Work Package 5

WP 5: In vitro and Ex vivo models of OA

Participants:

KU Leuven, Tigenix, Oxford, LUMC, deCode

Objectives:

To develop in vitro models for OA that will facilitate the investigation of the potential mode action of the identified genes associated with OA and look into models perceived as relevant to the disease process.
To systematically screen in vitro mostly primary cells and tissues of relevance to the joint and joint associated tissues such as chondrocytes/cartilage, meniscal cells, osteoblasts/bone and mesenchymal stem cells/synovium.

Status:

Osteoarthritis (OA) is the most common form of arthritis and a major cause of disability in Europe. In this disorder, the articular cartilage that covers the ends of the bones deteriorates. Symptoms of OA range from pain to disability. Various treatment options and lifestyle change can help alleviate the symptoms but as there are no drugs that can cure, reverse or halt OA, the current treatments are palliative rather than therapeutic. Surgery, including joint replacement, is used when everything else has failed.
TREAT-OA is a large multi-disciplinary project aiming at identifying diagnostic and predictive genetic markers for OA risk and progression and, ultimately, to find potential therapeutic targets. The approach followed is gene driven, whereby genes and biochemical markers which are consistently associated with risk and progression of OA are identified by genome-wide association analyses. Next, the roles of these genes of interest are studied to understand the molecular pathways involved in the disease. This is done by studying their expression and function in in vitro and in vivo models of OA. The functional analysis of the molecular pathways should ultimately lead to the identification of targets for pharmacological intervention. This workpackage aims to study gene expression and to perform in vitro studies.
During this first reporting period, the expression of several of the genes that were identified by linkage analysis in OA patient cohorts (deCode Genetics and ERGO, Rotterdam) or were selected by the consortium, was documented by means of quantitative PCR methods. This comprised the systematic screening of the expression pattern in primary cells of various joint tissues, such as articular chondrocytes, meniscal cells, synovial membrane-derived cells and osteoblasts.
Typical features of an OA joint is an imbalance in joint homeostasis, flairs of inflammation and dedifferentiation of cartilage cells towards a chondroprogenitor stage. Assays were implemented to mimic these features in vitro and used to study the expression dynamics of the genes of interest. Chondrocyte dedifferentiation, as assessed by a decrease in the synthesis of cartilage markers and a gradual loss of chondrogenic potential of the chondrocytes, was achieved by expanding primary human cells in monolayer culture for several passages. The inflammation and immune response, as encountered in an OA joint, was mimicked by adding pro-inflammatory cytokines (IL1, TNFa) to the cells.
This expression analysis resulted in an atlas of mRNA expression patterns of the genes under study. It revealed that, whereas most of the genes that were identified by genetic means are indeed reproducibly expressed at low levels throughout the various tissues analysed, a few genes (a.o. belonging to the family of transforming growth factors, a hormone-converting enzyme and a mediator of apoptosis) appear to be differentially expressed under the above described disease-mimicking in vitro conditions. Furthermore, the expression of some particular genes appeared to be dynamically controlled by environmental factors such as cytokines. This makes these genes to be prime candidates to be involved in the etiology of OA. Based on this selection criterion, these latter genes are selected for a detailed functional analysis by means of additional in vitro studies and in vivo animal models of OA. This will allow the investigation of the mode action of the identified proteins and provide a platform to screen for potential drugs that target the pathways involved.

Publications:

Wnt signaling and osteoarthritis. Luyten FP, Tylzanowski P, Lories RJ. Bone. 2009 Apr;44(4):522-7. Epub 2008 Dec 14.
Articular cartilage and biomechanical properties of the long bones in Frzb-knockout mice. Lories RJ, Peeters J, Bakker A, Tylzanowski P, Derese I, Schrooten J, Thomas JT, Luyten FP. Arthritis Rheum. 2007 Dec;56(12):4095-103.
Contemporary concepts of inflammation, damage and repair in rheumatic diseases. Luyten FP, Lories RJ, Verschueren P, de Vlam K, Westhovens R. Best Pract Res Clin Rheumatol. 2006 Oct;20(5):829-48.
Noggin haploinsufficiency differentially affects tissue responses in destructive and remodeling arthritis. Lories RJ, Daans M, Derese I, Matthys P, Kasran A, Tylzanowski P, Ceuppens JL, Luyten FP. Arthritis Rheum. 2006 Jun;54(6):1736-46.
Bone morphogenetic protein signaling in joint homeostasis and disease. Lories RJ, Luyten FP. Cytokine Growth Factor Rev. 2005 Jun;16(3):287-98.