Influence of canstatin on fibroblast-driven hypervascularisation in rheumatoid arthritis

Abstract

Objectives: Hypervascularisation is a dominant feature of the inflamed synovium in rheumatoid arthritis (RA). As RA synovial fibroblasts (RASFs) are key cells of synovial pathophysiology and are located adjacent to the aberrant endothelial cells (ECs), we hypothesised that this interaction might be responsible for the pathological hypervascularisation.

Methods: In the severe combined immunodeficiency (SCID) mouse model for RA, RASF-mediated helix-like vessel (HLV) formation was described and modulated by canstatin, an antiangiogenic collagen IV fragment that blocks the angiopoietin (ANGPT)/Tie2 pathway in EC. ANGPT2/CD31 and CXCL2 immunofluorescence on implants and human synovium was performed. RASF were stimulated with interleukin (IL)-1β once or repetitively and immunoassays, real-time polymerase chain reaction and RNA sequencing were performed. Two-dimensional (2D) tube formation and 3-dimensional spheroid-based assays using human umbilical vein ECs and fluorescent-stained RASF with/without canstatin, IL-11 and CXCL2 were evaluated.

Results: In SCID mice, RASF-specific HLV formation started early and increased until day 30. The number of HLV was significantly reduced by canstatin. Compared to osteoarthritis synovium, ANGPT2 was significantly upregulated in RA vessels. Repetitive RASF stimulation significantly decreased IL-6, IL-11 and CXCL-2 compared to RASF stimulated once with IL-1β. When RASF was stimulated once, CXCL2 and IL-11 were significantly reduced along with 2D tube formation, while repetitive stimulation significantly attenuated hypervascularisation. RNAseq revealed underlying pathways leading to altered tube formation.

Conclusions: We showed that RASF affect vascularisation in vitro and in vivo. The results support the idea that canstatin is able to alter the RASF pathological HLV formation. In the SCID mouse model, this was regulated on a molecular level mediated by ANGPT2 in a vascular endothelial growth factor A-independent manner, contributing significantly to one of the central aspects of RA pathophysiology.