Delta-like ligand 4 (DLL4) is a ligand for the Notch family of receptors. DLL4 is an important regulator of angiogenesis and DLL4 blockade promotes non-productive angiogenesis and delays tumour growth. The aim of this thesis was to investigate the effects of anti-DLL4 therapy in solid tumours in combination with a clinically relevant dose of ionising radiation (5 Gy; IR) and to analyse alterations in the Notch pathway induced by the treatments. Combining both treatments resulted in a greater than additive tumour growth delay in LS174T tumours, compared to either treatment alone. DLL4 blockade dysregulated vasculature and increased necrosis in LS174T and HCT-15 (DLL4-expressing and negative cell lines respectively) tumours within 3 days after treatment, but no changes were observed with IR alone. Additionally, combined IR and anti-DLL4 treatment of FaDu tumours (another DLL4-negative cell line) by our colleagues, also resulted in a supra-additive growth delay. These results show that combining IR with DLL4 blockade is an effective strategy for prolonging tumour growth delay and suggest that the stroma/vasculature provide the main therapeutic target for the anti-DLL4 therapy. Analysis of Notch pathway shows that IR upregulated Jag1 in tumour cells, and may inhibit Notch and downregulate DLL4 in the stroma. These changes may potentially affect tumour vessels and response to anti-DLL4 therapy. In vitro, anti-DLL4 therapy induced proliferation in quiescent contact-inhibited endothelial cells and also appeared to abrogate IR-induced inhibition of migration. These results suggest that DLL4 may be important in maintaining vessel quiescence and that IR may in part decrease migration through Notch signalling. Combining IR and DLL4 blockade to target tumour growth is an effective and well tolerated strategy and warrants further validation and refinement to be translated into clinical practice.

Background. Recent studies in cancer research have focused on the reciprocal interaction between cancer cells and their microenvironment. Tumour growth is angiogenesis dependent and the rate of angiogenesis correlates with a poor prognosis in many different cancers. We have shown that the rate of angiogenesis correlates with prognosis in Prostate Cancer (PC). We have also observed that the vasculature is involved during the involution of the prostate in rodents subsequent to hormonal ablation. Patients with metastatic PC are subjected to hormonal ablation therapy – a therapy unfortunately not curative. Our ambition is therefore to find means to enhance the effects of castration therapy of prostate tumours, possibly by a simultaneous inhibition of angiogenesis and of growth factors populating the tumour stroma. The angiopoietins are a family of growth factors that regulate angiogenesis by direct effects on endothelial cells in a context dependent manner. The purpose of this thesis was therefore to examine the role of the angiopoietins and the stroma in general in PC and to explore their potential as novel targets. Materials and Methods. We have had at our disposal access to clinical materials in the form of paraffin embedded samples from untreated PC patients with a long follow up. We have also used animal tumour models and in vitro cell culture systems followed by immunohistochemistry, in situ hybridization, western blotting, laser micro dissection, and quantitative real-time PCR for evaluation of the experiments. Results. In paper I, we found a significant correlation between high levels of angiopoietin 2 (Ang 2) and high vascular density, histological grade, metastases and poor prognosis in PC patients. In the second paper we found that the receptor for the angiopoietins, Tie 2, and the ligand Ang 1 mediated the decrease in vascular stability observed after castration treatment. This was not observed in prostate tumours subsequent to hormonal ablation (paper III), nor was there a decrease of other growth factor receptors. In summary (paper III), we found that a combined inhibition of the tumour stroma in terms of an inhibition of the PDGF-Rs by the use of Imatinib, and the vasculature in terms of a perturbed Tie 2 signalling, inhibited tumour growth. Finally, in paper IV, we found that Imatinib inhibited the castration induced influx of mast cells after castration therapy. The mast cells expressed high levels of FGF 2 and epiregulin, and inhibition of mast cell function inhibited tumour growth, by inhibiting angiogenesis. Conclusions. We have observed that the tumour stroma is of particular importance for tumour growth in PC. Targeting the tumour microenvironment, and in particular by a simultaneous inhibition of the vasculature and stroma, could prove beneficial for patients with advanced PC.

Radiotherapy plays a significant role in cancer treatment, and is thought to be curative by mainly killing tumor cells through damage to their genetic material. However, recent findings indicate that the tumor’s vascular blood supply is also a major determinant of radiation response. The goals of this thesis are to: (1) develop an experimental platform for small animals to deliver ionizing radiation and perform high-resolution optical imaging to treatment targets, and (2) use this toolkit to longitudinally monitor the response of tumors and the associated vasculature. The thesis has achieved: (1) customization of a novel micro-irradiator for mice, (2) technical development of an improved optical coherence tomography imaging system, (3) comprehensive experimental protocol and imaging optimization for optical microscopy in a specialized animal model, and (4) completion of a feasibility study to demonstrate the capabilities of the experimental platform in monitoring the response of tumor and vasculature to radiotherapy.

Radiotherapy plays a significant role in cancer treatment, and is thought to be curative by mainly killing tumor cells through damage to their genetic material. However, recent findings indicate that the tumor’s vascular blood supply is also a major determinant of radiation response. The goals of this thesis are to: (1) develop an experimental platform for small animals to deliver ionizing radiation and perform high-resolution optical imaging to treatment targets, and (2) use this toolkit to longitudinally monitor the response of tumors and the associated vasculature. The thesis has achieved: (1) customization of a novel micro-irradiator for mice, (2) technical development of an improved optical coherence tomography imaging system, (3) comprehensive experimental protocol and imaging optimization for optical microscopy in a specialized animal model, and (4) completion of a feasibility study to demonstrate the capabilities of the experimental platform in monitoring the response of tumor and vasculature to radiotherapy.

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth. We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP. Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model. The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.

The five discrete, but related studies presented in this thesis investigate several aspects of the physiology and biochemistry of whole animals, perfused and isolated tissues from fishes and other vertebrates. Important fundamental questions about tissue metabolism and energy supply and utilisation in relation to oxygen supply, in addition to applied questions relating to commercial harvesting and post-mortem muscle physiology were addressed. Oxyconformance of oxygen consumption (VO2) at low oxygen delivery rates was shown using an isolated, perfused salmon tail preparation, composed primarily of skeletal muscle. Addition of pig red blood cells to the perfusing solution at a haematocrit of 5 or 10%, increasing the capacitance, resulted in oxyregulation of VO2 by the tail tissues. Below c.60 ml O2.kg-1.h-1 of oxygen delivery, VO2 was delivery dependent. Above this value additional oxygen delivery did not increase VO2 of resting muscle above c.35 ml O2 kg-1.h-1. The preparation was validated by measuring mitochondrial activity using MTT and blood flow distribution to the red and white muscle using fluorescent microspheres. Evidence of both O2-independence of VO2 in the vasculature and strict O2-dependence of VO2 in striated muscles of fishes and a mammal is presented using isolated vascular tissue and an in vitro tissue slice model. VO2 by vessels from rat, salmon and hagfish showed varying degrees of independence between PO2s of 15-95 mmHg in vitro (1 mmHg = 0.133 kPa). Above and below these values, VO2 was highly PO2-dependent. VO2 by cardiac and skeletal muscles from rat, salmon, snapper and hagfish were shown to relate linearly to PO2 between zero and 125 mmHg. VO2 in these tissues was highly dependent on tissue type (cardiac, red and white muscle) which correlated with haem protein concentration. The increase in VO2 in muscle slice mitochondria uncoupled with FCCP and DNP ruled out diffusion-limitation as a constraint on VO2. Mitochondrial activity was constant over time and reoxygenation of the Ringer bathing the tissues after the initial run down in PO2 resulted in VO2 rates that were unchanged from the starting values, demonstrating that the tissues remained viable over time. ATP turnover in red muscle was significantly increased at 100 mmHg relative to 30 mmHg, and increased in both treatments from values at the start. Our data suggest that ATP supply and ATP demand were reduced in conjunction with falling PO2. The effects of hydrogen sulphide (H2S) (derived from Na2S) and isoeugenol exposure on activity, VO2 and ventilation frequency (Vf) in a teleost fish are reported. In the H2S treatment group (200 μM Na2S) both resting VO2 and Vf decreased after 30 minutes of exposure, concurrent with narcosis and a loss of equilibrium. These events corresponded with a significant fall in VO2 (33%) and Vf (20%) by 15 minutes, both declining further to a nadir of 40% of resting values at 30 minutes. After flushing, VO2 increased to resting levels, with Vf remaining significantly depressed until 30 minutes of recovery. Recovery was accompanied by regained mobility and equilibrium and significantly increased VO2 and Vf. Isoeugenol anaesthetised fish (0.011 g.L-1) reached stage 4-5 of anaesthesia accompanied by significant decreases in VO2 (45%) and Vf (25%) at 25 minutes, both parameters declining further to around 64% and 38% respectively by 35 minutes. Similar to H2S exposed fish, VO2 increased to resting values after flushing, followed by a significant rise in VO2. Likewise, Vf had risen to resting values post-flushing, subsequently increasing significantly during recovery. Overall, VO2 in relation to resting rate was reduced in the isoeugenol treated animals, while in H2S treated fish, exposure there was increased oxygen usage, possibly associated with a toxic effect. H2S significantly reduced cytochrome c oxidase activity in muscle and gill tissue in vitro between 69-79% at 20 μM and 77-97% at 200 μM Na2S, while isoeugenol had no effect on activity in any tissue. Calorimetric and biochemical profiles of anoxic, post-mortem white muscle from Chinook salmon subjected to rested and exhausted harvesting regimens at their acclimation temperature (10°C) are reported. Prior to harvest rested animals were anaesthetised with 0.012 g.L-1 isoeugenol without disturbance. The muscle of these animals had a high metabolic rate at the time of death, at around 400 μW.g-1, which declined rapidly over the first 12 hours to15 μW.g-1. Exhausted animals were forced to swim and were crowded before capture, resulting in an initial heat output of <10 μW.g-1. Heat output was significantly greater in the rested group at the time of death and for 7 hours post-mortem. In both groups there was an exothermic event, occurring between 4 and 6 hours post-mortem amounting to a rise of around 35 μW.g-1. A one-phase exponential decay model appropriately described the net heat output of the rested profile minus the exhausted data. Rested animals had significantly higher initial cut surface pH (7.5 vs 6.7), tissue glycogen (16 vs 2 μmol.g-1), creatine phosphate (18 vs 0.1 μmol.g-1), ATP (6 vs 3.5 μmol.g-1) and potential energy (30 vs 7 μmol.g-1) than the exhausted group, which had significantly elevated tissue concentrations of lactate (43 vs 18 μmol.g-1) and glucose (5 vs 2 μmol.g-1). Potential energy in the form of ATP, glycogen and creatine phosphate remained elevated for an extended period post-mortem in rested animals while catabolites further down the chain such as inosine, hypoxanthine and uric acid accumulated at similar rates in both groups. We examined the relationship between exogenous and endogenous H2S and oxygen partial pressure in isolated hagfish and lamprey vessels that exhibit profound hypoxic vasoconstriction (HVC). In myography studies, H2S (Na2S) dose-dependently constricted dorsal aortas (DA) and efferent branchial arteries but did not affect ventral aortas or afferent branchial arteries, which was similar to the effects produced by hypoxia. Sensitivity of H2S-mediated contraction in hagfish and lamprey DA was enhanced by hypoxia. HVC in hagfish DA was enhanced by the H2S precursor cysteine and inhibited by amino-oxyacetate (AOA), an inhibitor of the H2S-synthesising enzyme, cystathionine β-synthase, and unaffected by propargyl glycine, an inhibitor of cystathionine λ-lyase. Oxygen consumption (MO2) of hagfish DA was constant between a PO2 of 15 and 115•mmHg, decreased when PO2 <15•mmHg, and increased if PO2 exceeded 115•mmHg. 10 μmol.l-1 H2S increased and concentrations above 100 μmol.l-1 H2S decreased MO2. Consistent with the effects on HVC, cysteine increased and AOA and hydroxylamine, an inhibitor of pyridoxyl 5’-phosphate-dependent enzymes, decreased MO2. These data show that H2S is a monophasic vasoconstrictor of specific cyclostome vessels and because hagfish lack vascular NO, and vascular sensitivity to H2S was enhanced at low PO2, it is unlikely that H2S contractions are mediated by either an H2S-NO interaction or an oxidation product of H2S. These experiments provide additional support for the hypothesis that the metabolism of H2S is involved in oxygen sensing/signal transduction in vertebrate vascular smooth muscle. Together the findings of this thesis contribute to the understanding of oxygen utilisation and energetics in relation to oxygen supply in a number of tissues. These data further our understanding of respiratory physiology and may have practical applications in the seafood industry.

To date, studies into sex determination and gonadal development have focused on the regulatory mechanisms governing development of the male or female phenotype. However, the formation of the testis and ovary from the bipotential gonad also present a fascinating model of tissue organisation which has been largely overlooked. When seeking to understand tissue organisation during gonadal development, the formation of testis cords takes center stage. However, despite a growing understanding of the cellular events in testis development, a number of key questions about the formation of testis cords remain unanswered. Specifically, I aimed to investigate the role of cell migration in testis organization, and the structure and morphogenesis of testis cords in three dimensions. To address these aims experimentally, I investigated the early morphogenesis of testis cords and the dependence of cord formation on cell migration from the mesonephros. I found that virtually all of the migrating cells express endothelial markers, indicating that endothelial, not peritubular myoid cells underlie the dependence of cord formation on cell migration. Further, disruption of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. These data reveal that migrating endothelial cells are required for testis cord formation, consistent with increasing evidence of a broader role for vasculature in establishing tissue architecture during organogenesis. To address the question of cord structure and morphogenesis, I developed and applied a novel fluorescence-based three-dimensional modeling approach to show that Sertoli cells coalesce into irregular groups surrounding germ cells, and that these groups are remodeled to form highly regular toroidal loops, joined by a flattened plexus at the dorsal side. This plexus is punctured by blood vessels as they ingress from the mesonephros, and contracts during maturation to form part of the rete testis. Variation in cord number and position demonstrates that cord establishment is not a stereotypic process. However, a tightly regulated modeling mechanism must contribute to uniformity on cord diameter and orientation as these parameters remain consistent across samples of the same age. These data clarify questions of cord structure and organisation, establish that cord formation is a variable process, and demonstrate novel structural features within the network of testis cords. Finally, to investigate an in vivo model where vascularisation and cord formation may be disrupted, I analysed gonads from embryos lacking Cited2. Consistent with a previous study, I found that testis development was delayed in Cited2-/- gonads, but found that despite the reported transcriptional recovery after the delay, testis vascular and cord structure was permanently disrupted. To investigate the defects in cord formation I assayed cell migration and found that migration was not disrupted in XY gonads, or mesonephroi lacking Cited2. However, ectopic cell migration was observed in the XX gonad in a dose-dependent response to loss of functional Cited2 alleles. Correspondingly, the female pathway was initially delayed but rallied for a late recovery, implicating Sf1 in the initiation of ovarian differentiation. These data underscore the fragility of the molecular control of sex determination as absence of Cited2 in the male permanently disrupts testis morphology, whereas in the female, promoters of the male pathway are not sufficiently suppressed. From these studies I construct an integrated model of testis cord formation and conclude that testis cord formation is a novel form of tubulogenesis. This morphogenesis is unique and offers insights into cell and tissue organisation, vascular interactions in organogenesis, and mechanisms of tube formation. Further study of cord formation is likely to lead to a broader understanding of tissue morphogenesis during development.

BACKGROUND Heart disease and renal dysfunction are often mutually reinforcing conditions, although the factors underlying this relationship are not fully understood. Cardiac remodelling resulting from disease is partly caused by the reactivation of developmental programmes. By unravelling the mechanisms that drive kidney development and function, it may be possible to gain novel insight into remodelled kidney states that are linked to disease. In this study, we have investigated the interplay between renal and cardiovascular systems during nephrogenesis at the level of the blood filter. METHODOLOGY Owing to the optical transparency and rapid external development of the embryo, the zebrafish provides a research model for advanced imaging technologies, allowing us to visualise structures located deep within living specimens. Here, we combine deep-tissue live imaging and novel functional assays to study development and function of the pronephric kidney - the first and most basic kidney to form in the embryo. RESULTS Using two-photon excitation microscopy, we have successfully established methodology for performing, deep-tissue, time-lapse imaging in living embryos of the two primary cell types forming the kidney – endothelial and epithelial cells. Fluorescence angiograms were performed using supra-vital dye agents to visualise circulatory flow in relation to the pronephric vasculature and the process of blood-filtration. Observations from live imaging studies, supported by immunostaining, were used to create a comprehensive model of the developing glomerular morphology, and interactions at the endothelial-epithelial cell interface, where glomerular epithelial primordia merge around the vascular component of the pronephric kidney. To investigate renal function, we devised a novel assay of pronephric filtration, by tracking the accumulation of injected fluorescent tracers within the excreted filtrate of embryos. This allowed us to relate our time-lapse observations to maturation of the glomerulus, and the evolution of perm-selective function. Finally, we explored methods of mechanically obstructing blood-flow in order to investigate whether altered hemodynamic forces would influence pronephric development. We found that in those embryos with severely disrupted circulatory flow, the glomerular morphology was affected. CONCLUSIONS In summary, the combination of these techniques has allowed us to visualise the multi-cellular organisation of the pronephric kidney over time, which has previously been limited to primarily fixed-tissue approaches. A detailed model of pronephric development has been developed, which could ultimately be used to dissect the molecular mechanisms underlying embryonic kidney development.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22369号 / 医博第4610号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 大森 孝一, 教授 富樫 かおり, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ischemic strokes are a major cause of death and disability, yet efficacious pharmacotherapies remain limited. Although neuronal cell death during stroke is primarily induced via excessive Ca2+ influx through NMDARs following overactivation by uncontrolled glutamate release, antagonism of these receptors has been shown to be ineffective due to intolerable side effects. This thesis highlights a novel therapeutic strategy for stroke wherein NMDAR-mediated excitotoxicity is temporarily and dynamically mitigated via the initiation of a process termed “glycine induced NMDAR internalization” (GINI). While GINI occurs in vitro following application of high doses of glycine, achieving these levels of glycine in vivo has long been thought impossible as glycine transporters (GlyT1) maintain synaptic glycine levels well below saturating concentrations. Here, we show that GINI can be triggered in vivo when mice are administered a glycine transporter-1 antagonist (GlyT1-A) prior to stroke and that this strategy provides neuroprotection. Mice pre-treated with a GlyT1-A, which elevates glycine levels, exhibited significantly smaller stroke volumes, reduced cell death, and significantly minimized behavioural deficits following stroke induction by either photothrombosis (PT) or endothelin-1 (ET-1). Moreover, we observed preservation of vasculature function and morphology in the peri-infarct area. These data strongly suggest that elevating brain glycine levels with GlyT1-As should be considered as a novel pharmacotherapy for ischemic stroke.