Date of Award

Spring 2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Julie Ji

Second Advisor

Sherry Voytik-Harbin

Committee Chair

Julie Ji

Committee Co-Chair

Sherry Voytik-Harbin

Committee Member 1

Sungsoo Na

Committee Member 2

Patricia Gallagher

Abstract

Endothelial cells are the interface between hemodynamic fluid flow and vascular tissue contact. They actively translate physical and chemical stimuli into intracellular signaling cascades which in turn regulate cell function, and endothelial dysfunction leads to inflammation and diseased conditions. For example, atherosclerosis, a chronic vascular disease, favorably develops in regions of disturbed fluid flow and low shear stress. Apoptosis, or programmed cell death, must be properly regulated to maintain homeostasis in the vascular wall. The loss of apoptosis control, as seen in low shear stress regions, is implicated in various diseases such as atherosclerosis and cancer. Death-associated protein kinase, DAPK is a pro-apoptotic regulator for various cell types that is localized in the cell cytoskeleton and regulates changes in cytoplasm associated with apoptosis. Yet its role in endothelial cells remains unclear. DAPK is a positive regulator in tumor necrosis factor &agr; (TNF&agr;) induced apoptotic pathway, and DAPK expression is lost in cancer cells. In this project, we begin to assess the effect of shear stress on endothelial cell apoptosis and DAPK.^ The potential role of DAPK and its corresponding signaling pathway in endothelial mechanotransduction and the role of nuclear lamina is further evaluated.^ Using bovine aortic endothelial cells, we have shown that laminar shear stress modulates DAPK expression. Initially, our study examined the time-dependent effects of conditioning cells with shear stress on apoptosis triggered by TNF&agr;, oxidative stress, and serum depletion; and the corresponding role of endothelial DAPK. Pre-conditioning cells with shear stress for 6 hours prior to apoptosis induction, decreased downstream caspase 3/7 activity, an apoptosis signaling constituent. Similarly, we also observed a corresponding decrease in DAPK in pre-sheared cells exposed to TNF&agr;, H2O 2, or serum starvation. Post-conditioning cells with 6 hours of shear after exposure to stimuli confirmed the protective effect of laminar shear stress in the presence of each apoptotic inducer. Our data suggest that shear stress and apoptosis agents may have competing effects on DAPK expression, and shear stress suppresses apoptosis by regulating DAPK in a time-dependent manner.^ Additionally, we transitioned our study to endothelial cells on non-glass substrates, such as flexible silicone membrane normally used for cyclic strain studies. We have shown a link between shear stress and DAPK expression and apoptosis in cells on membrane. Along with biochemical and molecular signals, the hemodynamic forces that the cells experience are also important regulators of endothelial functions. We found that adding shear stress significantly suppressed TNF&agr; induced apoptosis in cells; while shearing cells alone also increased apoptosis on either substrate. These data suggest that shear stress induced apoptosis in endothelial cells via increased DAPK expression and activation as well as caspase-3/7 activity. Most in vitro shear stress studies utilize the conventional parallel plate flow chamber where cells are cultured on glass, which is much stiffer than what cells encounter in vivo. Other mechanotransduction studies have utilized the flexible silicone membrane as substrate, for example, in cyclic stretch studies. Thus, this study bridges the gap between shear stress studies on cells plated on glass to future studies on different stiffness of substrates or mechanical stimulation such as cyclic strain.^ The nuclear lamina plays an important role in nuclear envelope structural support and connect to the cytoskeletal network through various co-localized proteins. Similarly, lamin is an integral part of the apoptosis process due to its direct linkage to the cytoskeleton and nuclear membrane. Conversely, lamin deficient cells lack nuclear structural integrity and the role of lamin in stress response is not completely understood. Mutations in lamin lead to changes in nuclear membrane mechanics eventually altering downstream signal response to stress. Therefore, we also looked at the impact of lamin deficiency in both MEF and BAEC cells on DAPK activity and apoptosis. We investigated the normally positive effects of shear on lamin deficient cells. The loss of lamin expression made cells more susceptible to shear stress and increased overall cell turnover compared to sheared control cells. Also, our results suggest the loss of lamin A/C expression mitigates TNF&agr;–induced apoptosis and affects its associated signaling pathways.^ In summary, the work presented in this dissertation has highlighted the role of DAPK in both mechanotransduction and endothelial apoptosis. Ultimately, this study demonstrates a key role lamin plays in mechanotransduction and apoptosis in response to both chemical and mechanical stimulation. By understanding how cells regulate phenotypic and genotypic changes in response to stimulation, we can better address disease and develop effective therapeutics.

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