Abstract. Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation.We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue-an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain *85 % of caveolin and ,',,55 % of a GPI-linked marker protein, while they exclude I>98% of integral plasma membrane protein markers and t>99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules-including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap I-the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.

The enzyme 11β–hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11β-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11β-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11β-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11β-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

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 (nϭ20) contained 56Ϯ4 nmol total cholesterol/mg wet wt tissue, 38Ϯ2 nmol free cholesterol/mg, and 17Ϯ2 nmol cholesteryl ester/mg. Simvastatin (nϭ22) significantly (PϽ0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol-lowering activity.

SummaryTumor necrosis factor a, granulocyte colony-stimulating factor, granulocyte/macrophage colonystimulating 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 ofprotein . The CD14 on the surface ofPMN was shown to serve two biological functions. It bound particles coated with complexes oflipopolysaccharide (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 ofPMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin. BacterialLPS(endotoxin) is known to have profound physiological effects on PMN both in vivo and in vitro. Animals respond to intravenous LPS with a rapid fall in the number of circulating PMN and a concomitant accumulation ofPMN in the lungs (1) . Isolated PMN respond to LPS with increased adhesion to protein-coated surfaces (2), enhanced ability to mount an oxidative burst (3), and enhanced microbicidal powers (4) . Many of these responses to LPS appear to depend at least in part on enhanced function of the three members of the CD11/CD18 family of adhesion-promoting receptors (LFA1, CR3, and p150,95, also known as the 02 or leukocyte integrins) on the surface of PMN . Adhesion of PMN to endothelium requires the participation ofCDII/CD18 molecules (5-7), and blockade ofCD18 with mAbs prevents accumulation ofPMN in the lungs ofendotoxin-treated animals (8) . Adhesion ofPMN to protein-coated glass (5), enhanced oxidative burst in response to soluble agonists (9), and microbicidal activity (5) are also dependent on increased CD11/CD18 function. Here, we directly measure the effect of LPS on the adhesive function ofCR3 (CD11b/CD18) and describe a dramatic enhancement of its adhesive activity by complexes of LPS with proteins from the serum.Recent studies have described a novel mechanism by which mononuclear cells may respond to LPS (10, 11) . LPS is first bound by the serum protein LPS binding protein (LBP)l, and the resulting LPS-LBP complex is then recognized by CD14, a 55-kD glycoprotein that is strongly expressed on monocytes and macrophages. LBP and CD14 serve two physiological roles. These proteins act as opsonin and opsonic receptor, respectively, to promote the phagocytic uptake of bacteria or LPS-coated particles by macrophages (12). They also dramatically enhance the ability of mononuclear cells to synthesize TNF in response to endotoxin . Addition of LBP speeds the synthesis of TNF and enables a response to doses of LPS 100-fold lower than are otherwi...

Inhibition of HSD1 with MK-0916 was generally well tolerated in patients with T2DM and MetS. Although no significant improvement in FPG was observed with MK-0916 compared to placebo, modest improvements in A1C, body weight and blood pressure were observed.

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.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.

Peroxisome proliferator-activated receptor-␥ (PPAR␥) 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 11␤-hydroxysteroid dehydrogenase type 1 (11␤-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 PPAR␥ agonists markedly reduced 11␤-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 11␤-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its K d for binding PPAR␥ and EC 50 for inducing adipocyte differentiation thereby indicating that this action was PPAR␥-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11␤-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPAR␥-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPAR␥ agonist inhibition of 11␤-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPAR␥ affects the down-regulation of 11␤-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11␤-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 PPAR␥ antidiabetic agents may result, at least in part, from the down-regulation of 11␤-HSD-1 expression in adipose tissue.