Background/Purpose: Rheumatoid arthritis (RA) is characterized by enhanced blood vessel development in joint synovium. This involves the recruitment of endothelial progenitor cells (EPCs), allowing for de novo vessel formation and pro-inflammatory cell infiltration. Inhibitor of DNA Binding 1 (Id1) is a transcription factor unique to EPCs that influences cell maturation. We hypothesized that Id1 could be secreted and expressed in RA, and influence blood vessel growth. We also wanted to define the contribution of this transcription factor in RA, and correlate it with CXCL16 expression, as EPCs also prominently express the only known CXCL16 receptor, CXCR6.

Methods: Enzyme Linked Immunosorbant Assay (ELISA) and Polymerase Chain Reaction (PCR) was used to examine Id1 levels in synovial fluids (SFs) and endothelial cells (ECs) respectively. Immunohistology and immunofluorescence (IF) histology was used to determine the expression of Id1 in RA compared to osteoarthritis (OA) and normal (NL) synovial tissues (STs). We used Matrigel angiogenesis and human dermal microvascular EC (HMVEC) migration assays to determine if recombinant human (rhuId1) and/or RA SF immunodepleted of Id1 alters angiogenic activity. Finally, CXCR6 deficient (CXCR6^-/-) and wild-type (wt) C57BL/6 mice were primed to develop K/BxN serum induced arthritis and evaluated for joint swelling. Joint tissues from these mice were examined for Id1 and correlated with CXCR6 expression and arthritis development.

Results: ST samples immunostained for Id1 showed heightened expression in RA compared to OA and NL ST. By IF staining, we found significantly more Id1 in RA compared to OA and NL vasculature, showing that Id1 expressing cells, therefore EPCs, are most active in vascular remodeling in the RA synovium. We also detected significantly more Id1 in RA compared to OA and other arthritis SFs by ELISA that highly correlated with CXCL16 levels (p<0.05, n=10, r=0.64 Pearson’s Correlation). In vitro chemotaxis assays also showed that Id1 is highly chemotactic for HMVECs. Using in vitro Matrigel assays, we found that HMVECs form tubes in response to rhuId1 and sham depleted RA SF, and that Id1 immunodepleted from RA SF profoundly decreases tube formation. PCR showed that Id1 mRNA could be upregulated in both HMVECs and EPCs with tumor necrosis factor-α (TNF-α) or CXCL16, with highest amounts seen in EPCs in response to CXCL16. Finally, using the K/BxN serum induced arthritis model, we correlated EC CXCR6 with Id1 expression by immunohistochemistry. These findings were further validated by highly significant reductions in blood vessels and hemoglobin (Hb) content in joint tissues from K/BxN serum induced CXCR6^-/-mice.

Conclusion: Our data indicates that Id1 correlates with CXCL16 in RA SF and that CXCR6^-/- arthritic mice have notable reductions in Id1 expression and arthritis development, correlating with profound declines in vasculature and joint Hb content. We also found that Id1 is potently angiogenic, and can be upregulated in HMVECs and EPCs by TNF-α and especially CXCL16. These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand-receptor pair that can be highly correlated with Id1 expression, EPC recruitment, and blood vessel formation in the RA joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibitor-of-dna-binding-1-as-a-secreted-angiogenic-transcription-factor-in-rheumatoid-arthritis/