Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis

Saleh Ibrahim,Ewa Paleolog,Yvonne Raatz,Marc Feldmann

doi:10.1186/ar3922

Abstract

IntroductionDysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.MethodsCIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.ResultsAlthough arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1B antibody significantly reduced disease severity and progression in CIA mice.ConclusionsOur findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.

Highlights

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA)
Staining for CD31 revealed occasional blood vessels with distinct lumen in paws obtained from healthy mice, whereas arthritic paws displayed marked vascularity especially in areas of dense cellular infiltration and in regions of synovium invading the bone
The functional capillary density (FCD) was significantly decreased in arthritic tissue (P = 0.027, Mann Whitney test), with an FCD of 320.30 ± 9.76 cm/ cm2 in knee synovia of healthy mice compared to an FCD of 286.70 ± 8.23 cm/cm2 in arthritic mice (Figure 1B)

Summary

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). The vascular turnover in the arthritic synovium is increased, and synovial endothelial cells express markers of proliferation [5]. Synovial fluids from RA joints have been shown to promote endothelial cell migration and proliferation, and to induce vessel formation in an angiogenesis assay [7,8], which reflects an active, proangiogenic phenotype of the arthritic synovium. A number of angiogenic factors, expression of which is triggered by the hypoxic and inflammatory environment within the arthritic joint [9,10], are abundant in RA synovial tissue, including vascular endothelial growth factor (VEGF) [8,11], angiopoietins [10,12], hepatocyte growth factor (HGF) [13] and fibroblast growth factor (FGF)-2 [14]. During RA, VEGF seems to mediate its effects through its two tyrosine kinase receptors fms-like tyrosine kinase (FLT)-1 and kinase insert domain receptor (KDR; mouse homolog is fetal liver kinase (FLK)-1), and neuropilin (NRP)-1 [11,17]

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