11ß-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11ß-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11ß-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11ß-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11ß-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11ß-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11ß-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11ß-HSD1 inhibition. Taken together, our data suggest 11ß-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium.

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.

CCR5 blockade modulates inflammation and alloimmunity in primates.

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.

Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes.

The sphingosine-1-phosphate (S1P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S1P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S1P receptor 1 (S1P1)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S1P1-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S1P1, and treatment with FTY-P resulted in intense staining and colocalization of CD31, beta-catenin, and zona occludens 1 in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S1P1, whereas S1P1 was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S1P1 agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.

The effect of actinomycin-D on regeneration time inTubularia.

Actinomycin-D was administered to regenerating stem segments ofTubularia spectabilis to ascertain if the synthesis of RNA is necessary for the completion of hydranth development. Preliminary experiments have indicated that this drug suppresses a burst of H3-uridine uptake into the extractable RNA of these regenerates.Wound healing was unaffected by doses of this drug which significantly retarded subsequent hydranth differentiation.The degree of regenerative suppression in this animal increases as a direct function of the administered concentration of actinomycin-D.The period of greatest sensitivity to actinomycin-D occurs early in the regenerative period: hours 6-17 following hydranth amputation. Wittman (1969) has demonstrated a puromyein-sensitive peak of C14-leucine uptake into proteins synthesized at hour 25 or beyond. The authors conclude that the control of hydranth differentiation in regeneratingTubularia resides at the transcriptional level of gene action.

A small molecule very late antigen-4 antagonist can inhibit ovalbumin-induced lung inflammation.

A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.