Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Current therapeutic approaches for HCC including surgical resection and trans-arterial embolization (TAE) remain largely ineffective, underscoring the need for development of novel therapeutic strategies. Because HCC is high vascularized, continuous administration angiogenesis inhibitor using gene therapy approach may facilitate long-term blockade tumor vasculature, thereby perturbing the growth of HCC. Vasostatin 112 (VS112) encodes an alternatively spliced fragment of angiogenesis inhibitor vasostatin, which encompasses residues 1-64 and 133-180 of calreticulin. In this study, recombinant adenovirus encoding VS112 (Ad-VS112) was generated to evaluate its potential for suppression of orthotopic Novikoff hepatoma in syngenic Sprague-Dawley (SD) rats. Adenovirus-mediated VS112 overexpression significantly inhibited the migration and tube formation of endothelial cells, indicating the anti-angiogenic potency of VS112 gene delivery. However, VS112 overexpression had no influence on the viability of N1-S1 Novikoff hepatoma cells. To investigate the prophylactic effect of VS112 expression on hepatoma growth, N1-S1 cells were infected with Ad-VS112 or adenovirus encoding green fluorescent protein (Ad-GFP) then implanted into the liver of SD rats. After 14 days, rats implanted with VS112-expressing showed significantly reduced incidence and size of hepatoma compared with those implanted with Ad-GFP-infected cells. To investigate the therapeutic efficacy of VS112 gene delivery, the SD rats were implanted with N1-S1 cells on day 0, treated with adenovirus vectors (2 x 1010 plaques forming units) via intravenous route on day 1, then sacrificed on day 14 to monitor hepatoma growth. By measuring tumor weight, it was found that Ad-VS112-treated rats exhibited significantly decreased tumor burden compared with control groups, which was in accordance with their lower serum GOT level. Histological analysis revealed a significant reduction of vWF-positive blood vessels in Ad-VS112-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, the expression of pro-inflammatory nuclear factor kappa B (NF£eB) and cyclooxgenase II (COXII) was also effectively attenuated in Ad-VS112-treated hepatoma. In conclusion, prior or post VS112 gene delivery potently suppresses the growth of orthotopic hepatoma,thereby holding promises for future treatment of HCC.

Angiogenesis, the formation of new blood vessels from preexisting ones through creation of new vessel branch points by sprouting or vessel splitting, is an important part of tissue growth in both physiological processes like wound healing and pathological conditions such as cancer. Growth factors like VEGF-A, FGF-2 and PDGF-BB are involved in both types of angiogenesis. Screening for genes regulated by VEGF-A stimulation in endothelial cells revealed up regulation of the endothelial cell specific glycoprotein endocan. Endocan itself did not stimulate angiogenesis. VEGF was a specific inducer since FGF-2, PDGF-BB, HGF and EGF did not alter expression. The signaling molecule PI3K was a negative regulator of endocan expression. Endocan was expressed in tumor cells and vessels, suggesting that although endocan did not directly regulate angiogenesis it can serve as a marker for angiogenic tumors. In two models of wound healing angiogenesis, the chick extra-embryonal CAM assay and the mouse cornea assay, we observed that blood vessels grew into avascular areas as functional mural cell covered loops by elongation of preexisting vessels. Loop formation was simultaneous with contraction of the avascular matrix mediated by proto/myofibroblasts. Reducing the contractibility of the stroma reduced vessel ingrowth, showing that contraction was necessary for mediating and directing growth of the vascular loops. These findings suggest a model for biomechanical regulation of vascularization that is complementary to sprouting angiogenesis which is guided by gradients of growth factors. In defining the role of growth factors, in the CAM assay, we found that FGF-2 and PDGF-BB induced vessel ingrowth, while VEGF-A, EGF and HGF did not. TGF-beta reduced the effect of FGF-2. By use of specific receptor kinase inhibitors we found an absolute requirement VEGF- and PDGF-receptor activity for vascularization while FGF- and TGF-beta-receptor function was dispensable. This suggests that functional VEGF- and PDGF-receptors are needed for vessel elongation.

Long-term survival of implanted cells requires oxygen and nutrients, the need for which is met by vasculari- zation of the implant. The use of scaffolds with surface-attached heparin as anchoring points for angiogenic growth factors has been reported to improve this process. We examined the potential role of surface modification of gelatin scaffolds in promoting endothelial cell infiltration by using a unique macromolecular conjugate of heparin as a coating. Compared to other heparin coatings, this surface modification provides flexible heparin chains, representing a new concept in heparin conjugation. In vitro cell infiltration of scaffolds was assessed using a three-dimensional model in which the novel heparin surface, without growth factors, showed a 2.5-fold increase in the number of infiltrating endothelial cells when compared to control scaffolds. No additional improvement was achieved by adding growth factors (vascular endothelial growth factor and/or fibroblast growth factor-2) to the scaffold. In vivo experiments confirmed these results and also showed that the addition of angiogenic growth factors did not significantly increase the endothelial cell infiltration but increased the number of inflammatory cells in the implanted scaffolds. The endothelial cell-stimulating ability of the heparin surface alone, combined with its growth factor-binding capacity, renders it an interesting candidate surface treatment to create a prevascularized site prepared for implantation of cells and tissues, in particular those sensitive to inflammation but in need of supportive revascularization, such as pancreatic islets of Langerhans. / <p>De två sista författarna delar sistaförfattarskapet.</p>

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.

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.

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.

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.

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.

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.

The mechanisms behind many important aspects of blood- and lymphatic vessel formation have yet not been elucidated in detail. The primary objectives of this thesis have therefore been to study the effects of hypoxia, platelet-derived growth factor (PDGF) and vascular endothelial growth factors (VEGFs) on vascular development and function. In conditions of low oxygen pressure, hypoxia, the survival of the organism is critically dependent on the ability to compensate for the reduced oxygen levels by promoting blood vessel growth and oxygen-independent energy production. Many direct effects of hypoxia in cells are attributed to the induction of a family of hypoxia-inducible transcription factors (HIFs) which control the expression of specific target genes. We found that capillary endothelial cells (ECs) respond to hypoxia with upregulation of genes involved in growth and remodeling of blood vessels. On the other hand, vein ECs responded to hypoxia with increased expression of genes involved in lymphatic vessel growth. Using differentiating embryonic stem (ES) cells, we have shown that hypoxia upregulates expression of VEGF receptor-3 (VEGFR-3) on blood vascular ECs. Furthermore, we have provided evidence for a critical role of VEGFR-3 in hypoxia-induced blood vessel development. Activation of PDGF receptor-β (PDGFR-β) on early vascular progenitors in differentiating ES cells or in mice induces blood vessel differentiation, while negatively influencing early hematopoiesis. PDGFR-β expression on vascular progenitors may therefore play a role in guiding differentiation of the vascular lineages. We have investigated the usefulness of differentiating ES cells as a model to study early lymphatic development. Administration of VEGF-C and VEGF-A induced formation of lymphatic vessel-like structures that seemed connected to the blood vasculature, supporting the general view that lymphatic ECs are derived from blood vascular ECs. In summary, this thesis has provided new insights in the contribution of different growth factors in hematopoietic, blood- and lymphendothelial development.