Inflammation is important in both the pathogenesis and outcome of atherosclerosis. Plaques containing numerous inflammatory cells, particularly macrophages, have a high risk of rupture, whereas those with fewer inflammatory cells are at lower risk. The current ‘gold standard’ technique for imaging atherosclerosis is x-ray contrast angiography, which provides high-resolution definition of the site and severity of luminal stenoses, but no information about plaque inflammation. Quantification of plaque inflammation is desirable both to predict risk of plaque rupture and to monitor the effects of atheroma-modifying therapies. This is important since recent studies strongly suggest that HMG Co-A reductase inhibitors promote plaque stability by decreasing plaque macrophage content and activity without substantially reducing plaque size and therefore angiographic appearance. FDG is a glucose analogue that is taken up by cells in proportion to their metabolic activity. In this work, the central hypothesis was that plaque inflammation could be visualised and quantified non-invasively using FDG-PET. Initially, THP-1 monocytes and buffy-coat macrophages were stimulated with cellular activators, and the effect on deoxyglucose uptake was observed. It was demonstrated that both types of cell accumulated deoxyglucose in proportion to their metabolic activity. Next, FDG uptake was assessed in endarterectomy specimens from patients with symptomatic carotid disease. Autoradiography of excised plaques confirmed accumulation of deoxyglucose in macrophage-rich areas. Subsequently, co-registered FDG-PET imaging was performed in patients with transient ischaemic attack. FDG accumulated within carotid plaques, with significantly more FDG being taken up into symptomatic plaques than contralateral asymptomatic lesions. Finally, a rabbit model of atherosclerosis was established to investigate two related questions: firstly, whether an animal PET scanner (MicroPet) might detect atheroma, and secondly whether FDG-PET could image and perhaps quantify both atheroma progression and regression. Aortic atheroma was identified by FDG-PET, but full quantification was not possible, because the microPet system is currently unable to perform studies with attenuation correction. In summary, it has been shown, both in vitro and in vivo, that inflammation within atherosclerotic plaques can be successfully imaged by FDG-PET. In addition, pilot data from an experimental study of atherosclerosis in rabbits suggested that serial imaging with this technique might be useful for monitoring the effects of anti-atheroma drugs.

Statement of the problem: Many methods for surrogate outcome validation require individual patient data which is often inaccessible by clinical trialists. Methods: A review was performed to identify statistical methods for surrogate outcome validation that may be implemented using summary data from published clinical trials. The methods were used to evaluate carotid intima-media thickness (CIMT) as a surrogate outcome for cardiovascular events in a systematic review of randomized trials of interventions for atherosclerosis. Results: the review of methods identified five procedures. At two or more years of follow-up, there was a marginally significant association of CIMT with myocardial infarction and a statistically significant association with cardiovascular mortality. At ≥ four years of follow-up, a statistically significant, negative relationship was observed between CIMT and stroke. Conclusions: CIMT may be a valid surrogate outcome for myocardial infarction and cardiovascular mortality. Additional data is needed to evaluate CIMT in specific drug classes.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Introduction: Rheumatoid arthritis (RA) is a systemic autoimmune condition, characterised by an inflammatory arthritis. It is associated with a 50% increased risk of cardiovascular (CV) mortality. Chronic inflammation is thought to lead to accelerated atherosclerosis in RA. There is some evidence to suggest that patients have a more inflammatory, unstable atherosclerotic plaque phenotype. The impact of advances in RA treatment, on cardiovascular co-morbidity remains unclear. The aims of the current study were to employ non-invasive imaging techniques to test the hypothesis that RA patients have more inflammatory, unstable atherosclerotic plaques compared to unaffected individuals and that treatment of active arthritis would lead to alterations in plaque composition and inflammation. Secondary aims were to evaluate the association of clinical phenotype and potential serological biomarkers of CV risk with plaque presence and phenotype. Methods: A prospective pilot study of patients with active RA and age and sex matched controls was conducted. Subjects underwent clinical and serological evaluation, then carotid artery ultrasound was performed to screen for carotid plaque. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was performed on those with suitable plaque. A subgroup of patients had a carotid artery positron emission tomography (PET) scan. Patients were followed up with repeat clinical, serological and DCE-MRI assessments. The primary outcome evaluated was difference in plaque inflammation measured on DCE-MRI between patients and controls and in patients longitudinally. Secondary outcomes included differences in plaque composition on DCE-MRI, plaque inflammation on PET and the relationship of clinical, serological and imaging findings. Results: 130 patients and 52 controls were recruited and screened for carotid plaque. There was a higher prevalence of plaque on ultrasound in the patient group (53% vs 36%) and plaque was independently associated with high sensitivity c reactive protein (hsCRP). Carotid DCE- MRI data was analysed in 15 patients and 5 controls. There was no significant difference in plaque inflammation on DCE-MRI between the groups. However there was a significantly higher rate of plaque calcification in patients, despite similar plaque burden in both groups (73.3% vs 20%, p=0.038). All 15 patients exhibited features of high-risk plaque. Plaque inflammation was seen in all 13 patients in whom PET imaging was undertaken. No significant improvement in plaque inflammation was detected on DCE-MRI over time, which was in keeping with the lack of clinical improvement found in most cases. ConclusionsIncreased prevalence of atherosclerosis and differences in plaque phenotype were observed in this study and findings would support the hypothesis that patients have a more high-risk plaque phenotype. The high prevalence of calcified lesions in RA is a novel finding which warrants further investigation. The study was underpowered to detect significant changes in plaque inflammation, measured on DCE-MRI, between the groups and in patients over time. However, this study provides valuable data with which to plan a larger study to investigate the effects of anti-inflammatory therapy on atherosclerosis in RA in the future.

The formation of atherosclerotic plaques is linked to a change in vascular function, with evidence of endothelial dysfunction and the proliferation of the underlying vascular smooth muscle cells (VSMCs). Prior to plaque development, risk factors are present that are capable of altering vascular function and promoting disease progression. These risk factors include hypercholesterolaemia, obesity and inflammation. The specific mechanisms of these risk factors in the early stages of atherosclerotic disease development have yet to be fully explored and are likely to be closely interwoven. The aim of this thesis was to assess the effects of these atherosclerotic risk factors, with a primary focus on the direct action of hypercholesterolaemia, on vascular function prior to the development of overt atherosclerosis. After acute ex vivo cholesterol depletion and enrichment, a range of contractile and relaxant stimuli were applied to thoracic aortic rings of wildtype C57BL/6 mice. Cholesterol depletion significantly reduced contractility to phenylephrine (p<0.05) and serotonin (p<0.01). Acute cholesterol enrichment had no effect on vascular contractility, however, acetylcholine stimulated endothelial-dependent relaxation was significantly reduced (p<0.05).Feeding with either a standard chow or a high fat ‘western’ diet was undertaken for eight weeks in both ApoE-/- and C57BL/6 mice. The extent of atherosclerotic disease development was measured through en face lipid staining and histological analysis of aortae. Atherosclerosis was present in the aortic root and intercostal branches of chow and high fat fed ApoE-/- mice but not in diet-matched C57BL/6 mice. No atherosclerotic lesions were observed in the thoracic aortae. In addition, to allow the possibility for direct associations to be made between the associated risk factors of hypercholesterolaemia, obesity and inflammation, and vascular function, a phenotypic assessment of these characteristics was conducted. Wire myography was employed to assess the vascular function of thoracic aortic rings from chow and high fat diet fed ApoE-/- mice and their age and diet matched wildtype C57BL/6 controls. It was found that contractility to both phenylephrine and serotonin was significantly increased in chow fed ApoE-/- mice (both p<0.05). Further investigation into the mechanism, using intracellular calcium imaging and the indo-1 dye, concluded that VSMC store-operated calcium entry was not altered. The exact mechanism behind this increase in contractility is therefore still unknown and there was no clear relationship to the atherosclerotic risk factors assessed. In addition to altered contractility, endothelial-dependent relaxation was shown to be significantly enhanced in high fat fed C57BL/6 (p<0.01) and ApoE-/- mice (p<0.001). Enhanced endothelial-dependent relaxations were transient and sensitive to specific inhibition of cyclooxygenase-2 but not nitric oxide synthase. These changes were hypercholesterolaemia-independent but correlated with signs of obesity and inflammation. In summary, this investigation has demonstrated that vascular function was altered in the murine thoracic aorta prior to overt atherosclerotic plaque development. The implications for these observations relate to the possibility of masked early signs of vascular dysfunction and also the induction of compensatory mechanisms which may have amplified effects over a longer time course; possibly promoting the development and advancement of atherosclerotic disease.

Bibliography: [appendix A-1 to A-2]. / This thesis was motivated by the idea of employing non-invasive investigations of atherosclerosis using Magnetic Resonance Imaging (MRI). MRI has the advantage of being able to detect atheroma in blood vessels with no risk to the patient but is still limited in its application to large blood vessels by the low geometrical resolution obtainable. The capability of MRI to measure velocities as well leads to the idea of correlating atheroma dimensions with measured velocities downstream of the blockage. This thesis makes a first step towards obtaining results that can be applied in investigations of atherosclerosis employing MRI. The fluid dynamics of arterial blood flow, the medical procedure of diagnosing and treating atherosclerotic diseases, and the physical principle of MRI are investigated to find out "if' and "how" the correlation between a blockage and the resulting downstream velocities can contribute to the diagnosis of atheroma. Parallel to this background research, experimental and numerical modeling of atheroma is carried out. These two approaches use identical geometrical and fluid parameters to enable a direct validation of the results. An experimental test-rig is designed. Experiments with different types of blockages are performed. The measured flow parameters are pressure and velocity profiles in a crosssection of the modeled artery. A commercial software package is employed for the numerical simulation of blockages with similar geometries to those used in the experiments. The pressure and absolute velocities are again the derived parameters. Both approaches are validated with analytical results obtainable for flow without any blockages. Blockages are then inserted and the results are compared and analyzed for their potential to contribute to the medical application. The results obtained with the two models give good correspondence. The transitional length of the laminar pipe flow corresponds very well to the expectations. A laminar velocity profile is completely built up before the fluid enters a blockage. Blockages with a small flow area cause a high peak velocity and a large wake. Blockages that slightly reduce the flow area have only a small influence on the flow. The length of a blockage has only a secondary influence on the downstream velocity distribution, while the influence of the surface roughness of the blockage is small. The peak velocities and pressure loss caused by the different blockages give good correlation. The prediction of the diameter of the blockage from peak velocities measured with MRI is an improvement on that which is possible from the theory only. In particular, the results obtained in this thesis show that the true maximum velocities are significantly lower than those obtained with theoretical predictions. The change in the velocity profiles, due to angioplasty, is shown in a simplified form with the models. Typical values of reduced areas before and after such surgery, where the atheroma is squeezed against the arterial walls, are analysed. The influence of the post-surgery blockage on the flow is very small, while the pre-surgery blockage shows a dominant influence. A prominent wake exists downstream of a highly reduced flow area, and high velocities occur. A wake is a potential risk area for atherosclerosis, as low shear rates and high turbulence intensities are possible. The blockage with the less reduced area has almost no influence on the flow, and a wake is hardly formed. The influence of different shapes of atheroma, while having a similar reduced area, is also demonstrated. The perfectly symmetrical blockage has less negative influence on the flow than one which is highly asymmetrical. The asymmetrical blockage causes a larger wake and higher maximum velocities.

archives@tulane.edu / Rupture of atherosclerotic plaque can cause acute life-threatening events such as myocardial infarction and ischemic stroke; therefore, there is much interest in developing therapies aimed at increasing plaque stability. More stable lesions are characterized as having high collagen content and containing a large number of vascular smooth muscle cells (SMCs) of contractile/differentiated phenotype. In our previous studies using an apolipoprotein E-deficient (Apoe -/-) mouse model of atherosclerosis, we found that insulin-like growth factor-1 (IGF-1)-infusion not only reduced total plaque burden, but also increased collagen expression and the number of alpha-smooth muscle actin (αSMA)-positive cells in plaque. In this study, we identify cellular mechanisms responsible for these observations. We found that in human aortic smooth muscle cells (HASMCs) grown in culture, IGF-1 post-transcriptionally upregulated expression of the procollagen type I alpha-1 subunit (pro-α1(I)) as well as contractile proteins, αSMA and smooth muscle 22-alpha (SM22α), via a PI3K-dependent but Erk1/2- and mTOR-independent signaling mechanism. Furthermore, experiments using an inhibitor of collagen synthesis or a blocking antibody against the alpha2beta1-integrin (α2β1) suggested that interaction with collagen type I promotes HASMC contractile phenotype. To elucidate mechanisms underlying IGF-1 upregulation of collagen synthesis we investigated the effect of IGF-1 on the mRNA-binding protein, la ribonucleoprotein domain family member 6 (LARP6), which had been shown to bind a conserved stem-loop secondary motif in the 5'UTR of COL1a1 and COL1a2 mRNA. IGF-1 rapidly increased LARP6 expression in HASMCs leading to increased COL1a1 and COL1a2 mRNA bound LARP6 and increased synthesis of collagen type I. Mutation of the 5'stem-loop of Col1a1 mRNA (that inhibited binding by LARP6) or overexpression of a 5'stem-loop RNA molecular decoy (that sequesters LARP6) both prevented the ability of IGF-1 to increase pro-α1(I) synthesis as well as mature α1(I) expression in cultured medium. Furthermore, IGF-1-infusion in Apoe -/- mice increased LARP6 and pro-α1(I) expression in aortic lysates, and SMC-specific IGF-1-overexpression in transgenic mice robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, this work identifies LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, and uncovers key mechanisms whereby IGF-1 promotes atherosclerotic plaque stability. / 1 / Admin

Atherosclerosis is a chronic inflammatory and lipid disorder caused by the buildup of cholesterol-loaded cells of monocyte and muscle cell origin in the arterial intima. While the relationship between excess cholesterol and macrophage behavior is well observed, the molecular mechanisms linking the two remain unclear. Therefore, characterizing the pathways from changes in intracellular cholesterol to the resulting inflammatory output is key to understanding the behavioral changes observed in human macrophages in vitro. We identified that THP-1 macrophages acutely depleted of cholesterol increase the expression of JMJD3, an H3K27me3 demethylase. By using IL-10 as a marker for immune-modulating genes and TNF-α as a marker for pro-inflammatory genes, cholesterol-depleted THP-1 macrophages responded inconsistently to LPS and echinomycin, an inhibitor of HIF-1α, as determined by RT-qPCR and ELISA. Further studies investigating other regulators and outputs of macrophage behavior linked to cellular cholesterol modification are required.

IL-15 is a pleiotropic cytokine which influences a variety of immune and inflammatory responses. Our lab has previously demonstrated that IL-15 and IL-15Rα promote atherosclerosis. However, the mechanisms by which IL-15 affect atherosclerosis development were not fully defined. In this study, we reported that overexpression of the IL-15 gene resulted in an increase of granzyme B level in the atherosclerotic plaque of ApoE deficient mice. Furthermore, we observed that leukocytes-specific genetic deletion of IL-15Rα reduced the granzyme B level within atherosclerotic lesions from LDLr deficient mice. Collectively, our data shows one of the mechanistic pathways by which IL-15 promotes atherosclerosis development. Moreover, we tested the role of IL-15 in carotid artery disease. It has been reported previously that immunization of low density lipoprotein receptor deficient mice against IL-15, by inoculating them with bacteria harboring an IL-15 expression plasmid, led to reduced development of diet induced atherosclerosis in carotid arteries whose diameter was restricted to induce a hemodynamic stress. Others, however, reported that injection of wild type mice with an antibody against IL-15 triggered increased neointima formation in carotid arteries that were partially ligated. In our study, we found no differences in the amount of collar/diet induced atherosclerosis in control apoE KO mice and in IL-15/apoE dKO mice.Therefore,inactivation of IL-15 gene expression does not appear to affect the rapid onset of atherosclerosis in carotid arteries of ApoE KO mice induced by a combination of high fat diet and hemodynamic stress. / Thesis / Master of Science (MSc)

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