Angiogenesis, the process of new vessel formation, appears to be a central mechanism that underlies the development of haemangiomas. Recently, intralesional bleomycin injection was used to treat paediatric haemangiomas with very good results. The purpose of this study was to determine whether there was significant systemic circulatory spill-over of bleomycin in haemangioma patients treated with intralesional bleomycin to determine safety of use. Furthermore, in order to elucidate bleomycin’s mechanism of action in inducing haemangioma regression, this study aimed at determining the effects of bleomycin on aspects of angiogenesis, namely, endothelial cell migration, growth and apoptosis, and comparing these effects with those of drugs previously reported to inhibit various aspects of the angiogenic process (mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D). Lastly, the effects of bleomycin, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D were studied in an animal haemangioma model. A rapid and highly sensitive high performance liquid chromatographic (HPLC) method was developed. Blood samples were collected from four haemangioma patients before and after (over a 24 hour period) intralesional bleomycin (IB) therapy. As a control, blood samples were also collected at identical time intervals from four patients undergoing intravenous (IV) bleomycin chemotherapy for various malignant tumours. The HPLC method was used to quantitate bleomycin fractions in patient samples. The mean bleomycin concentration detected in plasma samples obtained from IB treated patients was 0.00 ìg/ml for both bleomycin A<Sub>2 and B2 over the 24-hour period following therapy. Plasma bleomycin A2 and B2 levels of 360.79 and 158.85 ìg/ml respectively were detected in samples obtained from cancer patients treated with bleomycin IV. These findings indicate that the low levels detected may translate to a significantly lesser risk of pulmonary fibrosis following IBI. The effect of drugs on endothelial cell migration was analyzed by wounding a confluent monolayer of cells and determining the number of cells that had migrated from the wound edge. Endothelial cell growth was determined in cells treated with various drug concentrations while apoptosis was examined using hematoxylin and eosin staining, DNA fragmentation assay and acridine orange staining. The effect of test drugs on in vitro angiogenesis was determined on endothelial cells induced to form capillary-like tubes in collagen gel. Test drugs were then evaluated for antitumour activity in an animal haemangioma model. Data demonstrated that test drugs inhibited endothelial cell migration, with the exception of mitomycin C. All test drugs induced a reduction in the percentage of viable endothelial cell in a dose-dependant manner, and also induced endothelial cell apoptosis. The drugs inhibited angiogenesis in vitro and inhibited tumour development in vivo with varying potency. In general, results from this study indicated that there was negligible systemic spill-over of bleomycin following IB administration in patients with haemangiomas, suggesting a much lesser risk of developing bleomycin-induced pulmonary fibrosis. This study also showed that test drugs inhibited angiogenesis in vitro and haemangioma development in vivo in a mouse model. Taken together, these observations demonstrate that bleomycin may inhibit haemangioma growth by inhibiting angiogenesis. In addition, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D may have potential in the treatment of haemangiomas of infancy, and should be investigated further in a murine haemangioma model to determine effective dose schedules. / Thesis (PhD)--University of Pretoria, 2009. / Physiology / unrestricted

L'hémangiome capillaire infantile (IH) est une tumeur vasculaire bénigne courante. Les formes sévères sont traitées depuis 2008 par propranolol bien que le mécanisme d’action reste à ce jour inexpliqué. La 1ère partie de ma thèse est une étude de tolérance chez les enfants traités par propranolol dans le cadre de l'ATU française. L'analyse de cette base de données prospective de 922 enfants a permis de confirmer la bonne tolérance du propranolol dans la population pédiatrique, et de cibler les situations à risque de complication. La 2e partie est une étude de la signalisation adrénergique à partir des tissus d'IH opérés. Tout d'abord, nous avons identifié par immunofluorescence les cellules exprimant les récepteurs béta-adrénergiques ADRB1, 2 et 3 : Nous avons observé une forte expression d'ADRB2 sur le mastocyte, et de manière plus modérée d'ADRB1 et 2 sur les vaisseaux. Ce profil d'expression était retrouvé quel que soit le degré d'involution de la tumeur, et était également observé sur des tumeurs vasculaires contrôles qui elles ne répondent pas au propranolol. Nous avons donc envisagé une activation des récepteurs ADRB propre aux IH prolifératifs. Cette hypothèse a été confirmée par la mise en évidence, de l'expression d'enzymes de synthèse des catécholamines et de marqueurs neuroendocrines sur les péricytes des IH prolifératifs, sur tissus inclus en paraffine et également péricytes d’IH mis en culture. La surexpression de HIF1-a sur ces cellules a conduit à tester l’effet du propranolol en hypoxie. Nous n’avons pas mis en évidence d'effet sur la prolifération cellulaire, mais par contre une inhibition de la sécrétion de VEGF induite par l’hypoxie. / Capillary infantile haemangioma (IH) is a common benign vascular tumour of infants. Severe forms are treated since 2008 by propranolol. However, its dramatic efficacy remains until now unexplained.The first part of my thesis is a safety study of children treated with propranolol as part of the French Compassionate Use Program. The prospective vigilance database, including 922 children, confirmed the safety of propranolol among the paediatric patients, and highlighted the circumstances at risk of complications.The second part is a study of adrenergic signaling in IH tissues. First, we identified by immunofluorescence the expression of beta-adrenergic receptors on IH cells. We observed a strong expression of ADRB2 on mast cells, and a moderate ADRB1 and 2 expression on vessels. This expression pattern was the same regardless of the degree of involution of the tumour, and has also been observed on tumour vascular controls which did not respond to propranolol. We therefore hypothesized that ADRB receptors may be activated in proliferative IH. We indeed detected the expression of catecholamine synthesis enzymes and of neuro-endocrine markers on pericytes in paraffin-embedded tissues, and also in IH pericytes in culture. We also detected HIF1-alpha overexpression and therefore explored propranolol effect during hypoxia. Propranolol had no effect on cell proliferation, but reduced hypoxia-induced secretion of VEGF-A.

Charakterisierung von Lebertumoren nach kontrastverstärkter Sonographie und digitaler Graustufenbestimmung Ziel: Die Charakterisierung fokaler Leberläsionen ist Bestandteil des klinischen Alltags und für Patienten von therapeutischer und prognostischer Relevanz. Auf diesem Gebiet wurde bisher die native Sonographie regulär eingesetzt. Eine sichere Artdiagnose bei unklaren Lebertumoren ist jedoch nur selten möglich. Die Einführung der kontrastmittelverstärkten Sonographie hat die differentialdiagnostischen Optionen erweitert. Ziel dieser Studie war es, den Nutzen des kontrastmittelverstärkten Ultraschalls und der anschließenden digitalen Graustufenanalyse bei fokalen Leberläsionen zu bewerten. Methodik: In einer prospektiven Studie wurde bei 50 Patienten mit CT oder MRT gesicherten Lebertumoren eine Sonographie des Oberbauches in nativer Sonographie und in Phaseninversionstechnik mit intravenöser Gabe des Ultraschallkontrastmittels SonoVue® durchgeführt. Nach Kontrastmittelgabe wurden über 120 s digitale Standbilder akquiriert. Mittels Software ermittelte man den dynamischen Graustufenverlauf für jeden einzelnen Tumor. Es folgte der Vergleich der einzelnen bildgebenden Modalitäten untereinander. Ergebnisse: Der Anteil der artdiagnostisch korrekten Zuordnungen belief sich in der CT bzw. MRT auf 78% und in der nativen Sonographie auf 60%. Mit Hilfe des kontrastmittelverstärkten Ultraschalls konnte er auf 86% gesteigert werden. Die digitale Graustufenanalyse lieferte für die einzelnen Tumorentitäten charakteristische Kurvenverläufe. Hierbei erwiesen sich die Zeitpunkte 20 s und 100 s nach Kontrastmittelgabe für die artspezifische Charakterisierung und für die Differenzierung in benigne und maligne Tumoren als optimal. Schlussfolgerung: Die kontrastmittelverstärkte Sonographie und deren digitale Graustufenbestimmung bilden eine Ergänzung der bisherigen Diagnostik von Lebertumoren und ermöglichen eine bessere Charakterisierung der Herde. Dies sollte in zusätzlichen Studien evaluiert werden. / Characterization of Hepatic Tumors with Contrast-enhanced Ultrasound and Digital Gray-Scale Analysis Purpose: The characterization of liver tumors is of therapeutic and prognostic relevance. Although ltrasound offers the opportunity to detect hepatic tumors, its previous techniques did not lead towards a definitve differentiation. The purpouse of this study was the evaluation of contrast enhanced ultrasound followed by quantitative digital analysis in patients with focal hepatic tumors. Materials and Methods: In a prospective stuy, 50 patients with liver tumors previously detected by CT or MRI were examined by ultrasound of the upper abdomen using conventional and phase inversion technique after intravenous application of an ultrasound contrast agent. Digital images were stored over 120 s and software powerd digital gray-scale curves were produced for each individual lesion. Results: While the percentage of tumors correctly characterised by CT/MRI amounted to 78%, the percentage increased from 60% using conventional ultrasound to 86% using contrast enhanced ultrasound including gray-scale analysis. Typical graphs were achieved for different tumor entities on digital gray-scale analysis. Time intervals at 20 and 100 seconds showed optimal for differantiation between particualar entities. Conclusion: Quantification of contrast enhanced ultrasound is an addition to the previous diagnostic procedure in hepatic tumors. It offers the possibility of an investigator-independent characterization of lesions and should be evaluated in further studies.