ABSTRACT
We report the design of novel, potent cPLA(2)α inhibitors that possess an α-methyl-2-ketothiazole that acts as a serine-reactive moiety. We describe the optimization of the series for potency and metabolic stability towards ketone reduction. This was achieved by attenuating the reactivity of the ketone using a combination of electronic and steric effects.
Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Group IV Phospholipases A2/antagonists & inhibitors , Ketones/chemistry , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Animals , Drug Stability , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , HL-60 Cells , Humans , Inhibitory Concentration 50 , Ketones/chemical synthesis , Ketones/pharmacology , Magnetic Resonance Spectroscopy , Molecular Structure , Oxidation-Reduction , Rats , Serine/chemistry , Thiazoles/chemistryABSTRACT
PURPOSE: The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy. METHODS: Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis. RESULTS: Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2. CONCLUSIONS: These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.
Subject(s)
Angiopoietin-1/pharmacology , Angiopoietin-2/pharmacology , Diabetic Retinopathy/metabolism , Pericytes/drug effects , Receptor, TIE-2/metabolism , Retinal Vessels/pathology , Animals , Annexin A5/metabolism , Apoptosis/drug effects , Blotting, Western , CD13 Antigens/metabolism , Cattle , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Pericytes/metabolism , Pericytes/pathology , Tumor Necrosis Factor-alpha/pharmacology , Up-RegulationABSTRACT
In this study, the phenotype of psoriatic keratinocytes and fibroblasts in reconstructed skin models was compared to those constructed from normal cells. Characterization of this model by immunohistochemistry showed that classical markers of keratinocyte differentiation exhibited similar patterns of distribution in the psoriatic models to those derived from normal cells and generally reflected in vivo observations. Some crucial differences, however, were observed between normal and psoriatic models when pro-inflammatory gene expression and keratinocyte proliferation were investigated. Notably, the chemokine receptor CXCR2 was overexpressed in the psoriatic models, and, moreover, was localized to the granular layer of keratinocytes as seen in psoriasis in vivo. Pro-inflammatory genes (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma], and interleukin 8 [IL-8]) were expressed at high levels in the psoriatic models, but were only minimally expressed in the normal models. Models derived from uninvolved psoriatic skin showed the same gene expression profile as those derived from involved skin along with an increased proliferation rate when compared to normal models. These results suggest that psoriatic individuals possess an inherent predisposition to develop the disease phenotype even in the absence of T cells. This study represents a comprehensive characterization of psoriatic human skin reconstructed in vitro, and demonstrates the potential of this model as a valuable tool in drug discovery.
