Session Information
Date: Sunday, November 8, 2015
Title: Cytokines, Mediators, Cell-cell Adhesion, Cell Trafficking and Angiogenesis
Session Type: ACR Concurrent Abstract Session
Session Time: 2:30PM-4:00PM
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. We have shown that Id1 exhibits pleiotropic activity, is highly expressed in rheumatoid arthritis (RA) synovial tissues (STs) and effusions, and is primarily fibroblast derived. Soluble Id1 acts as a potent inducer of angiogenesis and may contribute to vasculogenesis and angiogenesis by independent mechanisms. This suggests that fibroblast-like synoviocytes (FLS) produce Id1, which induces blood vessel growth after release from FLS through an unknown mechanism.
Methods: Histologic analysis of RA STs and K/BxN mice ankles was conducted to reveal the cell types expressing Id1 in these tissues and how Id1 expression correlates with disease severity. FLS from RA, osteoarthritis (OA), and normal (NL) STs were plated and cell supernatants were measured for Id1 expression by ELISA. Supernatants were subjected to ultracentrifugation to isolate and purify exosomes. Nanoparticle Tracking Analysis was used to quantify the size and concentration of particles within the exosome fractions. Whole and lysed (0.5% Triton X-100) exosome fractions were then measured for Id1. For signal transduction analysis, human dermal microvascular endothelial cells (HMVECs), endothelial progenitor cells, and FLS were plated and stimulated with human Id1 to assess the kinetics of protein phosphorylation. Finally, we confirmed the effects of Id1 signaling on angiogenesis using silencing RNA (siRNA) to inhibit HMVEC signaling pathways in the mouse Matrigel plug assay. Further studies of Id1 as a secreted factor included electroporation of RA FLS with a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 plasmid with a guideRNA targeting the Id1 gene. Successful expression of this plasmid will cause error-prone nonhomologous end joining (NHEJ) repair at the start codon of the Id1 gene, likely causing a nonsense mutation, resulting in an Id1 knockout cell.
Results: Histologic analysis revealed that Id1 expression correlated well with disease severity in RA STs and in K/BxN mouse ankles. We confirmed that >70% of particles in the exosome fraction of RA FLS supernatants were between 25-135 nm in diameter, which is the expected size of exosomes. We found that >80% of the Id1 released by RA FLS was encapsulated within these exosomes. Cell signaling assays showed the JNK pathway was consistently upregulated by Id1 in the cell types tested. Furthermore, we showed that inhibiting HMVEC associated JNK with siRNA reversed Id1 induced vessel formation in Matrigel plugs. Finally, we confirmed Cas9 mediated NHEJ at the start codon of the Id1 gene via Sanger sequencing and T7 endonuclease activity. These Id1 knockout FLS showed a 40% decrease in cell proliferation compared to sham transfected cells.
Conclusion: Id1 is a pleotropic molecule that has significant effects on angiogenesis, vasculogenesis, and FLS proliferation. Our data shows that Id1 is not only an important nuclear protein, but that it is also released from FLS, primarily in exosomes, thus expanding its role in the orchestration of inflammatory lesions through trans-cellular effects.
To cite this abstract in AMA style:
Edhayan G, Ha CM, Ohara RA, Isozaki T, Amin MA, Arbab AS, Campbell PL, Morgan R, Stinson WA, Friday SC, Fox DA, Ruth JH. Inhibitor of DNA Binding 1 As a Fibroblast Derived Inflammatory Angiogenic Agonist in Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/inhibitor-of-dna-binding-1-as-a-fibroblast-derived-inflammatory-angiogenic-agonist-in-rheumatoid-arthritis/. Accessed .« Back to 2015 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibitor-of-dna-binding-1-as-a-fibroblast-derived-inflammatory-angiogenic-agonist-in-rheumatoid-arthritis/