LXR ligand lowers LDL cholesterol in primates, is lipid neutral in hamster, and reduces atherosclerosis in mouse.

Liver X receptors (LXRs) are ligand-activated transcription factors that coordinate regulation of gene expression involved in several cellular functions but most notably cholesterol homeostasis encompassing cholesterol transport, catabolism, and absorption. WAY-252623 (LXR-623) is a highly selective and orally bioavailable synthetic modulator of LXR, which demonstrated efficacy for reducing lesion progression in the murine LDLR(-/-) atherosclerosis model with no associated increase in hepatic lipogenesis either in this model or Syrian hamsters. In nonhuman primates with normal lipid levels, WAY-252623 significantly reduced total (50-55%) and LDL-cholesterol (LDLc) (70-77%) in a time- and dose-dependent manner as well as increased expression of the target genes ABCA1/G1 in peripheral blood cells. Statistically significant decreases in LDLc were noted as early as day 7, reached a maximum by day 28, and exceeded reductions observed for simvastatin alone (20 mg/kg). Transient increases in circulating triglycerides and liver enzymes reverted to baseline levels over the course of the study. Complementary microarray analysis of duodenum and liver gene expression revealed differential activation of LXR target genes and suggested no direct activation of hepatic lipogenesis. WAY-252623 displays a unique and favorable pharmacological profile suggesting synthetic LXR ligands with these characteristics may be suitable for evaluation in patients with atherosclerotic dyslipidemia.

Expression of the cysteine protease legumain in vascular lesions and functional implications in atherogenesis.

OBJECTIVE: The present study was conducted to characterize the expression of the cysteine protease legumain in murine and human atherosclerotic tissues, and to explore the molecular mechanisms by which legumain may contribute to the pathophysiology of atherosclerosis. METHODS AND RESULTS: Using microarray analysis, legumain mRNA expression was found to increase with development of atherosclerosis in the aorta of aging Apolipoprotein E deficient mice while expression remained at low level and unchanged in arteries of age-matched C57BL/6 control mice. In situ hybridization and immunohistochemical analysis determined that legumain was predominantly expressed by macrophages in the atherosclerotic aorta, in lesions at the aortic sinus and in injured carotid arteries of Apolipoprotein E deficient mice as well as in inflamed areas in advanced human coronary atherosclerotic plaques. In vitro, M-CSF differentiated human primary macrophages were shown to express legumain and the protein could also be detected in the culture media. When tested in migration assays, legumain induced chemotaxis of primary human monocytes and human umbilical vein endothelial cells. CONCLUSIONS: Legumain is expressed in both murine and human atherosclerotic lesions. The macrophage-specific expression of legumain in vivo and ability of legumain to induce chemotaxis of monocytes and endothelial cells in vitro suggest that legumain may play a functional role in atherogenesis.

Characterization of the novel P-selectin inhibitor PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid] in vitro and in rodent models of vascular inflammation and thrombosis.

P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 microM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.

Discovery of phenyl acetic acid substituted quinolines as novel liver X receptor agonists for the treatment of atherosclerosis.

A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.