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Surgical skin flaps are frequently used in plastic and reconstructive surgery to repair acquired or congenital defects. Necrosis is a common complication associated with these flaps postoperatively as a result of inadequate blood supply. Stem cells are precursor cells with the potential to differentiate into more specialized cells. Angiogenic factors act to direct cellular differentiation and organization to form new vascular elements. Our theory was that the combination of angiogenic growth factors with stem cells derived from the subject preoperatively would augment neovascularization, thereby increasing blood supply to the flap, which may ultimately improve flap survival. In phase I, 40 Lewis rats were randomized into 4 groups of 10. Random dorsal skin flaps were elevated and treated at the same time. The first group was injected with only medium, the second with stem cells, the third with stem cells and angiogenic factors, and the fourth with angiogenic growth factors. Millimetric measurements of flap viability at 7 and 14 days did not show any statistically significant differences between the studied groups. In phase II, 24 rats were also randomized into 4 groups of 6, but this time were treated 1 week before flap elevation. The viability measurements showed an increased rate of viability in the group in which stem cells and the angiogenic factors were administered simultaneously (84.5% ± 3.2%) as compared with the unmodified control group (62.6% ± 7.3%) or to the groups in which only precursor cells (60.4% ± 7.9%) or angiogenic factors (62.3% ± 10.1%). Increased blood supply brought by these manipulations is believed translated to increased tissue flap survival. Punch biopsies showed that "green fluorescent protein"-labeled precursor cells was noted to form luminal structures in the treated flaps. The vascular cast of all flaps was filled with Mercox plastic resin. After euthanasia, the soft tissues of the harvested flaps were dissolved and the remaining vascular cast was weighted. The weight-based ratio of the vascular composition was determined. The flaps injected with both stem cells and angiogenic factors showed higher values. We conclude that the administration of bone marrow stem cells with angiogenic factors 1 week before flap creation improves the survival of ischemic random skin flaps.

Glioblastoma multiforme (GBM) is an extremely aggressive and almost always fatal brain tumor. GBM literature indicates defective mismatch repair (MMR) mechanisms are not involved in GBM tumorigenesis as in other tumors, and instigating mechanisms of GBM tumorigenesis remain unclear. GBM and neural progenitor (NPR) cells were exposed to three concentrations of H2O2 (0, 0.5, and 1.0 μM), cultured, and then harvested 0, 2, 4, and 6 days post-exposure; DNA from cells was amplified with microsatellite primers, investigating whether or not H2O2 exposure affected microsatellite instability (MSI) in target sequences. Three out of six markers showed significant MSI in the H2O2-exposed NPR cells. Our results suggest H2O2, which generates reactive oxygen species (ROS), correlated with MSI accumulation that occurred in NPR cells in specific DNA regions. Thus, gene expression analysis to assess normal and abnormal gene expression of GBM and NPR cellss is warranted.

The chick chorio-allantoic membrane (CAM) contains a complex vascular network commonly used to study angiogenesis. The application of chemical factors and oxygen barrier films onto this tissue can easily influence the process of angiogenesis. In this study, oxygen barrier film patches (Krehalon, polyvinylidene chloride, 12 μm thick, O2 transmission rate = 2.19 cm3•ml/100 in2•day•atm) were applied to areas of the CAM. Holes were made in the film and alginate beads incubated in various chemical factors were placed in the holes. After 24 and 48 hours of exposure to the alginate beads, images were taken of the tissue using a stereomicroscope and then processed using ImageJ software (from the National Institue of Health (NIH)). The images were analyzed with the Fractal Analysis plugin of ImageJ (also from NIH) using four parameters. These parameters are the number of vessel segments, the number of vessel bifurcations, the total length of the vessels, and the complexity of the vascular network. From these parameters, the chemical factors can be identified as promoting angiogenesis (pro-angiogenic), inhibiting angiogenesis (anti-angiogenic), or having no effect on angiogenesis (not angiogenic). For the angiogenic beads, significant results were found in at least one of the four parameters. SNAP and H2O2 gave pro-angiogenic responses while Angiotensin II, Losartan, and Adenosine were anti-angiogenic. To test the effect of an oxygen barrier film patch on angiogenesis, images were taken of the tissue under the film patch (virtual holes) and holes exposed to atmospheric oxygen. Analysis of the virtual holes compared to the control holes gave significant results for several of the film patches. These film patches are distinguished by the chemical that was tested on each of the films. The virtual holes containing Angiotensin II, Losartan, Adenosine, and H2O2 gave pro-angiogenic results while SNAP and L-NAME virtual holes were anti-angiogenic. Thus, the chemical factors and the oxygen barrier film patches did have an effect on angiogenesis in the CAM.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-11-22T18:16:35Z No. of bitstreams: 1 LUCIANA CARDOSO MARINHO.pdf: 2007688 bytes, checksum: 7b1d1ab427984f805bae729662ab0296 (MD5) / Made available in DSpace on 2016-11-22T18:16:35Z (GMT). No. of bitstreams: 1 LUCIANA CARDOSO MARINHO.pdf: 2007688 bytes, checksum: 7b1d1ab427984f805bae729662ab0296 (MD5) Previous issue date: 2016-08-10 / The latest research has shown that the angiogenic factors play an important role in the modulation of tumor angiogenesis process. Thus, this study aimed to analyze the influence of this process on the development of leukemias. a systematic review based on scientific articles that investigated the interference of angiogenic factors in cases of leukemia early and advanced stages was performed. The major evidence indicates that these factors, in special VEGF, are expressed intensely in malignant cells and may generate, for example, a hematologic malignancy. The regulation of expression of certain genes is also involved in abnormal proliferation, differentiation and apoptosis of these cells. The expression, function and regulation mechanism of angiogenic factors have been gradually elucidated in this review and in conclusion, it is noted that some of them have relation to the development and leukemias prognosis, and provide potential strategies for the treatment of patients with leukemia. / As últimas pesquisas demonstram que os fatores angiogênicos desempenham um importante papel na modulação do processo da angiogênese tumoral. Neste sentido, o presente estudo tem como objetivo a análise da influência deste processo no desenvolvimento de leucemias. Foi realizada uma revisão sistemática com base em artigos científicos que investigaram a interferência de fatores angiogênicos em casos de leucemias em estágios iniciais e avançados. As principais evidências indicam que estes fatores, em especial o VEGF, são expressos de forma intensa em células malignas e podem gerar, por exemplo, uma malignidade hematológica. A regulação de expressão de determinados genes também está envolvida na proliferação anormal, na diferenciação e apoptose destas células. A expressão, função e o mecanismo de regulação dos fatores angiogênicos foram elucidados gradualmente nesta revisão e como conclusão, salienta-se que alguns deles apresentam relação com o desenvolvimento e prognóstico de leucemias, além de fornecer possíveis estratégias para o tratamento de pacientes com leucemia.

Die Bedeutung der angiogenen Faktoren „soluble fms-like tyrosine kinase-1“ (sFlt-1) und „placental growth factor“ (PIGF) in der Pathogenese der Präeklampsie (PE) ist in den letzten Jahren hinreichend belegt worden. Bei Patienten mit manifester PE lassen sich dramatisch hohe sFlt-1- bzw. sehr niedrige PlGF-Werte nachweisen. Mittlerweile konnte in Studien die sFlt-1/PlGF-Ratio als sensitiver Parameter ermittelt werden. Ziel der vorliegenden Arbeit ist es, die Zusammenhänge zwischen klinischen Parametern der manifesten Präeklampsie und der sFlt-1/PlGF-Ratio zu spezifizieren. Dabei soll geprüft werden, ob bei manifester PE die Höhe der sFlt-1/PlGF-Ratio vor Entbindung klinische Bedeutung hat und mit Parametern des Schwangerschaftsausgangs in Beziehung steht.

京都大学 / 0048 / 新制・論文博士 / 博士(人間・環境学) / 乙第12951号 / 論人博第42号 / 新制||人||178(附属図書館) / 27||論人博||42(吉田南総合図書館) / 32250 / 三重大学大学院医学系研究科生命医科学専攻 / (主査)教授 石原 昭彦, 教授 神﨑 素樹, 准教授 久代 恵介, 准教授 月浦 崇, 教授 藤野 英己 / 学位規則第4条第2項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

The goals of the thesis were to, in pig models, (1) assess manganese-enhanced magnetic resonance imaging (MEMRI) in the characterization of acute and chronic ischemia-induced myocardial infarction (MI), (2) characterize cryoinjury-induced MI with contrast-enhanced MRI and optical methods, and (3) observe the effects of locally released angiogenic factors on the repair of cryoinjury-induced MI. Firstly, after acute MI was established by occlusion of the coronary artery branches, the pig hearts were isolated and mounted onto an ex vivo perfusion system inside a 7T magnet. After administration of MnCl2, T1-weighted MR images showed gradual enhancement of signal intensity within the normal myocardium, whereas the ischemic counterpart remained hypointense. During chronic MI progression, the intensity increased slowly after exposure to MnCl2 within the infarcted myocardium. Secondly, a new MI model was tested via direct 2-min contact of left ventricular epicardium with a liquid nitrogen-cooled aluminum bar. Subsequent in vivo Gd-enhanced MRI showed a uniform hypointense area (~10 mm in depth) surrounded by a hyperintense rim. Histology showed erythrocytes embolism within the cryolesion with a thin necrotic rim neighboring the normal myocardium. Four weeks later, the cryoinjured myocardium was replaced by scar tissue. Thirdly, in vivo MEMRI was tested on this cryoinjury model. After intravenous administration of MnCl2 via intermittent bolus or continuous infusion, normal myocardium showed prolonged hyperintense, which led to significant signal contrast between it and cryoinjured myocardium. Continuous infusion scheme minimized hemodynamic fluctuation. Finally, angiogenic therapy was assessed by anchoring of vascular growth factors-loaded alginate beads or adipose-derived stem cells (ADSCs)-loaded agarose patch on top of the cryoinjured myocardium. Gd-enhanced MRI revealed (1) growth of new tissue wrapping the growth factors-loaded alginate beads and (2) higher perfusion within the ADSCs-treated cryoinjured myocardium as compared with the growth factors-treated counterpart. Histological and fluorescent microsphere examination revealed that ADSCs induced more significant growth of mature microvasculature within the cryoinjured myocardium. These results indicate that MnCl2 could characterize MI ex vivo and in vivo. Epicardial implantation of ADSCs-loaded agarose hydrogel can induce angiogenesis within the cryoinjured myocardium, a form of MI with similar progression features as that induced by ischemia.

Hämangiosarkome, maligne endotheliale Neoplasien, werden bei den Haussäugetieren am häufigsten beim Hund beobachtet und treten bei diesem vor allem in Milz und rechtem Herzohr auf. Anhand ihrer Wachstumsmuster werden sie in kapilläre, kavernöse und solide wachsende Tumoren eingeteilt (PULLEY u. STANNARD 1990, HARRY u. PALEOLOG 2003). Die Prognose kaniner Hämangiosarkome der Milz ist schlecht, wobei in der Literatur nach Splenektomie mediane Überlebenszeiten von 19-240 Tagen angegeben werden (JOHNSON et al. 1989, OGILVIE et al. 1996, SPANGLER u. KASS 1997, WOOD et al. 1998) und auch Praktiker von sehr stark variierenden Überlebenszeiten berichten. Die Eignung verschiedener Parameter (z.B. adjuvante Chemotherapie, klinisches Staging) zur prognostischen Beurteilung wurde bereits von zahlreichen Autoren untersucht (BROWN et al. 1985, SORENMO et al. 1993, WOOD et al. 1998). Studien zum eventuellen Einfluss des Wachstumsmusters auf die Variabilität der Überlebenszeiten existieren bisher jedoch nicht. Darüber hinaus können Hämangiosarkome der Milz auch diagnostisch eine Herausforderung darstellen. Dies ist zum Einen bedingt durch die hohe Anfälligkeit der Milz gegenüber einer raschen Autolyse, zum Anderen erschwert die sehr variable Histomorphologie in einigen Fällen eine sichere Diagnosestellung. Mit dem immunhistologischen Nachweis klassischer Endothelzellmarker wie von Willebrand Faktor und CD31 kann in den meisten Fällen die Diagnose gesichert werden. Besonders bei schlecht differenzierten Hämangiosarkomen, erweisen sich die genannten Marker oftmals als z. T. unzuverlässig (VON BEUST et al. 1988, GAMLEM u. NORDSTOGA 2008). Bei rein solide wachsenden Neoplasien kann der Transkriptionsfaktor Fli-1 hilfreich bei der Diagnose sein (STEIGER et al. 2003). Pro- und anti-angiogene Faktoren steuern in einem streng regulierten, stufenweise ablaufenden Prozess die Entstehung neuer Blutgefäße aus präexistenten Gefäßen – die Angiogenese (KERBEL et al. 1998, CARMELIET et al. 1998, RAK et al. 2000, JOUSSEN et al. 2003, DISTLER et al. 2003). Diese Faktoren konnten u.a. in Neoplasien und deren versorgenden Gefäßen nachgewiesen werden (RESTUCCI et al. 2002, RESTUCCI et al. 2004). Ziel der Studie ist (I) die Untersuchung der Überlebenszeiten von Hunden mit lienalen Hämangiosarkomen unter Berücksichtigung des Wachstumsmusters und weiterer Parameter, (II) wird mittels vergleichender immunhistologischer Untersuchungen die Eignung klassischer Endothelzellmarker sowie angiogener Faktoren und deren Rezeptoren zum Nachweis neoplastischer Endothelzellen überprüft. Der Vergleich des Expressionsverhaltens der genannten Marker zwischen Hämangiosarkomen und nicht-neoplastischen Endothelzellen gibt eventuell Hinweise auf die biologische Wertigkeit der Neoplasien.

The goals of the thesis were to, in pig models, (1) assess manganese-enhanced magnetic resonance imaging (MEMRI) in the characterization of acute and chronic ischemia-induced myocardial infarction (MI), (2) characterize cryoinjury-induced MI with contrast-enhanced MRI and optical methods, and (3) observe the effects of locally released angiogenic factors on the repair of cryoinjury-induced MI. Firstly, after acute MI was established by occlusion of the coronary artery branches, the pig hearts were isolated and mounted onto an ex vivo perfusion system inside a 7T magnet. After administration of MnCl2, T1-weighted MR images showed gradual enhancement of signal intensity within the normal myocardium, whereas the ischemic counterpart remained hypointense. During chronic MI progression, the intensity increased slowly after exposure to MnCl2 within the infarcted myocardium. Secondly, a new MI model was tested via direct 2-min contact of left ventricular epicardium with a liquid nitrogen-cooled aluminum bar. Subsequent in vivo Gd-enhanced MRI showed a uniform hypointense area (~10 mm in depth) surrounded by a hyperintense rim. Histology showed erythrocytes embolism within the cryolesion with a thin necrotic rim neighboring the normal myocardium. Four weeks later, the cryoinjured myocardium was replaced by scar tissue. Thirdly, in vivo MEMRI was tested on this cryoinjury model. After intravenous administration of MnCl2 via intermittent bolus or continuous infusion, normal myocardium showed prolonged hyperintense, which led to significant signal contrast between it and cryoinjured myocardium. Continuous infusion scheme minimized hemodynamic fluctuation. Finally, angiogenic therapy was assessed by anchoring of vascular growth factors-loaded alginate beads or adipose-derived stem cells (ADSCs)-loaded agarose patch on top of the cryoinjured myocardium. Gd-enhanced MRI revealed (1) growth of new tissue wrapping the growth factors-loaded alginate beads and (2) higher perfusion within the ADSCs-treated cryoinjured myocardium as compared with the growth factors-treated counterpart. Histological and fluorescent microsphere examination revealed that ADSCs induced more significant growth of mature microvasculature within the cryoinjured myocardium. These results indicate that MnCl2 could characterize MI ex vivo and in vivo. Epicardial implantation of ADSCs-loaded agarose hydrogel can induce angiogenesis within the cryoinjured myocardium, a form of MI with similar progression features as that induced by ischemia.