Osteopontin: a novel inflammatory mediator of cardiovascular disease.

Osteopontin, also called cytokine Eta-1, is a multifunctional protein containing Arg-Gly-Asp-Ser (RGDS) cell-binding sequence. It interacts with alpha(v)beta1, alpha(v)beta3 and alpha(v)beta5 integrins and CD44 receptors. OPN is suggested to play a role during inflammation via the recruitment and retention of macrophages and T-cells to inflamed sites. OPN regulates the production of inflammatory cytokines and nitric oxide in macrophages. In this review, we will discuss diverse roles of OPN related to cardiovascular diseases, including atherosclerosis, valvular stenosis, hypertrophy, myocardial infarction and heart failure.

Microarray and gene-clustering analysis.

Human mast cells are capable of secreting a plethora of inflammatory mediators and cytokines that may play a pivotal role in innate immune and inflammatory responses. Activation of mast cells by antigen and immunoglobulin E (IgE) results in signaling, gene expression, and expression of inflammatory mediators. Although a variety of techniques have been used to evaluate mast cell biology, recent advances in molecular techniques have provided unprecedented tools to study these cells. The complimentary deoxyribonucleic acid (DNA) oligonucleotide microarray, or DNA-chip technology, allows simultaneous monitoring of gene expression, provides a format for identifying genes as well as changes in their activity on a whole genome scale, and potentially offers a global view of pathophysiologic changes. This chapter reviews the use of DNA-chip technology in studying the expression of genes (transcriptional profiling) in activated human mast cells obtained from cultured cord blood-derived mononuclear cells and comments on the use of bioinformatics on analysis of gene expression. The most powerful applications of transcriptional profiling involve identification of the common patterns of gene expression across many experiments using various gene-clustering analyses. Several techniques have been used for the analysis of gene-expression data including hierarchical clustering and self-organizing maps. In this chapter, a general laboratory protocol for array analysis currently being used in our laboratory and the use of bioinformatics is discussed. Although the focus is on Affymetrix oligonucleotide arrays, the techniques described are generally applicable to expression data generated using other array formats.

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling.

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.

Molecular regulation of interleukin-13 and monocyte chemoattractant protein-1 expression in human mast cells by interleukin-1beta.

Mast cells play pivotal roles in immunoglobulin (Ig) E-mediated airway inflammation, expressing interleukin (IL)-13 and monocyte chemoattractant protein-1 (MCP-1), which in turn regulate IgE synthesis and/or inflammatory cell recruitment. The molecular effects of IL-1beta on cytokine expression by human mast cells (HMC) have not been studied well. In this report, we provide evidence that human umbilical cord blood-derived mast cells (CBDMC) and HMC-1 cells express the type 1 receptor for IL-1. We also demonstrate that IL-1beta and tumor necrosis factor-alpha are able to induce, individually or additively, dose-dependent expression of IL-13 and MCP-1 in these cells. The induction of IL-13 and MCP-1 gene expression by IL-1beta was accompanied by the activation of IL-1 receptor-associated kinase and translocation of the transcription factor, nuclear factor (NF) kappaB into the nucleus. Accordingly, Bay-11 7082, an inhibitor of NF-kappaB activation, inhibited IL-1beta-induced IL-13 and MCP-1 expression. IL-1beta also induced IL-13 promoter activity while enhancing the stability of IL-13 messenger RNA transcripts. Dexamethasone, a glucocorticoid, inhibited IL-1beta-induced nuclear translocation of NF-kappaB and also the secretion of IL-13 from mast cells. Our data suggest that IL-1beta can serve as a pivotal costimulus of inflammatory cytokine synthesis in human mast cells, and this may be partly mediated by IL-1 receptor-binding and subsequent signaling via nuclear translocation of NF-kappaB. Because IL-1beta is a ubiquitously expressed cytokine, these findings have important implications for non-IgE-mediated signaling in airway mast cells as well as for innate immunity and airway inflammatory responses, such as observed in extrinsic and intrinsic asthma.

CD40-mediated activation of vascular smooth muscle cell chemokine production through a Src-initiated, MAPK-dependent pathway.

The interaction between CD40 ligand (CD154) expressed on activated T cells and its receptor, CD40, has been shown to play a role in the onset and maintenance of autoimmune inflammation. Recent studies suggest that CD154+T cells also contribute to the regulation of atherogenesis due to their capacity to activate CD40+cells of the vasculature, including vascular smooth muscle cells (VSMC). The present study evaluated the signalling events initiated through CD40 ligation which culminate in VSMC chemokine production. CD40 ligation resulted in the phosphorylation/activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38, but not c-jun N-terminal kinase. Inhibition of both ERK1/2 and p38 activity abrogated CD40 stimulation of IL-8 and MCP-1 production. CD40-mediated induction of chemokines also showed dependence on the Src family kinase activity. The Src kinase inhibitor, PP2, was found to inhibit CD40-induced phosphorylation of ERK1/2 as well as activation of IkappaB kinase. An evaluation of Src kinases that may be important in CD40 signalling identified Lyn as a potential candidate. These data indicate that CD40 signalling in VSMC activates a Src family kinase-initiated pathway that results in the induction of MAPK activities required for successful induction of chemokine synthesis.