Abstract 20139: Loss of Sam68 Attenuates Inflammatory Response in Injured Arteries and Enhances Recovery
Background: The role of Src-associated in mitosis 68 kDa (Sam68) protein in cardiovascular biology is unknown. We sought to determine whether Sam68 regulates vascular remodeling after injury.
Methods & Results: After carotid artery endothelial denudation injury, Sam68-/- mice displayed an accelerated re-endothelialization (P<0.05 at day 5 post-injury) and attenuated neointima formation (P<0.05, at day 14), which was associated with reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines (i.e., TNF-a, IL-1b, IL-6 and MCP-1) in the injured vessels. The improved carotid recovery in Sam68-/- mice was recapitulated in WT mice that had received Sam68-/- bone-marrow (BM) transplantation, and in our newly-generated CreLysM;Sam68fl/fl mice, suggesting that Sam68 impedes vascular recovery primarily by its function in macrophages. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-a-induced expression of pro-inflammatory cytokines and in the level of P-IKKαβ, P-IkBa (in cytosol) and P-P65 (in nucleus), which indicates attenuated NF-kB activation. These results were confirmed in peritoneal macrophages of Sam68-/- and WT mice. Next, Raw264.7 cells were treated with TNF-a and Vehicle, followed by Sam68 co-immunoprecipitation (co-IP) and mass-spec identification of Sam68-interacting proteins. We found that cytoskeleton protein Filamin A (FLNA) is a novel Sam68-interacting protein in macrophages in response to TNF-a treatment. Reverse co-IP and immunoblotting confirmed Sam68-FLNA interaction that requires the N-terminus of Sam68. Furthermore, analyses in Raw264.7 cells with FLNA knockdown confirmed that the role of Sam68 in TNF-a[[Unsupported Character - Codename ­]]-induced NF-kB signaling and pro-inflammatory cytokine expression was dependent on FLNA.
Conclusions: Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery, and this effect is attributable, at least partially, to the exaggerated NF-kB activity.
Author Disclosures: S. Han: None. J. Zhou: None. G. Qin: None.
- © 2016 by American Heart Association, Inc.