Current perspectives on the interleukin-1 family as targets for inflammatory disease.

Since the first description of interleukin-1 (IL-1) and the genesis of the field of cytokine biology, the understanding of how IL-1 and related cytokines play central orchestrating roles in the inflammatory response has been an area of intense investigation. As a consequence of these endeavours, specific strategies have been developed to target the function of the IL-1 family in human disease realizing significant impacts for patients. While the most significant advances to date have been associated with inhibition of the prototypical family members IL-1α/ß, approaches to target more recently identified family members such as IL-18, IL-33 and the IL-36 subfamily are now beginning to come to fruition. This review summarizes current knowledge surrounding the roles of the IL-1 family in human disease and describes the rationale and strategies which have been developed to target these cytokines to inhibit the pathogenesis of a wide range of diseases in which inflammation plays a centrally important role.

Liraglutide dictates macrophage phenotype in apolipoprotein E null mice during early atherosclerosis.

BACKGROUND: Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response. METHODS: Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE-/-) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE-/- mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 µg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE-/- mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry. RESULTS: Liraglutide decreased atherosclerotic lesion formation in ApoE-/- mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 µg/kg liraglutide treatment in ApoE-/- mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells. CONCLUSIONS: This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.

Assaying the efficacy of dual-antiplatelet therapy: use of a controlled-shear-rate microfluidic device with a well-defined collagen surface to track dynamic platelet adhesion.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

Prostaglandin F2alpha elevates blood pressure and promotes atherosclerosis.

Little is known about prostaglandin F(2alpha) in cardiovascular homeostasis. Prostaglandin F(2alpha) dose-dependently elevates blood pressure in WT mice via activation of the F prostanoid (FP) receptor. The FP is expressed in preglomerular arterioles, renal collecting ducts, and the hypothalamus. Deletion of the FP reduces blood pressure, coincident with a reduction in plasma renin concentration, angiotensin, and aldosterone, despite a compensatory up-regulation of AT1 receptors and an augmented hypertensive response to infused angiotensin II. Plasma and urinary osmolality are decreased in FP KOs that exhibit mild polyuria and polydipsia. Atherogenesis is retarded by deletion of the FP, despite the absence of detectable receptor expression in aorta or in atherosclerotic lesions in Ldlr KOs. Although vascular TNF(alpha), inducible nitric oxide enzyme and TGF(beta) are reduced and lesional macrophages are depleted in the FP/Ldlr double KOs, this result reflects the reduction in lesion burden, as the FP is not expressed on macrophages and its deletion does not alter macrophage cytokine generation. Blockade of the FP offers an approach to the treatment of hypertension and its attendant systemic vascular disease.

Analysis of the zebrafish proteome during embryonic development.

The model organism zebrafish (Danio rerio) is particularly amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. Unraveling the functional dynamics of such networks requires precise quantitation of protein expression during organismal growth, which is incrementally challenging with progressive complexity of the systems. In an approach toward such quantitative studies of dynamic network behavior, we applied mass spectrometric methodology and rigorous statistical analysis to create comprehensive, high quality profiles of proteins expressed at two stages of zebrafish development. Proteins of embryos 72 and 120 h postfertilization (hpf) were isolated and analyzed both by two-dimensional (2D) LC followed by ESI-MS/MS and by 2D PAGE followed by MALDI-TOF/TOF protein identification. We detected 1384 proteins from 327,906 peptide sequence identifications at 72 and 120 hpf with false identification rates of less than 1% using 2D LC-ESI-MS/MS. These included only approximately 30% of proteins that were identified by 2D PAGE-MALDI-TOF/TOF. Roughly 10% of all detected proteins were derived from hypothetical or predicted gene models or were entirely unannotated. Comparison of proteins expression by 2D DIGE revealed that proteins involved in energy production and transcription/translation were relatively more abundant at 72 hpf consistent with faster synthesis of cellular proteins during organismal growth at this time compared with 120 hpf. The data are accessible in a database that links protein identifications to existing resources including the Zebrafish Information Network database. This new resource should facilitate the selection of candidate proteins for targeted quantitation and refine systematic genetic network analysis in vertebrate development and biology.

Cyclooxygenases, thromboxane, and atherosclerosis: plaque destabilization by cyclooxygenase-2 inhibition combined with thromboxane receptor antagonism.

BACKGROUND: Antagonism or deletion of the receptor (the TP) for the cyclooxygenase (COX) product thromboxane (Tx)A2, retards atherogenesis in apolipoprotein E knockout (ApoE KO) mice. Although inhibition or deletion of COX-1 retards atherogenesis in ApoE and LDL receptor (LDLR) KOs, the role of COX-2 in atherogenesis remains controversial. Other products of COX-2, such as prostaglandin (PG) I2 and PGE2, may both promote inflammation and restrain the effects of TxA2. Thus, combination with a TP antagonist might reveal an antiinflammatory effect of a COX-2 inhibitor in this disease. We addressed this issue and the role of TxA2 in the promotion and regression of diffuse, established atherosclerosis in Apobec-1/LDLR double KOs (DKOs). METHODS AND RESULTS: TP antagonism with S18886, but not combined inhibition of COX-1 and COX-2 with indomethacin or selective inhibition of COX-2 with Merck Frosst (MF) tricyclic, retards significantly atherogenesis in DKOs. Although indomethacin depressed urinary excretion of major metabolites of both TxA2, 2,3-dinor TxB2 (Tx-M), and PGI2, 2,3-dinor 6-keto PGF(1alpha) (PGI-M), only PGI-M was depressed by the COX-2 inhibitor. None of the treatments modified significantly the increase in lipid peroxidation during atherogenesis, reflected by urinary 8,12-iso-iPF(2alpha)-VI. Combination with the COX-2 inhibitor failed to augment the impact of TP antagonism alone on lesion area. Rather, analysis of plaque morphology reflected changes consistent with destabilization of the lesion coincident with augmented formation of TxA2. Despite a marked effect on disease progression, TP antagonism failed to induce regression of established atherosclerotic disease in this model. CONCLUSIONS: TP antagonism is more effective than combined inhibition of COX-1 and COX-2 in retarding atherogenesis in Apobec-1/LDLR DKO mice, which perhaps reflects activation of the receptor by multiple ligands during disease initiation and early progression. Despite early intervention, selective inhibition of COX-2, alone or in combination with a TP antagonist, failed to modify disease progression but may undermine plaque stability when combined with the antagonist. TP antagonism failed to induce regression of established atherosclerotic disease. TP ligands, including COX-1 (but not COX-2)-derived TxA2, promote initiation and early progression of atherogenesis in Apobec-1/LDLR DKOs but appear unimportant in the maintenance of established disease.

EBP, a program for protein identification using multiple tandem mass spectrometry datasets.

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.