Original article
Smooth muscle cells in human atherosclerosis: Proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease

https://doi.org/10.1016/j.yjmcc.2012.11.002Get rights and content

Abstract

Smooth muscle cells (SMC) contribute to the development and stability of atherosclerotic lesions. The molecular mechanisms that mediate their properties are incompletely defined. We employed proteomics and in vitro functional assays to identify the unique characteristics of intimal SMC isolated from human carotid endarterectomy specimens and medial SMC from thoracic aortas and carotids. We verified our findings in the Tampere Vascular Study. Human atheroma-derived SMC exhibit decreased expression of mitochondrial proteins ATP Synthase subunit-beta and Aldehyde dehydrogenase 2, and decreased mitochondrial activity when compared to control SMC. Moreover, a comparison between plaque-derived SMC isolated from patients with or without recent acute cerebrovascular symptoms uncovered an increase in Annexin A1, an endogenous anti-inflammatory protein, in the asymptomatic group. The deletion of Annexin A1 or the blockade of its signaling in SMC resulted in increased cytokine production at baseline and after stimulation with the pro-inflammatory cytokine Tumor Necrosis Factor α. In summary, our proteomics and biochemical analysis revealed mitochondrial damage in human plaque-derived SMC as well as a role of Annexin A1 in reducing the production of pro-inflammatory mediators in SMC.

Highlights

► The proteome of human atheroma-derived SMC (AthSMC) has not been studied before ► AthSMC have decreased levels of ALDH2 and ATP5B ► AthSMC have decreased mitochondrial activity ► AthSMC from asymptomatic patients show decreased levels of ANXA1 ► ANXA1 signaling reduces cytokine production in SMC

Introduction

The seminal work of Campbell and Campbell demonstrated that smooth muscle cells (SMC) undergo ‘phenotypic modulation’ from a quiescent to a synthetic state within the atherosclerotic intima to accommodate an increased synthesis of extracellular proteins [1] that strengthen the fibrous cap [2]. Thus, the survival of SMC is essential to plaque stability [2]. Classical studies by Bennett and Schwartz have shown that cultured SMC isolated from atherosclerotic lesions display decreased rates of proliferation and increased senescence and apoptosis compared to medial SMC [3]. Importantly, apoptosis of SMC led to increased inflammatory cell infiltration, suggesting that the survival of SMC may modulate inflammation within atherosclerotic plaques [4]. The molecular mechanisms through which SMC can modulate the inflammatory response are currently unknown.

Proteomics approaches provide an unbiased assessment of the protein components of diseased tissues. Proteomics analyses in carotid tissue have generated biomarkers for cardiovascular events, such as osteopontin [5]. Only transcriptomic approaches have so far been applied to SMC from human atherosclerotic lesions [6], [7], while no study has thus far compared the proteome of SMC derived from symptomatic or asymptomatic carotid disease and control vascular tissue.

In order to identify qualitative protein changes in SMC in established human atherosclerosis, we performed a proteomics characterization of SMC derived from carotid artery stenosis and control vascular tissue. We herein describe that Annexin A1 (ANXA1) levels in SMC are modulated in carotid disease and that ANXA1 signaling blockade or its deletion increases cytokine and chemokine production by SMC. We also show that mitochondrial damage is a distinctive feature of SMC isolated from human atherosclerotic lesions.

Section snippets

Materials and methods

An expanded Materials and methods section is available in the Supplementary data.

AthSMC show evidence of phenotypic modulation

Both AthSMC and AoSMC appeared spindle-shaped and displayed the characteristic hill and valley pattern at confluence (Fig. S1A). Evaluation of the contractile markers smooth muscle α-actin (αSMA) and smooth muscle myosin heavy chain (SM-MHC) by PCR, immunofluorescence and immunoblotting demonstrated a decrease in αSMA and SM-MHC expression in AthSMC compared with AoSMC (Fig. S1B–D).

Eighty-four spots on the 2D gels were identified by Mass Spectrometry (Table SIII). The 2D gel analysis with

Discussion

SMC are important arbiters of the outcome of atherosclerotic plaque formation. Our study is the first to characterize the proteome of SMC derived from human atherosclerotic lesions (AthSMC), and it reveals that AthSMC exhibit signs of exposure to oxidative stress and mitochondrial damage when compared to control SMC (AoSMC). We also show that SMC isolated from asymptomatic patients are qualitatively different from SMC isolated from symptomatic patients, as they have an increased expression of

Conclusions

In established human atherosclerosis SMC acquire a distinct protein “signature” that suggests exhaustion of antioxidant mechanisms and mitochondrial damage. We identify biological differences between SMC extracted from patients with symptomatic and asymptomatic carotid disease, including the increased expression of Annexin A1 in SMC resident in asymptomatic carotid stenosis. Our findings underscore the protective role of SMC in advanced atherosclerosis and suggest Annexin A1 signaling as a

Disclosure statement

None declared.

Acknowledgements

This study was supported by European Commission (LSHM-CT-2006-037400; IMMUNATH and FP7/2007-2013; 201668; AtheroRemo), The Graham-Dixon Charitable Trust, Yrjö Jahnsson Foundation, Medical Research Fund of Tampere University Hospital (9M048) and The Finnish Cardiovascular Foundation.

References (31)

  • D.P.V. de Kleijn et al.

    Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular events

    Arterioscler Thromb Vasc Biol

    (2010)
  • S.K. Payeli et al.

    Prothrombotic gene expression profile in vascular smooth muscle cells of human saphenous vein, but not internal mammary artery

    Arterioscler Thromb Vasc Biol

    (2008)
  • Q.J. Zhang et al.

    Differential gene expression in vascular smooth muscle cells in primary atherosclerosis and in stent stenosis in humans

    Arterioscler Thromb Vasc Biol

    (2002)
  • M. Levula et al.

    Genes involved in systemic and arterial bed dependent atherosclerosis—Tampere Vascular study

    PLoS One

    (2012)
  • J.E. Cole et al.

    Unexpected protective role for Toll-like receptor 3 in the arterial wall

    Proc Natl Acad Sci USA

    (2011)
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    These authors contributed equally to this manuscript.

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