Endostatin, an angiogensis inhibitor, was discovered by Dr. Judah Folkman¡¦s group in 1997. From their series studies, they demonstrated that the angiogenesis inhibition approach, which abolished the formation of new blood vessels and led to starvation of cancer cells, is a safe, effective anticancer method without side effect and drug resistance. Phase clinical trial on endostatin was carried out in 1999 and completed in 2001, heralding the approaching of a new arsenal of cancer therapy drugs. Endostatin is also a proteolytic fragment (~20 kDa) from an extracellular protein, collagen XVIII. It potently inhibits endothelial cell proliferation and angiogenesis, but has no cytotoxic effects on other cells. Above all, cycled therapy of experimental cancer in rodents with endostatin led to tumor dormancy without drug resistance. However, the exact mechanism on how endostatin inhibited endothelial cells proliferation remains largely unknown. We have cloned mouse endostatin cDNA from mice liver by RT-PCR. After verification by DNA sequencing, endostatin cDNA was subcloned in to E. coli expression vector to express and generate large quantities of recombinant GST-fused endostatin. Unlike His-tagged endostatin, GST-endostatin is soluble and capable of inhibiting endothelial cell lines EA.hy926 with a half-maximal inhibition concentration (IC50) of 20 nM. In present study, we investigated whether GST-endostatin caused alterations in cytoskeleton in endothelial cells. By using a fluorescence dye to visualize the actin filament under confocal microscope, it was found that endostatin induced the corruption of actin network in endothelial cells. Western blot analysis revealed that GST-endostatin treatment caused downregulation of cytoskeleton proteins such as tubulin, vimentin and ECM-related signaling molecules such as focal adhesion kinase (FAK), mitogen activated protein kinse (MAPK), Erk in a dose-dependent manner. Moreover, GST-endostatin decreased the levels of cell survival factor such as AKT and NF-£eB. Since GST-endostatin induced sustained calcium rise, the effect of endostatin on protein kinase Cs (PKCs) were studied and revealed that endostatin reduced the levels of PKCK1¡BPKC eta¡BPKC iota and PKC lamda. Other than endothelial cell, the cytotoxicity of GST-endostatin in hepatoma cells were investigated since liver the primary expression site of collagen XVIII, precursor of endostatin. Unexpectedly, endostatin also inhibited the proliferation of hepatoma cells. Flow cytometry and nucleus staining indicated that GST-endostatin also induced apoptosis in hepatoma cells. Moreover, GST-endostatin exhibited differential cytotoxic effect against well-differentiated (such as HepG2, Hep3B) and poor differentiated (such as Mahlavu, Sk-hep-1) hepatoma cells that the IC50 for well differentiated hepatoma cells were 8-10 folds lower than for poor-differentiated cells. Above all, GST-endostatin inhibited the migration of SK-hep-1 and modulated the secretion of matrix-metalloproteinases (MMPs) by Mahlavu and SK-hep-1 cells. In summary, present study explored the role of alterations in cytoskeleon network in the cytotoxic mechanism of GST-endostatin. Moreover, the inhibitory effects of GST-endostatin on proliferation of hepatoma cells were reported for the first time.

Abstract Vasostatin, the N-terminal 180 amino acids domain of calreticulin, induces apoptosis in endothelial cells and inhibits angiogenesis. However, the mechanism underlying the apoptosis induce by vasostatin remains elusive. In the present study, we investigated the role of (1) Fas /FasL pathway, (2) oxidative stress, and (3) nitric oxide (NO) in the apoptotic mechanism of vasostatin in endothelial cells. Recombinant vasostatin was generated and shown to induce apoptosis of bovine aortic endothelial cells (BAEC) as demonstrated by flow cytometry analysis, nucleus staining, and DNA fragmentation assay. Vasostatin elevated the levels of Fas and its adaptor, FADD, in BAEC. Furthermore, vasostatin treatment increased the activities as well as the expression of active form of caspase-8 and caspase-3 in BAEC. However, pretreatment with either caspase-3 inhibitor or caspase-8 inhibitor alone was not sufficient to blockade the vasostatin-mediated apoptosis, suggesting the involvement of other pathways. Extensive screening using an array of caspase inhibitors further supported such notion. Oxidative stress is frequently involved in the apoptosis of endothelial cells. Previous studies indicated that vasostatin enhanced WST-1-derived formazan formation despite its cytotoxic effect, suggesting vasostatin treatment might enhance the production of superoxide. By measuring the level of superoxide anion in cultured media by cytochrome c reducing test, it was found that vasostatin treatment increased the production of superoxide anion in endothelial cells. Antioxidants such as NAC, GSH, BHA partially attenuated the vasostatin-mediated cytotoxicity and cell death in endothelial cells. Noteworthingly, adding allopurinol, inhibitor of xanthine oxidase, but not other oxidase inhibitors abrogated the cytotoxicity of vasostatin, indicating that xanthine oxidase could be the source of ROS produced by vasostatin relate with apoptosis. The elecctrophoretic mobility shift assays (EMSA) suggested that vasostatin treatment increased the NF£eB DNA binding activity. Western blot analysis indicated vasostatin increased the levels of NF£eB but decreased I£eB level, which seemed to coincide with the EMSA findings. NO plays an important role in endothelial function. To investigate the role of NO in the cytotoxicity by vasostatin, analyzed the levels of NO metabolites in cultured media of endothelial cells and found that vasostatin treatment increased NO release in time- dependent manners. The expression of eNOS, but not iNOS, in endothelial cells was upregulated by vasostatin. Besides, vasostatin treatment also increased the AP-1 binding activities. Moreover, NOS inhibitor, L-NAME, or NO scavenger, carboxy-PTIO, slightly attenuated the cytotoxic effects of vasostatin in endothelial cells. In addition to direct cytotoxicity, NO may react with superoxide (O2-) to form peroxynitrite (ONOO-), which attacked the intracellular protein and caused the cell damage. Indeed, we also detected a dose-dependent increment in the nitrotyrosination of cellular protein by vasostatin treatment. Taking together, these results indicate that vasostatin induces apoptosis in endothelial cells via multiple pathways. The interactions between these distinct pathways remain to be elucidated in the future.

Blood vessel growth is mediated by angiogenesis, which is defined as the formation of new blood vessel out of existing vessels, as well as vasculogenesis, a process that circulating progenitor cells contributes to adult neovascularization. Cilostazol, a commercially available drug holding antiplatelet and vasodilating effects, increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibiting the activity of phosphodiesterase 3. Interestingly, this chemical compound has a lot of cellular effects. In current work, we demonstrated that cilostazol promoted proliferation and migration of human umbilical cord vein endothelial cells (HUVECs), enhanced in-vitro vascular tube formation, and increased releasing of cAMP and NO from them. Furthermore, cilostazol activated eNOS and PI3-K/Akt signaling pathways. We also examined the angiogenic and vasculogenic effects of cilostazol in a murine hindlimb ischemia model. Our data showed that cilostazol enhanced angiogenesis and vasculogenesis with resultant flow recovery after murine hindlimb ischemia partly mediated by promoting mobilization of bone marrow-derived stem cells into circulation and increasing in situ expression of some proteins involved in angiogenesis. In addition, cilostazol significant increased colony forming unit of human endothelial progenitor cells. These results are unique and clinically significant with potential in translational therapy. According to our report, further preclinical and clinical studies of cilostazol on the other ischemic situations such as myocardial infarction will be justified.

Tumor stroma is known to affect tumor growth and metastasis. Inhibiting PDGF signaling, with the goal of depleting PDGFRβ+ stromal cells, is a putative therapeutic approach in this context. PDGFRβ is widely accepted as a pericyte marker and targeting PDGF signaling primarily affects pericytes. Pericyte-endothelial cell interactions modulate angiogenesis and vascular stability in developmental and pathological contexts. Owing to this, pericytes are speculated to be important regulators of tumor growth and metastasis, although their role is not clear.

Formation of functional collateral circulation, to repair blocked or damaged arterial blood flow, is an important process in amending adverse outcomes after acute coronary occlusion events. Inadequate capillary growth during pressure overloads impairs myocardial perfusion, often contributing to the progression of coronary heart disease and ischaemia. Considered to be the critical rate-limiting step in physiological angiogenesis, the binding of VEGF (vascular endothelial growth factor) to VEGFR (vascular endothelial growth factor receptors) is essential for the growth and repair of arteries. Conversely, VEGF mediated angiogenesis has also been shown to promote atherosclerosis through arterial wall thickening. However, an alternatively spliced soluble form of VEGFR-1 (sFlt-1) has been shown to inhibit VEGF activity. sFlt-1 binds and sequesters free extracellular VEGF and/or heterodimerizes with VEGFR preventing the angiogenic pathway occurring. As a result, the primary pathway of angiogenesis does not occur. In recent years this has led to debate over the nature of sFlt-1 in the VEGF system. However, the level of sFlt-1 found in cardiovascular disease (CVD) patients, as well as its stability in plasma, has allowed for current research into its involvement with ischemic disorders to take place. Enhanced T-cell activity that results in increased production of interferon-γ has been shown to have involvement in the pathogenesis of CVD. 7,8-dihydroneopterin (7,8 NP) production by monocytes and macrophages is primarily in response to stimulation by interferon-γ (IFN-γ) released by activated T-lymphocytes. When combined with neopterin, the oxidised product of 7,8 NP, the total neopterin is accounted for which is a measure of the total macrophage activation by interferon-γ. Therefore, the levels of total neopterin observed may reflect the level of cell-mediated immunity within individuals which could contribute to mortality post CVD event. Progression of coronary heart disease is often clinically silent, without signs or symptoms. For this reason, the ability of markers to monitor progression is a powerful tool for predicting cardiovascular risk and the level of preventative treatment required. This study shows, that in 514 stable post-ACS (MI or unstable angina) patients, above median baseline sFlt-1, total neopterin and 7,8 NP levels, were strong predictors of mortality over a median 5 year period. Furthermore, above median sFlt-1 levels were specifically predictive of CVD death (p=0.001). This suggests that sFlt-1, total neopterin and 7,8 NP may be useful markers for risk prediction in CVD patients, post-acute event, with potential to aid prognosis in previously diagnosed patients. In support of these findings, levels of sFlt-1 measured in plasma taken from patients, immediately prior to undergoing carotid endarterectomy procedures (n=27), were significantly raised in comparison to age and gender matched healthy controls (p<0.001). Furthermore, levels of sFlt-1 in patient and control groups were shown to be independent of both age and gender. Another aspect of the study, analysis of excised live plaque tissue from carotid endarterectomy patients, showed the presence of live inflammatory cell populations. Macrophages, in the plaque sections, could be stimulated in the presence of IFN-γ to produce significantly elevated (p<0.01) levels of the antioxidant 7,8 NP. Since bivariate analysis of 7,8 NP and sFlt-1, in plasma from the endarterectomy patients, yields a positive correlation (r=0.323, p<0.01), further analysis of live plaque may give insight into the association between inflammation and hypoxic up-regulation of sFlt-1. It is now generally accepted, in diseases with complex pathogenesis, that particular biomarkers are predominantly indicative of only a single variable in a wide range of contributing factors. The data generated in this study highlights the potential for sFlt-1, neopterin and 7,8 NP to be used as contributing biomarkers in the prognosis of patients suffering from CVD, which if confirmed, may have important clinical implications in the medical community.

Because of the severe toxicity and limiting side effects of traditional chemotherapy, there exists a critical need to develop better-tolerated, safer drugs to treat cancer. Recent advances in our understanding of the molecular mechanisms governing carcinogenesis have ushered in a new age in drug discovery and have enabled the design of much more sophisticated agents to treat cancer. This work describes two approaches to the development of novel, specifically targeted cancer therapeutics.The first approach involves the synthesis of a class of a new class of small molecules called epidithiodiketopiperazines (ETPs) designed to inhibit hypoxia-induced transcription. Specifically, these agents block the interaction of the transcription factor HIF-1&alpha; (hypoxia inducible factor-1&alpha;) and its required coactivator p300/CBP by inducing a structural change in p300 that renders it incapable of binding to HIF-1&alpha;. Preventing hypoxia-mediated transcription has the potential to stop the process of angiogenesis that is critical for sustained tumor growth and metastasis. Moreover, because HIF-1&alpha; also controls genes for energy production and matrix remodeling, ETPs may also halt metabolic adaptation and tumor progression. Our results show that ETPs prevent the association of HIF-1&alpha; and p300 and abrogate hypoxia signaling on both the transcriptional and translational levels in endogenous systems. In addition, they do not exhibit broad-spectrum cytotoxicity or global inhibition of the transcriptional response.The second approach addresses the validation of poly(ADP-ribose) glycohydrolase (PARG) as a new therapeutic target. This project describes studies aimed to further our understanding of the interaction between poly(ADP-ribose) polymerases (PARPs) and PARG with the ultimate goal of using this knowledge to design novel therapeutics. This portion of the dissertation involves a series of studies in mouse embryonic fibroblasts (MEFs) with genetic mutations in their PARP and PARG function. MEF cell lines containing a truncated form of PARG lacking the regulatory domain demonstrate over-activation of PARP-1, but not PARP-2. Additionally, deletion of the PARG regulatory domain impairs the DNA damage response to SSBs and DSBs and significantly increases cell death resulting from genotoxic stress. Taken together, these studies suggest a specific interaction between PARP-1 and the regulatory domain of PARG that is critical for proper PARP-1 function.

Low Dose Metronomic (LDM) chemotherapy, combined with VEGF pathway inhibitors, is a highly effective strategy to coordinately inhibit angiogenesis and tumor growth. We have tested the efficacies of daily oral LDM topotecan alone and in combination with pazopanib, in three pediatric extracranial solid tumors mouse models. We also investigated the effect of prolonged combination therapy with the combination on tumor behavior in a neuroblastoma mouse xenograft model. In-vitro dose-response study of topotecan and pazopanib was conducted on several cell lines. In-vivo antitumor efficacies of drugs, as single agents and combination, were tested in immunodeficient mice models. For studying the mechanisms of resistance to our therapy, a time-response study (28, 56 and 80 days) was conducted in SK-N-BE(2) xenografts model, treated in same way as earlier. In vitro, topotecan caused a dose-dependent decrease in viabilities of all cell lines, while pazopanib did not. In vivo, the combination of topotecan and pazopanib demonstrated significant anti-tumor activity compared to the respective single agents in all models. Reductions in the levels of viable Circulating Endothelial Progenitors and/or Circulating Endothelial Cells and tumor microvessel density were correlated with tumor response and therefore confirmed the antiangiogenic activity of the regimens. However, the combination also caused significantly higher myelotoxicity than single agents. Pharmacokinetic study did not reveal any interaction between the two co-administered drugs. In the time-response study, we found that only combination treated animals survived till 80 days. However, tumors in these animals started growing gradually after 50 days. Unlike single agents, all three durations of combination treatment significantly lowered tumor microvessel densities, compared to the control. However, tumors treated with the combination for 56 and 80 days had higher pericyte coverage. The combination increased the hypoxia, angiogenic expression and proliferative index and caused metabolic reprogramming of tumor cells. We conclude that the combination of LDM topotecan and pazopanib has superior efficacy than either single agents, which is attributed to superior antiangiogenic activity. However, prolonged treatment with the combination can have additive myelotoxicity and may encounter adaptive resistance associated with metabolic reprogramming and increased proliferation of the tumor cells.

A crucial requirement for the stepwise, continued growth of a brain tumor is the acquisition of a blood supply from the host, i.e. angiogenesis. The mechanisms of copper activity linked to neovascular and neoplastic growth are largely unknown. Copper ion was shown to be a cofactor for angiogenesis. / We tested the effect of copper depletion achieved by a low copper diet and a copper chelator D-penicillamine, on the intracerebral growth of two experimental brain tumors. We developed in in vivo brain tumor model using the VX2 carcinoma. Implantation of 5 $ times$ 10$ sp5$ VX2 carcinoma cells into the parietal lobe of normocupremic rabbits consistently yielded large hemorrhagic, necrotic, vascularized tumors. The cortical surface revealed numerous, hypertrophied, tortuous new vessels with feeding arteries and draining veins similar to the angioarchitecture of malignant human brain tumors. We report here that copper depletion prevents tumor neovascularization and restricts tumor growth of the VX2 carcinoma in the rabbit brain. Low copper diet and penicillamine are both necessary to achieve angiogenic inhibition. We also tested the effect of copper depletion on the 9L gliosarcoma. We observed that invasive growth of the tumor was blocked in rats depleted of copper. Electron microscopy revealed the absence of cytoplasmic extensions, including pseudopodia, by contrast, in normocupremic controls, cytoplasmic extensions, typical of mobile cells, invaded the surrounding neuropil. Our findings link the activity of copper in vascular and neoplastic growth. / We found an increase in the peritumoral brain water content in the copper depleted animals and that copper depletion by itself in nontumor implanted animal has no effect on brain water content. / Because of the ability to pharmacologically suppress capillary growth induced by the VX2 carcinoma, we could test the relative contribution of breakdown of the blood-brain barrier compared with that of angiogenesis in the appearance of contrast enhancement in computed tomographic examinations. We conclude from our data that tumor neovascularization, in our brain tumor model, is the key determinant for the appearance of contrast enhancement. / The same protocol used in the brain failed to prevent tumor neovascularization and growth of the VX2 carcinoma in the muscle of the rabbit thigh indicating the crucial role played by the milieu (muscle versus brain) for the growth of malignant tumor. In the same manner, lung metastases were not prevented.

Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された