Proteomic Characterisation of Vascular Smooth Muscle Cells in Atherosclerosis
Author(s)
Full, Louise Elizabeth
Type
Thesis
Abstract
During atherosclerosis, smooth muscle cells (SMC) migrate to the intima, losing their
contractile ability and reacquiring characteristics seen during development: migration, proliferation,
and synthesis of extracellular matrix – a process known as “phenotypic modulation”. I hypothesised
that these phenotypic and functional characteristics indicate underlying differences in protein
expression between intimal and medial SMC, which could constitute a characteristic signature
indicative of the diseased state.
I utilised 2-dimensional gel electrophoresis (2DE) and tandem mass spectrometry to compare
the cytosolic proteome of the human SMC from atherosclerotic lesions with that of non-diseased
SMC. Novel findings were validated by immunoblotting and immunohistochemistry. I detected
decreases in contractile proteins and an increase in the oxidised form of peroxiredoxins in carotid
atheroma-derived SMC - compared to non-diseased SMC. These changes indicate respectively loss of
contractile features and exposure to oxidative stress, and are in accordance with the existing literature.
I also identified significant decreases in the key mitochondrial proteins ATP synthase subunit-β and
aldehyde dehydrogenase-2 potentially linked to mitochondrial damage.
More notably, I described for the first time that differences in expression of members of the
annexin family distinguish SMC derived from stable atheroma and unstable atheroma. The antiinflammatory
protein Annexin I was significantly up regulated in SMC derived from stable plaques.
Annexin II, a protein involved in facilitating fibrinolysis, was significantly down regulated in SMC
derived from unstable plaques. These novel findings suggest that the failure of anti-inflammatory and
antithrombotic mechanisms provided by SMC might play a role in plaque instability.
Finally, I investigated the effect of the exposure to pro-atherogenic factors -cholesterol and the
proinflammatory cytokine tumour necrosis factor α (TNFα) - on the proteome of SMC. With this, I
was able to reproduce some of the features distinguishing atheroma-derived SMC, including the down
regulation of contractile proteins and signs of mitochondrial damage. In addition I uncovered features
of priming to challenge with TNFα in atheroma-derived SMC, potentially mediated by an increase of
the p75 receptor. In contrast, cholesterol loading of SMC induced changes primarily in endoplasmic
reticulum proteins, in line with current hypotheses in the literature of the activation of the Unfolded
Protein Response – a protective mechanism engaged in conditions of endoplasmic reticulum stress.
The proteome of SMC in advanced human atheroma has never been investigated before,
particularly in the context of an unstable plaque. My data reveal an intricate relationship between
inflammation, oxidative stress, mitochondrial damage and down regulation of mechanisms of
resolution of inflammation in SMC in the context of vascular disease and its complications.
contractile ability and reacquiring characteristics seen during development: migration, proliferation,
and synthesis of extracellular matrix – a process known as “phenotypic modulation”. I hypothesised
that these phenotypic and functional characteristics indicate underlying differences in protein
expression between intimal and medial SMC, which could constitute a characteristic signature
indicative of the diseased state.
I utilised 2-dimensional gel electrophoresis (2DE) and tandem mass spectrometry to compare
the cytosolic proteome of the human SMC from atherosclerotic lesions with that of non-diseased
SMC. Novel findings were validated by immunoblotting and immunohistochemistry. I detected
decreases in contractile proteins and an increase in the oxidised form of peroxiredoxins in carotid
atheroma-derived SMC - compared to non-diseased SMC. These changes indicate respectively loss of
contractile features and exposure to oxidative stress, and are in accordance with the existing literature.
I also identified significant decreases in the key mitochondrial proteins ATP synthase subunit-β and
aldehyde dehydrogenase-2 potentially linked to mitochondrial damage.
More notably, I described for the first time that differences in expression of members of the
annexin family distinguish SMC derived from stable atheroma and unstable atheroma. The antiinflammatory
protein Annexin I was significantly up regulated in SMC derived from stable plaques.
Annexin II, a protein involved in facilitating fibrinolysis, was significantly down regulated in SMC
derived from unstable plaques. These novel findings suggest that the failure of anti-inflammatory and
antithrombotic mechanisms provided by SMC might play a role in plaque instability.
Finally, I investigated the effect of the exposure to pro-atherogenic factors -cholesterol and the
proinflammatory cytokine tumour necrosis factor α (TNFα) - on the proteome of SMC. With this, I
was able to reproduce some of the features distinguishing atheroma-derived SMC, including the down
regulation of contractile proteins and signs of mitochondrial damage. In addition I uncovered features
of priming to challenge with TNFα in atheroma-derived SMC, potentially mediated by an increase of
the p75 receptor. In contrast, cholesterol loading of SMC induced changes primarily in endoplasmic
reticulum proteins, in line with current hypotheses in the literature of the activation of the Unfolded
Protein Response – a protective mechanism engaged in conditions of endoplasmic reticulum stress.
The proteome of SMC in advanced human atheroma has never been investigated before,
particularly in the context of an unstable plaque. My data reveal an intricate relationship between
inflammation, oxidative stress, mitochondrial damage and down regulation of mechanisms of
resolution of inflammation in SMC in the context of vascular disease and its complications.
Date Issued
2010-08
Date Awarded
2011-01
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Monaco, Claudia
Saklatvala, Jeremy
Wait, Robin
Creator
Full, Louise Elizabeth
Publisher Department
The Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)