The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.

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

Numerous studies have identified that elevated plasma concentrations of lipoprotein(a) [Lp(a)] are an emerging risk factor for a variety of atherothrombotic disorders. Apolipoprotein(a) [apo(a)], the unique glycoprotein component of Lp(a), consists of tandem repeats of a plasminogen kringle (K) IV-like domain, followed by sequences homologous to the plasminogen KV and protease domains. Apo(a)/Lp(a) has been consistently shown to regulate endothelial function and inhibit plasminogen activation. In the present study, we have demonstrated that apo(a), signaling via integrin alphaVbeta3, is the functional unit in Lp(a) to stimulate in vitro endothelial cell (EC) proliferation and migration, and activate focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPK) in cultured ECs. Both apo(a) and Lp(a) have also been shown to reduce the levels of active and total transforming growth factor (TGF)-beta in cultured EC medium in an integrin alphaVbeta3–dependent manner. Despite the stimulatory effects of apo(a) on EC proliferation and migration, we have further confirmed an inhibitory effect of apo(a) on EC in vitro angiogenesis using a fibrin gel tube formation assay. We have provided evidence proving apo(a) inhibits angiogenesis through inhibition of plasminogen activation, and this inhibitory effect is dependent on the presence of apo(a) KV domain. Lastly, apo(a) is shown to reduce the protein levels of annexin A2 and S100A10 in ECs, which implies another potential mechanism by which apo(a)/Lp(a) could impair plasminogen activation on cell surface. In summary, we have discovered the first complete outside-in signaling pathway elicited by apo(a)/Lp(a) in ECs and have built up a connection between the ability of apo(a) to inhibit plasminogen activation and its inhibition of angiogenesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-09-25 18:29:47.106

No description available.

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.

No description available.

Antiphospholipid syndrome (APS) is an autoimmune disorder defined as the persistent presence of antiphospholipid antibodies (aPL) associated with recurrent thrombotic events and/or fetal loss. The mechanisms for fetal loss in this syndrome have not yet been clearly explained, although several hypotheses based on experimental data have been put forward. It has been shown that proliferation of human umbilical vein endothelial cells (HUVECs) decreased significantly in cultures that contained sera positive for anticardiolipin antibody activity collected from patients with recurrent fetal loss. We explored the effect of ACAs on Vascular Endothelial Growth factor- A (VEGF-A) secretion of cultured HUVECs. VEGF appears to be the most endothelial cell-specific and unequivocal angiogenic factor. In vitro, VEGF causes endothelial cell proliferation and migration; in vivo, it is potently angiogenic and causes vascular permeability. We determined the effect of ACA IgG 40μg/ml and 80μg/ml on VEGF secretion using ELISA. The results were measured in mean ± SD and expressed in pg of VEGF/5 x 10⁴ cells. P ≤ 0.05 considered significant as compared to control. The results showed that ACA increases VEGF-A secretion and/or may bind at VEGF-A binding sites. This finding may be useful for finding therapies for patients with recurrent miscarriages. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science