The goal of this thesis was to produce an animal model that develops atherosclerotic plaque featuring plaque neovascularization leading to intraplaque hemorrhage and is suitable for noninvasive imaging studies. Several strategies were tested for their effectiveness in producing such plaques in the rabbit aorta, including: a high cholesterol diet, vascular endothelial growth factor injections, therapeutic contrast ultrasound, and balloon catheter injury. It was found that a combination of the high cholesterol diet and balloon injury was able to achieve plaque neovascularization in a manner dependent on circulating plasma cholesterol levels. In addition, a contrast-enhanced magnetic resonance imaging technique implemented in the animal model was able to detect plaque neovascularization and monitor its change over time in a single group of animals. In conclusion, an animal model was created where plaque neovascularization occurs in a predictable fashion and can be studied with non-invasive magnetic resonance imaging.

This thesis presents a high-resolution volumetric technique to measure the longitudinal relaxation time T1 in the vessel wall using MRI. The method of Variable Flip Angles (VFA) was applied using a new strategy for flip angle selection that allows measurement of T1 with high accuracy (< 10% mean error) and precision (T1-to-noise ratio > 10) over the wide range of anticipated values (300-3000ms) in the vessel wall. This strategy was validated in simulation, phantom and volunteer spinal cord experiments. Initial validation of vessel wall T1 measurements was performed in ex-vivo thoracic aorta samples from cholesterol-fed rabbits. For in-vivo vessel wall T1 mapping, the technique was augmented with spatial saturation bands for flow suppression and applied to the carotid arteries of three volunteers. Preliminary results from volunteers suggest that this approach may be useful in characterizing T1 changes associated with high-risk atherosclerotic disease.

The goal of this thesis was to produce an animal model that develops atherosclerotic plaque featuring plaque neovascularization leading to intraplaque hemorrhage and is suitable for noninvasive imaging studies. Several strategies were tested for their effectiveness in producing such plaques in the rabbit aorta, including: a high cholesterol diet, vascular endothelial growth factor injections, therapeutic contrast ultrasound, and balloon catheter injury. It was found that a combination of the high cholesterol diet and balloon injury was able to achieve plaque neovascularization in a manner dependent on circulating plasma cholesterol levels. In addition, a contrast-enhanced magnetic resonance imaging technique implemented in the animal model was able to detect plaque neovascularization and monitor its change over time in a single group of animals. In conclusion, an animal model was created where plaque neovascularization occurs in a predictable fashion and can be studied with non-invasive magnetic resonance imaging.

This thesis presents a high-resolution volumetric technique to measure the longitudinal relaxation time T1 in the vessel wall using MRI. The method of Variable Flip Angles (VFA) was applied using a new strategy for flip angle selection that allows measurement of T1 with high accuracy (< 10% mean error) and precision (T1-to-noise ratio > 10) over the wide range of anticipated values (300-3000ms) in the vessel wall. This strategy was validated in simulation, phantom and volunteer spinal cord experiments. Initial validation of vessel wall T1 measurements was performed in ex-vivo thoracic aorta samples from cholesterol-fed rabbits. For in-vivo vessel wall T1 mapping, the technique was augmented with spatial saturation bands for flow suppression and applied to the carotid arteries of three volunteers. Preliminary results from volunteers suggest that this approach may be useful in characterizing T1 changes associated with high-risk atherosclerotic disease.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

L'objectiu d'aquesta tesi doctoral és avaluar els efectes de les fraccions del vi (alcohòlica i no alcohòlica -principalment polifenols-) sobre els principals factors de risc cardiovascular en 67 homes amb un elevat risc de patir malalties cardiovasculars. Es va realitzar un assaig clínic, aleatoritzat i creuat en el qual tots els participants van rebre en un ordre aleatori les tres intervencions: 30 gr d'alcohol / dia en forma de vi negre, la mateixa quantitat de polifenols en forma de vi negre desalcoholitzat i 30gr d'alcohol / dia en forma de ginebra, durant 28 dies cada intervenció. Es van avaluar els efectes al final de cada una de les tres intervencions sobre els biomarcadors cel•lulars i sèrics d’inflamació relacionats amb l'aterosclerosi, el metabolisme glucídic, el perfil lipídic i la pressió arterial. El consum moderat d'alcohol (intervencions amb vi negre i ginebra) van incrementar la interleucina (IL) -10 i van disminuir la monocyte-derived chemoquine (MDC). Els polifenols del vi negre (intervencions amb vi negre i vi negre desalcoholitzat) van disminuir les concentracions sèriques de la intercellular adhesion molecule (ICAM) -1, E-selectina i IL-6, i també inhibiren l'expressió del lymphocyte function-associated antigen 1 ( LFA-1) en els limfòcits T i la de Mac -1, SLex (CD15) i CCR2 en la superfície dels monòcits. Tant l'etanol com els polifenols del vi negre varen disminuir les concentracions circulants de l’antigen i el lligand del CD40, CD40, IL-16, monocyte chemoprotein (MCP) -1 i vascular cellular adhesion molecule (VCAM) -1. La concentració de glucosa en dejú es va mantenir constant al llarg de l'estudi, mentre que la concentració d'insulina plasmàtica i, en conseqüència, l'índex HOMA va disminuir després del vi negre i el vi negre desalcoholitzat. El colesterol HDL, l'apolipoproteïna AI i A-II van augmentar després de les intervencions amb vi negre i ginebra. La lipoproteïna (a) plasmàtica només va disminuir després de la intervenció amb vi negre. A més, la pressió sistòlica i diastòlica només van disminuir significativament després de la intervenció amb vi negre desalcoholitzat, canvis correlacionats amb un augment de la concentració plasmàtica d'òxid nítric. La pressió sistòlica i diastòlica van disminuir i l'òxid nítric va augmentar, encara que no significativament, després de la intervenció amb vi negre i també comparat amb la ginebra encara que tampoc significativament. El consum moderat de ginebra no va tenir cap efecte en la pressió arterial ni en la concentració plasmàtica d'òxid nítric. Per tant, els polifenols del vi negre disminueixen l'expressió de molècules d'adhesió leucocitàries i la pressió arterial, així com també milloren la resistència a la insulina mentre que tant l'alcohol com els polifenols del vi negre modulen els mediadors inflamatoris solubles en homes d'alt risc cardiovascular, conferint al vi negre més efectes protectors sobre la patologia cardiovascular que altres begudes alcohòliques. / "Benefit / risk balance of polyphenols and alcohol content of wine: scientific basis of the effects of moderate wine consumption on the cardiovascular system" The objective of this thesis is to evaluate the effects of the fractions of the wine (alcoholic and non-alcoholic, mainly polyphenols) on major cardiovascular risk factors in 67 men with at high risk of cardiovascular disease. We performed a clinical, randomized, crossover in which all participants received in a random order the following three interventions: 30 g alcohol / day as red wine, the equivalent amount of dealcoholized red wine and 30g alcohol / day as gin, for 28 days each intervention. The effects at the end of the three interventions on serum and cellular biomarkers of inflammation associated with atherosclerosis, glucose metabolism, lipid profile and blood pressure were evaluated. Moderate consumption of alcohol (red wine interventions and gin) increased interleukin (IL) -10 and decreased monocyte-derived chemoquine (MDC). Polyphenols in red wine (red wine and dealcoholized red wine interventions) decreased serum concentrations of intercellular adhesion molecule (ICAM) -1, E-selectin and IL-6, and inhibited the expression of lymphocyte function-associated antigen 1 ( LFA-1) on T lymphocytes and Mac -1, sLex (CD15) and CCR2 on the surface of monocytes. Both ethanol and wine polyphenols decreased circulating levels of CD40a, CD40L, IL-16, monocyte chemoprotein (MCP) -1 and vascular cellular adhesion molecule (VCAM) -1. The fasting blood glucose concentration was constant throughout the study, whereas the concentration of plasma insulin and consequently the HOMA index decreased after the red wine and the dealcoholized red wine interventions. HDL cholesterol, apolipoprotein AI and A-II increased after the intervention with red wine and gin. Lipoprotein(a) decreased only after the intervention with red wine. Moreover, the systolic and diastolic pressure significantly decreased only after the intervention with dealcoholized red wine, changes correlated with an increased plasma concentration of nitric oxide. In conclusion, red wine polyphenols modulate the expression of leukocyte adhesion molecules, insulin resistance and blood pressure, while both alcohol and red wine polyphenols modulate the soluble inflammatory mediators in men at high cardiovascular risk, conferring to red wine greater protective effects on cardiovascular diseases than other alcoholic beverages.

As applications of nanotechnologies for life and health sciences get booming, magnetic nanovectors undergo a considerable development. Such composite structures made from polymer spheres encapsulating magnetic nanoparticles or from a nanoparticular magnetic core surrounded by an organic coverage exhibit a combination of physical, chemical and magnetic properties very appropriate for diagnostic by imaging such as Magnetic Resonance Imaging (MRI), or for therapy: targeted pharmaceutical vectorization, therapeutic hyperthermia... When such vectors exhibit a nanometric size, intravenous injection and easy spread in the body of the patients are allowed, while effects related to the specific surface area are increased. The present doctoral work was concerned by two important families of magnetic nanovectors: - nanospheres of biocompatible polymer having loaded a radioactivable compound for tumoral therapy and having encapsulated magnetite nanoparticles for diagnostic by MRI: a system for thera-diagnostic is thus obtained.- contrast agents for MRI of vascular or cerebral inflammation, consisting of a nanometric iron oxide (maghemite or magnetite) core i.e. ultrasmall superparamagnetic iron oxide - USPIO - surrounded by an organic coverage for targeting the affected region. These USPIO were used to study inflammation in the atherotic plaque of the aorta in a murine model.Most of the time, such nanovectors are administered to the patients in liquid suspensions by intravenous injection. It is thus crucial to characterize both the collective behaviour and the individual structure of the vectors in liquid suspension. On the other hand their interactions with the targeted regions in the body have to be understood. For this purposes, a multiscale approach of the structure and properties of such nanovectors has been developed, with structural studies carried out through innovative developments based on electron microscopies down to subnanometric resolution and correlated with physical properties. To achieve characterization of nanovectors in liquid media we have developed the application of Wet-STEM, a new mode in transmission of environmental scanning electron microscopy (ESEM), to image the internal structure of the magnetic nanoparticles in liquid suspension and image calculations by Monte Carlo simulations have shown that a nanometric resolution could be theoretically achieved. By the same technique, stability or tendency to flocculation in suspensions can be evidenced with respect to the collective behavior of different nanovectors.In a second step we have investigated the interactions of the nanoparticles with targeted regions. The biodistribution and biotransformation of the USPIO contrast agents in the tissular and cellular environments were investigated at increasing spatial resolution using different techniques. The biodistribution of a MRI contrast agent grafted with a fluorophore, in ex vivo samples from atherotic aorta and spleen were revealed by biphoton microscopy with a resolution of a few hundred nanometers, down to macrophage scale. Then preparation of ex vivo samples for transmission electron microscopy (TEM) was adapted from standard protocols especially with respect to staining after inclusion in resin. This way, the first high resolution HR(TEM) images and electron diffraction patterns of crystallized USPIO contrast agents in the aorta or the spleen of an atheromatous mouse were obtained. Combining such structural studies with measurement (using a SQUID setup) of magnetic properties, a longitudinal follow-up of USPIO nanoparticles injected in mice for MRI of the atherotic plaque has been completed for USPIO particles embedded in the aorta and the spleen: the results were interpreted in terms of agglomeration of the particles with a decreasing size depending on time after injection and found consistent with a model of in vitro degradation in acidic environment proposed to mimick the lysosomal metabolism.

With each heartbeat, major arteries experience circumferential expansion due to internal pressure changes. This pulsatile force is called cyclic strain and has been implicated in playing a pivotal role in the genetic regulation of vascular physiology and pathology. This dissertation investigates the hypothesis that in human umbilical vein endothelial cells (HUVEC), pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction. To determine expression and time-dependency of c-Myc in HUVEC, mRNA and protein expression of c-Myc under physiological (6-10% cyclic strain) and pathological conditions (20% cyclic strain) were studied. Both c-Myc mRNA and protein expression increased more than three-fold in HUVEC (P4-P5) cyclically-strained at 20%. This expression occurred in a time-dependent manner, peaking in the 1.5-2 hour range and falling to basal levels by 3 hours. Subsequently, the mechanism of c-Myc transcription was investigated by using specific inhibitors to modulate c-Myc transcriptional activation. These compounds, obtained from the University of Arizona Cancer Center, attenuated cyclic-strain-induced c-Myc transcription by about 50%. Having established this reduction in expression, it was investigated how these effects modulate downstream genes that are regulated by c-Myc. The results indicate that direct targeting of the c-Myc promoter may decrease stretch-induced gene expression of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA) and heat shock protein 60 (HSP60). These findings may help in the development of a novel therapeutic opportunity in vascular diseases.

It is hypothesized that exposing endothelial cells to steady or non-reversing pulsatile shear stress produces a healthy, anti-atherogenic endothelium, whereas a reversing pulsatile shear stress promotes an unhealthy, pro-atherogenic endothelium. To further investigate this hypothesis, a novel parallel plate flow chamber system was used to expose human endothelial cells to a pro-atherogenic reversing shear stress waveform designed to simulate the wall shear stress at the carotid sinus, a region prone to atherosclerosis. Cells exposed to this reversing shear stress were compared to cells exposed to high levels of steady shear stress (15 dynes/cm²), low steady shear stress (1 dyne/cm², the time-average of the carotid shear stress), and static culture conditions. Functional analysis confirmed previous findings that reversing shear stress increases cell proliferation and monocyte adhesion. Microarray results indicate that although there are unique sets of genes controlled by both low average shear stress and by reversing flow, more genes were controlled by low average shear stress. We propose that low-time average shear stress, and not fluid reversal/oscillation, may be the more significant mechanical force. The reversing shear stress system was also used to investigate two shear stress-responsive genes, CYP1A1 and CYP1B1. Both were maximally up-regulated at arterial steady shear stresses of at least 15 dynes/cm² and reversing pulsatile shear stress attenuated expression of both genes. Furthermore, AhR nuclear localization and CYP1A1 protein expression correlate with the flow patterns in the mouse aortic arch. The data strongly suggest that the AhR/CYP1 pathway promotes an anti-atherogenic phenotype in the endothelium. Changes in free zinc were measured under different shear stresses. High steady shear stress dramatically increases the levels of free zinc in endothelial cells as compared to cells grown in static culture. This increase in free zinc is attenuated under reversing shear stress and low steady shear stress, which correlates with an increase in zinc-binding metallothinein proteins and zinc exporter Znt-1. Overall, the findings provide further insight into endothelial responses to mechanical forces and may be important in understanding mechanisms of atherosclerotic development and localization to regions of disturbed flow.

Atherosclerosis is a multifactoral inflammatory disease that occurs in predisposed locations in the vasculature where blood flow is disturbed. In vitro studies have implicated reactive oxygen species as mediators of mechanotransduction leading to inflammatory protein expression and ultimately atherogenesis. While these cell culture-based studies have provided enormous insight into the effects of WSS on endothelial biology, the applicability to the in vivo setting is questionable. We hypothesized that low magnitude oscillatory WSS acts through reactive oxygen species (ROS) to increase expression of inflammatory cell adhesion molecules leading to the development of atherosclerotic lesions. The overall objective for this thesis was to develop an in vivo flow model that produces low magnitude oscillatory WSS which could be used to investigate the in vivo molecular mechanisms of mechanotransduction. We created a novel aortic coarctation model using a shape memory nitinol clip. The clip reproducibly constricts the aorta creating a narrowing of the lumen resulting in a stenosis. This mechanical constraint produces a region of flow separation downstream from the coarctation. We have characterized the coarctation in terms of the efficacy, pressure loss, and fluid dynamics. We then measured the endothelial response of shear sensitive redox and inflammatory markers. Lastly, we utilized genetically modified mice and mice treated with pharmacological inhibitors to investigate the mechanisms involved in the expression of WSS induced inflammatory and redox markers. We found that inducing a coarctation of the aorta using a nitinol clip uniquely created a hemodynamic environment of low magnitude oscillatory WSS without a significant change in blood pressure. Using this model we found that the in vivo endothelial phenotype associated with acutely disturbed flow was characterized by increased production of superoxide and increased expression of select inflammatory proteins. In comparison, the phenotype associated with chronically disturbed flow was characterized by a more modest increase in superoxide and increased levels of multiple inflammatory proteins. We determined that in regions of acutely disturbed flow in vivo, VCAM-1 expression was not modulated by reactive oxygen species. Additionally, p47 phox-dependent NADPH Oxidase activity does not have a functional role in WSS induced superoxide generation in the endothelium. In summary, we have created a novel murine model of low magnitude oscillatory WSS that can be used to investigate the in vivo molecular mechanisms associated with atherogenesis. While previous data obtained in vitro indicated that depletion of an individual ROS was sufficient to inhibit flow-induced inflammatory protein expression, our findings, to the contrary, showed that antioxidant treatment in vivo does not inhibit shear-dependent inflammatory protein expression. Our results suggest that atherogenesis in the in vivo environment is significantly more complicated than the in vitro environment and that parallel pathways and compensatory mechanisms are likely activated in vivo in response to WSS. These results could have significant implications in the efficacy of antioxidant treatment of atherosclerosis and could explain the complexity of results observed in clinical trials.