Microbubble ultrasound (US) contrast agents have been used to monitor the progression of anti-angiogenic chemotherapies. However, US backscatter measurements used in contrast imaging are inherently variable, given the presence of many microbubbles of random position and size. A model was developed to investigate the influence of US scanner and microbubble characteristics on these variable measurements. The Coefficient of Variation was used to measure variability. It was found that an optimum excitation frequency exists that minimizes this variability. In the case of DefinityTM, a 2.25 MHz centre-frequency pulse yielded a less variable measurement than at 5 MHz. Conversely, decreasing microbubbble concentration was found to significantly increase variability. Evidence suggests that microbubbles are no longer Rayleigh scatterers at sufficient low concentrations. Post-processing was found to aid in reducing measurement variability by averaging samples where microbubble positions are uncorrelated. As well, reduction can be achieved by averaging about a region-of-interest of uniform perfusion.

Inhibition of angiogenesis is an effective and low toxic therapeutic avenue for the treatment of cancer patients in addition to traditional interventions. Majority of current available angiogenesis inhibitors for cancer therapies are growth factor inhibitors and small molecule tyrosine kinase inhibitors. A number of endogenous proteins and/or proteolytic fragments of extracellular matrix proteins are shown to have the activity of inhibition of angiogenesis by directly targeting endothelial cells. Structural analyses have indicated that a common structure of anti-parallel β-sheet with a highly positively charged surface presents in many of those inhibitors. This common structural feature is critical for the maintenance of their anti-angiogenic function. With this structural information, we have designed and developed a new class of anti-angiogenic proteins by integrating the short anti-parallel β-sheet forming sequences of endogenous anti-angiogenic proteins into a stable host protein, the extracellular domain-1 of cluster of differentiation 2 molecule (CD2D1). 1D 1H NMR spectra analyses indicated that the designed anti-angiogenic protein (ref to as ProAgio) folded as a β-sheet structure similar to that of the parental protein, CD2D1. ProAgio inhibited the growth of human umbilical vein cells (HUVECs) without affecting the growth of epithelial cells, suggesting a specific effect to endothelial cells. ProAgio effectively reduced endothelial tubules formed by the co-culture of HUVECs and PC3 cells on matrix gel in vitro. The designed anti-angiogenic protein was further site-specifically PEGylated in order to improve PK/PD properties and reduce immunogenicity. Examinations with PC3 xenografts showed that both ProAgio and the PEGylated ProAgio dramatically inhibited tumor growth. Immunofluorescence staining analyses of the endothelial marker CD31 indicated dramatic decreases in tumor vessels in lengths and branching points. Histological and immunofluorescence staining analyses of tissue slices of major organs indicated that there were no pathological damages to the tissue structure or disruption of normal vessels associated with the treatment of our designed anti-angiogenic agent. Overall, our studies developed a novel anti-angiogenesis agent that may have great clinical potentials. Our concept of protein design can be extended to the development of other novel protein drugs.

Microbubble ultrasound (US) contrast agents have been used to monitor the progression of anti-angiogenic chemotherapies. However, US backscatter measurements used in contrast imaging are inherently variable, given the presence of many microbubbles of random position and size. A model was developed to investigate the influence of US scanner and microbubble characteristics on these variable measurements. The Coefficient of Variation was used to measure variability. It was found that an optimum excitation frequency exists that minimizes this variability. In the case of DefinityTM, a 2.25 MHz centre-frequency pulse yielded a less variable measurement than at 5 MHz. Conversely, decreasing microbubbble concentration was found to significantly increase variability. Evidence suggests that microbubbles are no longer Rayleigh scatterers at sufficient low concentrations. Post-processing was found to aid in reducing measurement variability by averaging samples where microbubble positions are uncorrelated. As well, reduction can be achieved by averaging about a region-of-interest of uniform perfusion.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21715号 / 薬科博第106号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 山下 富義, 教授 髙倉 喜信, 講師 樋口 ゆり子 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

L’angiogenèse est une composante majeure de l’agressivité des tumeurs malignes comme le glioblastome (GBM). Pourtant le traitement des patients par l’Avastin, un anticorps bloquant du VEGF ne leur confère qu’une augmentation limitée de la durée de survie sans progression. Les mécanismes de récurrence tumorale sont extrêmement complexes. Les glioblastomes sont en effet des tumeurs particulièrement hétérogènes sur le plan génétique, il existe très peu de marqueurs moléculaires d’expression fiables. La contribution à la récidive des potentiels angiogéniques, infiltrants, et souches est difficile à modéliser. Notre laboratoire s’intéresse à la caractérisation de nouveaux modulateurs de l’angiogenèse, dont certains pourraient contribuer à la croissance tumorale, indépendamment ou en aval du VEGF chez les patients traités par l’Avastin. Nous avons étudié l'expression de deux gènes candidats, surexprimés de façon significative dans les GBM et dont l'expression semble liée à l'angiogenèse tumorale : DPY19L1 et KIF20A. Nous avons identifié DPY19L1 comme marqueur pronostique du GBM. Ce gène est exprimé dans les cellules musculaires lisses, où il pourrait participer à la résistance de la tumeur aux anti-angiogéniques, en interagissant avec la voie thrombospondine/TGFβ. KIF20A quant à lui est exprimé dans les cellules souches tumorales et semble impliqué dans la vascularisation et la résistance tumorale. Dans un second temps, nous avons étudié la façon dont les GBM échappent aux traitements anti-angiogéniques, tel que l’Avastin, par la mise en place d'un système d'étude in vitro et in vivo, basé sur l’utilisation de cellules de patients atteints de GBM, ayant la capacité de pousser sous forme de neurosphères. Les cellules xénogreffées chez la souris immunodéfisciente permettent le développement d’une tumeur très invasive, co-optive et insensible aux traitements anti-angiogéniques. Ces tumeurs vont donc permettre d’étudier ce mode de vascularisation participant activement à la récidive de la tumeur chez les patients traités avec l’Avastin afin de développer des traitements contrecarrant ce mécanisme. Dans ce modèle, seul l’un des gènes candidats définis au début de ce travail, DPY19L1, semble participer à la croissance tumorale. / Angiogenesis is a major element driving malignancy of tumors like glioblastoma (GBM). However, Avastin,a neutralizing antibody directed against VEGF, provides only a limited therapeutical benefit in terms ofprogression free survival. The mechanisms of recurrence are complex due to extreme heterogeneity ofglioblastoma at the genetic and tissular levels. There is a lack of diagnosis and prognosis markers for GBM.The relative contribution of the angiogenic, infiltrative, and stem potentials to tumor relapse is difficult tomodel. Our laboratory aims at characterizing new modulators of tumor vascularization, some of whichcould contribute to the tumor growth and resistance, independently or downstream VEGF in patientstreated with Avastin. We have studied the expression of two candidate genes, significantly overexpressedin GBM and which expression seems to be linked to tumor vascularization: DPY19L1 and KIF20A. Weidentified DPY19L1 as a prognosis marker of GBM. This gene is expressed in smooth muscle cellsspecifically in tumoral tissue, where it could participate to tumor resistance to anti-angiogenics, byinteracting with the thrombospondin/TGFβ pathway. KIF20A is expressed in glioma stem cells and seemsto be implicated in the vascularization and tumor resistance. Next, we have studied the way by whichGBM resist to anti-angiogenics such as Avastin, by the development of an in vitro and in vivo modelsystem, based on GBM cells cultured as neurospheres. When xenografted in immunodeficient mice, thesecells induce the growth of very invasive, co-optive tumors which are insensitive to angiogenesis inhibitors.These tumors will allow investigating alternative modes of vascularization which are actively involved intumor recurrence in patients treated with Avastin, namely co-option and transdifferentiation and theirmolecular regulation. In this model, one of the candidate genes defined at the beginning of this study,DPY19L1, seems to be implied in tumor growth and specifically labels tumor cells with co-optive andtransdifferenciating properties.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20810号 / 医博第4310号 / 新制||医||1025(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 清水 章, 教授 萩原 正敏, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules.</p><p>Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement.</p><p>We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. </p><p>Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.</p>

Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules. Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement. We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.

New chimeric inhibitors targeting the epidermal growth factor (EGFR) and histone deacetylases (HDACs) were synthesized and tested for antineoplastic efficiency in solid cancer (prostate and hepatocellular carcinoma) and leukemia/lymphoma cell models. The most promising compounds, 3BrQuin-SAHA and 3ClQuin-SAHA, showed strong inhibition of tumor cell growth at one-digit micromolar concentrations with IC50 values similar to or lower than those of clinically established reference compounds SAHA and gefitinib. Target-specific EGFR and HDAC inhibition was demonstrated in cell-free kinase assays andWestern blot analyses, while unspecific cytotoxic effects could not be observed in LDH release measurements. Proapoptotic formation of reactive oxygen species and caspase-3 activity induction in PCa and HCC cell lines DU145 and Hep-G2 seem to be further aspects of the modes of action. Antiangiogenic potency was recognized after applying the chimeric inhibitors on strongly vascularized chorioallantoic membranes of fertilized chicken eggs (CAM assay). The novel combination of two drug pharmacophores against the EGFR and HDACs in one single molecule was shown to have pronounced antineoplastic effects on tumor growth in both solid and leukemia/lymphoma cell models. The promising results merit further investigations to further decipher the underlying modes of action of the novel chimeric inhibitors and their suitability for new clinical approaches in tumor treatment.