Original articleSam68 impedes the recovery of arterial injury by augmenting inflammatory response
Introduction
Rapid expanding endovascular techniques, including angioplasty and stenting, are important options for patients with coronary and peripheral arterial disease. However, the procedure-associated injuries can result in adverse vascular remodeling characterized by impaired endothelial recovery, neointima hyperplasia, and loss of arterial lumen (i.e., restenosis); and currently restenosis remains one of the major barriers to the full success of these techniques [1]. Although the exact mechanisms of restenosis are not completely understood, experimental evidence suggests that vascular inflammatory response plays a critical role. Arterial injury first activates cytokine gene expression in monocytes, macrophages and/or vascular smooth muscle cells (VSMCs), evoking secondary, self-sustaining autocrine and paracrine growth factor and cytokine expression [[2], [3], [4]]. This cytokine-growth factor cascade appears to contribute to VSMC proliferation and migration, resulting in neointima hyperplasia [5]. Mononuclear phagocytes are important contributors in these processes, in part via secretion of pro-inflammatory cytokines and chemokines.
Among various pro-inflammatory cytokines, TNF-α is perhaps the most potent one. It represses post-injury re-endothelialization and promotes VSMC proliferation [6]. The pro-inflammatory activities of TNF-α are primarily mediated by TNF-α receptor 1 (TNFR1), which initiates a pro-survival pathway through the activation of the transcription factor NF-κB [7]. It is known that NF-κB plays a central role in the cellular response to pro-inflammatory stimuli and in the expression of pro-inflammatory cytokines. Repression of NF-κB activation has been suggested and tested an effective approach for restenosis prevention [8,9]. TNF-α stimulation of cells leads to formation of an early complex I in the membrane within minutes, which result in NF-κB activation [10]. The membrane complex I is composed of TNFR, TNFR1-associated DEATH domain protein (TRADD), receptor-interacting protein (RIP), TNFR-associated factor 2 (TRAF2), inhibitor of apoptosis proteins (cIAPs), and IkappaB kinases (IKKs). The TRAF2 is a prototypical member of the TRAF family and is crucial for TNF-α induction of NF-κB activation [11,12]. Notably, TNF-α mediated NF-κB signaling is critically dependent on cytoskeleton. For example, it has been shown that TRAF2 interacts with Filamin A (FLNA), an actin cross-linking protein; in the FLNA-deficient cells, TNF-α fails to activate NF-κB [13].
Src-associated-in-mitosis-68-KD (Sam68) is an RNA binding protein and Src kinase substrate. It can act as an adaptor protein and involve in the regulation of cellular metabolism, nuclear export and stability of RNAs [14]. However, the role of Sam68 in cardiovascular biology has not been studied. Interestingly, emerging evidence suggests that Sam68 is a necessary component of the TNFR early complex I and contributes to TNF-α–induced NF-κB activation in MEF, T cells and fibroblast-like synoviocytes [[15], [16], [17]]. Specifically, Sam68 has been shown to interact with TRAF2, and its deficiency impairs the maintenance of the recruited TRAF2 at TNFR [15]. However, it is unknown whether Sam68 mediated regulation of TNF-α/NF-κB signaling involves cytoskeleton and whether it plays a role in vascular injury response.
In this study, we found that upon TNF-α stimulation, Sam68 interacts with FLNA to bridge TRAF2, thus TNFR complex I, with cytoskeleton to sustain NF-κB signaling in macrophages. Genetic deletion of Sam68 in mice significantly decreases NF-κB activation in macrophages, ameliorates inflammatory response and adverse remodeling of carotid artery from endothelial denudation injury.
Section snippets
Materials and methods
The authors declare that all supporting data are available within the article and its online Supplementary Materials files. The Major Resource Table is given as an online-only Supplementary Materials file.
Deletion of Sam68 leads to accelerated re-endothelialization and attenuated neointima hyperplasia and macrophage infiltration in the injured carotid artery
To understand the role of Sam68 in the vascular response to injuries, we induced wire-mediated endothelial denudation injury in the left carotid artery of Sam68−/− and WT mice. Re-endothelialization and neointima formation were evaluated at serial time points post-injury by Evan's Blue perfusion with en face photograph and by cross-section H.E. staining of neointima thickness, respectively. While the initial areas of injury were similar between the two groups, Sam68−/− mice demonstrated a
Discussion
In this report, we have provided compelling evidence that Sam68 is an essential component of the TNF-α/NF-κB signaling during vascular inflammatory response to denudation injury. Upon TNF-α stimulation in macrophages, Sam68 interacts with TRAF2 of the TNFR complex and the cytoskeleton protein FLNA to enhance NF-κB activation. Genetic deletion of Sam68 in mice or in the transplanted BM cells significantly reduces NF-κB signaling, inflammatory cytokine expression and macrophage infiltration in
Sources of funding
This work was supported by the National Institute of Health (R01 Grants# HL093439, HL113541, HL131110, HL138990 to G.Q.; HL142291 to H.Q & G.Q.); American Diabetes Association (Grant# 1-15-BS-148 to G.Q.); American Heart Association (Grant# 19TPA34910227 to G.Q.; 15POST25340008 to S.H.; 16POST29820001 to L.Y.; 18POST34070088 to S.X.; 18PRE34080358 to E.Z.; and 19CDA34630052 to A.Q.).
Author contributions
S.H., S.X., J.Z., and G.Q. conceived and designed the project. S.H., S.X., J.Z., A.Q, W.M., C.B., H.L, D.B., L.Y., and E.Z. acquired and analyzed data. S.R. provided key materials, Q.L., S.J., T.C.Z., P.K., C.Z., and J.Z. assisted data interpretation. S.H., S.X. and G.Q. wrote the manuscript.
Declaration of Competing Interest
The authors declare no competing financial interests.
Acknowledgement
We thank Dr. Chi Wai Eric So (The Institute of Cancer Research, Sutton, UK) for providing us with Myc-Sam68 plasmid.
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Equal contributions.