Imaging of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of PET Tracers for Vascular Inflammation

Atherosclerosis is a vascular inflammatory disease causing coronary artery disease, myocardial infarct and stroke, the leading causes of death in Finland and in many other countries. The development of atherosclerotic plaques starts already in childhood and is an ongoing process throughout life. Rupture of a plaque and the following occlusion of the vessel is the main reason for myocardial infarct and stroke, but despite extensive research, the prediction of rupture remains a major clinical problem. Inflammation is considered a key factor in the vulnerability of plaques to rupture. Measuring the inflammation in plaques non-invasively is one potential approach for identification of vulnerable plaques. The aim of this study was to evaluate tracers for positron emission tomography (PET) imaging of vascular inflammation. The studies were performed with a mouse model of atherosclerosis by using ex vivo biodistribution, autoradiography and in vivo PET and computed tomography (CT). Several tracers for inflammation activity were tested and compared with the morphology of the plaques. Inflammation in the atherosclerotic plaques was evaluated as expression of active macrophages. Systematic analysis revealed that the uptake of 18F-FDG and 11C-choline, tracers for metabolic activity in inflammatory cells, was more prominent in the atherosclerotic plaques than in the surrounding healthy vessel wall. The tracer for αvβ3 integrin, 18Fgalacto- RGD, was also found to have high potential for imaging inflammation in the plaques. While 11C-PK11195, a tracer targeted to receptors in active macrophages, was shown to accumulate in active plaques, the target-to-background ratio was not found to be ideal for in vivo imaging purposes. In conclusion, tracers for the imaging of inflammation in atherosclerotic plaques can be tested in experimental pre-clinical settings to select potential imaging agents for further clinical testing. 18F-FDG, 18F-galacto-RGD and 11C-choline choline have good properties, and further studies to clarify their applicability for atherosclerosis imaging in humans are warranted.

Conventional and novel cardiovascular risk factors in young Finns and their associations with structural and functional vascular changes of subclinical atherosclerosis. The Cardiovascular Risk in Young Finns Study

Background: Atherosclerosis begins in early life progressing from asymptomatic to symptomatic as we age. Although substantial progress has been made in identifying the determinants of atherosclerosis in middle to older age adults at increased cardiovascular risk, there is lack of data examining determinants and prediction of atherosclerosis in young adults. Aims: The current study was designed to investigate levels of cardiovascular risk factors in young adults, subclinical measures of atherosclerosis, and prediction of subclinical arterial changes with conventional risk factor measures and novel metabolic profiling of serum samples. Subjects and Methods: This thesis utilised data from the follow-ups performed in 2001 and 2007 in the Cardiovascular Risk in Young Finns study, a Finnish population-based prospective cohort study that examined 2,204 subjects who were aged 30-45 years in 2007. Subclinical atherosclerosis was studied using noninvasive ultrasound measurements of carotid intima-media thickness (IMT), carotid arterial distensibility (CDist) and brachial flow-mediated dilation (FMD). Measurements included conventional risk factors and metabolic profiling using highthroughput nuclear magnetic resonance (NMR) methods that provided data on 42 lipid markers and 16 circulating metabolites. Results: Trends in lipids were favourable between 2001 and 2007, whereas waist circumference, fasting glucose, and blood pressure levels increased. To study the stability of noninvasive ultrasound markers, 6-year tracking (the likelihood to maintain the original fractile over time) in 6 years was examined. IMT tracked more strongly than CDist and FMD. Cardiovascular risk scores (Framingham, SCORE, Finrisk, Reynolds and PROCAM) predicted subclinical atherosclerosis equally. Lipoprotein subclass testing did not improve the prediction of subclinical atherosclerosis over and above conventional risk factors. However, circulating metabolites improved risk stratification. Tyrosine and docosahexaenoic acid were found to be novel biomarkers of high IMT. Conclusions: Prediction of cardiovascular risk in young Finnish adults can be performed with any of the existing risk scores. The addition of metabonomics to risk stratification improves prediction of subclinical changes and enables more accurate targeting of prevention at an early stage.

Metabolic syndrome – early cardio-metabolic, vascular and hepatic changes

Background: Metabolic syndrome (MetS) is a combination of several cardio-metabolic risk factors including obesity, hyperglycemia, hypertension and dyslipidemia. MetS has been associated with increased levels of apolipoprotein B (apoB) and low-density lipoprotein oxidation (OxLDL) and with an increased risk of cardiovascular disease and non-alcoholic fatty liver disease. Aims: To establish the relation of apoB and OxLDL with the MetS development and to determine the status of MetS as a risk factor for adverse liver changes and for subclinical atherosclerosis. Subjects and Methods: The present thesis is part of the two large scale population-based, prospective, observational studies. Cardiovascular Risk in Young Finns study was launched in 1980 including 3,596 subjects aged 3-18 years. Thereafter follow-up studies have been conducted regularly. In the latest follow-ups that were performed in 2001 (N=2,283) and 2007 (N=2,204), non-invasive ultrasound studies were introduced to the study protocol to measure subclinical atherosclerosis i.e. carotid intima-media thickness (IMT), carotid artery distensibility (Cdist) and brachial flow-mediated dilatation (FMD). Alanine-aminotransferase (ALT) and gammaglutamyltransferase (GGT) were measured in 2007 to assess liver function. The Bogalusa Heart Study is a long-term epidemiologic study of cardiovascular risk factors launched in 1972 in a biracial community of Bogalusa, Louisiana, USA. Total of 374 youths (aged 9-18 years at baseline in 1984-88) who underwent non-invasive ultrasound studies of the carotid artery as adults, were included in the analyses of the present thesis. Results: The odds ratios (95% confidence intervals) for MetS incidence during a 6-year follow-up by quartiles of apoB were 2.0(1.0-3.8) for the second quartile, 3.1(1.7-5.7) for the third quartile and 4.2(2.3-7.6) for the fourth quartile. OxLDL was not independently associated with incident MetS. Youth (aged 9-18 years) with MetS or with high body mass index were at 2-3 times the risk of having MetS, high IMT, and type 2 diabetes 24-years later as adults. IMT increased 79±7μm (mean±SEM) in subjects with MetS and 42±2μm in subjects without the MetS (P<0.0001) during 6- years. Subjects who lost the MetS diagnosis during 6-year follow-up had reduced IMT progression compared to persistent MetS group (0.036±0.005vs.0.079±0.010 mm, P=0.001) and reduced Cdist change compared to incident MetS group (-0.12±0.05vs.-0.38±0.10 %/mmHg, P=0.03) over 6-year follow-up. MetS predicted elevated ALT (β±SEM=0.380±0.052, P<0.0001 in men and 0.160±0.052, P=0.002 in women) and GGT (β±SEM=0.240±0.058, P<0.0001 in men and 0.262±0.053, P<0.0001 in women) levels after 6-years. Conclusions: These findings suggest that apoB may give additional information on early metabolic disturbances predisposing MetS. MetS may be used to identify individuals at increased risk of developing atherosclerosis and non-alcoholic liver disease. However, recovery from the MetS may have positive effects on liver and vascular properties.

PV-1: Leukocyte trafficking molecule and vascular marker

The distinction between lymphatic vessels and blood vessels is a crucial factor in many studies in immunology, vascular biology and cancer biology. They both share several characteristics and perform related, though different functions. They are equally important for the performance of the human immune system with the continuous recirculation of leukocytes from the tissues via lymphatics to the blood vessels and back into the tissue presenting the link between both systems. This study was undertaken to elucidate the differences in the gene expression between primary blood- and lymphatic endothelial cells as well as the two immortalized cell lines HMEC-1 (human microvascular endothelial cell line 1) and TIME (telomerase immortalized microvascular endothelial cell line). Furthermore, we wanted to investigate the mystery surrounding the identity of the antigen recognized by the prototype blood vascular marker PAL-E. In the last step we wanted to study whether the PAL-E antigen would be involved in the process of leukocyte migration from the bloodstream into the surrounding tissue. Our results clearly show that the gene expression in primary blood endothelial cells (BEC), lymphatic endothelial cells (LEC) and the cell lines HMEC-1 and TIME is plastic. Comparison of a large set of BEC- and LEC datasets allowed us to assemble a catalog of new, stable BEC- or LEC specific markers, which we verified in independent experiments. Additionally, several lines of evidence demonstrated that PAL-E recognizes plasmalemma vesicle associated protein 1 (PV-1), which can form complexes with vimentin and neuropilin-1. Finally, numerous in vitro and in vivo experiments identify the first function of the protein PV-1 during leukocyte trafficking, where it acts as regulatory molecule.