<p>One of today’s most widely spread health problems is urinary incontinence, affecting 60-80% of the US population from age 15 and up. Treatment based on the possibility to implant a scaffold seeded with the patients’ own urine-derived stem cells, hUSC, to regenerate the damaged muscle tissue, would prove effective. A main challenge in regenerating new tissue from cell-seeded scaffolds is the limited cell survival due to insufficient oxygen diffusion to the center of the scaffold. Ways of enhancing cell survival, and thereby, proliferation and differentiation, is by hypoxic preconditioning of the cells or implantation in an oxygen-release material. Hypoxic preconditioning has shown to enhance proliferation as well as the expression of vascular endothelial growth factor, VEGF, in for example human bone marrow derived stem cells, hBMSC. VEGF is involved in the establishment of vasculature structures and an upregulation of its expression may therefore help promote quicker angeogenisis, increasing the oxygen supply and the cell survival. Oxygen-release materials have shown to enhance cell survival and growth both <em>in vitro</em> and <em>in vivo</em>.<em></em></p><p>This study aims to investigate the effect of hypoxia on hUSC, during 9 days of hypoxic culturing (2.0% ± 0.1% O₂) with and without oxygen-release material (PLGA 75:25 with 5 w% CPO) <em>in vitro</em>. hBMSC, and human smooth muscle cells, hSMC, have been used as control groups. Cell proliferation, morphology, differentiation, production of VEGF, and expression of hypoxia inducible factor HIF-1α have been studied.</p><p>According to the results, combining hypoxic preconditioning of hUSC with implantation in oxygen-release material could be an effective way to regenerate muscular tissue. Hypoxic preconditioning enhanced cell proliferation, production of VEGF, and HIF-1α expression. The increase of VEGF and HIF-1α would promote vascularization when implanted. The oxygen-release material showed possible promotion of cell differentiation, which would augment the hUSCs’ myogenic differentiation, while supplying oxygen until the tissue’s vascular structure has been established.</p>

<p>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.</p><p>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. </p><p>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.</p><p>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.</p><p>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. </p><p>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. </p><p> 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. </p>

<p>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. </p><p>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. </p><p>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. </p><p>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.</p><p>In summary, this thesis has provided new insights in the contribution of different growth factors in hematopoietic, blood- and lymphendothelial development. </p>

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Les organismes vivants sont en intime relation avec leur environnement et sont constamment influencés par de nombreux facteurs chimiques et physiques. Parmi les facteurs physiques présents dans notre environnement, les forces mécaniques, y compris la gravité, les radiations, dont les ultraviolets, et les champs électromagnétiques constituent les trois pôles principaux de nos travaux de doctorat. Des outils biologiques, cellulaires et moléculaires ont été développés afin dévaluer le rôle des RhoGTPases dans les altérations morphologiques, prolifératives et phénotypiques induites par la perte du vecteur gravité au cours de vols spatiaux. Au cours du vol de la capsule spatiale inhabitée FOTON-M3, nous avons pu mettre en évidence que la suppression de Rac1 par ARN interférentiel permettait de contrecarrer les effets délétères de la microgravité sur larchitecture du cytosquelette suggérant que cette molécule de signalisation participe à la réception et à la réactivité à la gravité. RhoA et Cdc42 ne semblent pas impliqués. Nous avons également développé un modèle expérimental dinduction de flux calcique par des peptides mimétiques de la matrice extracellulaire activant les intégrines destiné à être expérimenté au cours de vols paraboliques. Au cours de nos travaux visant à évaluer les effets biologiques des champs électromagnétiques, nous avons observé que les EMF de très basse fréquence (450µT-50Hz) naffectent ni les signaux calciques induits par des concentrations élevées de sérum ou des peptides mimétiques de la matrice extracellulaire, ni lexpression des gènes régulés par les UV-B. Ils sont cependant capables de soutenir les oscillations calciques induites par une concentration sub-optimale de sérum, sans toutefois réguler de manière évidente les voies de signalisation contrôlant la prolifération. Lirradiation par les UV-B dun grand nombre de cellules, primaires, immortalisées et tumorales induit, par épissage alternatif du préARNm du VEGF-A, lexpression dun nouveau variant, le VEGF111. Celui-ci est constitué de la combinaison des exons 1-4 et de lexon 8. Cette nouvelle forme de VEGF-A contient donc les sites de fixation aux VEGF-R1 et R-2 et est pro-angiogène in vitro sur les cellules endothéliales et les cellules souche embryonnaires et in vivo chez la souris. Labsence des exons 6 et 7 codant pour la liaison aux protéines de la matrice extracellulaire lui confère une diffusibilité tissulaire. Une de ses caractéristiques remarquable est sa résistance à la dégradation en raison de labsence du site de clivage par la plasmine et les MMPs. Ce nouveau variant est également induit par diverses substances génotoxiques dont les agents chimiothérapeutiques. Les mécanismes régulant lexpression du VEGF111 dépendent des voies de signalisation ATM/ATR, p53 et MAPKinases. La double personnalité de ce nouveau facteur pro-angiogène, néfaste par son induction potentielle au cours de traitements anti-cancéreux mais bénéfique par son utilisation dans le traitement de pathologies ischémiques particulièrement pertinente en cas dactivités protéolytiques élevées, ouvre un champ considérable dinvestigations. ----------------------------------------------------------------------------------------------------- Living organisms interact with their environment and are constantly influenced by various chemical and physical factors. Among the physical factors present in our environment, mechanical forces, including gravity, radiations, among which ultraviolet radiations, and electromagnetic fields constitute the three main poles of our research. Biological, cellular and molecular tools have been developed with the aim to evaluate the role of RhoGTPases in the morphological, proliferative and phenotypic alterations induced by the loss of gravitational field experienced during space flight. During the flight of the unmanned FOTON-M3 capsule, we have demonstrated that the suppression of Rac1 by small interference RNA was able to counteract the deleterious effects of microgravity on the cytoskeleton architecture. This suggests that this signaling molecule participates to the reception and reaction to gravity. RhoA and Cdc42 do not seem to be implicated. We have also developed an experimental model of induction of intracellular calcium ions fluxes by mimetic peptides of the extracellular matrix activating integrins to be used in parabolic flights. During our investigations aimed at evaluating the biological effects of electromagnetic fields, we observed that EMF of very low-frequency (450µT-50Hz) do not affect neither the calcium signals induced by high concentrations of serum or extracellular matrix mimetic peptides, nor the expression of genes regulated by UV-B. They are however able to sustain calcium oscillations induced by a sub-optimal concentration of serum but without disturbing the cellular proliferation rate. Irradiation by UV-B of a large number of cells, primary, immortalized and tumoral, induces, by alternative splicing of the VEGF-A pre-mRNA, the expression of a new variant, the VEGF111. This isoform is made of a combination of exons 1-4 and exon 8. This new VEGF-A variant contains therefore the binding sites to VEGF-R1 and R-2 and proved to be proangiogenic in vitro for endothelial and ES cells and in vivo in mice. The absence of exons 6 and 7 coding for the heparin binding sites confers it with tissue diffusibility. One of its striking characteristics is its resistance to degradation due to the absence of the cleavage site by plasmin and MMPs. This new variant is also induced by a series of genotoxic agents, including chemotherapeutic drugs. The mechanisms controlling the VEGF111 expression depend on the ATM/ATR, p53 and MAPKinases signaling pathways. The dual faces of VEGF111, detrimental by its potential induction during anti-cancer therapy but beneficial by its use for managing ischemic pathologies, mostly relevant when associated with high proteolytic activities, opens a considerable field of investigations.

Malignant glioma consists of a group of diseases where the localisation and the nature of the disease makes treatment an extreme challenge. Two important biological features of malignant glioma cells are their infiltrative growth and their ability to induce angiogenesis. Glioma cells migrate extensively behind the blood-brain barrier and infiltrate the surrounding brain making radical treatment with surgery and radiotherapy almost impossible. The aims of this thesis were to investigate factors of importance for glioma cell migration and angiogenesis and to evaluate if an anti-angiogenesis approach alone or in combination with current treatment modalities could inhibit glioma growth. For this purpose we used the BT4C orthotopic rat glioma model and investigated treatment effects of the vascular endothelial growth factor (VEGF) receptor-2 and epidermal growth factor (EGF) receptor tyrosine kinase inhibitor ZD6474 alone or in combination with temozolomide or radiotherapy. Altered protein expression pattern after anti-angiogenesis treatment was measured using a mass-spectrometric proteomic method, followed by multivariate data-analysis. The tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and VEGF showed altered temporal and spatial mRNA expression during glioma progression. In early stages of tumour progression the expression was found throughout the tumour while in later stages, the expression was more predominant in the invasive tumour border. ZD6474 in monotherapy significantly inhibited tumour growth in the BT4C glioma model. The effect was further enhanced when combined with radiotherapy or temozolomide. Using mass-spectrometric methods an altered protein expression pattern after ZD6474 treatment was observed implicating the possibility to use proteomic methods for finding predictive biomarkers for anti-angiogenesis treatment. In conclusion, this thesis demonstrates a co-expression of factors important for glioma growth and angiogenesis and that treatment with an angiogenesis inhibitor has additive effects on glioma growth when combined with radiotherapy and chemotherapy. Finally, an altered protein expression pattern after anti-angiogenesis treatment is evident and detectable. Hopefully this work will contribute to and encourage further research to reach a better understanding of how to combine and evaluate different treatment approaches in malignant glioma.

Background: Glioblastoma is the most common and aggressive type of glioma and associated with poor prognosis. Apart from ionizing radiation and some rare genetic disorders, few aetiological factors have been identified for primary brain tumours. Inverse associations to asthma and low IgG levels for varicella zoster virus have in previous studies indicated that the immune system may play a role in glioma development. Little is known about prognostic factors in glioma. Previous studies have shown an association between age, Karnofsky performance status, O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation, and prognosis. Polymorphisms in different low penetrance genes have in some studies been associated with glioma prognosis. Material and methods: In paper I, we analysed IgG levels for four different viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and adenovirus (Ad), in prediagnostic blood samples from 197 cases with glioma and 394 controls collected from three large cohorts: the Northern Sweden Health and Disease Study; the Malmö Diet and Cancer Study; and the Diet, Cancer and Health cohort from Copenhagen. ELISA was used to measure IgG levels and for EBV response to both the nuclear antigen (EBNA1) and the viral capsid antigen (VCA) was measured, for VCA using immunoflourescence. IgG levels were divided into quartiles and binary logistic regression was used to compare the quartiles in cases and controls. All odds ratios were adjusted for age, sex, and cohort. In paper II-IV, we studied 176 glioblastoma cases from Sweden and Denmark. We collected treatment and follow-up data on the cases. We genotyped 30 tagging SNPs in EGF, 89 in EGFR, 27 in VEGFR2, and 17 in VEGF. We also studied 1458 SNPs in 136 DNA repair genes. Hazard ratios were calculated using Cox regression; the major allele was set as categorical variable and all HR were adjusted for age, sex, country, and treatment. For the DNA repair gene results, we adjusted the p-values for multiple testing. Significant findings were confirmed in separate datasets. Results and Discussion: We found a trend towards higher IgG VZV levels in controls compared to glioma cases, especially when restricting the analyses to only include glioma cases with at least 2 years between blood sample and diagnosis. This finding might indicate that there is an aetiological and not a disease-related association. This confirms previous findings and support that a strong immune system can detect and inhibit growth of small cancer clusters. In EGF, we found seven SNPs in one haplotype block that were significantly associated with glioblastoma survival. Four of the SNPs were available for confirmation; however, none reached statistical significance. One explanation could be age differences in the different cohorts. In EGFR, four SNPs associated with survival were found; however, as 89 polymorphisms were tested this was the expected outcome by chance. In VEGF and VEGFR2, we found two SNPs associated with glioblastoma survival, but they could not be confirmed in the separate dataset, and due to multiple testing, were considered to be false positives. Among the DNA repair genes, we found nine SNPs in three genes-MSH2, RAD51L1 and RECQL4-associated with glioblastoma survival after confirmation and adjustment for age, sex, country, and treatment. After adjusting for multiple testing, one SNP in MSH2 and one in RECQL4 remained significant. Conclusions: Our studies provide additional knowledge to the aetiological and prognostic factors important for glioma, emphasising the possible importance of immune function mechanisms. We found limited evidence for the role of genetic variants in glioma progression genes, and some for DNA repair variants as prognostic factors for glioblastoma survival.

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.

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.

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.