Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

Cardiovascular disease, and myocardial infarction (MI) in particular, remains a major burden in the developed world today. In fact, the remodeling process, which follows the initial ischemic episode of MI, is a major determinant of heart failure. Although several key mechanistic pathways involving cell growth and death have been identified, there is limited knowledge surrounding the role of the innate immune response as a positive or negative regulator of cardiac remodeling. Recent data strongly support a role for key regulatory components within the toll-like receptor (TLR) family as potent modulators of cardiac remodeling post-MI. It has been demonstrated that targeted gene knockdown of TLR4, as well as downstream adaptor proteins and kinases, significantly improve cardiac function and overall survival. While the well-known NF-κB transcriptional factor that is downstream to TLR4 signaling has been linked to remodeling, there has been no evidence thus far describing a role of the parallel interferon regulatory factor-3 (IRF3) signaling cascade in any facet of this process. Several key findings suggest that IRFs contribute to both cell growth and apoptosis, thus providing an appealing, and novel target for interrogation. In this thesis I describe how IRF3 contributes to maladaptive remodeling post-MI. In my first set of experiments, I demonstrate that IRF3 is acutely upregulated within the cardiomyocyte following MI and that this response contributes to excessive apoptosis post-MI. A targeted deletion of the IRF3 gene enhances cardiac function, decreases infarct size, and improves survival following MI. In the second set of experiments I demonstrate that IRF3 attenuates angiogenesis at the ischemic border zone by upregulating the expression of thrombospondins. I have shown that IRF3 deficiency, which liberates endogenous anti-angiogenic signals, promotes angiogenesis following ischemic injury. These data suggest that IRF3 is a potent regulator of cardiac remodeling and may be an effective therapeutic target to ameliorate maladaptive cardiac repair post-MI.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.

Osteocytes make up the largest cell population in bone and are believed to be the main mechanosensory bone cells. During mechanical disuse and overuse, osteocyte viability is compromised and is found to be co-localized with increased osteoclastic bone resorption. Osteoclasts are recruited to remodel sites of apoptosis or bone microdamage; however, it is unclear whether the apoptotic or neighbouring healthy osteocytes are responsible for targeted bone remodeling. I hypothesized that apoptotic osteocytes are: (a) directly responsible for initiating bone remodeling by recruiting osteoclast precursors and directing osteoclast differentiation, and (b) indirectly responsible by signaling to nearby healthy osteocytes that, in turn, regulate osteoclastogenesis. In this in vitro study, apoptotic osteocytes were found to increase osteoclast precursor migration and osteoclast formation. Inhibition of the osteoclastogenic protein, receptor activator of nuclear factor kappa B ligand (RANKL), in conditioned medium abolished the osteoclastogenic effect of apoptotic osteocytes. Healthy osteocytes surrounded by apoptotic regions were modeled by applying apoptotic osteocyte conditioned medium to healthy osteocytes. These cells also promoted osteoclastogenesis, and had increased expression of macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Inhibition of these factors abrogated the pro-osteoclastic effect of healthy osteocytes conditioned by apoptotic osteocytes. These findings support the hypothesis that apoptotic osteocytes directly and indirectly, by signaling to nearby healthy osteocytes, initiate osteoclastogenesis. One limitation of our and other conventional in vitro models is the lack of real-time cell communication and physiologically-relevant mechanical environment. Using a microfluidics approach, a miniature fluid shear delivery system was created for in vitro osteocyte cultures. The purpose of this microsystem was to increase control of the cell microenvironment for subsequent integration into scalable screening platforms or co-culture systems for studying osteocyte mechanobiology under physiological loading conditions. Fluid shear stress was periodically applied without external pumping using a deflecting elastomer membrane, where up to 2 Pa of oscillating shear stress was possible by manipulating membrane dimensions. Osteocyte culture, viability and calcium response were demonstrated in the microdevice. Further studies should attempt to characterize calcium signaling in osteocytes which, using a conventional macro-scale system, was found to dependent on cell-cell communication.

In this study, we investigated the role of Ang II in pathological vascular remodeling. We sought to determine whether the humoral or the mechanical effects of Ang II are the dominant factor driving the remodeling process. The following experimental groups were used: control group (untreated mice), mice treated with an angiotensin receptor blocker (Candesartan, 0.5 mg/kg/day,SQ), an ACE inhibitor (Captopril, 6 mg/kg/day), and a calcium-channel blocker (Amlodipine, 7.5 mg/kg/day). All mice (n=6 per experimental group) were from the C57Bl/6 background. The carotid ligation model was implemented to study the differences in vascular remodeling. Additionally, multiple time points (7-, 14-, and 21-days post-surgery) were used to track the progression of remodeling. In Day-7 analysis, all three treatment groups yielded similar remodeling patterns as evidenced by a significant reduction in neointimal area, medial thickening and hypertrophy compared with the control group. Histomorphometric analysis of carotid sections collected 1mm below the ligation demonstrated that the Amlodipine group had 26% reduction in total vessel area, Candesartan a 36% reduction, and Captopril a 28% reduction (p less than 0.05 in all groups compared with Control), as well as a parallel 38-40% drop in medial thickness. In Day-14 analysis, no significant differences between the Controls and treatment groups were observed, although differences were emerging between the treatment groups. Candesartan was found to reduce the extent of negative remodeling observed between the 7- and 14-day Control data, whereas the Captopril group did not exhibit this trend. All treatment groups exhibited less neointimal formation than Controls, similar to Day-7. By the 21-day time point, the Captopril group underwent positive remodeling, resembling the Candesartan and Amlodipine groups. Although total vessel area was analogous among all groups, neointimal areas were significantly decreased in the treatment groups. Blood pressure plays a pivotal role in the modulation of vascular remodeling in response to mechanical injury. Although intermediate timepoint analysis suggests that humoral aspects of ACE inhibition or angiotensin-receptor blockade yielded unique effects on the overall vessel caliber, upon reaching the late, 21-day time point, the mechanical factors became predominant. These data support the importance of blood pressure control in the attenuation of pathological vascular remodeling.

In order to create informed predictive models that capture artery dependent responses during atherosclerosis progression and the long term response to hypertension, one needs to know the structural, biochemical and mechanical properties as a function of time in these diseased states. In the case of hypertension more is known about the mechanical changes; while, less is known about the structural changes over time. For atherosclerotic plaques, more is known about the structure and less about the mechanical properties. We established a congruent multi-scale model to predict the adapted salient arterial geometry, structure and biochemical response to an increase in pressure. Geometrical and structural responses to hypertension were then quantified in a hypertensive animal model. Eventually this type of model may be used to predict mechanical changes in complex disease such as atherosclerosis. Thus for future verification and implementation we experimentally tested atherosclerotic plaques and quantified composition, structure and mechanical properties. Using the theoretical models we can now predict arterial changes in biochemical concentrations as well as salient features such as geometry, mass of elastin, smooth muscle, and collagen, and circumferential stress, in response to hemodynamic loads. Using an aortic coarctation model of hypertension, we found structural arterial responses differ in the aorta, coronary and cerebral arteries. Effects of elevated pressure manifest first in the central arteries and later in distal muscular arteries. In the aorta, there is a loss and then increase of cytoskeleton actin fibers, production of fibrillar collagen and elastin, hyperplasia or hypertrophy with nuclear polypoid, and recruitment of hemopoeitic progenitor cells and monocytes. In the muscular coronary, we see similar changes albeit it appears actin fibers are recruited and collagen production is only increased slightly in order to maintain constant the overall ratio of ~55 percent. In the muscular cerebral artery, despite a temporary loss in actin fibers there is little structural change. Contrary to hypertensive arteries, characterizing regional stiffness in atherosclerotic plaques has not been done before. Therefore, experimental testing on atherosclerotic plaques of Apolipoprotein E Knockout mice was performed and revealed nearly homogenously lipidic plaques with a median axial compressive stiffness value of 1.5 kPa.

During the last years, biomechaniccs, the understanding of the very complex mechanical behavior of living tissue, becomes a very important field of research of the wide bravch of mechanichs. Thia study is very important, for instance , to improve the design of implants, with the important social and economical impact. one of important challenges in implant design is to model the complex material behavior of the bone. Remodeling of the bone due to the lodaing is one of the essential behaviors of the bone which can cause for the dental implants, marginal bone loss accuring particallary in the first years of function. On the other hand the finite element method(FEM) has become the most extended tool for engineers to analyse the mechanical behavior of the products which usualy have a complex material behavior and complicated geometries. The aim of this study is to model the remodeling behavior of the bone using FEM and to use the new bone material method to evaluate the implant performance for some commercial implants, taking account of the bone adaptation.

Hypertension is a risk factor for many cardiovascular and cerebrovascular diseases such as atherosclerosis and stroke. It is therefore important to understand the effect of hypertension on temporal growth and remodeling of arteries. In this study, experimental hypertension was induced in the mini-pig by aortic coarctation. Basilar arteries and aortas were collected for analysis over an eight week period of hypertension with specimens from normotensive animals serving as controls. Changes in mechanical properties of the basilar artery were evaluated by in vitro pressure-diameter tests on intact cylindrical segments at their in situ length. The basilar arteries from hypertensive animals became less distensible, reflecting increases in both structural and material stiffness, compared to their normotensive counterparts. The circumferential stress rapidly returned toward its homeostatic value by increasing the wall thickness within two weeks. Immunohistochemistry, which is capable of illustrating the localization and distribution of protein expression, was performed to examine changes in wall constituents in the aorta. The increased medial thickness observed in hypertensive pigs compared to normotensive pigs was due to hyperplasia of smooth muscle cells (SMCs) and accumulation of extracellular matrix proteins, which were accompanied by the phenotypic modulation of SMCs. The increased interlamellar thickness, collagen fibers, and the thickness of elastic lamina found in the inner media of hypertensive animal may be associated with the gradient of stress decreasing into the outer media. SMC proliferation, if any, was found evenly distributed across the media, however. In cases showing increased proliferation and matrix protein synthesis, the SMC contractile markers were down-regulated whereas the SMC synthetic markers were up-regulated. While the aortic intima appeared normal in the normotensive animals, neointima formation, which may predispose the vessel to atheroma formation, was found in the hypertensive animals. Immunohistochemistry of Hsp47 and procollagen revealed that the endothelial cells (ECs) may produce collagen, specifically type I collagen in response to hypertension and contribute to the thickened intima. In addition, lectin staining for ECs markers and immunostaining for eNOS suggested that endothelial cells may transdifferentiate into intimal SMCs. These findings suggested an alternative role that ECs may play in hypertension-induced atherogenesis.

The evolution of biomechanical behavior of arteries plays a key role in the onset and progression of cardiovascular disease. Biomechanical behavior is governed by the content and organization of the key structural constituents (e.g., collagen, elastin, and smooth muscle) and vessel geometry. The evolution of biomechanical behavior of arteries is governed by biologically-mediated synthesis, degradation, and reorganization of these key structural constituents. A hallmark goal in biomechanics is quantifying the relationship between the microstructure of tissues and their mechanical response throughout tissue growth and remodeling; this will provide a crucial link in understanding the tissue level effects of biological processes involved in disease and normal growth Fibulin-5 (fbln5) is an ECM protein that binds tropoelastin and interacts with integrins. Arteries from fbln5 knockout mice lack functional elastic fibers and provide a system for investigating the link between an artery's microstructure and its mechanical response. The overall goal of this project was to develop multi-scaled theoretical and experimental frameworks to quantify the relationship between microstructural content and organization and tissue level material properties of arteries from fbln5 null mice and littermate controls and to quantify the effects of fbln5 on the in vivo maturation of mouse carotid arteries. We found significant differences in the mechanical properties of carotid arteries of fbln5 null mice, and these differences were correlated with altered extracellular matrix organization. We also developed a microstructurally-motivated 3-dimensional constrained mixture model for vascular growth and remodeling. Using physiological rates of constituent growth and turnover, the model captured the salient findings found in the literature. Incorporating experimentally measured fiber angle data into constitutive relations yielded greater predictive accuracy. This dissertation incorporates experimental data quantified at the micro (microstructural-level fiber distributions) and macro (tissue-level mechanical response) scale and incorporates these data into microstructurally motivated constitutive relations. The use of structurally motivated constitutive relations and experimentally measured microstructural data provides a foundation for future work in further understanding the relationship between processes governing microstructure and the tissue level effects of disease and normal growth.

Major advances in highly active antiretroviral therapies (ARVs) have extended the lives of people living with HIV, but there still remains an increased risk of death by cardiovascular diseases (CVD). HIV proteins and ARVs have been shown to contribute to cardiovascular dysfunction with effects on the different cell types that comprise the arterial wall. In particular, HIV-1 transactivating factor, Tat, is a cationic polypeptide that binds to endothelial cells, inducing a range of responses that have been shown to contribute to vascular dysfunction. It is well established that hemodynamics also play an important role in endothelial cell mediated atherosclerotic development where upon exposure to low or oscillatory shear stress, such as that found at branches and bifurcations, endothelial cells contribute to proteolytic vascular remodeling, by upregulating cathepsins, potent elastases and collagenases. The results of this work demonstrate that upregulation of cathepsins in vivo and in vitro is caused by a synergism between pro-atherogenic shear stress and HIV-1 proteins, elucidates pathways that are activated by HIV-1 Tat and pro-atherogenic shear stress - leading to cathepsin-mediated ECM degradation, and identifies cathepsins as novel biomarkers to monitor the adherence of patients on efavirenz- and tenofovir-containing antiretroviral regimens.