Doctor of Philosophy / This thesis describes work done at the Institute of Dental Research in Sydney between February of 1986 and January 1990. The broad subject of the work is the role of vascular endothelial cells (ECs) in chronic inflammation. Periodontitis has been used as an example of chronic inflammatory disease, and provides the focus for this study of endothelial biology. In Chapter 1, aspects of the endothelial literature which provide relevant background information for work described in later chapters are reviewed. In Chapter 2, literature relating to aetiology and pathogenesis of chronic inflammatory periodontal disease is discussed. To maintain relevance of literature reviews to experimental work, each subsequent chapter contains a small literature review of material relating to the subject of the specific chapter. Early laboratory work is described in Chapter 3, and consisted of a morphological survey of the vascular changes occurring in gingival tissues with development of chronic periodontitis. Expansion of the vasculature and appearance of phenotypically specialised high endothelial cells (HECs), were associated with progression of the disease. Vessels with HECs and had a similar appearance to those known to be responsible for lymphocyte recirculation described in lymphoid tissues and chronic inflammatory sites. In the course of performing this survey, a perivascular hyaline material was noted surrounding capillaries close to the bacterial plaque irritant. The incidence, distribution, extent, ultrastructre and immuo-histochemistry of this material was more closely investigated, and the possible pathogenesis and significance of the material discussed in Chapter 4. In Chapter 5, the ultrastructural, histochemical and functional properties of gingival HECs are described, and compared with the well characterised HECs of rat lymph nodes. It was found that periodontal vessels were very similar to those in rat lymph nodes, with the exception however, that the gingival vessels appeared to exchange polymorphonuclear leukocytes almost exclusively, while vessels with HECs in lymph nodes and other locations are known as sites of lymphocyte recirculation. This observation indicated that the function of HECs requires further investigation, with particular regard to the synthetic activity of the cells. HECs were consistently alkaline phosphatise (AP) negative. The negative association between leukocyte emigration and AP activity (APA), as well as evidence in the literature illustrating both the wide substrate specificity of this enzyme and the importance of phosphorylation in the control of protein activation, suggested that AP could play a role in regulating leukocyte emigration. A pre-requisite for the investigation of this possibility, is the identification of a rich source of the identical iso-enzyme of AP to what is present in ECs. In Chapter 6, the sensitivity of endothelial AP to a panel of inhibitors is compared with that of a number of tissues for which isoenzyme has been identified. Endothelial AP was identified as the liver/bone/kidney isoenzyme. This allows the use of kidney tissue as a relevant source of AP for use in further study of the role of this enzyme in EC biology. It was clear that in order to study both the synthetic activity of HECs, as well as the role of AP in the control of leukocyte emigration, a method for obtaining high density primary cultures of HECs had to be established. Chapter 7 describes work done towards the development of such a culture system. The availability in the latter phase of the work of suitable probe for the technique of the in-situ hybridization allowed the possibility of testing the hypothesis that HECs are important cytokine producers. It was felt that this would provide some basis for the further study of those cells in-virtro. This work is described in the appendix. The general discussion in Chapter 8, summarises the work, and develops potential areas of study arising from the finding of this thesis.

Doctor of Philosophy / This thesis describes work done at the Institute of Dental Research in Sydney between February of 1986 and January 1990. The broad subject of the work is the role of vascular endothelial cells (ECs) in chronic inflammation. Periodontitis has been used as an example of chronic inflammatory disease, and provides the focus for this study of endothelial biology. In Chapter 1, aspects of the endothelial literature which provide relevant background information for work described in later chapters are reviewed. In Chapter 2, literature relating to aetiology and pathogenesis of chronic inflammatory periodontal disease is discussed. To maintain relevance of literature reviews to experimental work, each subsequent chapter contains a small literature review of material relating to the subject of the specific chapter. Early laboratory work is described in Chapter 3, and consisted of a morphological survey of the vascular changes occurring in gingival tissues with development of chronic periodontitis. Expansion of the vasculature and appearance of phenotypically specialised high endothelial cells (HECs), were associated with progression of the disease. Vessels with HECs and had a similar appearance to those known to be responsible for lymphocyte recirculation described in lymphoid tissues and chronic inflammatory sites. In the course of performing this survey, a perivascular hyaline material was noted surrounding capillaries close to the bacterial plaque irritant. The incidence, distribution, extent, ultrastructre and immuo-histochemistry of this material was more closely investigated, and the possible pathogenesis and significance of the material discussed in Chapter 4. In Chapter 5, the ultrastructural, histochemical and functional properties of gingival HECs are described, and compared with the well characterised HECs of rat lymph nodes. It was found that periodontal vessels were very similar to those in rat lymph nodes, with the exception however, that the gingival vessels appeared to exchange polymorphonuclear leukocytes almost exclusively, while vessels with HECs in lymph nodes and other locations are known as sites of lymphocyte recirculation. This observation indicated that the function of HECs requires further investigation, with particular regard to the synthetic activity of the cells. HECs were consistently alkaline phosphatise (AP) negative. The negative association between leukocyte emigration and AP activity (APA), as well as evidence in the literature illustrating both the wide substrate specificity of this enzyme and the importance of phosphorylation in the control of protein activation, suggested that AP could play a role in regulating leukocyte emigration. A pre-requisite for the investigation of this possibility, is the identification of a rich source of the identical iso-enzyme of AP to what is present in ECs. In Chapter 6, the sensitivity of endothelial AP to a panel of inhibitors is compared with that of a number of tissues for which isoenzyme has been identified. Endothelial AP was identified as the liver/bone/kidney isoenzyme. This allows the use of kidney tissue as a relevant source of AP for use in further study of the role of this enzyme in EC biology. It was clear that in order to study both the synthetic activity of HECs, as well as the role of AP in the control of leukocyte emigration, a method for obtaining high density primary cultures of HECs had to be established. Chapter 7 describes work done towards the development of such a culture system. The availability in the latter phase of the work of suitable probe for the technique of the in-situ hybridization allowed the possibility of testing the hypothesis that HECs are important cytokine producers. It was felt that this would provide some basis for the further study of those cells in-virtro. This work is described in the appendix. The general discussion in Chapter 8, summarises the work, and develops potential areas of study arising from the finding of this thesis.

Throughout much of the blood circulatory system, supply vessels (arteries and arterioles) are situated adjacent to corresponding draining vessels (veins and venules), which flow in the opposite direction. In this dissertation, mathematical models are developed to describe diffusive exchange of heat, oxygen and inert gases between such paired countercurrent blood vessels and surrounding tissue. In preliminary analyses, exchange between a single vessel and surrounding tissue is considered. The concept of equilibration length is developed. Then a well-known solution for two-dimensional diffusion between two vessels situated in an infinite domain is presented. This provides a basis for developing semianalytic solutions for two vessels situated in a cylindrical tissue region with Dirichlet or zero-flux conditions at the outer boundary. A general approach is then developed for obtaining semianalytic solutions for domains with non-circular cross-sections, and applied to the case of a rectangular domain. The governing equations for paired blood vessels are then solved to obtain the axial variation of temperature or concentration for a variety of cases, including Dirichlet and zero-flux boundary conditions, with and without deposition or consumption of heat or gas. For the Dirichlet case, the equilibration length is compared to that for a single vessel, showing that equilibrium is achieved more rapidly when a single vessel is replaced by two vessels with the same diameter as the single vessel. For the zero-flux case, particular solutions to the full three-dimensional diffusion equation in the tissue are obtained from the two-dimensional solutions. The total transport (convective and diffusive) in the axial direction is evaluated, with and without consumption/deposition, and the results are interpreted in terms of an enhanced diffusivity. Finally, the complementary roles of convection and diffusion in mass and heat transport in the axial direction are considered. It is shown that as vessel diameter decreases, countercurrent exchange eventually results in a reduction of convective transport. Axial diffusion becomes significant at approximately the same range of diameters. This finding is interpreted in terms of the efficiency by which a branching network can transport heat and mass to its extremities.

Liver kinase B1 (LKB1), a serine/threonine kinase, is responsible for the activation of AMP-activated protein kinase (AMPK), the master regulator of energy metabolism. LKB1/AMPK signaling pathway possesses a wide range of biological functions in regulating cell cycle progression, cell polarity, senescence and inflammation. In cultured endothelial cells, the pro-senescence function of LKB1/AMPK signaling pathway has been observed. However, the mechanisms by which LKB1 is regulated in endothelial cells remain largely uncharacterized. Furthermore, little is known about the effects of activated endothelial LKB1/AMPK signaling pathway on vascular and energy homeostasis. The present study aimed to investigate the upstream molecular mechanisms regulating LKB1 protein stability during endothelial senescence and the downstream pathophysiological effects of hyperactivated AMPK signaling in endothelial cells. In cultured model of cellular senescence, the lysine (K) 64 residue of LKB1 was found to be crucial for mediating its pro-senescence activities. The protein stability and intracellular localization of LKB1 mutant with K64 replaced by arginine (R) was different from the wild type protein. K64R exhibited enhanced effects on promoting endothelial senescence. Moreover, mutation of this residue attenuated the binding to HERC2, a newly identified E3 ubiquitin ligase for LKB1, in turn preventing its ubiquitination and degradation. Using a transgenic mouse model that selectively over-expresses a constitutively active AMPK α1 subunit (EC-AMPK) in endothelial cells, the influence of hyperactivated AMPK signaling on metabolic and vascular functions was investigated. Under standard chow condition, the metabolic phenotypes were similar between wild type and EC-AMPK mice; under high fat diet condition, EC-AMPK mice showed more severe obesity-induced fatty liver injury. Selective activation of AMPK in endothelial cells caused vascular and hepatic inflammation. Cyclooxygenase-2 (COX-2) was found to be the mediator for the pro-inflammatory functions of AMPK in vascular endothelial cells and facilitated to the development of obesity-induced fatty liver injury in EC-AMPK mice. Evaluation using isolated arteries revealed an increased systolic blood pressure and abnormal endothelial function in EC-AMPK miceunder high fat diet. AMPK activation in endothelium of the blood vessel could not block vascular remodeling associated with dietary obesity. Taken in conjunction, the above findings suggest that continuous activation of LKB1/AMPK signaling elicits adverse effects on both energy and vascular homeostasis. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Wong Chi Ming. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 182-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

O objetivo desse trabalho foi a avaliação da atividade elétrica uterina em ratas Wistar prenhes antes e após a eletroacupuntura e a influência do sistema nervoso central e dos circuitos elétricos biologicamente fechados. A avaliação da atividade elétrica cerebral e uterina foi realizada por meio de experimentos crônicos (n=16, 8 prenhes e 8 não prenhes) e agudos (n=11, 4 prenhes e 7 não prenhes). As ratas prenhes receberam a eletroacupuntura nos pontos Sanyinjiao e Zusanli entre 17 a 19 semanas de prenhez. A fase de experimentos agudos consistiu de lesão na medula espinhal no nível de T1 (2 ratas, uma prenhe e a outra não), denervação do útero utilizando álcool absoluto (3 ratas, uma prenhe e duas não), eletroestimulação de artéria isolada do rabo (2 ratas não prenhes) e experimentos in vitro (4 ratas, duas prenhes e duas não). A atividade elétrica uterina de repouso foi semelhante nas ratas prenhes (1,87 +/- 2,35 eventos / 3 minutos) e não prenhes (2,31 +/- 1,57 eventos / 3 minutos, p>0.1). Após a eletroacupuntura o número de eventos aumentou de 1,87 +/- 2,35 / 3 minutos para 28,06 +/- 17,27 / 3 minutos; p<0.01. Verificou-se uma correlação entre o aumento da atividade elétrica cerebral (córtex e hipocampo) e da atividade uterina em 3 ratas (2 prenhes e 1 não prenhe). Nem lesões na medula espinhal, nem a denervação do útero nem a realização dos experimentos in vitro modificou o aumento da atividade uterina. A eletroestimulação da artéria isolada do rato alcançou o útero mas parou após o clampeamento da artéria. Portanto, a atividade elétrica uterina é semelhante nas ratas prenhes e não prenhes. A atividade elétrica uterina aumenta após 90 minutos de eletroacupuntura em ratas Wistar prenhes, provavelmente através dos circuitos elétricos biologicamente fechados. O sistema nervoso não é necessário para esse fenômeno. / The objective of this work was the evaluation of uterine electric activity in pregnant Wistar rats before and after electroacupuncture stimulation and the influence of central nervous system and biologically closed electric circuits on uterine electric activity. The evaluation of brain electric activity and uterine electric activity was studied by means of chronic experiments (n=16, 8 pregnant and 8 non pregnant rats). In pregnant rats electroacupuncture was performed at acupoints Sanyinjiao and Zusanli between 17 and 19 days of pregnancy. The acute experiments phase was lesions in spinal cord at T1 level (2 rats, one pregnant and one non pregnant), denervation of uterus using absolute alcohol (3 rats, one pregnant and two non pregnant), electric stimulation of isolated tail artery (2 animals, non pregnant) and in vitro experiment (2 pregnant rats and 2 non pregnant rats). The uterine activity was similar in pregnant (1,87 +/- 2,35 events / 3 minutes) and non pregnant rats (2,31 +/- 1,57 events / 3 minutes, p>0.1). After electroacupuncture, the number of events rises for 1,87 +/- 2,35 / 3 minutes to 28,06 +/- 17,27 / 3 minutes; p<0.01 (pregnant rats). In 3 rats (1 non-pregnant and 2 pregnants), was observed correlation between the rise of cerebral activity (cortex and hipoccampus) and the uterine activity. Neither lesions in spinal cord nor denervation of uterus and in vitro experiments modified the rising of uterine activity. The electric stimulation of isolated tail artery reached the uterus but stopped after clamping the artery. Therefere, the basal uterine electric activity is similar in pregnant and non pregnant Wistar rats; uterine activity rises after 90 minutes of electroacupuncture in Wistar pregnant rats, probably by biologically closed electric circuits and the nervous system is not necessary for this phenomenon.

O objetivo desse trabalho foi a avaliação da atividade elétrica uterina em ratas Wistar prenhes antes e após a eletroacupuntura e a influência do sistema nervoso central e dos circuitos elétricos biologicamente fechados. A avaliação da atividade elétrica cerebral e uterina foi realizada por meio de experimentos crônicos (n=16, 8 prenhes e 8 não prenhes) e agudos (n=11, 4 prenhes e 7 não prenhes). As ratas prenhes receberam a eletroacupuntura nos pontos Sanyinjiao e Zusanli entre 17 a 19 semanas de prenhez. A fase de experimentos agudos consistiu de lesão na medula espinhal no nível de T1 (2 ratas, uma prenhe e a outra não), denervação do útero utilizando álcool absoluto (3 ratas, uma prenhe e duas não), eletroestimulação de artéria isolada do rabo (2 ratas não prenhes) e experimentos in vitro (4 ratas, duas prenhes e duas não). A atividade elétrica uterina de repouso foi semelhante nas ratas prenhes (1,87 +/- 2,35 eventos / 3 minutos) e não prenhes (2,31 +/- 1,57 eventos / 3 minutos, p>0.1). Após a eletroacupuntura o número de eventos aumentou de 1,87 +/- 2,35 / 3 minutos para 28,06 +/- 17,27 / 3 minutos; p<0.01. Verificou-se uma correlação entre o aumento da atividade elétrica cerebral (córtex e hipocampo) e da atividade uterina em 3 ratas (2 prenhes e 1 não prenhe). Nem lesões na medula espinhal, nem a denervação do útero nem a realização dos experimentos in vitro modificou o aumento da atividade uterina. A eletroestimulação da artéria isolada do rato alcançou o útero mas parou após o clampeamento da artéria. Portanto, a atividade elétrica uterina é semelhante nas ratas prenhes e não prenhes. A atividade elétrica uterina aumenta após 90 minutos de eletroacupuntura em ratas Wistar prenhes, provavelmente através dos circuitos elétricos biologicamente fechados. O sistema nervoso não é necessário para esse fenômeno. / The objective of this work was the evaluation of uterine electric activity in pregnant Wistar rats before and after electroacupuncture stimulation and the influence of central nervous system and biologically closed electric circuits on uterine electric activity. The evaluation of brain electric activity and uterine electric activity was studied by means of chronic experiments (n=16, 8 pregnant and 8 non pregnant rats). In pregnant rats electroacupuncture was performed at acupoints Sanyinjiao and Zusanli between 17 and 19 days of pregnancy. The acute experiments phase was lesions in spinal cord at T1 level (2 rats, one pregnant and one non pregnant), denervation of uterus using absolute alcohol (3 rats, one pregnant and two non pregnant), electric stimulation of isolated tail artery (2 animals, non pregnant) and in vitro experiment (2 pregnant rats and 2 non pregnant rats). The uterine activity was similar in pregnant (1,87 +/- 2,35 events / 3 minutes) and non pregnant rats (2,31 +/- 1,57 events / 3 minutes, p>0.1). After electroacupuncture, the number of events rises for 1,87 +/- 2,35 / 3 minutes to 28,06 +/- 17,27 / 3 minutes; p<0.01 (pregnant rats). In 3 rats (1 non-pregnant and 2 pregnants), was observed correlation between the rise of cerebral activity (cortex and hipoccampus) and the uterine activity. Neither lesions in spinal cord nor denervation of uterus and in vitro experiments modified the rising of uterine activity. The electric stimulation of isolated tail artery reached the uterus but stopped after clamping the artery. Therefere, the basal uterine electric activity is similar in pregnant and non pregnant Wistar rats; uterine activity rises after 90 minutes of electroacupuncture in Wistar pregnant rats, probably by biologically closed electric circuits and the nervous system is not necessary for this phenomenon.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerotic lesions of human beings are common diagnosed in regions of arte- rial branching and curvature. The prevalence of atherosclerosis is usually associated with hardening and ballooning of aortic wall surfaces because of narrowing of flow path by the deposition of fatty materials, platelets and influx of plasma through in- timal wall of Aorta. High Wall Shear Stress (WSS) is proved to be the main cause behind all these aortic diseases by physicians and researchers. Due to the fact that the atherosclerotic regions are associated with complex blood flow patterns, it has believed that hemodynamics and fluid-structure interaction play important roles in regulating atherogenesis. As one of the most complex flow situations found in cardio- vascular system due to the strong curvature effects, irregular geometry, tapering and branching, and twisting, theoretical prediction and in vivo quantitative experimental data regarding to the complex blood flow dynamics are substantial paucity. In recent years, computational fluid dynamics (CFD) has emerged as a popular research tool to study the characteristics of aortic flow and aim to enhance the understanding of the underlying physics behind arteriosclerosis. In this research, we study the hemo- dynamics and flow-vessel interaction in patient specific normal (healthy) and dilated (diseased) aortas using Ansys-Fluent and Ansys-Workbench. The computation con- sists of three parts: segmentation of arterial geometry for the CFD simulation from computed tomography (CT) scanning data using MIMICS; finite volume simulation of hemodynamics of steady and pulsatile flow using Ansys-Fluent; an attempt to perform the Fluid Structure Simulation of the normal aorta using Ansys-Workbench. Instead of neglecting the branching or smoothing out the wall for simplification as a lot of similar computation in literature, we use the exact aortic geometry. Segmen- tation from real time CT images from two patients, one young and another old to represent healthy and diseased aorta respectively, is on MIMICS. The MIMICS seg- mentation operation includes: first cropping the required part of aorta from CT dicom data of the whole chest, masking of the aorta from coronal, axial and saggital views of the same to extract the exact 3D geometry of the aorta. Next step was to perform surface improvement using MIMICS 3-matic module to repair for holes, noise shells and overlapping triangles to create a good quality surface of the geometry. A hexahe- dral volume mesh was created in T-Grid. Since T-grid cannot recognize the geometry format created by MIMICS 3-matic; the required step geometry file was created in Pro-Engineer. After the meshing operation is performed, the mesh is exported to Ansys Fluent to perform the required fluid simulation imposing adequate boundary conditions accordingly. Two types of study are performed for hemodynamics. First is a steady flow driven by specified parabolic velocity at inlet. We captured the flow feature such as skewness of velocity around the aortic arch regions and vortices pairs, which are in good agreement with open data in literature. Second is a pulsatile flow. Two pulsatile velocity profiles are imposed at the inlet of healthy and diseased aorta respectively. The pulsatile analysis was accomplished for peak systolic, mid systolic and diastolic phase of the entire cardiac cycle. During peak systole and mid-systole, high WSS was found at the aortic branch roots and arch regions and diastole resulted in flow reversals and low WSS values due to small aortic inflow. In brief, areas of sudden geometry change, i.e. the branch roots and irregular surfaces of the geom- etry experience more WSS. Also it was found that dilated aorta has more sporadic nature of WSS in different regions than normal aorta which displays a more uniform WSS distribution all over the aorta surface. Fluid-Structure Interaction simulation is performed on Ansys-WorkBench through the coupling of fluid dynamics and solid mechanics. Focus is on the maximum displacement and equivalent stress to find out the future failure regions for the peak velocity of the cardiac cycle.

Indiana University-Purdue University Indianapolis (IUPUI) / The passive properties of the venous wall are important in the development of venous pathology. Increase in venous pressure due to retrograde flow (reflux) and obstruction of venous flow by intrinsic and extrinsic means are the two possible mechanisms for venous hypertension. Reflux is the prevailing theory in the etiology of venous insufficiency. The objective of this thesis is to quantify the passive biomechanical response and structural remodeling of veins subjected to chronic venous reflux and hypertension. To investigate the effects of venous reflux on venous mechanics, the tricuspid valve was injured chronically in canines by disrupting the chordae tendineae. The conventional inflation-extension protocol in conjunction with intravascular ultrasound (IVUS) was utilized to investigate the passive biomechanical response of both control common iliac veins (from 9 dogs) and common iliac veins subjected to chronic venous reflux and hypertension (from 9 dogs). The change in thickness and constituent composition as a result of chronic venous reflux and hypertension was quantified using multiphoton microscopy (MPM) and histological evaluation. Biomechanical results indicate that the veins stiffened and became less compliant when exposed to eight weeks of chronic venous reflux and hypertension. The mechanical stiffening was found to be a result of a significant increase in wall thickness (p < 0.05) and a significant increase in the collagen to elastin ratio (p < 0.05). After eight weeks of chronic reflux, the circumferential Cauchy stress significantly reduced (p < 0.05) due to wall thickening, but was not restored to control levels. This provided a useful model for development and further analysis of chronic venous insufficiency and assessment of possible intervention strategies.