Sulei Xu

Date of Graduation

2014

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Pingnian He

Committee Co-Chair

Robert W Brock

Committee Member

Gregory M Dick

Committee Member

Fred L Minnear

Committee Member

Geert W Schmid-Schonbein

Committee Member

Stanley D Yokota

Abstract

Mechanical forces have been indicated to play important roles in the regulation of inflammatory cell interaction with endothelium resulting in localized leakage formation and contributing to many disease-associated microvascular dysfunctions. However, most of the mechanical force related studies were conducted in vitro. The underlying mechanisms are still controversial. There is a need to investigate how shear stress regulates the endothelial cell (EC) signaling and related vascular barrier function using intact microvessels with experimental conditions closely replicating in vivo situations. The overall aim of my dissertation is to understand the molecular and cellular mechanisms of how shear stress and nitric oxide (NO) regulate microvessel function under physiological and pathological conditions. Studies were conducted on individually perfused intact rat mesenteric venules.;It is well known that shear stress is one of most important regulators in stimulating endothelial cells to produce NO. NO, in addition to being a potent vasodilator, has also been considered a "double edged sword"-mediator in inflammation. Under basal conditions, it prevents leukocyte and platelet adhesion, whereas under inflammatory conditions, the inflammatory mediator-induced excessive NO production contributes to permeability increases. In Chapter 2, we investigated the roles of endothelial basal NO production in leukocyte adhesion and adhesion-induced changes in microvessel permeability. The results indicated that the application of the eNOS specific inhibitor, caveolin-1 scaffolding peptide (CAV), caused reduction of basal NO and promoted ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. Also, CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.;In Chapter 3, we investigated the roles of shear stress (SS) in the regulation of microvessel permeability and its related EC signaling involving blood cells in individually perfused intact microvessels. Our results demonstrated that in response to a sudden change of SS, transient shear magnitude-dependent increases in EC [Ca2+]i occurred only in vessels perfused with whole blood or perfusate containing RBCs, which was correlated with EC gap formation illustrated by fluorescent microsphere accumulation. Carbenoxolone, a Pannexin 1 inhibitor, significantly reduced shear magnitude-dependent ATP release from RBCs and also abolished SS-induced increases in EC [Ca 2+]i and EC gap formation. Meanwhile, both plasma and whole blood perfusion induced shear magnitude-dependent NO production and eNOS-Ser 1177 phosphorylation.;It is unknown how EC sense SS, but the Glycocalyx (GCX), a layer of proteoglycans covering the endothelium, has been implicated as a mechanical sensor for changes in SS in vitro. The objective of chapter 4 is to identify the changes in GCX in microvessels of streptozotocin-induced diabetic rats and evaluate the associated changes in sensing SS and SS-induced NO production in individually perfused venules of diabetic rats. Our results indicated that the impaired GCX in diabetic microvessels enhances EC response to mechanical force and potentiates NO production and EC responses to ATP, resulting in enhanced endothelial gap formation.;Advances in micromanufacturing and microfluidic technologies have enabled a variety of insights into biomedical sciences while curtailing the high experimental costs and complexities associated with animals and in vivo studies. In Chapter 5, we presented and discussed our research work in creating engineered microvessels using a microfluidic platform and demonstrated the formation of the microvascular network in vitro and validated the key features that have been observed in microvessels in vivo. In our future studies, this may provide us a novel platform for studying spatial and temporal change of shear stress in the regulation of microvessel function in a close in vivo situation.;In conclusion, we revealed the role of shear stress and NO in the regulation of endothelial cell signaling and microvessel permeability in vivo, involving blood and non-blood components. The results also suggest the potential in using a microfluidic device in studying the physiological microvessel function.

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