Browsing by Subject "endothelium"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Effect of Vascular Heterogeneity, Aging, and Exercise Training on eNOS ? Associated Protein:Protein Interactions(2014-04-14) Luttrell, Meredith JoyEndothelial dysfunction is a major risk factor for the development of cardiovascular diseases, and aging is associated with a gradual decline in endothelial function. Furthermore, endothelial dysfunction in arteries and arterioles supplying skeletal muscle has been implicated in the decline in exercise capacity with aging. Defined as an imbalance between the production and degradation of nitric oxide (NO), limited NO bioavailability is the hallmark characteristic of endothelial dysfunction. Production of NO is controlled by the enzyme endothelial nitric oxide synthase (eNOS), which is regulated in part by post-translational protein modifications. The purpose of this research was to examine the effect of vascular heterogeneity, aging, and endurance exercise training on eNOS-associated protein:protein interactions. Caveolin-1 (Cav1) is a negative regulator of eNOS activity, so that NO production is suppressed when Cav1 is bound to eNOS. Conversely, calmodulin (CaM) and heat-shock protein 90 (Hsp90) are positive regulators of eNOS activity, thus increasing eNOS activity and NO production when either are bound to eNOS. Co-immunoprecipitation was used to determine protein:protein interactions among eNOS and its regulatory proteins throughout the arterial network, from the aorta to third order skeletal muscle arterioles. Results show that eNOS-associated protein:protein interactions vary throughout the arterial network, and Cav1:eNOS and CaM:eNOS interactions are altered with aging. Additionally, endurance exercise training has no effect on the protein:protein interactions examined. In conclusion, eNOS regulation via protein:protein interactions appears to be vessel-specific, and aging has a heterogeneous effect on protein:protein interactions throughout the arterial network.Item Microvascular endothelial response to cocaethylene exposure: morphological and molecular observations(2004-11-02) Danyel Hermes Tacker; Anthony O. Okorodudu, Ph.D.; Robert H. Glew, Ph.D.; Norbert K. Herzog, Ph.D.; M. Tarek Elghetany, M.D.; Kathryn A. Cunningham, Ph.D.; Hal K. Hawkins, M.D., Ph.D.Cocaethylene (CE) is an active metabolite of cocaine and ethanol and is a toxicant of physiological relevance due to the high rate of cocaine and ethanol co-exposure (~80%) in cocaine abusers. It has prolonged action and increased potency on known physiological targets relative to the effect of cocaine. Since pathology in cocaine abusers is typically chronic and systemic, and CE persists in the body three to five times longer than cocaine, a link between CE and systemic disease in cocaine abusers was proposed. Consequently, this dissertation contains the studies that were used to test the hypothesis that the microvascular endothelium is a target tissue that is central in the pathogenic mechanism of cocaine-associated systemic disease, and that endothelial injury after CE exposure would result in dysregulation and altered barrier function due to changes in intracellular second messengers and signaling. To test this hypothesis, an in vitro model of CE exposure in human dermal microvascular endothelial cells (HMEC-1) was developed. Four Aims were designed to compartmentalize various components of the endothelial response to CE. The Aims included an array of methods to address cellular toxicity and dysfunction, including classical cytotoxicity and viability assays (Aim One), microscopic and electrical analyses of monolayer integrity (Aim Two), molecular analysis of second messengers, signaling molecule phosphorylation, and transcription factor DNA binding activity (Aims Three and Four). Aim One experiments demonstrated a lack of overt endothelial cytotoxicity caused by CE. Aim Two morphological analysis of endothelial intercellular borders and barrier integrity showed that CE exposure in the endothelial monolayers resulted in increased permeability, and hence a decrease in barrier integrity. These changes were observed temporally with alterations in cytosolic and total cellular free calcium ion (Aim Three), inositol 1,4,5 trisphosphate, and phosphorylated p38 mitogen-activated protein kinase concentrations, as well as changes in DNA binding activity and dimer composition of nuclear factor-kappaB (Aim Four). The observed changes suggest a distinct alteration of endothelial cell and monolayer function consistent with increased vascular permeability in vivo. Potential pathological outcomes of such effects include inflammation, vasculitis, systemic disease, and organ failure.Item The Synergic Effects of Flow and Sphingosine 1-Phosphate on Sprouting Angiogenesis Into Three-Dimensional Collagen Matrices(2012-07-16) Kang, Ho JinThe vascular endothelium continually senses and responds to both biochemical and mechanical stimuli to regulate vascular function in health and disease. The purpose of this dissertation was to understand the molecular mechanisms by which endothelial cells (ECs) respond to sphingosine 1-phosphate (S1P) and fluid wall shear stress (WSS) to initiate angiogenesis. To accomplish this, a novel cell culture system was developed to study the combined effects of S1P and WSS on inducing EC invasion into three-dimensional (3-D) collagen matrices. EC invasion required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable with S1P combined with pro-angiogenic growth factor stimulation. The extent of EC invasion depended on the magnitude of WSS in a biphasic manner, with the greatest induction occurring at 5.3 dyn/cm2 WSS. Several proteins have been implicated in EC invasion, including calpain, Akt, vimentin, p21-activated kinase (PAK), and membrane type 1-matrix metalloproteinase (MT1-MMP). Interestingly, activations of calpain and MT1-MMP and phosphorylations of Akt, PAK, and vimentin coincided with, and were required for, S1P- and WSS- induced EC invasion. Further, inhibitors of calpain, MT1-MMP, Akt and PAK all attenuated invasion induced by WSS and S1P. Calpain inhibition reduced Akt phosphorylation, vimentin cleavage, and MT1-MMP membrane translocation, suggesting that calpain regulates MT1-MMP via Akt phosphorylation and vimentin remodeling. Akt inhibition also completely blocked MT1-MMP membrane translocation and decreased phosphorylation of PAK and vimentin. In summary, these results suggest a new molecular pathway by which the combination of S1P and WSS stimulates EC invasion through calpain, Akt, PAK and vimentin to regulate activation and membrane translocation of MT1-MMP in 3-D collagen matrices.