Coronary heart diseases are a significant cause of death among both men and women in the industrialized world; thus finding ways to detect factors which cause coronary heart diseases is worthy challenge for researchers. Ultrasound elastography system (50MHz) for measuring mechanical properties of arteries was developed as part of this thesis. Ability to discriminate between various tissue types was demonstrated using fresh and modified porcine coronary arteries, which closely models the plaque in human atherosclerosis. Elastographic measurements agreed well with uniaxial mechanical testing over a range of compression moduli.

Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

<p>Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes.</p><p>The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems.</p><p>Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis.</p><p>A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner.</p><p>Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression.</p><p><b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling</p>

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 103-119). Also available on the World Wide Web.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 81 p. Vita. Includes abstract. Includes bibliographical references (p. 44-48).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Highly antiretroviral therapies (HAART) have been implemented to slow the progression of the human immunodeficiency virus (HIV). Although these new advances in the medications for HIV-positive patients have contributed in longer life expectancy, comorbidities, such as cardiovascular disease, still cause higher number of deaths among HIV-positive patients than in the regular population. Because of the intrinsic inflammation caused by the HIV virus, atherogenesis is more likely to occur and is driven by infected macrophages. These macrophages are known to secrete cathepsins, but infection causes the macrophages not to perform their function properly as an immune agent. I hypothesize that antiretroviral drugs play an important role in arterial remodeling by affecting cells within the artery and causing an alteration of cathepsin activity, leading to an increased risk of atherosclerosis in HIV patients. To test this hypothesis, we incubated THP-1 monocytes with antiretroviral drugs efavirenz and tenofovir individually to observe any changes in cathepsin activity. These lysates were analyzed through multiplex cathepsin zymography and quantified through densitometry. We found that our hypothesis held true for efavirenz and tenofovir in THP-1 monocytes, which caused decreased cathepsin K activity compared to vehicle controls. Still, stimulation of peripheral blood mononuclear cells (PBMCs) with efavirenz and tenofovir caused differential effects. Together, our data suggest that the HAART interaction with monocytes that are physiologically relevant to our system possibly contributes to the advancement of atherogenesis in HIV+ patients.

Atherosclerosis is a complex cardiovascular disease characterized by chronic progressive inflammation of the arteries. The progression of atherosclerosis from fatty streak to advance atherosclerotic plaque involves the development of a necrotic core region consists of cholesterol, lipids, calcium (Ca²⁺), dead cells and other cellular debris. Macrophage infiltrations occurred in all stages of atherosclerotic progression and they are abundantly found in atherosclerotic plaques. Oxidised low density lipoprotein (oxLDL) plays a vital role in the initiation and development of atherosclerosis. OxLDL is present within atherosclerotic plaque and has been shown to be cytotoxic to various types of cells including macrophages. This research initially examined the cytotoxic effects of copper oxidised LDL on U937, human monocytes and HMDM cells. As expected oxLDL was cytotoxic; causing rapid, concentration and time dependent cell viability loss in all types of cells examined. Examination of the cell morphology showed that oxLDL caused a necrotic like cell death characterised by cell swelling and lysis. Flow cytometric assay coupled with propidium iodide (PI) staining of necrotic cells was compared to MTT reduction assays of cell viability. The flow cytometric technique had the advantage over the MTT reduction assay of being rapid and showing both the live and dead cell levels at an individual cell level. The progression of oxLDL-induced cell death correlated with the rapid increased in intracellular ROS production in the cytosol and the mitochondria. Immunoblotting results showed that oxLDL induced NADPH oxidase (NOX) activation and increased p47phox expression. This suggests NOX as the generator of reactive oxygen species (ROS) induced by oxLDL in these cells. However, apocynin and VAS2870, the two NOX inhibitors used in this study, were unable to inhibit the ROS generation caused by the oxLDL. This suggests that either these inhibitors are unable to inhibit the targeted NOX or other sources of ROS might exist and contributed to the overall increase in oxidative flux. OxLDL caused a rapid increase in cytosolic Ca²⁺ level. This was contributed by the extracellular Ca²⁺ source as well as Ca²⁺ mobilisation from the intracellular stores such as endoplasmic reticulum (ER). OxLDL-induced intracellular Ca²⁺ increase also correlated with the increase in intracellular ROS. Nevertheless, blocking of oxLDL-induced intracellular Ca²⁺ elevation by Ca²⁺ chelator, EGTA, did not reduce intracellular ROS generation. Accordingly, this suggests that oxLDL-induced intracellular Ca²⁺ increase is not the cause for oxLDL-induced cell death. Additionally, oxLDL may also initiate a Ca²⁺-independent cell death pathway. The excess cytosolic Ca²⁺ taken up by the mitochondria may be detrimental and could result in mitochondrial Ca²⁺ overload. This will increase mitochondrial ROS production and initiate mitochondrial permeability transition (MPT) pores opening. Consequently, this could collapse the mitochondrial membrane potential ( m) due to the rupture of outer mitochondrial membrane (OMM) and resulted in cell death. Blocking of Ca²⁺ uptake into the mitochondria by ruthenium red protected cells from oxLDL-mediated cell death, possibly by reducing ROS production and preventing MPT activation. This study also demonstrated the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP protected cells from oxLDL-induced intracellular ROS generation and cell viability loss. Intracellular Ca²⁺ increase was also reduced by 7,8-NP in particular after 3 hours incubation with oxLDL. The action of 7,8-NP was better than that of apocynin in protecting U937 cells from oxLDL suggests its potential ability to scavenge ROS from various sources. Studies have implicated the involvement of H₂S in various biological processes including atherosclerosis. Thus, the disruption of H₂S homeostasis may contribute to the progression of atherosclerosis. Slow releasing H₂S molecules (H₂S donors) have been developed for a controlled and stable delivery of H₂S to cells. In this study, specific H₂S donors, including one which targets the mitochondria, were found to be protective against oxLDL-induced cell death in U937, human monocytes and HMDM cells. Although the exact mechanism is yet to be elucidated, these H₂S donors were shown to block the elevation of intracellular Ca²⁺ and ROS production mediated by oxLDL. Therefore, these H₂S donors could be the potential candidates for future development of therapeutics in treating atherosclerosis.

No description available.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types.