Compelling evidence indicated that major risk factors for atherosclerosis such as oxidatively modified low density lipoprotein (oxLDL), high glucose, and reactive oxygen species promote endothelial cell apoptosis and thereby may contribute to the initiation of atherosclerotic lesion formation. Using fast protein liquid chromatography (FPLC), plasma LDL from familial hypercholesterolemic (FH) patients were separated into five subfractions, L1¡VL5. Among them, L5 subfraction was highly electronegative and suppressed DNA synthesis in cultured bovine aortic endothelial cells (BAEC) and stimulated mononuclear cell adhesion to cultured endothelial cells in vitro. Because impaired angiogenesis plays an important role in the pathogenesis of atherosclerosis, the anti-angiogenic functions of LDL subfractions from FH subjects were examined. Subconfluent BAEC (6 to 10 passages) maintained in DMEM containing 10% serum were treated with LDL subfractions at a dose of 20 £gg/ml, and the effects on anti-angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, and secretion of matrix metalloproteinase (MMP) were determined. Similar to Cu2+ ox-LDL, FH-L4 and FH-L5 inhibited cell proliferation to 80.9¡Ó2.4% (p<0.05) and 58.5¡Ó4.3% of control (p<0.001), respectively, while the other FH (L1-L3) and all subfractions isolated from normocholesterolemic (N) subjects had negligible effects. Similarly, FH-L4 and -L5, but not FH-L1 to -L3, retarded cell migration to 326.9 ¡Ó 19.4 (p<0.05) and 215¡Ó16 cells (p<0.001 with the control values of 402¡Ó34 cells), respectively. FH-L5 induced almost 20% of BAEC to undergo apoptosis; FH-L4 caused very mild effects, and other subfractions did not affect apoptosis In addition, FH-L4 and -L5 perturbed tube formation by BAEC in culture (5.8¡Ó0.2 and 3.4¡Ó0.4, respectively, versus control 8.5¡Ó1.5 tubes). Finally, FH-L4 and -L5 inhibited secretion of MMP-2 by BAEC (72.7¡Ó6.9 and 18.9¡Ó4.8% of control, respectively). The results demonstrate that FH-L5 potently affects multiple processes that are vital to normal angiogenesis, FH-L4 had milder effects, and other FH and N subfractions had negligible effects. In turn, these effects in vitro on processes pivotal to angiogenesis are consistent with potential effects of ox-LDL on endothelial dysfunction during atherogenesis in vivo.

The impact of percutaneous renal artery angioplasty and stenting (PTRAS) for treatment of atherosclerotic renal artery stenosis (ARAS) is not fully understood, especially in patients with chronic kidney disease (CKD). We performed a retrospective cohort study of patients with significant ARAS and moderate to severe chronic kidney disease (estimated GFR 15-60 ml/min/1.73m2) who were treated medically or with PTRAS. The primary endpoint of this study was change in renal function over the first year after treatment. Secondary endpoints included hemodynamic outcomes, antihypertensive medication doses, end stage renal disease (ESRD), and death. We reviewed all patients with a diagnosis of significant ARAS and impaired GFR treated between 1997-2007 in the Veterans Affairs Connecticut Healthcare System (VACHS). A total of 118 patients met inclusion criteria (71 medical treatment, 47 PTRAS), with an average follow-up of 34 months. The students t-test was used to compare baseline characteristics, as well as renal and hemodynamic endpoints between the two treatment groups. The cohort had a mean age of 73 ± 9 years and average baseline GFR of 37.2 ± 14.9 ml/min/1.73m2. Demographic, clinical and laboratory characteristics at baseline were similar between the two groups, with the exception of higher diastolic blood pressure in the stent group at baseline (75 versus 70 mmHg, p=0.028). No statistically significant difference was found between the two treatment groups for any renal endpoints. After a steady decline in GFR in both the medical treatment and stent groups during the 12 months preceding diagnosis (-4.2 versus -4.0 ml/min/1.73m2, p=0.911), GFR stabilized in both groups over the year following diagnosis (decline in GFR of -1.6 versus -1.4 ml/min/1.73m2, p=0.938). Multivariate models did not reveal an association between treatment modality and percent change in GFR during follow-up. No difference was found in blood pressure outcomes at 12 months between the medical and stent groups. Antihypertensive therapy, measured in defined daily doses (DDDs), was significantly higher in the medical treatment group at 12 months (4.5 versus 3.5 DDDs, p=0.048), but lost significance thereafter. In addition, the number of deaths was significantly higher in the stented group on univariate analysis, although this did not remain significant on multivariable Cox analysis. No difference was found between treatment groups in the development of ESRD. These data suggest that, among patients with ARAS and CKD, medical therapy and renal artery stenting are comparable in stabilizing renal function.

Thesis (Ph.D.) -- University of Texas at Arlington, 2008.

Application of the nonlinear optical microscopy (NLOM) for investigation of biological samples has, to date, primarily focused upon the qualitative analysis of images. The general consensus is that the nonlinear optical (NLO) techniques provide enough bio- chemical information when compared to, for example, visible light microscopy. Herein, it is presented a detailed study where a set of tools for quantitative extraction of infor- mation from NLO images were developed and tested for the analysis of complex tissue assemblies. Two-photon excited autofluorescence (TPEF), second-harmonic generation (SHG), and coherent anti-Stokes Raman scattering (CARS) were used for the charac- terization of atherosclerotic plaques. Our NLO-based image analysis of animal arteries affected by atherosclerotic plaque accumulation revealed that images of the healthy regions of the artery can be readily distinguished by marked differences in morphology, due to a fluorescent signal generated from the presence of generally intact elastic layer. Regions affected by lesions were dominated by lipid-rich cells and collagen fibers; the elastic layer was disrupted and the presence of fluorescent particles were also detected. Next, the potential of using information extracted from NLO images lead us to the development of a new optical index for plaque burden (OIPB). Through the OIPB, it was possible to investigate and to classify the plaque severity regarding the already established and currently used definition during clinical analyses. Extrapolating to and anticipating future applications, several methods for extracting specific information from images acquired by each NLOM modality were developed and tested. Texture analysis, particle-specific features, fractal analysis and directionality of components within the images were successfully adapted and tailored to better extract relevant information from the NLO images. Even though the methods presented in this thesis were mostly tested in images from arterial plaques, there is strong evidence that all tools presented here are capable of tracking changes that occur in many medical conditions and applications.

The leading cause of death among women is coronary heart disease (CHD), a multifactorial disease with polygenic heritability estimated at 50%. Polymorphisms in the family with sequence similarity 5, member C’ ( FAM5C) gene have been associated with myocardial infarction (MI), and one single-nucleotide polymorphism (SNP) has partially accounted for linkage in an acute coronary syndrome subset. The linkage peak on FAM5C corresponds directly with a quantitative trait locus for the inflammatory biomarker monocyte chemoattractant protein 1, as well as a linkage peak to metabolic syndrome (MetS). Metabolic syndrome increases the risk of developing CHD, and MI has been positively associated with elevated inflammatory biomarkers. This study was designed as a descriptive pilot gene association study. The purpose was to investigate the variability of the FAM5C SNP (rs10920501) in a cohort of women with documented CHD. It also examined the association between the variability of the FAM5C SNP (rs10920501), MetS, inflammatory markers, and the association with early onset CHD in the presence or absence of MI. A subset of 91 women was derived from an earlier study of women randomized to either a gender-tailored or traditional cardiac rehabilitation program. The results indicated the T allele of FAM5C SNP rs10920501 has a strong protective effect in women with a history of MI. Women with a history of MI and the heterozygous (AT) genotype had a mean age of onset of CHD at 62 years, compared to the homozygous wild type (AA) with a mean age of onset at 55 years, (F (3, 34) = 5.00, p < .01). No women in this study with the homozygous variant (TT) had an MI, further demonstrating the protective effective of the T allele. The genotype of FAM5C SNP rs10920501 explains approximately seven percent of the variability of age of onset of CHD in women who have had an MI, while holding body mass index (BMI) and smoking history constant. There was no significant relationship between FAM5C SNP (rs109320501) and MetS or any inflammatory biomarkers in this sample. In conclusion, FAM5C remains a gene of interest in a complex disease process.

These studies investigated the effect of physical inactivity (prolonged sitting) and physical activity (walking, standing, and moderate intensity exercise) on postprandial plasma triglyceride concentration (PPTG). In the first study, we evaluated the effect of low intensity intermittent walking at ~25% VO₂max (WALK) and energy-matched moderate intensity running at ~65% VO₂max (RUN) on PPTG, compared to a sitting control (SIT). RUN reduced incremental area under the curves for plasma triglyceride concentration (TG AUC[subscript I]), compared to WALK by 17.3% (p = 0.04) and SIT by 27% (p [less than] 0.001). The reduced TG AUC[subscript I] in RUN was accompanied by enhanced whole body insulin sensitivity, compared to WALK and SIT (for both, p [less than] 0.05). Whole body postprandial fat oxidation at rest following a high fat test meal intake was enhanced in RUN by 31% (P [less than] 0.001) and to a lesser extent in WALK by 8.4% (p [less than] 0.005), compared to SIT. In the second study, we evaluated 1) the effect of 2 days of prolonged sitting on PPTG, and 2) the effect of 4 days of SIT on the ability of an acute bout of exercise to reduce PPTG, compared to the same days of active walking and standing with calorically balanced diet (WALK+B). To distinguish the effect of prolonged sitting from the excess calorie effect, we had a sitting condition with calorically balanced diet (SIT+B) in addition to a sitting condition with hypercaloric diet (SIT+H). Following 2 days of respective food and activity control, WALK+B was lower in TG AUC[subscript T] by 21.3% and AUC[subscript I] by 17.4%, compared to SIT+H (for both, p [less than] 0.005). WALK+B was lower than SIT+B for TG AUC[subscript T] by 17.7% (p = 0.165) and AUC[subscript I] by 23.5% (p = 0.145) although statistical significance was not achieved. Remarkably, an acute exercise following 4 days of either SIT+H or SIT+B failed to reduce both TG AUC[subscript T] and AUC[subscript I], compared to SIT+B in HFTT1. The same exercise following 4 days of WALK+B, however, reduced both TG AUC[subscript T] by 29% and TG AUC[subscript I] by 32% in HFTT2, compared to SIT+B in HFTT1 (for both, p [less than] 0.02). Further, both SIT conditions reduced relative whole body fat oxidation in favor of increases in carbohydrate oxidation, compared to WALK+B by more than 40% in both HFTT1 and HFTT2. Taken together, our data suggest that 1) exercise intensity plays an independent role with higher intensity being more effective than lower intensity exercise in reducing PPTG, and 2) prolonged sitting with excess energy intake amplifies PPTG and prolonged sitting impairs the ability of an acute bout of moderate intensity exercise to reduce PPTG. This emphasizes the importance of regular participation in moderate-to-vigorous intensity exercise and reducing sitting time by increasing non-exercise physical activities (i.e., walking and standing) for the favorable postprandial metabolic health from the individual and public health perspectives. / text

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Atherosclerosis is a chronic disease characterized by the formation of plaque in hemodynamically unstable regions of arteries. The disease involves complicated molecular and cellular processes including inflammation, the immune system, low density lipoprotein, cytokines, and many other components. As such, the degree of disease is difficult to determine, and the clinical outcomes that stem from the disease are hard to predict. Current imaging techniques lack specificity for the plaques likely to cause clinical consequences such as heart attack or stroke. Consequently, a new and molecularly selective contrast agent formulation is necessary for accurate imaging of plaque and to aid in the determination of the correct patient-specific treatment. To that end, a stealth biodegradable particle was designed containing a high payload of contrast agent that is targetable to specific states of plaque development. The core material used in creation of the particle was the FDA-approved poly(lactide-co-glycolide) (PLGA), with carboxylic acid termini. The polymer was used in a modified water-in-oil-in-oil double emulsion method to form particles of sizes ranging from approximately 50 nm to 20 [mu]m, of near‐spherical shape, and with smooth surfaces. The PLGA particles were loaded with up to 30% Gd-DTPA, an FDA-approved contrast agent used with magnetic resonance imaging (MRI). As an adjunct, to enable visualization of individual particles in vitro, particles were alternatively loaded with rhodamine 6G, a fluorescent agent. The PLGA particles were surface functionalized with poly(ethylene glycol) (PEG) with a primary amine end group. The acid group of the PLGA and PEG-linked amine were coupled through an amide bond using carbodiimide chemistry. The presence of PEG on the surface of particles was confirmed using electron microscopy, 1H NMR, and zeta potential. The other end of the PEG chain terminated in a carboxylic acid that was subsequently used for coupling to a monoclonal antibody against the cell surface markers of inflammation and atherosclerosis, vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule-1 (ICAM-1). Particles with conjugated antibodies successfully attached to, entered, and distributed throughout cells in vitro. / text

Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed world. Atherosclerosis and the rupture of vulnerable atherosclerotic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been identified as a pathological inflammatory process. Targeting atherosclerotic drug therapies to inflammatory markers has emerged as an important and growing research area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of development. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antibody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. The IM7 conjugated immunoniosome has been shown to bind to endothelial and synovial lining cells in vitro. The preliminary research is involved with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability. A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polymer groups without prior derivatization. Subsequent research tested the resulting 'immunoniosome's' ability to bind to target antigens with selectivity and specificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and scanning electron microscopy. In vivo uptake of vesicles at sites of inflammation is size dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles. Further analysis of the efficacy of the system looked at live cell uptake of the immunoniosomes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component was modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellular uptake capabilities in inflammatory cells was produced by this research using a novel surfactant-antibody linker.

Abstract Cardiovascular disease is one of the leading causes of death in Canada. Atherosclerosis is considered the primary cause for cardiovascular disease. Optical coherence tomography (OCT) provides a means to minimally invasive imaging and assessment of textural features of atherosclerotic plaque. However, detecting atherosclerotic plaque by visual inspection from Optical Coherence Tomography (OCT) images is usually difficult. Therefore we developed unsupervised segmentation algorithms to automatically detect atherosclerosis plaque from OCT images. We used three different clustering methods to identify atherosclerotic plaque automatically from OCT images. Our method involves data preprocessing of raw OCT images, feature selection and texture feature extraction using the Spatial Gray Level Dependence Matrix method (SGLDM), and the application of three different clustering techniques: K-means, Fuzzy C-means and Gustafson-Kessel algorithms to segment the plaque regions from OCT images and to map the cluster regions (background, vascular tissue, OCT degraded signal region and Atherosclerosis plaque) from the feature-space back to the original preprocessed OCT image. We validated our results by comparing our segmented OCT images with actual photographic images of vascular tissue with plaque. / October 2015