The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term. Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue. The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta. No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

The genus Bartonella comprises emerging pathogens that are causative agents of a wide range of clinical manifestations such as cat scratch disease, bacillary angiomatosis, and Carrion’s disease. All species are transmitted by blood-sucking arthropods and infect erythrocytes and endothelial cells of hosts. Carrion’s disease is a bi-phasic infection caused by Bartonella bacilliformis which is characterized by hemolysis of infected erythrocytes followed by invasion of the vascular endothelium. This provokes pronounced cellular proliferation, angiogenesis and skin eruptions called verruga peruana. Endothelial cells are thought to be the primary niche wherein bacteria reside between inoculation and erythrocyte infection. This study aims to elucidate some of the endothelial factors involved during the verruga peruana phase of Carrion’s disease. In order to adhere to and invade human microvascular endothelial cells (HMEC-1), B. bacilliformis engages a family of cell receptors called integrins. We used anti-integrin antibodies to show that the primary integrin involved is the fibronectin receptor á5â1, although the vitronectin receptor áVâ3 also plays a minor role. We show B. bacilliformis invasion is also dependent on integrin ligands, fibronectin and vitronectin as antibodies against these proteins decreased invasion and attachment, whereas pre-treatment of the bacteria with these molecules enhanced infection of endothelial cells. Bacterial uptake requires various host cytoplasmic signaling pathways to work in tandem, and our study identified three mitogen activated protein kinases involved. Apart from MAPKs, phosphotidylinositol 3 kinase plays a role during invasion and cell survival. PI3K inhibitors blocked bacterial internalization and B. bacilliformis infected cells showed accelerated apoptosis. Lastly, microarray analysis was performed to study the gene expression profile of B. bacilliformis infected HMEC-1 cells. Numerous molecules of the integrin signaling pathways are involved, suggesting integrins as the major receptor recruited for the successful infection by B. bacilliformis. In summary this is the first study to demonstrate the role of integrins as B. bacilliformis receptors and integrin ligands as facilitators of infection. Gene expression analysis suggests the possibility that integrin mediated signaling pathways are the key modulators of cellular alterations during B. bacilliformis infection. This hypothesis is supported by the identification of some members of the integrin signaling pathway necessary for B. bacilliformis entry into endothelial cells.

Sprouting endothelial cells (ECs) use soluble and insoluble cues to guide migration and expand the existing vascular network to meet changing trophic needs of the tissue during angiogenesis. A noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique was used to optically image endothelial sprouting morphogenesis in three dimensional (3D) collagen matrices with simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Sprout advancement and lumen expansion companying with ECM alteration were the synergistic results of membrane-associated matrix metalloproteinase and cell traction evidenced by proteinase inhibition and Rho-associated kinase (p160ROCK) inhibition experiments. These physical EC-ECM interactions suggest that ECM mechanical properties may influence angiogenic responses. In a 3D angiogenesis model, we measure angiogenic responses as a function of collagen matrix stiffness by inducing collagen cross-linking with microbial transglutaminase (mTG). Collagen matrices stiffen with both mTG treatment and incubation time as evidenced with biaxial mechanical test results and collagen TPF intensity increases with mTG treatment and that the ratio of TPF/SHG correlates with biaxial tested mechanical stiffness. SHG and optical coherence microscopy (OCM) are further used to show that other physical properties of the matrix do not change with mTG treatment, thus providing the same density but different stiffness with which to measure angiogenic responses. Stiffer matrices promote angiogenesis with more invading sprouts that invade deeper. No differences in lumen size were observed between control and mTG stiffened 3D cultures, but there was evidence of greater matrix remodeling in stiffer gels using NLOM. Results of this study show angiogenic responses are enhanced with increasing stiffness and suggest that these properties may be used in tissue engineering and regenerative medicine applications to engineer angiogenesis.

A CLCA protein (CL for chloride channel and CA for calcium) cloned from porcine ileum was expressed and characterized. The regulatory behavior, inhibitor sensitivity, and functional properties of chloride conductance associated with the expression of pCLCA1 cDNA were investigated in non-epithelial NIH/3T3 fibroblasts and in an epithelial Caco-2 cell line. These properties were also investigated in freshly isolated retinal pigment epithelial (RPE) cells and in primary cultures of these cells which express an endogenous cCLCA1. In NIH/3T3 fibroblasts, the chloride efflux induced by pCLCA1 was directly activated by calcium. A and C kinase agonists were without effect. The electrogenic nature of chloride efflux was confirmed by detection of outwardly rectified chloride currents. Selected anion channel blockers inhibited both the pCLCA1 agonist-induced current and chloride efflux. The inhibitors also reduced Ussing chamber short circuit current and chloride efflux from primary RPE cultures. However, these same agents did not inhibit chloride efflux in fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive chloride channel. The expression of pCLCA1 increased cAMP/A kinase-dependent chloride ion release from fibroblasts and Caco-2 cells expressing CFTR. These pleiotropic effects of CLCA protein expression suggested that the protein may regulate the activity of chloride conductance, rather than functioning as a primary ion transporter. This putative regulatory behavior was further investigated in Caco-2 cells. The rate of 36Cl efflux and the amplitude of currents in patch clamp studies after activation of A kinase or intracellular Ca2+ mobilization was significantly increased in freshly passaged Caco-2 cells expressing pCLCA1. However, 36Cl efflux and short circuit Ussing chamber studies in polarized Caco-2 cells provided evidence that both endogenous and pCLCA1-dependent Ca2+-sensitive chloride conductance were lost from 14 day post-passage cells. cAMP-dependent chloride conductance continued to be modulated by pCLCA1 expression in differentiated 14 day post-passage Caco-2 cells, demonstrating the retention of pCLCA1 effects in these mature cells. We conclude that pCLCA1 expression enhances the sensitivity of endogenous chloride channels to both natural agonists, Ca2+and cAMP, but that it lacks inherent Ca2+-dependent chloride channel activity.

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, in vitro approaches for functional assessments have been relatively limited. Herein, we describe the guidance of primary endothelial cells (ECs) in an agarose hydrogel scaffold that is chemically patterned with an immobilized concentration gradient of vascular endothelial growth factor 165 (VEGF165) using multiphoton laser patterning of VEGF165. This is the first demonstration of this patterning technology to immobilize proteins; and the first demonstration of immobilized VEGF165 to guide endothelial cell growth and differentiation in 3D environments. It is particularly compelling that this 3D hydrogels provide an excellent biomimetic environment for stem cell niche, thereby offering a new approach to study stem cell biology. In this thesis, we focused on the retinal stem cell niche, investigating cellular interactions between retinal stem and progenitor cells (RSPCs) and endothelial cells (ECs). By using this 3D in vitro model, we demonstrated the synergistic interactions between RSPCs and ECs wherein RSPCs migrated into 3D gels only in the presence of ECs and RSPCs stabilized EC tubular-like formations. Moreover, we characterized the contact-mediated effects of ECs on RSPC fate in terms of proliferation and differentiation.

The endothelium plays an essential role in the regulation of vascular homeostasis and a state of endothelial dysfunction, which develops in the presence of cardiovascular risk factors, may contribute to the development and progression of cardiovascular disease. As such, the measurement of endothelial function, beyond being an experimental tool, may serve as an important tool to complement current risk assessment algorithms in the identification of high-risk patients. Flow-mediated dilation (FMD) is a non-invasive measure of peripheral conduit artery endothelial function that holds great promise. Presently, FMD suffers from methodological heterogeneity and a poor understanding of the various biological components involved in eliciting the dilatory response to a given shear stimulus. We compared both traditional and alternative methods of arterial diameter characterization with regards to their repeatability, nitric oxide-dependency, and their sensitivity in distinguishing between normal and dysfunctional endothelial responses. Our findings emphasize the importance of continuous arterial diameter measurement and suggest that the time to peak FMD is not a useful adjunctive measure of the FMD response. Given that endothelial dysfunction may be of clinical importance, strategies to correct it or prevent it from occurring may be of benefit. The 3-hydroxy-3-methylglutaryl coenzyme A inhibitors are agents that have demonstrated marked cholesterol-independent, endothelial-protective effects. We investigated the ability of rosuvastatin and atorvastatin to protect against endothelial dysfunction associated with ischemia and reperfusion (IR) injury, and chronic nitrate therapy. Using the FMD technique, we demonstrated, for the first time in humans, that acute rosuvastatin administration protects against IR-induced conduit artery endothelial dysfunction. Additionally, we demonstrated that this effect likely occurred by a cyclooxygenase-2-dependent mechanism, which may provide mechanistic insight into the observed cardio-toxicity with cyclooxygenase-2 inhibitors. In contrast, we observed that this endothelial-protective effect was lost upon sustained rosuvastatin administration, which may have important implications regarding the generation of sustained cardioprotective phenotypes. Finally, we demonstrated that atorvastatin co-administration prevented the development of tolerance and endothelial dysfunction associated with continuous transdermal nitroglycerin therapy in humans, likely through an antioxidant mechanism. Future studies are needed in disease patients to determine whether the concept of nitrate tolerance needs reconsideration in the presence of vascular-protective agents.

Many studies have demonstrated that there is a strong relationship between endothelial dysfunction and oxidative stress and have demonstrated also that hyperglycemia is associated with increased generation of oxidative stress and atherosclerotic vascular diseases, but we do not know how hydrogen peroxide (H2O2) and high glucose (HG) could affect calcium regulatory proteins of human vascular endothelial cells (HUVECs) in vitro. In the present study, we have examined the acute effect of H2O2 (100 M) and the effect of chronic exposure to HG concentration (35 mM) on the calcium regulatory system of human vascular endothelial cells using fluorescence imaging microscopy (fura-2). In this study, we tested the hypothesis that calcium regulatory proteins (SERCA-ATPase and PMCA-ATPase pumps and the NCX exchanger) of ECs have different sensitivities to H2O2 and high glucose concentration. We also tested the hypothesis that calcium regulatory proteins could be potential targets of ROS at the early stage of vascular disease. The results of this study showed that both H2O2 and high glucose induced significant delay in calcium removal time (CRT). The study of H2O2 showed that the delay in CRT was due to partial inhibition of SERCA-ATPase and the sodium calcium exchanger (NCX) activity and the effect of H2O2 on CRT was reversible. In contrast, the PMCA-ATPase pump was resistant to inhibition by H2O2. Furthermore, H2O2 induced a 40 ± 6.5 % reduction in endoplasmic reticulum refilling. The second part of the study showed that exposure of ECs to HG concentration for 10 days induced a significant delay in CRT and this delay was due to partial blockade of the SERCA-ATPase pump. Blockade of PMCA-ATPase pump with vanadate showed a further delay in CRT. We conclude that: 1- Both H2O2 and HG affected components of the calcium removal system with different sensitivities; 2- H2O2 and HG did not show any inhibitory effects on the PMCA-ATPase pump; 3- The effect of H2O2 on CRT was reversible; 4- The effect of HG on CRT could be due to increased production of H2O2; 5- The calcium regulatory proteins of ECs could be potential targets for ROS during the early stage of a cardio-vascular disease such as diabetes mellitus.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.