Mandar N. Dave

Functional genomic analysis in arthritis-affected cartilage : yin-yang regulation of inflammatory mediators by alpha 5 beta 1 and alpha V beta 3 integrins

Osteoarthritis-affected cartilage exhibits enhanced expression of fibronectin (FN) and osteopontin (OPN) mRNA in differential display and bioinformatics screen. Functional genomic analysis shows that the engagement of the integrin receptors α5β1 and αvβ3 of FN and OPN, respectively, have profound effects on chondrocyte functions. Ligation of α5β1 using activating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory mediators such as NO and PGE2 as well as the cytokines, IL-6 and IL-8. Furthermore, up-regulation of these proinflammatory mediators by α5β1 integrin ligation is mediated via induction and autocrine production of IL-1β, because type II soluble IL-1 decoy receptor inhibits their production. In contrast, αvβ3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1β, NO, and PGE2 (triggered by α5β1, IL-1β, IL-18, or IL-1β, TNF-α, plus LPS) in a dominant negative fashion by osteoarthritis-affected cartilage and activated bovine chondrocytes. These data demonstrate a cross-talk in signaling mechanisms among integrins and show that integrin-mediated “outside in” and “inside out” signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of osteoarthritis.

Functional Genomic Analysis of Type II IL-1β Decoy Receptor: Potential for Gene Therapy in Human Arthritis and Inflammation

Gene expression arrays show that human epithelial cells and human arthritis-affected cartilage lack detectable amounts of mRNA for IL-1 antagonizing molecules: IL-1Ra and IL-1RII, but constitutively express IL-1. Functional genomic analysis was performed by reconstituting human IL-1RII expression in various IL-1RII-deficient cell types to examine its antagonist role using gene therapy approaches. Adenovirus-expressing IL-1RII when transduced into human and bovine chondrocytes, human and rabbit synovial cells, human epithelial cells, and rodent fibroblasts expressed membrane IL-1RII and spontaneously released functional soluble IL-1RII. The IL-1RII+ (but not IL-1RII−) cells were resistant to IL-1β-induced, NO, PGE2, IL-6, and IL-8 production or decreased proteoglycan synthesis. IL-1RII inhibited the function of IL-1 in chondrocytes and IL-1- and TNF-α-induced inflammatory mediators in human synovial and epithelial cells. IL-1RII+ chondrocytes were more resistant to induction of NO and PGE2 by IL-1β compared with IL-1RII− cells incubated with a 10-fold (weight) excess of soluble type II IL-1R (sIL-1RII) protein. In cocultures, IL-1RII+ synovial cells released sIL-1RII, which in a paracrine fashion protected chondrocytes from the effects of IL-1β. Furthermore, IL-1RII+ (but not IL-1RII−) chondrocytes when transplanted onto human osteoarthritis-affected cartilage in vitro, which showed spontaneous release of sIL-1RII for 20 days, inhibited the spontaneous production of NO and PGE2 in cartilage in ex vivo. In summary, reconstitution of IL-1RII in IL-1RII− cells using gene therapy approaches significantly protects cells against the autocrine and paracrine effects of IL-1 at the signaling and transcriptional levels.

Osteopontin : An intrinsic inhibitor of inflammation in cartilage

OBJECTIVEnTo identify extracellular and intraarticular matrix components that are differentially expressed in normal and osteoarthritis (OA)-affected cartilage and to investigate their functions with respect to regulation of mediators of inflammation.nnnMETHODSnDifferential-display reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of a pool of messenger RNA (mRNA) from 10 human OA cartilage samples and 5 normal cartilage samples was performed using arbitrary primers. Confirmatory analysis of the up-regulated transcripts of fibronectin (FN) and osteopontin (OPN) was performed by RT-PCR of individual RNA samples from a separate set of donors. The effect of recombinant OPN (or anti-OPN antiserum) on chondrocyte function was examined by analyzing the spontaneous or interleukin-1 (IL-1)-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) from human OA-affected cartilage under ex vivo conditions.nnnRESULTSnUp-regulation (300-700%) of FN and OPN mRNA was observed in human OA-affected cartilage as compared with normal cartilage. Functional analysis of the role of OPN in OA cartilage showed that 1) Addition of 1 microg/ml (20 nM) of recombinant OPN to human OA-affected cartilage under ex vivo conditions inhibited spontaneous and IL-1beta-induced NO and PGE2 production, and 2) neutralization of intraarticular OPN with anti-OPN antiserum augmented NO production.nnnCONCLUSIONnThe data indicate that one of the functions of intraarticular OPN, which is overexpressed in OA cartilage, is to act as an innate inhibitor of IL-1, NO, and PGE2 production. These findings suggest that the production of pleiotropic mediators of inflammation that influence cartilage homeostasis, such as NO and PGE2, is regulated by the interaction of chondrocytes with differentially expressed proteins within the extracellular matrix.

Functional Genomics Approaches in Arthritis

The post-genomic era of functional genomics and target validation will allow us to narrow the bridge between clinically correlative data and causative data for complex diseases, such as arthritis, for which the etiological agent remains elusive. The availability of human and other annotated genome sequences, and parallel developments of new technologies that allow analysis of minute amounts of human and animal cells (peripheral blood cells and infiltrating cells) and tissues (synovium and cartilage) under different pathophysiological conditions, has facilitated high-throughput gene mining approaches that can generate vast amounts of clinically correlative data. Characterizing some of the correlative/causative genes will require reverting to the hypothesis-driven, low throughput method of complementary experimental biology using genomic approaches as a tool. This will include in silico gene expression arrays, genome-wide scans, comparative genomics using various animal models (such as rodents and zebrafish), bioinformatics and a team of well trained translational scientists and physicians.For the first time, the ‘genomic tools’ will allow us to analyze small amounts of surgical samples (such as needle biopsies) and clinical samples in the context of the whole genome. Preliminary genomic analysis in osteoarthritis has already resurrected the debate on the semantic issues in the definition of inflammation. Further analyses will not only facilitate the development of unbiased hypotheses at the molecular level, but also assist us in the identification and characterization of novel targets and disease markers for pharmacological intervention, gene therapy, and diagnosis.

Model protocol to study pharmacogenomics in inflammatory diseases: Human rheumatoid arthritis

Pharmacogenomics is a revolution in molecular medicine, especially in view of the development of microarray technologies and proteomics to monitor gene and protein expression. The ability to monitor up to 60,000 potential parameters in a clinical setting gives a whole new meaning to 1) toxic effects, 2) side effects, 3) primary and secondary targets, and 4) drug resistance and nonresponders during a clinical trial. Pharmacogenomics may set a new standard to monitor the effects of drugs such as NSAIDS or disease‐modifying drugs in diseases such as arthritis. The present article outlines a pharmacogenomics clinical protocol that is in progress, a study that will address some of the above questions. Drug Dev. Res. 49:29–33, 2000.