Mujing Yan

11β-Hydroxysteroid Dehydrogenase Type 1 Gene Knockout Attenuates Atherosclerosis and In Vivo Foam Cell Formation in Hyperlipidemic apoE−/− Mice

Background Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. Methodology/Principal Findings To examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE−/− background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1−/−/apoE−/− mice vs. 11βHSD1+/+/apoE−/− mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1−/−/apoE−/− mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1−/−/apoE−/− mice. Bone marrow transplantation from 11βHSD1−/−/apoE−/− mice into apoE−/− recipients reduced plaque area 39–46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1+/+/apoE−/− and 11βHSD1−/−/apoE−/− mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1−/−/apoE−/− mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1−/−/apoE−/− mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1−/−/apoE−/−-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. Conclusions These findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

Background P2Y6, a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y6 deficiency on atherosclerosis. Methodology/Principal Findings Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y6 receptors, showed that exogenous expression of P2Y6 induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y6-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y6 and in acute peritonitis models of inflammation. To evaluate the role of P2Y6 in atherosclerotic lesion development, we used P2Y6-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y6 receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y6xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y6 deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. Conclusions P2Y6 receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y6 deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y6 in vascular disease pathophysiologies, such as aneurysm formation.

(D)-2-tert-Butoxycarbonylamino-5,5-difluoro-5-phenyl-pentanoic acid: synthesis and incorporation into the growth hormone secretagogues.

The first enantioselective synthesis of (D)-2-tert-butoxycarbonylamino-5,5-difluoro-5-phenyl-pentanoic acid 3 was achieved. The incorporation of the titled compound into growth hormone secretagogue (GHS) compounds resulted in new analogs 10 and 16, both of which had significantly increased in vitro potency. The compound 10 also showed improved in vivo efficacy as well as pharmacokinetic properties in rat models.

Design and synthesis of tetrazole-based growth hormone secretagogue: the SAR studies of the O-benzyl serine side chain.

The structure-activity relationship of the O-benzyl serine side chain was investigated based on the tetrazole-based growth hormone secretagogue BMS-317180 (2). The ortho position of the benzyl moiety was found to be favorable for introduction of substituents. A series of ortho-substituted compounds were synthesized with improved in-vitro and in-vivo activity. Among them, the biphenyl compound 2p shows twofold improvement in potency compared to its parent compound BMS-317180 (2).

Tetrazole based amides as growth hormone secretagogues

A novel series of N1 substituted tetrazole amides were prepared and showed to be potent growth hormone (GH) secretagogues. Among them, hydroxyl containing analog 31 displayed excellent in vivo activity by increasing plasma GH 10-fold in an anesthetized IV rat model.

Optimization of 1H-tetrazole-1-alkanenitriles as potent orally bioavailable growth hormone secretagogues.

1H-tetrazole-1-alkanenitrile SR-9g exhibits a >10-fold in vivo potency enhancement over the lead nitrile 1 and has acceptable oral bioavailability in rats and dogs. An enantiospecific synthesis of 1H-tetrazole-1-alkanenitrile nitriles 9 has been developed.