Steven S. Mundt

11β-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice

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.

Deficiency in Inducible Nitric Oxide Synthase Results in Reduced Atherosclerosis in Apolipoprotein E-Deficient Mice

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.

Induction of 11β-hydroxysteroid dehydrogenase type 1 but not -2 in human aortic smooth muscle cells by inflammatory stimuli

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.

Depot‐specific Regulation of the Conversion of Cortisone to Cortisol in Human Adipose Tissue

Objective: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue.

Phenylcyclobutyl triazoles as selective inhibitors of 11β-hydroxysteroid dehydrogenase type I

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-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.

4-Methyl-5-phenyl triazoles as selective inhibitors of 11β-hydroxysteroid dehydrogenase type I

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.

Simvastatin has anti-inflammatory and anti-atherosclerotic activities independent of plasma cholesterol-lowering

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 <0.02) 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.

Substituted phenyl triazoles as selective inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1.

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1 (HSD1). They were shown to be active in the mouse in vivo pharmacodynamic model (PD) for HSD1 but exhibited a potent off-target activation of the Pregnane X Receptor (PXR). SAR studies and synthesis of analogs that led to the discovery of a selective HSD1 inhibitor are described in detail.

Deficiency in sPLA2 does not affect HDL levels or atherosclerosis in mice

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.

Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11β-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11β-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.