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.

Wir untersuchten Liquor- und Serumproben von 40 an einem shuntversorgten Hydrocephalus erkrankten Kindern auf die Konzentration der Zytokine bFGF, TGF-β1, VEGF, IL-6, IGF-1 und Leptin sowie deren Korrelation mit dem Risiko von Shuntinsuffizienzen. Dabei konnten wir die Hypothese bestätigen, dass erhöhte Konzentration der fibrogenen Zytokine bFGF und TGF-β1 im Serum bzw. Liquor mit einem erhöhten Risiko für operationspflichtige Shuntinsuffizienzen durch Obstruktion des Schlauchsystems einhergehen, und dass diese Komplikationen mit steigenden Zytokinkonzentrationen umso eher eintreten. Außerdem war bFGF im Liquor von Kindern, die zum Abnahmezeitpunkt an einer Shuntdysfunktion durch Obstruktion oder Einwachsen des Shunts litten, signifikant höher als bei Kindern, die zum Zeitpunkt der Abnahme keine Shuntdysfunktion aus eben genannten Gründen hatten. Des Weiteren fanden wir Konzentrationsunterschiede für IL-6 im Liquor zwischen den einzelnen Ursachen der Erkrankung, wobei das Zytokin am höchsten bei Tumorpatienten war, gefolgt von posthämorrhagischem und postmeningitischem Hydrocephalus, und am niedrigsten bei Kindern mit kongenitaler ZNS-Fehlbildung.

<p>Development requires a delicate balance of proliferation and differentiation. Too little proliferation can result in dysfunctional tissues, while prolonged or heightened proliferation can result in tumor formation. This is clearly seen with the granule neuron precursors (GNPs) of the cerebellum. Too little proliferation of these cells during development results in ataxia, whereas too much proliferation results in the cerebellar tumor medulloblastoma. While these cells are known to proliferate in response to Shh, it is not clear what controls the differentiation of these cells in vivo.</p><p> Previous work from our lab has identified basic fibroblast growth factor (bFGF) as a candidate differentiation factor for these cells. In this thesis, I characterize some of the cellular and molecular mechanisms involved in FGF-mediated inhibition (FMI) of Shh-induced GNP proliferation. In addition, I employ FGFR knockouts and a bFGF gain-of-function mouse to determine whether FGF signaling is necessary and/or sufficient for differentiation of GNPs during cerebellar development. Finally, the question of whether bFGF can be effective as a therapeutic agent for in vivo tumor treatment is tested in a transplant model.</p><p> These experiments indicate that FGF signaling is neither necessary nor sufficient for GNP differentiation during cerebellar development. However, transplanted tumors are potently inhibited by bFGF treatment. Furthermore, FMI is shown to occur around the level of Gli2 processing in the Shh pathway, implying that such a treatment has promise to be widely effective in treatment of Shh-dependent medulloblastomas.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18133号 / 医博第3853号 / 新制||医||1001(附属図書館) / 30991 / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂田 隆造, 教授 瀬原 淳子, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

No description available.

Traumatic brain injury (TBI) induces a robust cellular proliferative response among neural stem/progenitor cells (NS/NPCs) in the dentate gyrus of the hippocampus. This proliferative effect is thought to contribute to the innate cognitive recovery observed following TBI. Inhibition of hippocampal neurogenesis impairs cognitive function. Furthermore, enhancement of injury-induced hippocampal neurogenesis via intraventricular administration of basic fibroblast growth factor (bFGF) improves cognitive function in animals following TBI. In this experiment, we investigated the direct association between injury-induced hippocampal neurogenesis and cognitive recovery utilizing an antimitotic agent, arabinofuranosyl cytidine (Ara-C). In this study, adult rats received a moderate lateral fluid percussion injury (LFPI). Immediately following injury, Ara-C with or without bFGF was infused into the lateral ventricle via an osmotic mini-pump for 7 days. To label dividing cells animals received daily single injections of 5-bromo-2'-deoxyuridine (BrdU) at 2-7 days post-injury. To examine the effect of Ara-C on cell proliferation, a group of animals was sacrificed at 1 week following injury. Brain sections were immunostained for BrdU and cell type specific markers, and the number of BrdU+ cells in the hippocampus was assessed by stereology. To examine the effect of inhibition of injury-induced cell proliferation on cognitive recovery, animals were assessed on Morris water maze tasks (MWM) either at 21 to 25 days or 56-60 days post-injury. We found that post-injury Ara-C treatment significantly reduces injury-induced cell proliferation in the DG and abolishes the innate cognitive recovery on MWM performance at 56-60 days post-injury. Additionally, Ara-C diminishes bFGF enhanced cell proliferation in the DG and cognitive recovery following TBI. These results support the causal relationship between injury-induced hippocampal neurogenesis and cognitive functional recovery. Our studies suggest that the post-TBI neurogenic response is an endogenous repair mechanism that contributes to the restoration of hippocampal function post-injury.

Peripheral vascular diseases such as peripheral artery disease (PAD) and critical limb ischemia (CLI) are growing at an ever-increasing rate in the Western world due to an aging population and the incidence of type II diabetes. A growing economic burden continues because these diseases are common indicators of future heart attack or stroke. Common therapies are generally limited to pharmacologic agents or endovascular therapies which have had mixed results still ending in necrosis or limb loss. Therapeutic angiogenic strategies have become welcome options for patients suffering from PAD due to the restoration of blood flow in the extremities. Capillary sprouting and a return to normoxic tissue states are also demonstrated by the use of angiogenic cytokines in conjunction with bone marrow cell populations. To this point, it has been determined that spatial and temporal controlled release of growth factors from vehicles provides a greater therapeutic and angiogenic effect than growth factors delivered intramuscularly, intravenously, or intraarterialy due to rapid metabolization of the cytokine, and non-targeted release. Furthermore, bone marrow cells have been implicated to enhance angiogenesis in numerous ischemic diseases due to their ability to secrete angiogenic cytokines and their numerous cell fractions present which are implicated to promote mature vessel formation. Use of angiogenic peptides, in conjunction with bone marrow cells, has been hypothesized in EPC mobilization from the periphery and marrow tissues to facilitate neovessel formation. For this purpose, controlled release of angiogenic peptides basic fibroblast growth factor (FGF-2) and granulocyte-colony stimulating factor (G-CSF) was performed using tunable ionic gelatin hydrogels or fibrin scaffolds with ionic albumin microspheres. The proliferation of endothelial cell culture was determined to have an enhanced effect based on altering concentrations of growth factors and method of release: co-delivery versus sequential. Scaffolds with these angiogenic peptides were implanted in young balb/c mice that underwent unilateral hindlimb ischemia by ligation and excision of the femoral artery. Endpoints for hindlimb reperfusion and angiogenesis were determined by Laser Doppler Perfusion Imaging and immunohistochemical staining for capillaries (CD-31) and smooth muscle cells (alpha-SMA). In addition to controlled release of angiogenic peptides, further studies combined the use of a fibrin co-delivery scaffold with FGF-2 and G-CSF with bone marrow stem cell transplantation to enhance vessel formation following CLI. Endpoints also included lipophilic vascular painting to evaluate the extent of angiogenesis and arteriogenesis in an ischemic hindlimb. Tissue engineering strategies utilizing bone marrow cells and angiogenic peptides demonstrate improved hindlimb blood flow compared to BM cells or cytokines alone, as well as enhanced angiogenesis based on immunohistochemical staining and vessel densities.

Wir untersuchten Liquor- und Serumproben von 40 an einem shuntversorgten Hydrocephalus erkrankten Kindern auf die Konzentration der Zytokine bFGF, TGF-β1, VEGF, IL-6, IGF-1 und Leptin sowie deren Korrelation mit dem Risiko von Shuntinsuffizienzen. Dabei konnten wir die Hypothese bestätigen, dass erhöhte Konzentration der fibrogenen Zytokine bFGF und TGF-β1 im Serum bzw. Liquor mit einem erhöhten Risiko für operationspflichtige Shuntinsuffizienzen durch Obstruktion des Schlauchsystems einhergehen, und dass diese Komplikationen mit steigenden Zytokinkonzentrationen umso eher eintreten. Außerdem war bFGF im Liquor von Kindern, die zum Abnahmezeitpunkt an einer Shuntdysfunktion durch Obstruktion oder Einwachsen des Shunts litten, signifikant höher als bei Kindern, die zum Zeitpunkt der Abnahme keine Shuntdysfunktion aus eben genannten Gründen hatten. Des Weiteren fanden wir Konzentrationsunterschiede für IL-6 im Liquor zwischen den einzelnen Ursachen der Erkrankung, wobei das Zytokin am höchsten bei Tumorpatienten war, gefolgt von posthämorrhagischem und postmeningitischem Hydrocephalus, und am niedrigsten bei Kindern mit kongenitaler ZNS-Fehlbildung.

Wounds in the mouth, occurring after oral surgery, take time to heal. No ointment can be added to help with the healing process because mouth saliva will constantly wash it away. In order to combat this problem, we propose engineering a normal flora microbe to grow at the site of injury and secrete a recombinant growth factor to promote healing of the damaged tissue. Our goal is to have the yeast Pichia pastoris produce human basic fibroblast growth factor (bFGF), which aids in cellular proliferation. P. pastoris is a good choice for this application because not only is it considered generally recognized as safe (GRAS) by the FDA, but it is a eukaryote that is able to perform posttranslational modifications and secrete large amounts of recombinant protein. Previous studies have shown that a strain of P. pastoris can be engineered to express bFGF from a methanol-sensitive promoter. The study also showed that the bFGF, which was purified from the yeast’s extracellular medium, was able to promote the growth of NIH/3T3 cells (mice fibroblasts). Because we needed the P. pastoris to express the bFGF in glucose –based tissue culture medium in the presence of mammalian cells, we expressed the bFGF from the constitutive promoter GAP promoter. Along with optimizing and characterizing expression of bFGF, we also investigated the effect of the recombinant protein on mammalian cell growth using both scratch ad MTS assays. In addition, the effects of the yeast being co-cultured with mammalian cells was studied. Our results provide a basis for how a recombinant protein can be clinically used to improve wound healing in the mouth using a yeast strain to produce and secrete a growth factor at the site of injury.

The angiogenic and cardioprotective effects of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) in the ischemic myocardium have been studied, but expression and release of endogenous FGF-2 and VEGF during myocardial ischemia are poorly understood. In addition, nitric oxide synthase isoforms eNOS and iNOS may play a role in myocardial ischemia. The purpose of this study was to investigate the release of FGF-2 and expression of FGF-2, VEGF, eNOS, and iNOS in the normal and ischemic heart. In Phase I, serum FGF-2 levels in patients undergoing treadmill stress test were measured to investigate correlation between serum FGF-2 levels and presence of ischemic heart disease. The study found that serum FGF-2 in ischemia-positive and ischemia-negative patients was not significantly elevated after treadmill stress test, and serum FGF-2 levels did not differ significantly between ischemia-positive and ischemia-negative patients. In Phase II, FGF-2 levels in coronary effluent from isolated perfused rabbit hearts subjected to low-flow ischemia was measured. Results suggest that FGF-2 is released into the coronary effluent of isolated perfused hearts over time and that this release may be elevated in ischemic (50% flow) hearts. Furthermore, the present study indicates that FGF-2 is released immediately after surgical isolation and instrumentation of the isolated heart. A linear model was developed to describe the release of FGF-2 from the isolated heart as a function of the coronary flow rate Q : [special characters omitted]where t = time and Q = 1 and 3.01 for normal and 50% flow rates respectively. In Phase III, effect of acute low-flow ischemia on FGF-2, VEGF, eNOS, and iNOS mRNA expression was measured in isolated perfused hearts using RT/PCR. Preliminary results indicate that FGF-2, VEGF, and iNOS mRNA expression is upregulated and eNOS expression is decreased in ischemic hearts suggesting that these growth factors play a role in short-term response of the myocardium to ischemia. The results of this study suggest that FGF-2, VEGF, and iNOS mRNA expression are increased, eNOS expression is decreased, and FGF-2 is released in response to low-flow ischemia in the isolated perfused heart.