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THU0072 Gastrin-Releasing Peptide (GRP) and RC-3095, a GRP Receptor Antagonist, Regulates Arthritic Mice Synovial Fibroblasts
  1. V.S. Clarimundo1,
  2. M. Farinon1,
  3. C. Nör1,
  4. L.I. Filipin2,
  5. P.S. Gulko3,
  6. R.M. Xavier4,
  7. P.G. Oliveira1
  1. 1Universidade Federal do Rio Grande do Sul, Porto ALegre
  2. 2Centro Universitário La Salle, Canoas, Brazil
  3. 3Icahn School of Medicine, New York, United States
  4. 4Reumatologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Abstract

Background Rheumatoid Arthritis (RA) is characterized by invasion of fibroblast-like synoviocytes (FLS) into de articular cartilage and bone erosion leading to progressive joint destruction. FLS invasiveness correlates with articular damage in RA, yet little is known about this regulation. Gastrin-releasing peptide (GRP) is a functional homologue of bombesin, and its receptor signaling is involved in several functions, including the inflammatory response. GRP and its receptor (GRPR) have been found in synovial membrane and fluid of RA patients. RC-3095 is an antagonist of GRPR.

Objectives To evaluate the presence and role of gastrin-releasing peptide and its receptor on FLS from arthritic mice.

Methods FLS were isolated from the ankle joints of DBA/1J mice with collagen-induced arthritis. Expression of GRPR protein in FLS was analyzed by immunocitochemistry and western blot (WB), and qPCR was used to measure expression levels of GRPR and GRP. Viability of FLS treated with GRP (0.1, 1 and 10 μM) or RC-3095 (0.05 to 10μM) was assessed by MTT assay in 24h and 48h. FLS (n=6) invasion was measured using a Matrigel-coated transwell invasion system in the presence of GRP (10 μM), RC-3095 (1μM) or GRP+RC-3095 (GRP 10 μM and RC 1μM) over 24 hours, in duplicate. Cytosol and culture supernatant GRP was measured by ELISA after treatment with GRP (10 μM), RC-3095 (1 μM) or GRP+RC-3095 (GRP 10 μM and RC-3095 1μM).

Results GRPR protein and mRNA was present in FLS. GRP mRNA was also detected. RC-3095 and GRP were not cytotoxic for FLS in the used concentrations. GRP increased FLS invasion by nearly two-fold (5356±767) compared with untreated FLS (2888±386) (p<0,02), whereas RC-3095 reversed that effect (1722±271) (p<0,005). GRP+RC-3095 (2670±499) decreased FLS invasion compared with GRP (p<0,0001). RC-3095 (206±10,55) increased GRP levels in supernatant comparing with GRP (334±61,26) (p<0,02) and with GRP+RC-3095 (211±12,39) (p<0,03). In cytosol no statistical difference was observed.

Conclusions This is the first study to identify the presence and activity of the GRP-GRPR pathway in FLS. These cells expressed the GRPR receptor and secreted GRP. RC-3095 was able to decrease FLS invasion while GRP increased. These findings suggest that interference with the GRP pathway is a potential therapeutic target on FLS for treatment to rheumatoid arthritis using RC-3095 for future clinical trials.

References

  1. Grimsholm O, et al. Neuropeptides. 2008.

  2. Laragione T, Gulko PS. Mol Med. 2012.

  3. Oliveira PG, et al. Arthritis Rheum. 2011.

Acknowledgements FIPE, CNPq, CAPES

Disclosure of Interest None declared

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