The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Historically, the angiogenesis field has advanced faster and farther than the field of lymphangiogenesis. The discovery of lymphatic markers and the emerging evidence implicating the lymphatic system as a central player in a variety of pathological conditions has attracted research interest and driven the field forward. Research efforts have produced the observation that regulators of the blood endothelium are frequently members of the same protein families of regulators of the lymphatic endothelium. More importantly, these regulators do not act discretely, restricting their regulatory activities to one endothelial cell (EC) type. Two examples of regulators that behave in this manner are the VEGF and the Angiopoietin families of proteins, which have cell-type-dependent effects on EC processes such as migration, proliferation and survival. The study of these regulators therefore requires an in vitro EC system capable of accommodating the simultaneous characterization of the signaling pathways downstream of these shared molecular regulators in venous, arterial and lymphatic endotheliums. To build such an in vitro system, I isolated and validated lymphatic, venous, and arterial ECs derived from vessels of bovine mesentery. The proteomes of the three cell types were comparatively studied using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric identification. The three cell types were used in a subtractive immunization scheme for the production of a monoclonal antibody selectively reactive to a potentially novel surface protein marker of lymphatic ECs. The studies recorded herein all share the common goal of identifying and characterizing unique molecular signatures that distinguish lymphatic ECs from blood ECs, and that may underline the cellular biology of the lymphatic endothelium as distinct from the blood endothelium.

Factor VIII (FVIII) is an essential blood-clotting protein and mutations in the FVIII gene are the cause of hemophilia A, a severe inherited bleeding disorder. FVIII synthesis has been observed in discreet endothelial sub-populations including liver sinusoidal endothelial cells and in selected microvascular beds. The mechanistic basis for this differential expression is unknown. Differences in shear stress are believed to play an important role in determining endothelial heterogeneity. In this study, we have evaluated the effect of various shear stress conditions on FVIII expression in blood outgrowth endothelial progenitor cells (BOECs) with an in vitro flow system. Under static conditions, BOECs do not express FVIII. In contrast, after exposure to laminar shear stress for 48 hrs, a significant increase in FVIII expression was documented by qRT-PCR, regardless of the magnitude of shear stress studied (1, 5, 15 and 30 dynes/cm2). To determine the effect of prolonged shear stress, laminar flow was applied over 120 hrs and FVIII mRNA levels returned to static levels. Induction of gene expression by laminar shear stress followed by repression after longer durations is common to other pro-coagulant genes induced by non-laminar or oscillatory flow (eg. tissue factor). BOECs exposed to 15 dyne/cm2 of shear stress, oscillating every 0.5 sec for 120 hrs, had FVIII mRNA levels 4.7-fold that of cells in static conditions. This was significantly higher than FVIII expression in BOECs exposed to 15 dyne/cm2 of laminar shear stress for the same duration. Expression of KLF2, a transcription factor that suppresses endothelial pro-coagulant gene expression under laminar shear stress, was significantly reduced in BOECs exposed to oscillatory as opposed to laminar shear stress. Finally, in BOECs exposed to oscillatory shear stress, FVIII protein was synthesized and co-localizes with its carrier protein VWF in Weibel-Palade bodies. These studies show that shear stress is a significant regulator of FVIII expression in BOECs, that FVIII expression is inversely correlated with that of KLF2, and that FVIII protein co-localizes with VWF in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-03-04 17:00:27.994

<p>The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin &#945;5&#946;1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.</p> / Dissertation

Angiogenesis is a fundamental process in reproduction and wound healing. Angiogenesis is also indispensable for solid tumor growth and metastasis, and also associated with angiogenic diseases. Beta-endorphin (£]-EP), derived from its precursor pro-opiomelancortin (POMC), is well known for its role in nociception and immune regulation. However, the function of morphine and £]-EP during angiogenesis remains characterization. One previous study indicated that morphine inhibited the proliferation and hypoxia-induced vascular endothelial growth factor (VEGF) release of endothelial cells. Contrastingly, another report found that morphine via Ras/PI3k/MAPK/ERK signaling promotes the survival and angiogenesis in endothelial cells. Besides, endogenous opioid peptides stimulated angiogenesis in chicken allantoic membrane assay through opioid receptors. Thus, the function and mechanism of £]-EP and opioid receptors in angiogenesis are controversial. This study evaluated the culture effects of £]-EP and morphine on angiogenesis . It was found that £]-EP stimulated the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Morphine at a high dose inhibited the proliferation, migration, and tube formation of endothelial cells. In the ex vivo rat aortic ring assay, £]-EP enhanced, whereas morphine perturbed, the microvessel sprouting. We also confirmed the expression of MOR¡ADOR¡AKOR opioid receptor in endothelial cells. Application of naloxone, a selective opioid antagonist, and neutralizing antibodies of MOR abolished the angiogenic effect of £]-EP and morphine. Thus £]-EP and morphine exert the pro- and anti-angiogenic effect via MOR, respectively .Besides, £]-EP can be regarded as a novel angiogenic factor.

The use of intravascular stents to restore blood flow through restricted vessels in patients with coronary artery disease has become the preferred method for treating a variety of lesion locations and pathologies. As new stent configurations and coatings are developed, a great need exists for high-throughput preclinical evaluation techniques that can interface human tissue with three-dimensional devices. Thus, the goals of this dissertation research were 1) to develop an in vitro blood vessel mimic composed of human cells for preclinical evaluation of intravascular devices, and 2) to utilize the mimic to assess neointimal responses to implanted stents.Experiments in support of these goals were broken into four specific aims. The first aim was to develop an in vitro human blood vessel mimic based on techniques for creating tissue engineered vascular grafts. The second aim was to determine the feasibility of utilizing this vessel mimic for bare metal stent evaluation. The third aim was to use the in vitro vessel to evaluate the cellular response to protein-coated stents. The fourth aim was to take advantage of the ability to control the in vitro vessel environment in order to evaluate the effect of shear rate on the neointimal response to implanted stents.Human blood vessel mimics were created by sodding fat-derived microvascular endothelial cells onto expanded polytetrafluoroethylene grafts and cultivating the vessels in bioreactor systems. This resulted in the development of a luminal lining of endothelial cells with sub-endothelial smooth muscle and mesenchymal cells. Deployment and assessment of bare metal stents within blood vessel mimics supported the feasibility of using the model for stent evaluation, and demonstrated that cell coverage of the device surface could be observed and measured. Protein-modified stents were created by submerging devices in enriched medium, and following implantation in the blood vessel mimic exhibited increased cell coverage and increased tissue thickness as compared with bare metal stents. Finally, an increase in shear rate lead to decreased neointimal coverage of implanted bare metal and modified stents. Overall, this dissertation demonstrates that in vitro human blood vessel mimics can be created and utilized for preclinical device evaluation.

Kawasaki disease (KD) is a childhood vasculitis with a predilection for the coronary arteries (CA). The etiology of KD is unknown; however, superantigens (SAg) have been implicated. SAg-activated T cells undergo massive proliferation followed by apoptosis; conversely, in KD these T cells may persist and target the CAs. Enhanced costimulation can rescue SAg-activated T cells from apoptosis, and Toll-like receptor 2 (TLR2) enhances costimulation. In a murine model of KD, TLR2-deficient mice are disease resistant, and evidence suggests preferential expression of TLR2 at the CA. Results from this study demonstrate that TLR2 is rapidly expressed in the heart following disease induction, and that TLR2 is expressed differentially in various arteries. The aorta, from which the CAs branch off, expressed the highest TLR2 levels. A microvascular endothelial cell line was shown to function as an APC following TLR2 stimulation, supporting the proliferation of SAg-activated T cells and their rescue from apoptosis.

Vascular endothelial cells (VECs) maintain vascular integrity by regulating the passage of solutes, macromolecules, and cells between the vascular and perivascular space and are critical in a wide number of physiological processes, such as the delivery of nutrients and oxygen to surrounding tissues, leukocyte trafficking, angiogenesis, and tissue repair. VEC permeability is regulated, at least in part, by VE-cadherin-based adherens junctions that coordinate inter-VEC contacts and communicate the strength of these interactions to the cell via the actin cytoskeleton. Although the ubiquitous second messenger, cyclic adenosine 3'€™, 5'€™-monophosphate (cAMP), has been shown to reduce VEC permeability, the molecular basis of this effect is currently unclear. Herein, we report that cAMP and its two effectors, cAMP-dependent protein kinase A-II (PKA-II) and exchange protein activated by cAMP-1 (EPAC1), improve barrier function and differentially coordinate this effect through both VE-cadherin and actin cytoskeletal structures. We have also identified cyclic nucleotide phosphodiesterase (PDE) 4 as the major PDE regulating VEC barrier function. Through the use of cAMP-elevating agents and RNAi-mediated knockdown of PKA-Cα, EPAC1 and PDE4D, we have identified a dominant role for EPAC1 in VEC permeability as well as recognized PDE4D as a potential adaptor protein VE-cadherin-based complexes. Our results are consistent with previous reports of a role for both PKA and EPAC1 in controlling VE-cadherin mediated barrier function and additionally provide novel insight into the differential roles that PKA, EPAC1 and PDE4D play in stabilizing VEC barrier function. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-09-18 16:09:59.12

Kawasaki disease (KD) is a childhood vasculitis with a predilection for the coronary arteries (CA). The etiology of KD is unknown; however, superantigens (SAg) have been implicated. SAg-activated T cells undergo massive proliferation followed by apoptosis; conversely, in KD these T cells may persist and target the CAs. Enhanced costimulation can rescue SAg-activated T cells from apoptosis, and Toll-like receptor 2 (TLR2) enhances costimulation. In a murine model of KD, TLR2-deficient mice are disease resistant, and evidence suggests preferential expression of TLR2 at the CA. Results from this study demonstrate that TLR2 is rapidly expressed in the heart following disease induction, and that TLR2 is expressed differentially in various arteries. The aorta, from which the CAs branch off, expressed the highest TLR2 levels. A microvascular endothelial cell line was shown to function as an APC following TLR2 stimulation, supporting the proliferation of SAg-activated T cells and their rescue from apoptosis.

No description available.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by excessive proliferation of pulmonary vascular cells, such as endothelial cells (ECs). Hereditary (H) PAH is mainly caused by ―loss-of-function‖ mutations in the gene coding for the bone morphogenetic protein type II receptor (BMPR2). However, the mechanisms by which these mutations cause PAH remain unclear. The hypothesis of this thesis was that BMPR2 mutations produce an imbalance in EC protein expression and/or activity that is integrally related to the development of abnormalities in lung vascular function and structure in HPAH. Patient-specific blood-outgrowth endothelial cells (BOECs) expanded ex vivo from peripheral blood mononuclear cells from patients with HPAH and healthy subjects were used to examine the consequences of BMPR2 mutations on the BOEC protein expression profile as well as on their functionality. Functional analyses of the BOECs revealed that HPAH-derived BOECs are more susceptible to apoptosis and more proliferative compared with healthy controls. Protein isolates of BOECs from patients with HPAH and from healthy subjects were subjected to 2-D gel electrophoresis and stained for total proteins and phosphoproteins, and to a quantitative computerassisted analysis. Differentially regulated proteins were identified by mass spectrometry (LC-MS/MS). Of the 416 total proteins detected under basal conditions, 11 were significantly downregulated in HPAH-derived BOECs and 11, including the translationally controlled tumor protein (TCTP), were upregulated. TCTP has previously been shown to be involved in systemic arteriolar remodeling, inflammation and growth. Therefore, the potential role of TCTP in PAH was studied in vivo in the SU5416 rat model of severe angioproliferative PAH. Immunofluorescence staining revealed high expression of TCTP in arteriolar ECs of PAH lungs tightly localized to proliferating cells within occlusive intimal lesions; whereas, only minimal TCTP expression was seen in vascular ECs of normal lungs. Similarly, abundant TCTP immunostaining was also seen in human PAH lung sections, again associated with complex vascular lesions. In BOECs, TCTP was found to participate in cell growth and survival. These data suggest that TCTP could play an important role in PAH by mediating pro-survival and growth signaling in vascular cells, contributing to occlusive pulmonary vascular remodeling triggered by EC apoptosis.