Introduction: RAGE activation plays a pivotal role in the pathogenesis of diabetic vasculopathy, however the signal transduction mechanism downstream of RAGE is not fully understood. Yoshimoto et al. previously established the RAGE-overexpressed vascular smooth muscle cell line, RAGE-A10, and found that its slow growth rate and increased monocyte chemotactic protein-one (MCP-1) mRNA expression were analogous to the phenomenon of senescence. Senescent cells display characteristic morphology, including increased senescence-associated ß-galactosidase (SA-ßG) activity, increased proinflammatory status and a slow growth rate.
Aim: This study was undertaken to investigate the possible cellular senescence of RAGE-A10 and determine its potential signal transduction mechanism(s).
Method: Four rat VSMC cell lines (A10) expressing human RAGE and control cell lines pMX-A10 were cultured. SAßG staining cells were counted by light microscopy. Enzyme-linked immunosorbent assay measured MCP-1 pro-inflammatory protein levels. RAGE cells were treated for 72 h with ligand S100B; NAD(P)H oxidase inhibitor, apocynin; inhibitor of IκB phosphorylation, Bay11074; MEK1 inhibitor, U0126; and Src inhibitor, PP2, all known to be involved in diabetic vasculopathy, Flow cytometry examined cell cycle distribution.
Results: RAGE-A10 cells demonstrated increased SA-ßG activity, significantly increased protein expression of MCP-1 (P<0.05) with ligand stimulation and a slower growth rate, due to delayed G1/S phase cell cycle progression. NF-κB, MAPK and non-receptor tyrosine kinase Src involvement in ligand-induced MCP-1 expression in RAGE-A10 was clearly shown. However increased SA-ßG activity and reduced growth rate appear to be mediated by a ligand-independent signaling pathway.
Conclusion: Our findings demonstrate that RAGE-overexpressed A10 cell lines exhibit characteristic hallmarks of senescence, represented by increased SA-ßG activity, increased proinflammatory status and a slower rate of cell proliferation, via an as yet unknown signal transduction pathway(s). Further investigation into mechanisms downstream of RAGE may provide clearer insight into potential therapeutic targets against RAGE and senescence in the treatment of diabetic vasculopathy.