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Title: Risk stratification in atherosclerotic cartoid stenosis
Author: Shalhoub, Joseph
ISNI:       0000 0004 2710 2330
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Introduction: Key trials and a Cochrane systematic review in asymptomatic carotid stenosis have highlighted the need to identify a high-risk subgroup of patients with carotid stenosis who may benefit from intervention. Traditionally, this risk stratification has considered structural imaging and clinical factors. However, using only these approaches, still a significant number of patients are missed. Biological attributes are acknowledged as key determinants of thrombo-embolic events. Functional and hybrid structural-functional imaging, and circulating biomarkers allow exploration of plaque biology non-invasively, in vivo. The importance of innate immunity in atherosclerosis is now established, with a recent interest in macrophage phenotypic polarisation in atherosclerosis supported by in vitro and experimental data, with the hypothesis of an M1 macrophage predominance associated with unstable plaques. The emergence of systems biology has been seen to facilitate understanding of biological pathways and generate hypotheses, although the utility of this approach for the examination of human atherosclerosis tissue has not been fully explored. Aims: (i) To employ functional imaging to probe carotid atherosclerosis in vivo; (ii) to assess the plaque microenvironment in determination of the balance of macrophage populations in unstable compared with stable atherosclerosis; (iii) to investigate whether late phase (LP-) contrast enhanced ultrasound (CEUS) reflects plaque biological features; (iv) to examine the utility of systems biology techniques in distinguishing symptomatic from asymptomatic carotid atherosclerosis tissue, and in hypothesis generation; and (v) to evaluate a putative biomarker for carotid atherosclerosis and plaque vulnerability. Methods: Patients with carotid stenosis, both symptomatic and asymptomatic, have undergone systematic collection of data, fresh carotid endarterectomy (CEA) specimens, and plasma. Thirty-two patients with 36 carotid stenoses underwent 11C-PK11195 PET/CT. Thirty-seven patients had dynamic (D-) and LP-CEUS carotid imaging. CEA specimens were assessed by immunohistochemical techniques, as well as atheroma cell culture with supernatant multi-analyte profiling (MAP). MAP data was subject to Ingenuity Pathway Analysis. CEA specimens were further examined using systems biology methodologies: transcriptomics with Affymetrix Human Exon 1.0 ST arrays; proteomics and lipidomics by liquid chromatography (LC) coupled to tandem triple quadrupole mass spectrometry (MS); and metabolite profiling by nuclear magnetic resonance and LC-MS. Furthermore, venous and arterial plasma was quantified for the lysozyme, a putative biomarker in carotid atherosclerosis. Results: 11C-PK11195 PET allowed the non-invasive quantification of intraplaque inflammation in patients with carotid stenoses and, when combined with CTA, provided an integrated assessment of plaque structure, composition and biological activity. 11C-PK11195 PET/CT distinguished between recently symptomatic vulnerable plaques and asymptomatic plaques with a high positive predictive value. D-CEUS and LP-CEUS (at a cut-off of zero) was able to distinguish symptomatic and asymptomatic plaques. Atheroma cell culture and supernatant MAP revealed that symptomatic human atherosclerotic carotid disease is associated with a cytokine and chemokine pattern consistent with the predominance of pro-inflammatory M1-type macrophage polarisation. Furthermore, IFNγ signatures are observed, including the novel finding of CCL20 with its significant elevation in symptomatic atherosclerosis. MAP of supernatants from patients who had undergone ipsilateral carotid LP-CEUS revealed significantly higher levels of IL6, MMP1 and MMP3, as well as greater CD68 and CD31 immunopositivity, in those with high (≥0) compared with low (<0) LP-CEUS signal. This suggests that LP-CEUS was able to reflect plaque biology. Transcriptomic analysis was able to clearly separate stenosing plaque and intimal thickening, as well as unstable and stable atherosclerosis, finding differential expression and alternative splicing of interferon regulatory factor 5 between stenosing plaque and intimal thickening. Proteomic analysis of the salt extract fraction from carotid atherosclerotic plaques identified 2,470 proteins implicated in 33 bio-molecular functions and having their origins previously described in 14 different cellular compartments. There were 159 proteins which, based upon the number of assigned spectra, were significantly different between symptomatic and asymptomatic atherosclerosis. Through lipidomic analysis, 150 lipid species from 9 different classes were identified, of which 24 were exclusive to atherosclerotic plaques. A comparison of 28 carotid endarterectomy specimens revealed differential lipid signatures of symptomatic compared with asymptomatic lesions, as well as stable and unstable plaque areas. Similarly, LC-MS metabolite profiling of organic plaque extract was able to separate symptomatic from asymptomatic atherosclerosis. Arterial and venous plasma lysozyme levels were seen to distinguish individuals with carotid atherosclerosis from matched control subjects. Furthermore, arterial plasma lysozyme levels were significantly higher in patients with symptomatic than asymptomatic carotid stenosis. Conclusions: These findings support the use of hybrid structural-functional imaging, and the utility and use of a systems biology approach in identifying significantly different and biologically relevant variations in atherosclerosis tissue, and in hypothesis generation for further study. The data presented concurs with recent reports in the literature linking the lipidic/organic component of atherosclerosis with the generation of a pro-inflammatory plaque microenvironment prone to lesion development, instability and the complications thereof. The importance of innate immunity has been highlighted with the demonstration of a predominance of M1 macrophage polarisation and evidence of Th17/IL17 signalling in unstable atherosclerosis. It is hoped that this work will contribute to the ongoing refinement of multi-factorial risk stratification in carotid atherosclerosis.
Supervisor: Davies, Alun ; Monaco, Claudia Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral