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.

The engineering of human vascular grafts is an intense area of study since there is crucial need for alternatives to native vein or artery for vascular surgery. This current study sought to prove that a tissue engineered blood vessel (TEBV) 1mm in diameter could be developed from human smooth muscle cells and that endothelial progenitor cells (EPCs) could be cultured and used to endothelialize these grafts. This project had four specific aims: the isolation and characterization of EPCs, the seeding of a novel scaffold with EPCs and exposure to physiologic shear stress in vitro, the development of TEBV from human smooth muscle cells that are strong enough to implant in vivo, and the in vivo implantation of TEBV into the rat aortic model with a comparison of EPC seeded TEBVs pretreated with shear stress and unseeded TEBVs. The results yielded isolation of four EPC lines and a flow system design capable of seeding EPCs onto a novel scaffold with preliminary studies indicating that it is capable of exposing the EPCs to physiologic shear stress, although further studies require more optimization. The development of mechanically strong TEBV was highly successful, yielding TEBVs comparable to native vessels in collagen density and burst pressure, but with much lower compliance. Current implantation studies indicated that unseeded TEBV grafts implanted into the rat aorta without anticoagulation is highly thrombogenic. However, anticoagulation using Plavix may be capable of maintaining graft patency. These TEBVs did not rupture or form aneurysm in vivo and the future completion of the in vivo studies are likely to demonstrate the high potential of these grafts.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 140-156). Also available in print.

Atherosclerosis is an inflammatory disease of the arterial walls and is the major cause of heart attack and stroke. Atherosclerosis is localized to curves or branches in the vasculature where disturbed blood flow alters endothelial cell (EC) gene expression and induces EC dysfunction. Epigenetics controls aberrant gene expression in many diseases, but the mechanism of flow-induced epigenetic gene regulation in ECs via DNA methylation has not been well studied until very recently. The goal of this project was to determine how the DNA methylome responds to flow, causes altered gene expression, and regulates atherosclerosis development. Here, we found that d-flow increases DNA Methyltransferase 1 (DNMT1) expression in ECs, and we hypothesized that this causes a shift in the EC methylome and transcriptome towards a pro-inflammatory, pro-atherosclerotic gene expression program, and further that this leads to atherosclerosis development. To test this hypothesis, we employed both in vitro and in vivo experimental approaches combined with genome-wide studies of the transcriptome and DNA methylome according to the following three specific aims: 1) to elucidate the role of DNA Methyltransferase 1 in EC function, 2) to uncover the DNA methylation-dependent EC gene expression response to flow, and 3) to discover and examine master regulators of EC function that are controlled by DNA methylation. The work presented here has resulted in new knowledge about the epigenetic EC shear response, details the previously unstudied EC methylome, and implicates specific loci within the genome for additional studies on their role in EC biology and atherosclerosis. This work provides a foundation for novel and more targeted therapeutic strategies for CVD.

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.

While cellular levels of arginine greatly exceed the apparent Km for endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production is limited by availability of arginine. Results from this work have provided a unique understanding of endothelial NO production, showing that arginine regeneration, that is the recycling of citrulline back to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL), defines the essential source of arginine for NO production. Using RNA interference analysis, selective reduction of AS expression was shown to directly correspond with a diminished capacity of endothelial cells to produce NO, despite saturating levels of arginine in the medium. In addition, the viability of AS siRNA-treated endothelial cells was compromised due to apoptotic cell death.AS expression was also investigated in response to two major vascular effectors. Tumor necrosis factor (TNF)-alpha; which is known to impair endothelial NO production, was shown to provoke a dose-dependent reduction of AS expression that corresponded to a decrease in NO production. Furthermore, TNF-alpha was shown to suppress AS expression through a NFkappaB mediated pathway, which involves three essential Sp1 elements in the proximal AS gene promoter. On the other hand, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, troglitazone and ciglitazone, which are known to elicit a vascular protective response against TNF-alpha effects, were shown to coordinately induce NO production and AS expression via a PPARgamma response element in the distal AS gene promoter. Importantly, these PPARgamma agonists were shown to restore AS expression and NO production following down-regulation by TNF-alpha, consistent with their vascular protective properties.

published_or_final_version / Anatomy / Master / Master of Philosophy

<p>The adaptive response of vascular endothelial cells to shear stress alteration induced by global hemodynamic changes is an essential component of normal endothelial physiology in vivo; and an understanding of the transient regulation of endothelial phenotype during adaptation will advance our understanding of endothelial biology and yield new insights into the mechanism of atherogenesis. The objective of this study was to characterize the adaptive response of arterial endothelial cells to acute increases in shear stress magnitude and frequency in well-defined in vitro settings. Porcine endothelial cells were preconditioned by a basal level shear stress of ±15dynes/cm^2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ±15 dynes/cm^2) or frequency (2 Hz) was then applied. Endothelial permeability to bovine serum albumin was measured and gene expression profiling was performed using microarrays at multiple time points during a period of 6 hours after the shear stress alteration. The instantaneous endothelial permeability was found to increase rapidly in response to the acute increase in shear stress magnitude. Endothelial permeability nearly doubled after 40 minutes exposure to the elevated shear magnitude, and then decreased gradually. However, less dependency of endothelial permeability on shear stress frequency was observed. Endothelial permeability increased slowly from 120 minutes to 6 hours after exposure to the elevated shear frequency, but the increase was not statistically significant and was relatively small (1.2 fold increase at 6 hours). The transcriptomics studies identified 86 genes that were sensitive to the elevated shear magnitude and 37 genes sensitive to the elevated frequency. A significant number of the identified genes are previously unknown as sensitive to shear stress. The acute increase in shear magnitude promoted the expression of a group of anti-inflammatory and anti-oxidative genes; while the acute increase in shear frequency upregulated a set of cell-cycle regulating genes and angiogenesis genes. The adaptive response of global gene expression profile to the elevated shear magnitude is found to be triphasic, consisting of an induction period, an early adaptive response (ca. 45 minutes) and a late remodeling response. However, no apparent temporal regulation pattern of global gene expression was found during the adaptation to the elevated shear frequency. The results from this dissertation suggest that endothelial cells exhibit a specific phenotype during the adaptive response to changes in shear stress; and the transient phenotype is different than that of fully-adapted endothelial cells and may alter arterial atherosusceptibility.</p> / Dissertation