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Title: Understanding the role of angiogenesis in rheumatoid arthritis : gene expression profiling and functional analysis of angiogenic markers in murine arthritis models
Author: Raatz, Yvonne
ISNI:       0000 0004 2688 2769
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Angiogenesis is believed to represent an early event in rheumatoid arthritis. However, the contribution of different factors involved in the angiogenic process during various stages of arthritis is still unclear. The aim of this study was to identify genes that are differentially expressed in arthritic joint tissue, particularly genes with a role in angiogenesis. Three different mouse models of collagen induced arthritis (CIA) were applied: namely, CIA induced by heterologous collagen in DBA/1 and C57BL/6 mice, as well as the chronic CIA model in DBA/1 mice induced by homologous collagen. Total RNA was extracted from mouse paws and applied to perform gene expression analysis. Therefore two different real-time RT-PCR approaches were employed: a pathway focused Angiogenesis Profiler Array analysing simultaneously the expression of 84 genes, and gene specific RT-PCR. Immunohistochemistry for CD31 (a well-known endothelial cell marker) and intravital fluorescence microscopy were applied to analyse the microvessel density in arthritic paws and knees, respectively. To assess the ability of arthritic joint homogenates to induce angiogenesis, different in vitro and in vivo angiogenesis assays (endothelial chemotaxis assay, matrigel plug assay) were employed. Immunohistochemical analysis revealed that highly inflamed synovial tissue of mouse paw joints contains large numbers of CD31 positive vessels. Although arthritic synovium showed regions of high vascular density, non-vascularised regions could be identified. Further, no significant difference in the functional capillary density in arthritic and healthy knee synovium was observed. Interestingly, there was a trend towards lower values in arthritic joints. This finding suggests that neovascularisation occurs in CIA, but pannus growth outpaces new vessel formation. The different CIA models displayed distinct mRNA expression signatures. However, arthritic tissue from DBA/1 and C57BL/6 mice did not exhibit a distinctive pro-angiogenic expression profile. For example, increased mRNA expression could be observed for VEGF receptors (FLT-1, FLK-1, NRP-1, NRP-2), midkine, HGF, IGF-1, ANG-1, ANG-2, TIE-2, whereas expression of VEGF/A and FGF-1 mRNA did not change during the course of acute CIA. The mRNA levels of TSP-1 and endostatin, both involved in angiogenesis inhibition, were also up-regulated during CIA, indicative of a balanced regulation of pro-angiogenic and antiangiogenic factors. Overall, in the acute models, a simultaneous induction of different angiogenic factors could be observed, which might indicate a concerted regulation of blood vessel formation in the arthritic synovium. Gene expression pattern in chronic CIA (induced by homologous collagen) varied between individual mice, reflecting the heterogeneous clinical features. But none of the analysed genes was markedly altered. Indeed, only a few animals exhibited changes in transcript levels of IL-1[beta], TNF[alpha], IL-6, HGF, IGF-1, midkine, leptin, and ANG-2. Although this requires further analyses, one might speculate that this is due to the fact that long-standing (burned-out) lesions tone down their pro-inflammatory and pro-angiogenic environment. A constant increase in HGF and NRP-1 mRNA levels suggested that both might be important key players during the development of CIA. In a series of consecutive experiments, two different therapeutic strategies were applied to block these molecules in acute CIA, namely the administration of a HGF antagonist, NK4, and an anti-NRP-1 antibody, respectively. Both inhibited angiogenesis induced by arthritic paw homogenates in vitro or in vivo. Treatment with either NK4 or anti-NRP-1 significantly attenuated the severity and progression of CIA. However, histological examination demonstrated that this clinical improvement was not necessarily associated with a reduction in synovial vascularity. These observations suggest that inhibiting NRP-1 or HGF signalling alone is not sufficient to treat arthritis.
Supervisor: Feldmann, Marc Sponsor: Marie Curie Training Network EURO-RA ; Kennedy Institute of Rheumatology
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral