title: RGD peptide-induced cell death of chondrocytes and synovial cells.
abstract: BACKGROUND
  Small peptides including the Arg-Gly-Asp (RGD) motif have been used in studies on cell-extracellular matrix (ECM) attachment due to their ability to disturb integrin-mediated attachment on the cell surface. As another biological action of RGD peptides, several reports have shown that RGD peptides are incorporated into cytoplasm and induce apoptosis by direct activation of caspase-3. This study evaluated the effect of RGD peptides on chondrocytes and synovial cells and studied the involvement of caspases.
  
  
  METHODS
  Chondrocytes and synovial cells were isolated and cultured from the knee joints of New Zealand White rabbits. Cells were incubated in serum-free medium with peptides (RGD, RGDS, GRGDSP, GRGDNP, RGES), and the survival rates were evaluated. The rate of apoptotic cells was measured by flow cytometry in cells treated with RGDS, GRGDSP, and RGES. Caspase-3, -8 and -9 activity was measured in cells treated with RGDS and GRGDSP. Osteochondral explants harvested from rabbits were also incubated with RGD peptides (RGDS, GRGDSP, and GRGDNP), and the survival rate of chondrocytes was evaluated.
  
  
  RESULTS
  The survival rate of cultured chondrocytes was significantly decreased in the GRGDSP- and GRGDNP-treated groups. The survival rate of synovial cells was significantly decreased with four of the RGD peptides (RGD, RGDS, GRGDSP, and GRGDNP) at 5 mM, and in the RGDS- and GRGDSP-treated groups at 1 mM. Flow cytometric assay revealed increases of apoptotic chondrocytes with GRGDSP and increases of apoptotic synovial cells with RGDS and GRGDSP. Caspase-3 was activated in chondrocytes treated with GRGDSP and it was also activated in synovial cells treated with RGDS and GRGDSP. Caspases-8 and -9 were not activated in chondrocytes or in synovial cells. The survival rate of chondrocytes in explants decreased in the superficial layer with all three RGD peptides (RGDS, GRGDSP, and GRGDNP) and in the middle layer with GRGDSP.
  
  
  CONCLUSIONS
  RGD peptides induced apoptosis in cultured chondrocytes as well as in cells in cartilage explants and synovial cells, presumably through direct activation of caspase-3.
authors.affiliation: Department of Orthopaedic Surgery, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Matsuki
authors.forename: Takahisa
authors.initials: T
authors.lastname: Sasho
authors.forename: Koichi
authors.initials: K
authors.lastname: Nakagawa
authors.forename: Masamichi
authors.initials: M
authors.lastname: Tahara
authors.forename: Kaori
authors.initials: K
authors.lastname: Sugioka
authors.forename: Nobuyasu
authors.initials: N
authors.lastname: Ochiai
authors.forename: Shuhei
authors.initials: S
authors.lastname: Ogino
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Wada
authors.forename: Hideshige
authors.initials: H
authors.lastname: Moriya
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes; D009842:Oligopeptides / Q000502:physiology*; D010003:Osteoarthritis / Q000503:physiopathology; D011817:Rabbits; D013583:Synovial Membrane / Q000166:cytology*
chemical_list: D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D053148:Caspase 3