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Vascular Bioengineering Laboratory |
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Summary of Research Interests | |
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The Vascular Bioengineering Laboratory is focused on determining the effect of flow-induced mechanical forces on:
The long-term objective of our research is to:
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Current Projects | |
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In I/RP, the production of reactive oxygen species (ROS; or free radicals) from vascular ECs during RP is thought to play a critical role in tissue injury. The injury is attributed partially to peroxynitrite (ONOO-), a product of the reaction between nitric oxide (NO) and superoxide radicals (O2-.), and to the small GTPase Rac1 that activates the O2-.-producing NADPH oxidase. In vitro studies have used hypoxia (H)/reoxygenation (RO) in static ECs to simulate I/RP, ignoring possible flow effects on the cellular response. We and others have shown, however, that the onset of steady laminar shear stress triggers NO and O2-. generation, ONOO- formation and redox-sensitive gene expression. Thus, this study aims to quantify the endogenous ROS production and investigate the role of ROS on EC dysfunction following exposure of ischemic (hypoxic and low/no flow) ECs, previously acclimatized to shear stress, to the onset of laminar shear stress concurrently with oxygen readmission (reperfusion). It is hypothesized that at RP, ECs will produce ROS of different levels/time profiles resulting in different extents of dysfunction compared to RO. We are currently building a system that will expose cultured ECs to I/RP, where ischemic cells will be exposed to flow simultaneously with the beginning of RO. Production rates of NO and O2-. will be measured by chemiluminescence or electron spin resonance spectroscopy during RP vs. RO. EC dysfunction after in vitro I/RP will be quantified by assaying for lipid peroxidation, apoptosis and activation of the pro-apoptotic transcription factor NF-κB. While measuring each marker of dysfunction, inhibitors of key ROS sources and ROS scavengers will help us identify the ROS responsible for the EC injury.
Increasing evidence suggests that activation of the complement (C) cascade in plasma plays a key role in the pathogenesis of atherosclerosis, vascular injury after I/RP, and injury to cardiac allografts. Immediately following transplantation, graft failure may result from alloantigen-independent mechanisms (I/RP injury) or alloantigen-dependent mechanisms (preformed alloantibodies). Alloantigen-dependent events may lead to rejection within 1-3 months after transplantation or chronic graft dysfunction. In each case, alloantigen-dependent or independent events activate complement, resulting in production of fluid phase (C3a, C5a) and membrane-bound (C3b, C5b-9 or membrane attack complex, MAC) complement proteins, which collectively promote leukocyte chemotaxis and adhesion, cell activation and even lysis. Studies have shown that blockade of complement activation protects allografts from macrophage infiltration and delays rejection. We have recently shown that exposure of antibody-sensitized HUVECs to human serum, and not to heat-inactivated human serum, leads to C3b deposition on the plasma membrane. It is our plan to study the expression of EC surface receptors for leukocytes on complement-activated ECs, and the resultant adhesive events between complement-activated ECs and a suspension of leukocytes using an in vitro flow adhesion assay. By perfusing whole blood with fluorescently-labeled platelets at venous or low arterial shear rates, the deposition of platelets on complement-activated ECs may be quantified by analyzing the images collected during the flow.
The final common step in homotypic platelet aggregation, regardless of the stimulus, involves the interaction of adhesive proteins, such as fibrinogen and von Willebrand factor (vWF), with platelet GP IIb/IIIa (αIIbβ3). Studies have identified the pivotal role of GP IIb/IIIa receptors in coronary thrombosis. Hence, this integrin receptor has emerged as a rational therapeutic target in the management of acute coronary syndromes. Intravenous administration of the anti-GP IIb/IIIa monoclonal antibody c7E3 (abciximab) in patients undergoing angioplasty was shown to reduce the incidence of ischemic events at the 30-day primary end point. Abciximab also demonstrated efficacy when given in combination with thrombolytic therapy. We studied the effect of abciximab on recombinant tissue-type plasminogen activator (rt-PA)-induced lysis of preformed platelet-fibrin substrates under flow. When platelets within platelet-fibrin substrates were organized in platelet-rich regions, the substrates showed a nonuniform mode of lysis, where fibrin between thrombi lysed first and thrombi themselves lysed at a slower rate. Platelet pretreatment with c7E3 Fab was found to abolish the formation of lytic-resistant thrombi and platelet-protected fibrin zones and to facilitate rt-PA-mediated thrombolysis. Recent studies have shown that reactive oxygen species (ROS) produced by platelets contribute to platelet activation. These studies offer a rationale to investigate the intracellular signaling leading to activation, explore antioxidant agents as modulators of platelet activation in experimental models of ischemia/reperfusion, and assess the relative contribution of EC-derived vs. platelet-derived ROS in thrombosis following ischemia/reperfusion.
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Resources | |
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The Vascular Bioengineering Laboratory consists of 750 sq. ft. for benchtop work and a darkroom. The lab is equipped with two Dell PCs and a digital image processor. It also contains a laminar flow hood for endothelial cell tissue culture, a Nikon phase contrast microscope with epifluorescence capabilities, a 5% CO2 incubator, a low range centrifuge, a refrigerator with freezer, equipment for biochemical studies (gel electrophoresis apparatus), and a basal media flow recirculation setup (with capabilities to monitor both flow rate and oxygen tension). Within our building (Davis Heart and Lung Research Institute), we have access to: tissue culture core facilities; autoclave; cold room; UV/VIS spectrophotometer; Coulter counter; -80° freezer; microplate reader; film exposure apparaturs; flow cytometer; fluorometer/luminometer; blood gas analyzer; Electron Paramagnetic Resonance (EPR) spectroscopy core; and gene microarray core facilities. | |
Currently Associated with the Vascular Bioengineering Lab | |
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B. Rita Alevriadou, Ph.D., Principal Investigator
Michael P. Burns, Ph.D.,
Postdoctoral Fellow
Zhaosheng (John) Han, Ph.D.,
Postdoctoral Fellow
Charles I. Jones III, B.S.,
BME M.S. Candidate
Guru Meenakshisundaram, MS, BME PhD candidate
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Last updated: 10/04/2004
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