Gillian Cockerill

Novel aspects of the pathogenesis of aneurysms of the abdominal aorta in humans

Aneurysm of the abdominal aorta (AAA) is a particular, specifically localized form of atherothrombosis, providing a unique human model of this disease. The pathogenesis of AAA is characterized by a breakdown of the extracellular matrix due to an excessive proteolytic activity, leading to potential arterial wall rupture. The roles of matrix metalloproteinases and plasmin generation in progression of AAA have been demonstrated both in animal models and in clinical studies. In the present review, we highlight recent studies addressing the role of the haemoglobin-rich, intraluminal thrombus and the adventitial response in the development of human AAA. The intraluminal thrombus exerts its pathogenic effect through platelet activation, fibrin formation, binding of plasminogen and its activators, and trapping of erythrocytes and neutrophils, leading to oxidative and proteolytic injury of the arterial wall. These events occur mainly at the intraluminal thrombus–circulating blood interface, and pathological mediators are conveyed outwards, where they promote matrix degradation of the arterial wall. In response, neo-angiogenesis, phagocytosis by mononuclear cells, and a shift from innate to adaptive immunity in the adventitia are observed. Abdominal aortic aneurysm thus represents an accessible spatiotemporal model of human atherothrombotic progression towards clinical events, the study of which should allow further understanding of its pathogenesis and the translation of pathogenic biological activities into diagnostic and therapeutic applications.

Plasma tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9: novel indicators of left ventricular remodelling and prognosis after acute myocardial infarction

AIMSnMatrix metalloproteinase (MMP) activity is central to the development of left ventricular (LV) remodelling and dysfunction after acute myocardial infarction (AMI). We assessed the relationships with LV structure and function and outcome, of tissue inhibitors of metalloproteinase-1 (TIMP-1) and MMP-9, and compared with N-terminal pro-B-type natriuretic peptide (NTproBNP).nnnMETHODS AND RESULTSnWe studied 404 patients with AMI. Primary outcome measures were the associations of TIMP-1, MMP-9, and NTproBNP with death or heart failure, and with LV dimensions, function and remodelling (ΔLVEDV, change in LV end-diastolic volume between discharge and follow-up). Cut-off concentrations for prediction of death or heart failure were identified from receiver operator characteristic (ROC) curves. In multivariable analysis, TIMP-1 and NTproBNP had predictive value for LV ejection fraction pre-discharge (TIMP-1 P = 0.023; N-BNP P = 0.007) and at follow-up (TIMP-1 P = 0.001; N-BNP P = 0.003). MMP-9, TIMP-1, and NTproBNP correlated directly with LV volumes. MMP-9 (P = 0.005) and TIMP-1 (P = 0.036), but not NTproBNP, correlated with ΔLVEDV. For the combined endpoint of death or heart failure the area under the ROC curve was 0.640 for MMP-9, 0.799 for NTproBNP and 0.811 for TIMP-1. Patients with TIMP-1 > 135 ng/mL (P < 0.001) or NTproBNP >1472 fmol/mL (P < 0.001) had increased risk of endpoint. Consideration of both NTproBNP and TIMP-1 further improved risk stratification.nnnCONCLUSIONnTIMP-1 and MMP-9 correlate with echocardiographic parameters of LV dysfunction and remodelling after AMI and may identify patients at risk of subsequent LV remodelling and adverse prognosis.

Circulating Stromelysin-1 (MMP-3): A novel predictor of LV dysfunction, remodelling and all-cause mortality after acute myocardial infarction

Changes to cardiac matrix are central to ventricular remodelling after acute MI and matrix metalloproteinase expression is implicated in this process. We investigated the temporal profile of MMP‐3 and its relationship to LV dysfunction and prognosis following AMI.

Whole genome-expression profiling reveals a role for immune and inflammatory response in abdominal aortic aneurysm rupture.

OBJECTIVESnThis study used the whole transcriptome approach to investigate the role of genes involved in immune and inflammatory response at the site of aneurysm rupture.nnnMATERIALS AND METHODSnRupture site and paired anterior sac biopsies (internal control) of ruptured abdominal aortic aneurysms (AAAs) (n=10) were analysed with Affymetrix Human Genome U133A plus 2.0 microarray. Twenty-one differentially expressed genes were selected for validation using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR).nnnRESULTSnA total of 139 genes (123 upregulated, 16 downregulated) at the aneurysm rupture site were differentially expressed (>2.5-fold, P<0.005). Immune and inflammatory responses (Gene Ontology Classification) were frequently associated with the differentially expressed genes. Genes with immune and inflammatory functions that were confirmed, by QRT-PCR, to be overexpressed at the aneurysm rupture site were interleukins-6 and -8 (IL-6 and -8), Selectin E (SELE), prostaglandin-endoperoxidase synthase 2 (COX2) and prokineticin 2 (PROK2). IL-6 (pro-immune) and IL-8 (pro-immune and pro-inflammatory) have previously been linked to aneurysm rupture; and SELE and COX2 (pro-inflammatory) have previous associations with aneurysm development but not rupture.nnnCONCLUSIONSnThe differential expression of genes involved in immune and inflammatory responses was confirmed at AAA rupture site. These genes may represent novel targets for treatment of aneurysms.

A Randomised Placebo-controlled Double-blind Trial to Evaluate Lipid-lowering Pharmacotherapy on Proteolysis and Inflammation in Abdominal Aortic Aneurysms

OBJECTIVESnModulation of abdominal aortic aneurysm (AAA) expansion by HMG-CoA reductase inhibitors (statins) might be linked to reducing IL-6 and MMP-9, which may be consequent on reducing plasma cholesterol. Ezetimibe is a novel cholesterol absorption inhibitor used in combination with statins. This pilot study compared the biological effects of ezetimibe combination therapy with simvastatin alone on parameters relevant to aneurysm expansion including cytokines and proteolytic enzymes.nnnDESIGNnRandomised placebo-controlled double-blind trial.nnnMATERIALS & METHODSnEighteen patients scheduled for elective open AAA repair were randomised to simvastatin 40 mg plus ezetimibe 10 mg (n = 9), or simvastatin 40 mg plus placebo (n = 9), for 32.5 days (IQR 28-50.5) until the day of surgery. Total concentrations of TNF-α, IL-1β, IL-6, IL-8, IL-10, MMPs-1, -2, -3, -8, -9, -12, -13, TIMP-1 and -2 were measured in plasma, aortic wall homogenates and tissue culture explants.nnnRESULTSnTwo patients in the placebo arm did not undergo open repair precluding aortic samples. Ezetimibe was associated with a significant reduction in aortic wall MMP-9 (p = 0.02) and aortic wall IL-6 (p = 0.02), associated with a reduction in plasma lipids.nnnCONCLUSIONSnThese results suggest that ezetimibe combination therapy reduces aortic wall proteolysis and inflammation, key processes that drive AAA expansion. A larger RCT is justified focussing on aneurysm growth rates in small AAA.

Genome Wide Association Studies: identifying the genes that determine the risk of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a multifactorial disease with a strong genetic component. Aside from the major environmental risk factor of tobacco use, and the unmodifiable risk factors of advancing age and male gender, there is compelling evidence for the genetic contribution to the pathogenesis of AAA, with up to ten-fold increased risk in first degree relatives of those with AAA. However, it is widely accepted that AAA is unlikely to be associated with a single gene, and that multiple genetic factors are responsible. This makes identifying the genes involved in AAA challenging, as conventional approaches that have been successful for monogenic diseases such as cystic fibrosis and Huntingtons disease are not appropriate. One approach has been through hypothesis-driven association studies, whereby the frequency of a common variant in a candidate gene is compared between a group of unrelated affected individuals and unaffected agematched controls. The major limitation of this approach is that the selection of appropriate candidate genes relies on knowledge of the pathological processes underlying the disease in question, and since this is currently unclear for AAA, the choice of genes studied to date has been made on a ‘best guess’ basis. These studies have recently been reviewed and the genes that have been studied include inflammatory mediators, tissue proteases and their inhibitors, those involved in endothelial and smooth muscle cell biology and those coding for components of the reninangiotensin system. While such approaches have often given inconsistent results in different laboratories, metaanalysis of the available data has shown some evidence for modest risk effects associated with common variants in the genes for Angiotensin Converting Enzyme (Odds Ratio (OR) 1.33, Methylenetetrahydrofolate reductase (OR 1.14)), and matrix metalloprotease-9 (OR 1.09). A complementary approach has recently become available due to advances in genomic investigation and bioinformatics that have allowed significant proportions of the common variation in the human genome to be identified and recorded. These variations consist mainly of single nucleotide

The role of proteomic research in vascular disease

INTRODUCTIONnArterial diseases including atherosclerosis, intimal hyperplasia and aneurysms have been shown to be a product of genotype and environment. Gene expression pathways rely on protein translation to generate target effects. As a result of alternative splicing and post-translational modifications, one gene does not code for a single protein but for many. Proteomic studies allow quantification of these proteins in a biological system and determination of altered protein expression in disease. Proteomics is a powerful and expanding field of investigation which in combination with other omics may enhance understanding of disease pathophysiology and/or identify biomarkers of vascular disease. This review describes the methodology of protein mining and provides an insight into the valuable contributions already made by proteomics to vascular surgery.nnnMETHODSnMEDLINE and EMBASE databases were searched for relevant articles.nnnRESULTSn118 relevant articles were identified. These were subdivided into categories based on the aspect of protein research they reported. The subheadings include methodology, atherosclerosis, intimal hyperplasia, aortic disease and biomarkers.nnnCONCLUSIONSnDisease processes classified as genetic are functionally proteomic. Equally disease pathophysiology is the result of, or leads to alternate protein expression. Understanding the proteome will clarify the pathophysiology of disease. The translation of these findings to clinical practice impacts diagnosis, staging and treatment of disease processes. Biomarker discovery will enable earlier diagnosis of unstable atherosclerotic plaques, it will allow identification of aneurysms more likely to rupture and stratify risk. Proteomic research has enormous potential to modulate many aspects of patient care.

Proteomics and Pitfalls in the Search for Potential Biomarkers of Abdominal Aortic Aneurysms

Proteomics is evolving as an important research technique in cardiovascular disease. We present exploratory research for a systemic biomarker of abdominal aortic aneurysm (AAA) in serum. Forty patients, 20 with large AAAs and 20 matched controls, were prospectively recruited. Serum was harvested, enriched, and mined for differential protein expression. Difference in gel electrophoresis using a two-dimensional platform, cyanine labeling, and Progenesis SameSpots software identified protein spots with significantly altered intensity. Liquid chromatography mass spectrometry aligned to the Seaquest protein database characterized proteins of interest, and 436 protein spots were demonstrated from the 20 processed gels. Thirteen spots of interest, demonstrating fold change (1.7–4) between the two patient cohorts and consistent significant differential expression (analysis of variance, p ≤ .003), were picked for identification. Four of 13 spots were identified according to their tandem mass spectra. These were fragments of serum albumin, hemoglobin, and apolipoprotein C-II precursor. Identified spots represented proteins highly abundant in serum, not candidate biomarkers. Issues of variability surrounding serum harvest, processing, enrichment, and the challenge of identifying minimally expressed proteins currently limit this avenue of research. No proteins identified in this study had the biologic plausibility to represent a possible biomarker of aneurysmal disease. The tissue proteome may be a more rewarding approach for preliminary investigation of plausible biomarkers.

The Long-term Effects of Open and Endovascular Aneurysm Repair on Circulating Interleukin-6

Interleukin-6 (IL-6) is associated with abdominal aortic aneurysm (AAA) development and is an independent risk factor for cardiovascular mortality. We tested the hypothesis that aneurysm repair reduces circulating IL-6 by comparing concentrations in patients with large AAA awaiting repair (n=50) with patients having undergone open (n=34) or endovascular (n=66) repair. Only open repair was associated with a significant reduction in IL-6 (p=0.025). These results suggest that AAAs remain biologically active following endovascular repair. Aneurysm-derived IL-6 may have serious implications for cardiovascular health, and attention should be directed to modifying cardiovascular risk factors in these patients, even after successful aneurysm repair.

Comparative proteomics reveals a systemic vulnerability in the vasculature of patients with abdominal aortic aneurysms

INTRODUCTIONnAbdominal aortic aneurysms (AAA) are associated with inflammation, apoptosis, and matrix degradation. AAA tissue represents the end stage of disease, limiting its utility in identification of factors culpable for initiation of aneurysm development. Recent evidence suggests that AAAs are a local representation of a systemic disease of the vasculature. Morphologic and molecular changes, comparable to those found in the aneurysm wall, have been demonstrated in veins from patients with AAAs. Changes in the vascular tissue proteome of patients with AAAs were investigated, using inferior mesenteric vein (IMV), to gain insight into early molecular changes contributing to AAA development.nnnMETHODSnIMV was harvested from 16 patients with AAA and 16 matched controls. Whole IMV lysates were subjected to 2-D difference in gel electrophoresis (2D-DIGE) with quantitative densitometry. Protein spots differentially expressed in AAA were identified using mass spectrometry. Differential protein expression was validated by Western blotting and localized to cell type by immunohistochemistry (IHC).nnnRESULTSnDecreased levels of prohibitin (AAA, 2.00 ± 1.37; controls, 3.81 ± 1.39; 1.9-fold change; P = .02) AAA (7.33 ± 3.9; controls, 14.5 ± 5.6; 2-fold change; P = .001), along with relative increases in a cleaved fragment of vimentin (AAA, 12.9 ± 9; controls, 6.9 ± 4.7; 2-fold change; P = .11) were identified in AAA patients. All proteins were localized to the vascular smooth muscle cells.nnnCONCLUSIONSnProteins important in combating the injurious effects of oxidative stress and modulating the response to inflammation appear reduced in the vasculature of patients with AAA. These changes may represent early events in AAA formation. Enhancing expression of these proteins might offer a novel therapeutic avenue to inhibit AAA development.