid metabolism.

Here, we characterize the actions of MK-0767, a dual ligand of the nuclear receptors peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma. In cell-based assays, MK-0767 produced potent activation of human PPARgamma and PPARalpha with a gamma:alpha potency ratio of approximately 2. The dual agonist induced high affinity interactions of PPARalpha and PPARgamma with the transcriptional coactivator CBP in vitro. In ob/ob mice, MK-0767 normalized hyperglycemia and hyperinsulinemia with equal or greater potency and efficacy than pioglitazone. Treatment of hamsters with MK-0767 produced substantial reductions in blood cholesterol and triglycerides. In dogs, MK-0767 reduced serum cholesterol levels with a potency more than 10-fold greater than simvastatin. The efficacies of MK-0767 and simvastatin were additive when given together. We conclude that MK-0767 is a potent dual PPARalpha/gamma agonist with robust insulin sensitizing and hypolipidemic activities.

Background — Statins exert antiinflammatory and antiproliferative actions independent of cholesterol lowering. To determine whether these actions might affect neointimal formation, we investigated the effect of simvastatin on the response to experimental angioplasty in LDL receptor–deficient (LDLR −/− ) mice, a model of hypercholesterolemia in which changes in plasma lipids are not observed in response to simvastatin. Methods and Results — Carotid artery dilation (2.5 atm) and complete endothelial denudation were performed in male C57BL/6J LDLR −/− mice treated with low-dose (2 mg/kg) or high-dose (20 mg/kg) simvastatin or vehicle subcutaneously 72 hours before and then daily after injury. After 7 and 28 days, intimal and medial sizes were measured and the intima to media area ratio (I:M) was calculated. Total plasma cholesterol and triglyceride levels were similar in simvastatin- and vehicle-treated mice. Intimal thickening and I:M were reduced significantly by low- and high-dose simvastatin compared with vehicle alone. Simvastatin treatment was associated with reduced cellular proliferation (BrdU), leukocyte accumulation (CD45), and platelet-derived growth factor–induced phosphorylation of the survival factor Akt and increased apoptosis after injury. Conclusions — Simvastatin modulates vascular repair after injury in the absence of lipid-lowering effects. Although the mechanisms are not yet established, additional research may lead to new understanding of the actions of statins and novel therapeutic interventions for preventing restenosis.

The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.

Secretory non-pancreatic phospholipase A(2) (sPLA(2)) has been implicated in inflammation and has been found in human atherosclerotic lesions. To test the effect of sPLA(2) deficiency on atherosclerosis, C57BL/Ks mice (apoE(+/+) and PLA(2)(++) were bred with C57BL/6 apoE knockout mice which are sPLA(2)(--) due to a spontaneous mutation. Sibling pairs of mice (apoE(--)/sPLA(2)(++) and apoE(--)/sPLA(2)(--)) on high fat Western diets were dissected at 22 weeks. In vitro enzyme assays confirmed higher serum sPLA(2) activity in the sPLA(2)(++) compared to sPLA(2)(--) for both sexes, while sPLA(2)(--) males had slightly higher serum cholesterol and phospholipids. Analysis of lipoprotein profiles by FPLC showed no effect of sPLA(2) genotype on any measured parameters. Atherosclerosis was quantitated by assaying cholesterol in aortic extracts. Male sPLA(2) trended slightly higher than sPLA(2)(++) with no statistical significance. Female sPLA(2)(++) and sPLA(2)(--) mice showed no significant differences in any of the measured parameters. These results suggest that the endogenous mouse sPLA(2) gene does not significantly affect HDL or atherosclerosis in mice.

Two ATP-binding cassette transporter proteins, ABCA1 and ABCG1, may mediate an active efflux of cellular cholesterol and phospholipids. They are ubiquitously expressed and are subject to regulation by cholesterol loading or by treatment with agents that activate the nuclear hormone receptor LXR. Earlier studies in both primates and non-primates reported that treatment with endotoxin (bacterial lipopolysaccharide, LPS) reduces plasma levels of HDL cholesterol. To determine if such HDL reduction correlates with a change in ABCA1 or ABCG1 expression, their expressions were measured in THP-1 monocytes and mice treated with LPS. LPS treatment leads to a rapid, dose-dependent increase of ABCA1 but not ABCG1 mRNA expression. Analysis of mouse livers showed that LPS treatment decreases expression of CYP7A, another target gene of LXR. When THP-1 cells were transfected with the ABCA1 promoter construct (−928 to +101 bp), promoter activity was significantly increased by treatment of 22(R)-hydroxycholesterol but not by LPS. Together, these studies show that LPS regulates ABCA1 expression through an LXR-independent mechanism. Further studies showed that treatment with Rhodobacter sphaeroiders LPS, an LPS antagonist, or PD169316, a specific p38 MAP kinase inhibitor, prevented the induction of ABCA1 by LPS. Therefore, this suggests that both transport of LPS from the plasma membrane to an intracellular site and activation of p38 MAP kinase are involved in the LPS-mediated induction of ABCA1.

A rapid in vitro assay was developed for monitoring protein-mediated cholesterol monomerization from bile acid aggregates. This assay uses a fluorescent cholesterol analog, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3 beta-ol (NBD-cholesterol), which was shown to be absorbed by hamster in a fashion similar to cholesterol. The fluorescence of aggregates of NBD-cholesterol was strongly quenched in 2.5 mM of taurocholic acid. Addition of proteins from enterocytes of hamster small intestine led to a time- and dose-dependent dequenching of NBD-cholesterol fluorescence. Comparable dequenching can be detected with SDS and appears to involve monomerization of the NBD-cholesterol. Purification of enterocyte extract by sequential chromatography revealed an approximately 140-kDa protein complex (p140) able to mediate the monomerization of NBD-cholesterol. Each p140 complex mediated monomerization of 2.7 NBD-cholesterol molecules. The p140 complex appeared to be formed by dimerization of two approximately 58-kDa molecules since SDS-PAGE revealed a single dominant band at 58 kDa (p58). Protein sequence analysis suggested that p58 is protein-disulfide isomerase (PDI), and this conclusion was confirmed by cloning of hamster PDI, and detection of PDI enzyme activity in the purified fraction. Additional studies with either pure PDI or lysates of cells transfected with hamster PDI showed that PDI by itself was not sufficient for monomerizing cholesterol. Further, despite a similar mobility on SDS-PAGE (approximately 58 kDa), the p140 complex appeared approximately 45-kDa larger than pure PDI (approximately 95 kDa) when analyzed by a gel-filtration chromatography. The p140 complex may thus contain an unidentified molecule(s) in addition to PDI that may contribute importantly to cholesterol monomerization.

The LXR nuclear receptors are intracellular sensors of cholesterol excess and are activated by various oxysterols. LXRs have been shown to regulate multiple genes of lipid metabolism, including ABCA1 (formerly known as ABC1). ABCA1 is a lipid pump that effluxes cholesterol and phospholipid out of cells. ABCA1 deficiency causes extremely low high density lipoprotein (HDL) levels, demonstrating the importance of ABCA1 in the formation of HDL. The present work shows that the acetyl-podocarpic dimer (APD) is a potent, selective agonist for both LXRalpha (NR1H3) and LXRbeta (NR1H2). In transient transactivation assays, APD was approximately 1000-fold more potent, and yielded approximately 6-fold greater maximal stimulation, than the widely used LXR agonist 22-(R)-hydroxycholesterol. APD induced ABCA1 mRNA levels, and increased efflux of both cholesterol and phospholipid, from multiple cell types. Gas chromatography-mass spectrometry measurements demonstrated that APD stimulated efflux of endogenous cholesterol, eliminating any possible artifacts of cholesterol labeling. For both mRNA induction and stimulation of cholesterol efflux, APD was found to be more effective than was cholesterol loading. Taken together, these data show that APD is a more effective LXR agonist than endogenous oxysterols. LXR agonists may therefore be useful for the prevention and treatment of atherosclerosis, especially in the context of low HDL levels.

Fenofibrate is a potent hypolipidemic agent that lowers plasma lipid levels and may thus decrease the incidence of atherosclerosis. Here we investigated the molecular mechanism of fenofibrate’s hypolipidemic action by characterizing its in vivo effects on the expression of mRNAs and the activities of pivotal enzymes in cholesterol and triglyceride metabolism in the hamster. Treatment of hamsters with fenofibrate led to a dose-dependent reduction in serum cholesterol concentrations. Studies on the incorporation of [14C]acetate and [14C]mevalonate into cholesterol suggested that this effect occurs primarily through inhibition of cholesterol biosynthesis at steps prior to mevalonate. Fenofibrate decreased levels of hepatic enzyme activities and mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase. A potential mechanism for transcriptional regulation of these enzymes is via SREBP-2 that we found to be suppressed 2-fold by fenofibrate. Fenofibrate also lowered circulatory triglyceride levels. In keeping with the effect, we observed strong suppression of fatty acid synthase, acetyl-CoA carboxylase and apolipoprotein C-III mRNA and stimulation of lipoprotein lipase and acyl-CoA oxidase mRNA in the liver of fenofibrate-treated hamsters. These observations suggest that the effect of fenofibrate on triglyceride metabolism is likely to be a result of both decreased fatty acid synthesis and increased lipoprotein lipase and acyl-CoA oxidase gene expression in the liver. Surprisingly, alterations in lipoprotein lipase, acyl-CoA oxidase, acetyl-CoA carboxylase, and apolipoprotein C-III could not be observed in hamster hepatocytes incubated with fenofibric acid in vitro. These observations raise the possibility that changes in these genes may be secondary to the metabolic alterations occurring in animals but not in cultured cells and thus that the effect of fenofibrate on these genes may be indirect.

ATP-binding cassette transporter A1 (ABCA1) mediates an active efflux of cholesterol and phospholipids and is mutated in patients with Tangier disease. Expression of ABCA1 may be increased by certain oxysterols such as 22(R)-hydroxycholesterol via activation of the nuclear hormone receptor liver X receptor (LXR). In searching for potential modulators of ABCA1 expression, we have studied the effects of various mevalonate metabolites on the expression of ABCA1 in two human cell lines, THP-1 and Caco-2 cells. Most of the tested metabolites, including mevalonate, geranyl pyrophosphate, farnesyl pyrophosphate, and ubiquinone, failed to significantly change the expression levels of ABCA1. However, treatment with geranylgeranyl pyrophosphate resulted in a dose- and time-dependent reduction of ABCA1 expression. Geranylgeranyl pyrophosphate appears to reduce ABCA1 expression via two different mechanisms. One of these mechanisms is by acting directly as an antagonist of LXR since it reduces the interaction between LXR alpha or -beta with nuclear coactivator SRC-1. Another mechanism appears to involve activation of the Rho GTP-binding proteins since treatment of Caco-2 cells with inhibitors of geranylgeranyl transferase or the Rho proteins significantly increased the expression and promoter activity of ABCA1. Further studies showed that mutations in the DR4 element of the ABCA1 promoter completely eliminate the inducible activities of these inhibitors. These data indicate that activation of the Rho proteins may change the activation status of LXR.

Dyslipidemia, a major risk factor for cardiovascular disease, may be directly linked to diabetic hyperglycemia and insulin resistance. An appropriate dyslipidemic animal model that has diabetes would provide an important tool for research on the treatment of diabetic dyslipidemia. Ten days of high fat feeding in golden Syrian hamsters resulted in a significant increase in insulin resistance and baseline serum lipid levels accompanied by a pronounced dyslipidemia. Thirteen days of treatment with fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) selective agonist, produced a dose-dependent decrease in serum lipid levels. The pattern observed was characterized by lowered very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) and raised high-density lipoprotein (HDL) cholesterol in a fashion similar to that seen in man. Diabetic conditions were also significantly improved by fenofibrate with a normalization of impaired glucose tolerance and an improvement of insulin sensitivity during an oral glucose tolerance test. These data suggest that fenofibrate may correct not only the dyslipidemia but also the insulin resistance caused by a high fat diet, and the high fat fed hamster may be a good animal model for research on the treatment of diabetic dyslipidemia with PPARalpha selective agonists.

The nuclear receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1, ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload.

Previous studies have suggested that the terminal complex of complement may contribute to the pathogenesis of atherosclerosis. C5b-9 complexes colocalize with the extracellular lipid in the aortic intima of hypercholesterolemic rabbits, and C6-deficient rabbits develop less atherosclerosis than controls. To test the role of complement in atherosclerosis in a different animal model, C5 deficient (C5def) mice were cross-bred with atherosclerosis susceptible apoE(-/-) mice, generating mice deficient in both apoE and C5 and control apoE(-/-) mice. Progeny were typed for C5 titer and serum cholesterol levels. Both male and female mice were fed a high fat diet from weaning until 22 weeks of age. At that time there were no significant differences in plasma cholesterol or triglycerides between apoE(-/-) control and apoE(-/-)/C5def groups. Morphometric analysis of the aortic root lesions gave mean (+/-SEM) lesion areas for male apoE(-/-) and apoE(-/-)/C5def mice of 468,176 +/- 21,982 and 375,182 +/- 53,089 microm(2), respectively (n = 10 each, P value = 0.123). In female apoE(-/-) mice (n = 5), the mean lesion area was 591,981 +/- 53,242 microm(2), compared to 618,578 +/- 83,457 microm(2) for female apoE(-/-)/C5def mice (n = 10) (P value = 0.835). Thus neither male nor female mice showed a significant change in lesion area when C5 was not present. In contrast to the case in the hypercholesterolemic rabbit, activation of the terminal complex of complement does not play a major role in the development of atherosclerosis in apoE(-/-) mice.

11β-hydroxysteroid dehydrogenases (11β-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11β-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11β-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11β-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor γ, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11β-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11β-HSD1 activity by up to 10-fold. IFN-γ, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11β-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11β-HSD1 and low levels of 11β-HSD2. The expression of 11β-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11β-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes catalyze the interconversion of active glucocorticoids (GC) with their inert metabolites, thereby regulating the functional activity of GC. While 11beta-HSD type 1 (11beta-HSD1) activates GC from their 11-keto metabolites, 11beta-HSD type 2 (11beta-HSD2) inactivates GC. Here we report that both of these enzymes are expressed in human aortic smooth muscle cells (SMC), and that 11beta-HSD1 is more abundant and is differentially regulated relative to 11beta-HSD2. Stimulation of SMC with IL-1beta or TNFalpha led to a time- and dose-dependent increase of mRNA levels for 11beta-HSD1, while 11beta-HSD2 mRNA levels decreased. Parallel enzyme activity studies showed increased conversion of 3H-cortisone to 3H-cortisol but not 3H-cortisol to 3H-cortisone, demonstrating 11beta-HSD1 in SMC acts primarily as a reductase. A similar increase of 11beta-HSD1 mRNA expression was also found in human bronchial SMC upon stimulation, indicating the regulatory effect is not limited to vascular smooth muscle. Additional parallel studies revealed a similar pattern of induction for 11beta-HSD1 and monocyte chemoattractant protein-1, a well-defined proinflammatory molecule. These data suggest 11beta-HSD1 may play an important role in regulating inflammatory responses in the artery wall and lung.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been shown to play an important role in the regulation of expression of a subclass of adipocyte genes and to serve as the molecular target of the thiazolidinedione (TZD) and certain non-TZD antidiabetic agents. Hypercorticosteroidism leads to insulin resistance, a variety of metabolic dysfunctions typically seen in diabetes, and hypertrophy of visceral adipose tissue. In adipocytes, the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) converts inactive cortisone into the active glucocorticoid cortisol and thereby plays an important role in regulating the actions of corticosteroids in adipose tissue. Here, we show that both TZD and non-TZD PPARgamma agonists markedly reduced 11beta-HSD-1 gene expression in 3T3-L1 adipocytes. This diminution correlated with a significant decrease in the ability of the adipocytes to convert cortisone to cortisol. The half-maximal inhibition of 11beta-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its K(d) for binding PPARgamma and EC(50) for inducing adipocyte differentiation thereby indicating that this action was PPARgamma-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11beta-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARgamma-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARgamma agonist inhibition of 11beta-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARgamma affects the down-regulation of 11beta-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11beta-HSD-1 in adipose tissue. This decrease in enzyme expression correlated with a significant decline in plasma corticosterone levels. In sum, these data indicate that some of the beneficial effects of PPARgamma antidiabetic agents may result, at least in part, from the down-regulation of 11beta-HSD-1 expression in adipose tissue.

Matrix metalloproteinase-9 (MMP-9) may play an important role in the development of inflammatory bowel disease (IBD). However, the cellular source of MMP-9 in the inflamed mucosa of IBD remains unclear. Here we report that MMP-9 mRNA is expressed in CaCO-2 cells, an intestinal epithelial cell line, and that its expression is upregulated by inflammatory stimuli. Stimulation of CaCO-2 cells with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) led to a dose-dependent increase in expression and secretion of MMP-9. In contrast, bacterial lipopolysaccharide (LPS) failed to induce expression or secretion of MMP-9, suggesting that an inflammatory reaction leading to cytokine release is a necessary step for the induction of MMP-9 release in intestinal epithelial cells. Additional studies show that induction of MMP-9 mRNA peaked at 16 h of IL-1beta stimulation, whereas expression of monocyte chemoattractant protein-1 (MCP-1) and IL-8 both peaked at 3 h of stimulation. Treatment of CaCO-2 cells with rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, significantly reduced secretion of MMP-9, indicating that agents that activate PPAR-gamma may have therapeutic use in patients with IBD.

Abstract —Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56±4 nmol total cholesterol/mg wet wt tissue, 38±2 nmol free cholesterol/mg, and 17±2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly ( P

11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects.