This thesis investigated the predictive and the prognostic powers of angiogenesis related markers in both operable and inoperable non-small cell lung cancer (NSCLC) patients. In the first and second study, we investigated the serological fractions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in 2 cohorts of patients with either operable or inoperable NSCLC. Regarding operable NSCLC, we demonstrated significant correlations between VEGF and tumour volume and overall survival. Regarding bFGF, significant correlations with recurrent disease and survival were demonstrated. VEGF and bFGF correlated to each other and with platelet counts. In multivariate analysis, bFGF proved to be a significantly independent prognostic factor. Regarding inoperable NSCLC, we demonstrated that patients with elevated bFGF levels before any treatment and during chemotherapy had a significantly poorer survival. During chemotherapy, each rise of one unit of bFGF (ng/L) corresponded to a 4 times increased risk of death. Regarding VEGF, elevated levels after radiotherapy corresponded with better survival. All prognostic information demonstrated in this study concerned patients with a, co-sampled, normal platelet count. In the third study, three putative markers, HER-2, EGFR and COX-2, suitable for targeted therapies in resected NSCLC were investigated in a panel of 53 tumours and further investigated for a possible correlation with microvessel density. We demonstrated that HER-2 and COX-2 were mainly expressed in adenocarcinomas, whereas EGFR was only expressed in squamous cell carcinomas. COX-2 showed a trend towards a correlation with microvesssel density. The expression profile, HER-2+/EGFR-, was significantly correlated to poorer survival. In the fourth study, a predictive model for recurrences consisting of p53, CD34 and CD105, and circulating serum fractions of VEGF and bFGF, was investigated. The two endothelial markers correlated with each other. CD105 expression correlated with p53 expression. No other significant correlations between markers could be demonstrated. A significant correlation between p53 overexpression and recurrent disease was demonstrated. The mutational status could not confirm the immunohistochemical correlation between p53 and recurrences. In conclusion, the present thesis demonstrates that the angiogenic factors VEGF and bFGF analysed in sera have both predictive and prognostic information when measured in operable and inoperable NSCLC. Since HER-2 is overexpressed in NSCLC and linked with prognostic information, this marker might be a suitable target for therapy in NSCLC. Furthermore, in patients with operable NSCLC, p53 expression status was linked with recurrent disease and mean MVD.

Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4) disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of N-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified N-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs). Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn2+-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups. The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.

Tissue neovascularization in postnatal life occurs during post-traumatic tissue healing, neoplastic growth and in the endometrium during the reproductive cycle of females. Although embryonic angiogenesis has been intensively studied, far less is known about tissue revascularization and vessel remodeling in adults due to methodological difficulties. In the current studies, we developed a novel in vivo model of neovascularization that is performed on the chicken chorioallantoic membrane (CAM). A provisional matrix placed on the CAM was vascularized in response to FGF-2. In order to distinguish new from pre-existing vessels, the matrix was separated from the CAM by a nylon grid. Techniques to visualize the three dimensional structure of vascular networks and a method for rapid and semi-automated quantification were developed. This novel model allowed us to study the effects of potential inhibitors of tissue vascularization and their effects on the pre-existing vasculature. We found that while fumagillin or inhibition of MEK and Src inhibited only neovascularization, addition of cortisol or wortmannin was toxic to pre-existing vessels. The CAM model allowed intravital observations during extended periods of time, which together with immunohistochemical analysis revealed a novel mechanism of tissue vascularization. Tensional forces generated by myofibroblast-mediated contraction of the provisional matrix, induced and directed ingrowth of vascular tissue. During the initial stages of vascularization, the vascular network was recruited from the surrounding tissue through a non-angiogenic mechanism by elongation and enlargement of pre-existing vessels, which were moved as vascular loops with constant functional circulation. Ingrown vessels were remodeled, presumably through intussusception, fusion and pruning. The CAM model was validated by observations of neovascularization associated with healing of the injured mouse cornea.

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.

The cardiovascular system is the first functional organ to develop during embryogenesis. As the embryo reaches above a certain size, passive diffusion of gases and nutrients is no longer compatible with efficient growth. During embryogenesis, endothelial progenitor cells (angioblasts) are recruited from the primitive streak mesoderm and instructed to express vascular endothelial growth factor receptor-2 (VEGFR-2). This thesis examines the roles played by VEGFR-2 in the events through which a subpopulation of embryonic stem (ES) cells differentiate into endothelial cells and form the vasculature. We show that ES cells gene targeted for VEGFR-2 (flk1-/-) develop immature endothelial cells (ECs), precursors, when differentiated in vitro as embryoid bodies (EBs). The flk1-/- ECs are unresponsive to VEGF-stimulation and consistently fail to form vessels. However, when co-cultured with wild type ES cells in chimeric EBs, flk1-/- endothelial precursors are guided by wild type ECs to form transient, chimeric vascular structures. Use of lentivirus in an add-back approach allowed reconstitution of VEGFR-2 expression in flk1-/- ES cells, and rescue of vasculogenesis and sprouting angiogenesis. We propose that recruitment to the endothelial lineage is not dependent on VEGFR-2, although this receptor tyrosine kinase appears indispensible for EC integrity, survival and for differentation of endothelial precursors into mature ECs formating a stable vasculature. Neuropilin-1 (NRP1) and heparan sulfate proteoglycans (HSPGs) function as co-receptors for VEGFs. The co-receptors influence, qualitatively and quantitatively, the intracellular signal relayed by VEGFR-2 but it is unclear how. We examined the contribution of NRP1 to VEGFR-2 signaling in EB cultures, in zebrafish and in mice. Only NRP1-binding VEGFs were able to promote sprouting angiogenesis and formation of properly branched vascular tubes, supported by pericytes. Downstream of VEGFR-2/NRP1 activation, we identified recruitment of p38MAPK in signal transduction regulating sprouting angiogenesis.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on factors mediating angiogenesis, the development of new blood vessels, in normal and malignant breast tissue. In this thesis we have investigated the effects of estradiol, progesterone, and the nonsteroidal anti-estrogen tamoxifen on vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) in normal human breast tissue, endothelial cells, and breast cancer. We have applied the technique of microdialysis to provide in situ sampling of estradiol and VEGF in tumors and normal breast tissue of breast cancer patients in vivo. Furthermore, we present a novel method of culturing normal human breast tissue ex vivo. Our results suggest a pro-angiogenic effect of estradiol and an anti-angiogenic effect of tamoxifen in the breast. Estradiol increased extracellular levels of VEGF in normal human breast tissue and breast cancer cells in vitro. In addition, estradiol decreased sVEGFR-1 in breast cancer cells and indirectly increased VEGFR-2 in endothelial cells. Compared to estradiol treatment alone, estradiol + tamoxifen increased sVEGFR-1 and decreased VEGF in breast cancer cells in vitro. Furthermore, estradiol + tamoxifen decreased tumor VEGF levels and tumor vasculature in human breast cancer xenografts in vivo. In breast cancer patients, a significant correlation was found between in vivo levels of estradiol and VEGF sampled by microdialysis in normal human breast tissue, suggesting that estradiol may be a potent regulator of VEGF in the breast in vivo. Tumor levels of VEGF were significantly higher than in normal breast tissue in vivo, supporting the role of VEGF in tumor angiogenesis. For studies of normal human breast, whole breast tissue may be cultured in vitro for up to one week with preserved morphology. Using this method, estradiol, and not progesterone, appears to be the main sex steroid regulator of extracellular VEGF in normal breast tissue. In conclusion, the data suggest that sex steroids and tamoxifen exert pro- and anti-angiogenic effects in normal breast tissue and breast cancer.

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.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.

Esophageal carcinoma is a malignancy with a poor prognosis and is the sixth cause of cancer related death worldwide. In Sweden approximately 400 new cases are diagnosed every year. The aim of this present thesis was to investigate predictive factors for esophageal carcinoma patients.126 esophageal carcinoma patients admitted to the department of Oncology at the University Hospital in Uppsala between 1990-2000 were investigated with focus on known and potential prognostic factors. Performance status and stage of the disease were the only independent prognostic factors (p-values <0.001). Angiogenic factors VEGF and bFGF were correlated to platelet and leukocyte counts and VEGF was correlated to tumor volume (p=0.04) whereas bFGF was not (p=0.08) in pre-treatment serum samples from 42 esophageal carcinoma patients. The use of the angiogenic factors as prognostic factors, prior to therapy in patients with esophageal carcinoma, according to the results from the present study, seems limited. HER-2 overexpression was seen in 17% of 97 investigated esophageal tumor samples. In squamous cell carcinoma patients, HER-2 overexpression correlated with poorer survival (p=0.035), whereas in adenocarcinoma patients, HER-2 status did not. HER-2 overexpression seems to be associated with poorer survival in esophageal carcinomas, especially in patients with squamous cell esophageal carcinoma. Telomerase activity was detected in all esophageal cell lines, with a broad range of activity levels. No correlation was found between telomerase activity levels and sensitivity to investigated cytotoxic drugs. We therefore conclude that basal telomerase activity level is not a key determinant of sensitivity to standard cytotoxic drugs in esophageal carcinoma cell lines. The virus HPV-16 was detected in 16 % of the patients; no other type HPV was detected. HPV-16 infection had no significant effect on survival (p=0.72). Our results did not show that HPV-16 increases survival or improve therapy response in patients with esophageal carcinoma.