Background/Purpose: Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Id1 has shown pleiotropic effects: important in vasculogenesis in endothelial progenitor cells (EPCs), and important in angiogenesis in mature endothelial cells (ECs). Our group was the first to report that rheumatoid arthritis (RA) synovial fluid contains elevated amounts of Id1, and histologic analysis of RA synovial tissue (ST) revealed that Id1 is highly expressed in the vasculature of RA. We later found that the primary source of Id1 in STs were activated fibroblasts. Once released, Id1 acts as a potent inducer of angiogenesis, suggesting that Id1 may contribute to vasculogenesis as well as angiogenesis by independent mechanisms. This suggests that hyperproliferating fibroblasts produce Id1 that induces blood vessel growth. What is still unknown is the method of release of Id1 by synovial fibroblasts, as well as the potential importance of Id1 in other rheumatic diseases such as scleroderma (SSc).

Methods: Synovial fibroblasts from RA, osteoarthritis (OA), normal (NL), and dermal fibroblasts from NL and SSc patients were plated and cultured without cytokines or stimulated with tumor necrosis factor-α (TNF-α), CXCL16, Interleukin-17 (IL-17), transforming growth factor-β (TGF-β), or platelet-derived growth factor (PDGF). Supernatants were measured for Id1 expression by ELISA. Fibroblast supernatants were subjected to rate zonal centrifugation to isolate and purify exosomes. Whole and lysed (0.5% Triton X-100) exosome fractions were also measured for Id1 by ELISA. For signal transduction analysis, human dermal microvascular endothelial cells (HMVECs), EPCs, and synovial fibroblasts were plated and stimulated with human Id1. Western blot analysis was used to determine the kinetics of protein phosphorylation in cell lysates. Finally, we assessed the effects of Id1 signaling on angiogenesis using silencing RNA (siRNA) to inhibit HMVEC signaling pathways in the mouse Matrigel plug assay.

Results: NL and RA synovial fibroblasts increased Id1 production with stimulation by TGF- β. Similarly, dermal fibroblast supernatants from NL and SSc patients showed a marked increase in Id1 production after stimulation with PDGF and TGF-β. We assessed the role of exosomes to determine the mechanism of Id1 transport outside the cell. We found that Id1 is encapsulated by fibroblast exosomes and that >80% of the Id1 released by RA synovial fibroblasts is contained within exosomes. Cell signaling assays following stimulation by recombinant human Id1 showed the JNK pathway was upregulated in HMVECs, EPCs, and RA synovial fibroblasts, while P38 phosphorylation was increased in only EPCs. Furthermore, we show that inhibiting HMVEC associated JNK with siRNA reverses Id1 induced HMVEC vessel formation in Matrigel plugs.

Conclusion:  Id1 is a pleotropic molecule that has significant effects on angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also that it can be released from fibroblasts in exosomes, thus expanding its role in the orchestration of inflammatory lesions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inflammatory-properties-of-inhibitor-of-dna-binding-1-as-a-unique-fibroblast-derived-nuclear-protein/