Roles of nitric oxide and shear stress in the regulation of microvessel permeability in intact rat mesenteric venules

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

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10–30min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system

Two kinds of implementations of sobel edge detection algorithm based on field programmable gate array

In this paper,two kinds of edge detection scheme based on Field Programmable Gate Array(FPGA) are realized and analyzed by using the Sobel operator.In view of the design which utilizes the intellectual property(IP) cores embedded in the Quartus II has some disadvantages such as more resources are consumed、process is relatively slow and so on,an improved scheme which improves the first design's shortcomings is proposed.Simulation and verification results of these two schemes which are combined with Matlab and Modelsim show that the improved Sobel operator scheme is better than the previous scheme designed by using IP cores embedded in the Quartus II,achieving a good image detection and reducing errors

In Vitro Recapitulation of Functional Microvessels for the Study of Endothelial Shear Response, Nitric Oxide and [Ca2+]i

Microfluidic technologies enable in vitro studies to closely simulate in vivo microvessel environment with complexity. Such method overcomes certain constrains of the statically cultured endothelial monolayers and enables the cells grow under physiological range of shear flow with geometry similar to microvessels in vivo. However, there are still existing knowledge gaps and lack of convincing evidence to demonstrate and quantify key biological features of the microfluidic microvessels. In this paper, using advanced micromanufacturing and microfluidic technologies, we presented an engineered microvessel model that mimicked the dimensions and network structures of in vivo microvessels with a long-term and continuous perfusion capability, as well as high-resolution and real-time imaging capability. Through direct comparisons with studies conducted in intact microvessels, our results demonstrated that the cultured microvessels formed under perfused conditions recapitulated certain key features of the microvessels in vivo. In particular, primary human umbilical vein endothelial cells were successfully cultured the entire inner surfaces of the microchannel network with well-developed junctions indicated by VE-cadherin staining. The morphological and proliferative responses of endothelial cells to shear stresses were quantified under different flow conditions which was simulated with three-dimensional shear dependent numerical flow model. Furthermore, we successfully measured agonist-induced changes in intracellular Ca2+ concentration and nitric oxide production at individual endothelial cell levels using fluorescence imaging. The results were comparable to those derived from individually perfused intact venules. With in vivovalidation of its functionalities, our microfluidic model demonstrates a great potential for biological applications and bridges the gaps between in vitro and in vivo microvascular research

Convergence Analysis of the LDG Method for Singularly Perturbed Reaction-Diffusion Problems

We analyse the local discontinuous Galerkin (LDG) method for two-dimensional singularly perturbed reaction–diffusion problems. A class of layer-adapted meshes, including Shishkin- and Bakhvalov-type meshes, is discussed within a general framework. Local projections and their approximation properties on anisotropic meshes are used to derive error estimates for energy and “balanced” norms. Here, the energy norm is naturally derived from the bilinear form of LDG formulation and the “balanced” norm is artificially introduced to capture the boundary layer contribution. We establish a uniform convergence of order k for the LDG method using the balanced norm with the local weighted L2 projection as well as an optimal convergence of order k+1 for the energy norm using the local Gauss–Radau projections. The numerical method, the layer structure as well as the used adaptive meshes are all discussed in a symmetry way. Numerical experiments are presented

Convergence Analysis of the LDG Method for Singularly Perturbed Reaction-Diffusion Problems

We analyse the local discontinuous Galerkin (LDG) method for two-dimensional singularly perturbed reaction–diffusion problems. A class of layer-adapted meshes, including Shishkin- and Bakhvalov-type meshes, is discussed within a general framework. Local projections and their approximation properties on anisotropic meshes are used to derive error estimates for energy and “balanced” norms. Here, the energy norm is naturally derived from the bilinear form of LDG formulation and the “balanced” norm is artificially introduced to capture the boundary layer contribution. We establish a uniform convergence of order k for the LDG method using the balanced norm with the local weighted L2 projection as well as an optimal convergence of order k+1 for the energy norm using the local Gauss–Radau projections. The numerical method, the layer structure as well as the used adaptive meshes are all discussed in a symmetry way. Numerical experiments are presented