Traumatic spinal cord injury (SCI) is a devastating condition for which there is no effective clinical treatment. Neuroprotective molecules that minimize tissue loss have shown promising results; however systemic delivery may limit in vivo benefits due to short systemic half-life and minimal passage across the blood-spinal cord barrier. To overcome these limitations, an injectable intrathecal delivery vehicle comprised of hyaluronan and methylcellulose (HAMC) was developed, and previously demonstrated to be safe and biocompatible intrathecally. Here, HAMC was determined to persist in the intrathecal space for between 4-7 d in vivo, indicating it as an optimal delivery system for neuroprotective agents to reduce tissue degeneration after SCI. HAMC was then investigated as an in vivo delivery system for two neuroprotective proteins: erythropoietin (EPO) and fibroblast growth factor 2 (FGF2). Both proteins demonstrated a diffusive release profile in vitro and maintained significant bioactivity during release. When EPO was delivered intrathecally with HAMC to the injured spinal cord, reduced cavitation in the tissue and significantly improved neuron counts were observed relative to the conventional delivery strategies of intraperitoneal and intrathecal bolus. When FGF2 was delivered intrathecally from HAMC, therapeutic concentrations penetrated into the injured spinal cord tissue for up to 6 h. Poly(ethylene glycol) modification of FGF2 significantly increased the amount of protein that diffused into the tissue when delivered similarly. Because FGF2 is a known angiogenic agent, dynamic computed tomography was developed for small animal serial assessment of spinal cord hemodynamics. Following SCI and treatment with FGF2 from HAMC, moderate improvement of spinal cord blood flow and a reduction in permeability were observed up to 7 d post-injury, suggesting that early delivery of neuroprotective agents can have lasting effects on tissue recovery. Importantly, the entirety of this work demonstrates that HAMC is an effective short-term delivery system for neuroprotective agents by improving tissue outcomes following traumatic SCI.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

Thesis (Ph.D.). / Written for the Division of Experimental Medicine, Dept. of Medicine. Title from title page of PDF (viewed 2008/07/23). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 117-139).

Rupture of the anterior cruciate ligament (ACL) of the knee leads to chronic joint instability and reduced range of motion while the long term results are marred by a high prevalence of degenerative joint disease especially osteoarthritis. Bundles of collagen threads have been widely investigated for the repair of torn ACL, but are limited by insufficient tissue ingrowth to repopulate and completely regenerate these grafts. We have developed a novel in vitro method of characterizing fiber-based thread matrices by probing their ability to promote tissue ingrowth from a wound margin as a measure of their ability to promote repopulation and regeneration. This method is useful in the optimization of thread scaffolds, and is sensitive enough to distinguish between subtle variations in biopolymer chemistry and organization. Furthermore, this method was used to characterize the effects of crosslinking on the cell outgrowth and correlated the findings with the mechanical properties of collagen threads. The results suggest that crosslinking is required to achieve sufficient mechanical properties for high stress applications such as ACL replacement, but regardless of technique, crosslinking attenuated the cell outgrowth properties of the threads. To improve the regenerative capacity of these scaffolds, novel fibrin microthread matrices were developed with a similar morphology to collagen threads and sufficient mechanical strength to be incorporated in composite thread scaffold systems. These fibrin microthreads were loaded with FGF-2, a potent mitogen and chemotactic agent that works synergistically with fibrin in regulating cell signaling and gene expression. Increases in fibroblast migration and proliferation in FGF-2-loaded fibrin threads were successfully demonstrated with the concomitant promotion of oriented, aligned, spindle-like fibroblast morphology. These results suggest that fibrin-FGF-2 microthreads have distinct advantages as a biomaterial for the rapid regeneration of injured tissues such as the ACL.

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-06-06Bitstream added on 2014-06-13T19:23:26Z : No. of bitstreams: 1 ruiz_kgs_dr_arafo.pdf: 357284 bytes, checksum: 93541a495590ca3b701f6cf9ef876131 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi avaliar o efeito da combinação do fator de crescimento de fibroblastos básico (b-FGF) e fator de transformação de crescimento beta (TGF- beta) na proliferação, expressão de genes para colágeno tipos I e III, metaloproteases -1 e -2 e TIMPs 1, 2 e 3, e na síntese destes próprios fatores de crescimento pelas células do ligamento periodontal de humanos. A avaliação da proliferação celular foi realizada após os períodos de 24 e 48h na presença dos fatores de crescimento, através da mensuração do nível de MTS reduzido a formazan pelas células viáveis, e a síntese de b-FGF e TGF-b pelo teste ELISA empregando a técnica sandwich. Conclui-se que as associações do bFGF e do TGF-b atuaram de maneira dose-dependente no estímulo à proliferação celular, o aumento na expressão de genes para colágeno e TIMPs e redução para as metalproteases e modulação da síntese deles próprios. / The aim of this study was to evaluate the effect of association of basic fibroblast growth factor and transforming growth factor beta on the proliferation, expression of colagen type I e III, matrix metalloproteinases-1 e -2 e TIMPs 1, 2 e 3, and on the modulation of the syntheses oh these growth factors by humans periodontal ligament cells. The cellular proliferation was evaluated to 24 and 48 hours of incubation by the colorimetric method. The synthese of bFGF and TGF-ß was verificated by ELISA method, using a sandwich techinique, and mRNA expression by Real Time - PCR. In conlusion, the associations of bFGF e TGF-ß influenced of dose-dependent manner on the cellular proliferation, on the increasing of the expression to collagen and TIMPs, and on the reduction to metaloproteinases and, the modulation of these own synthesis.

Orientador: Ricardo Samih Georges Abi Rached / Banca: Silvana Regina Perez Orrico / Banca: Regina Maria Barretto Cicarelli / Banca: Francisco Humberto Nociti Junior / Banca: Márcio Zafallon Casati / Resumo: O objetivo do presente estudo foi avaliar o efeito da combinação do fator de crescimento de fibroblastos básico (b-FGF) e fator de transformação de crescimento beta (TGF- beta) na proliferação, expressão de genes para colágeno tipos I e III, metaloproteases -1 e -2 e TIMPs 1, 2 e 3, e na síntese destes próprios fatores de crescimento pelas células do ligamento periodontal de humanos. A avaliação da proliferação celular foi realizada após os períodos de 24 e 48h na presença dos fatores de crescimento, através da mensuração do nível de MTS reduzido a formazan pelas células viáveis, e a síntese de b-FGF e TGF-b pelo teste ELISA empregando a técnica "sandwich". Conclui-se que as associações do bFGF e do TGF-b atuaram de maneira dose-dependente no estímulo à proliferação celular, o aumento na expressão de genes para colágeno e TIMPs e redução para as metalproteases e modulação da síntese deles próprios. / Abstract: The aim of this study was to evaluate the effect of association of basic fibroblast growth factor and transforming growth factor beta on the proliferation, expression of colagen type I e III, matrix metalloproteinases-1 e -2 e TIMPs 1, 2 e 3, and on the modulation of the syntheses oh these growth factors by humans periodontal ligament cells. The cellular proliferation was evaluated to 24 and 48 hours of incubation by the colorimetric method. The synthese of bFGF and TGF-ß was verificated by ELISA method, using a "sandwich" techinique, and mRNA expression by Real Time - PCR. In conlusion, the associations of bFGF e TGF-ß influenced of dose-dependent manner on the cellular proliferation, on the increasing of the expression to collagen and TIMPs, and on the reduction to metaloproteinases and, the modulation of these own synthesis. / Doutor

INTRODUÇÃO: Os níveis séricos de fósforo (P) têm sido associados a doenças cardiovasculares e mortalidade em pacientes com doença renal crônica e na população geral. Estudos in vitro demonstram que altas concentrações de fósforo extracellular são capazes de induzir calcificação vascular e disfunção endotelial. O Fibroblast Growth Factor 23 (FGF-23) é um hormônio fosfatúrico e foi relacionado à presença de aterosclerose em pacientes idosos. OBJETIVO: O objetivo deste estudo foi investigar as relações entre P, FGF-23 e outros atores do metabolismo mineral e a ocorrência de doença arterial coronariana em pacientes com função renal preservada. MÉTODOS: Duzentos e noventa pacientes clinicamente estáveis com indicação de cineangiocoronariografia eletiva e clearance de creatinina superior a 60 ml/min/1.73 m2 foram submetidos à Tomografia Computadorizada Multislice para avaliação da calcificação coronariana e coleta de sangue para dosagens bioquímicas. A calcificação coronariana foi quantificada através do Escore de Agatston (EA) e os Escores de Friesinger e Gensini foram calculados para quantificar a obstrução coronariana. RESULTADOS: A média de idade dos pacientes foi 58,1± 9,3 anos, 81% eram hipertensos e 35,5% diabéticos. Os pacientes foram divididos em grupos de acordo com o EA utilizando-se como ponto de corte o valor de 10 Unidades Hounsfield (HU). O P sérico foi maior no grupo de pacientes com EA > 10 HU (3,63 0,55 vs 3,49 0,52mg/dL; p=0,019). Cada 1 mg/dL de elevação no P sérico associou-se a um aumento de 92% no risco de apresentar o EA > 10HU [Odds Ratio (OR) =1,92, CI 1,56-3,19; p=0,01]. Quando os pacientes foram divididos de acordo com a mediana do Escore de Friesinger (4 pontos), o grupo com valores superiores à mediana apresentou P sérico maior (3,6 0,5 vs. 3,5 0,6 mg/dl; p=0,04) e FGF-23 menor (mediana 40,3 pg/mL intervalo interquartil 24,1-62,2 vs. 45,7 pg/mL intervalo interquartil 31,7-76,1; p=0,01) quando comparado àquele com valores menores ou iguais a 4. Pacientes no tercil mais alto do escore de Gensini também apresentaram P sérico mais elevado que os demais (p<0,05). Nas análises de regressão logística uni e multivariadas, cada 1 mg/dL de elevação no P sérico implicou em um aumento de 74% no risco de apresentar o Escore de Friesinger superior à mediana (OR 1,74, CI 1,06- 2,88; p=0,03) e o FGF-23 sérico foi preditor negativo do Escore de Friesinger (OR 0,26, CI 0,11-0,63; p=0,002) Os níveis séricos de cálcio e paratormônio não mostraram associação com a presença de doença coronariana. CONCLUSÃO: Em pacientes com suspeita de doença arterial coronariana e função renal preservada, o fósforo sérico foi preditor da presença de calcificação e obstrução coronariana e houve uma associação negativa entre o FGF-23 sérico e a presença de obstrução coronariana. / INTRODUCTION: Serum phosphorus (P) has been associated with cardiovascular diseases and mortality in chronic kidney disease patients and in the general population. In vitro studies suggest that excessive phosphorus induces vascular calcification and endothelial dysfunction. Fibroblast growth factor 23 (FGF-23) is a phosphaturic hormone and has been correlated to atherosclerosis in the community. AIM: This study intended to investigate the associations between P, FGF-23 and other mineral metabolism players and coronary artery disease in patients with preserved renal function. METHODS: Two-hundred ninety patients with a creatinine clearance higher than 60ml/min/1,73m2 undergoing elective coronary angiography were submitted to Multislice Computed Tomography in order to evaluate coronary calcification and blood was collected for biochemical analyses. Coronary artery calcification was quantified using the Agatston Score (AS). Friesinger (FS) and Gensini Scores (GS) were calcutalet to quantify coronary obstruction. RESULTS: Considering the whole population, mean age was 58.1±9.3 anos, 81% were hypertensive and 35.5% were diabetics. Patients were divided according to AS using the value of 10 Hounsfield Units (HU) as the cutoff.point. Serum phosphorus was higher in patients with an AS > 10HU when compared to the group with an AS 10 HU (3.63 0.55 vs 3.49 0.52mg/dL, p=0.019). Each 1 mg/dL of elevation in the serum phosphorus implied a 92% additional risk of presenting an AS > 10 HU [Odds Ratio (OR) =1.92, CI 1.56-3.19; p=0.01]. Patients were also divided using the median Friesinger score (4 points) as the cutoff value. Serum phosphorus was higher (3.6 0.5 vs. 3.5 0.6 mg/dl, p=0.04) and intact FGF-23 was lower (median 40.3 interquartile range 24.1-62.2 pg/mL vs. 45.7 interquartile range 31.7- 76.1 pg/mL, p=0.01) in the FS > 4 group. Patientis in the higher Gensini Score tertile presented elevated serum phosphorus when compared to the other groups (p<0,05). In the uni and multivariate logistic regression analyses, a rise of 1 mg/dL of serum phosphorus carried a 74% increase in the risk of having a FS higher than 4 (OR 1.74, CI 1.06-2.88; p=0.03) and FGF-23 was a negative predictor of FS (OR 0.26, CI 0.11-0.63; p=0.002). Serum calcium and parathormone were not associated with the presence of coronary artery disease. CONCLUSIONS: In patients with suspected coronary artery disease and preserved renal function, phosphorus was predictive of both coronary artery calcification and obstruction. There was a negative association between FGF-23 and coronary obstruction

Introdução: Doador de rim em vida é uma importante fonte de órgão para os pacientes portadores de doença renal crônica (DRC). Os doadores experimentam uma redução abrupta da taxa de filtração glomerular (TFG) e adaptações ao metabolismo mineral demandam estudos nesta população. Nós avaliamos prospectivamente esta adaptação em doadores de rim em vida. Métodos: Entre janeiro de 2010 a agosto de 2011, no hospital das Clínicas de São Paulo e na Universidade de Miami, realizamos a avaliação prospectiva do metabolismo mineral e da função renal por 1 ano em 74 doadores de rim em vida. Medimos a taxa de filtração glomerular (TFG), fósforo (Pi), cálcio (Ca), paratohormônio (PTH), fibroblast Growth Factor 23 (FGF23) e a fração de excreção do fósforo (FePO4) no pré-operatório e nos dias 1, 2, 14, 180 e 360 do pós-operatório. Resultados: Observamos uma redução, aproximadamente, de 40% da TFG nos dois primeiros dias após a cirurgia. No décimo quarto dia após a nefrectomia, observamos o início da recuperação da TFG, chegando ao máximo da recuperação com 1 ano, quando se atingiu 68,6% da função renal se comparado com o dia anterior a doação (75,3 ml/min/1,73m2, p < 0,001). O cálcio sérico apresentou seu nadir no dia 1 (7,99 mg/dL; p < 0,01) e o Pi sérico atingiu seu nadir no dia 2 (2,61 mg/dL; p < 0,01). Já no dia 14, os valores de Ca e Pi retornaram aos valores basais tendo o fósforo evoluído novamente com valores inferiores ao basal no último dia de seguimento (3,36mg/dL; p < 0,001). FGF23 e PTH apresentaram elevação no D1 (111,0144,6 percentil 25-75: 16-63 RU/ml 64,9 30,3pg/mL; p < 0,01). Os valores de FGF23 se mantiveram elevados até o final do estudo enquanto que o PTH retornou aos valores de base no segundo dia e, a partir de então, manteve sem diferença do valores basais até o último dia de estudo. FePO4 elevou de 11,45,2% para 15,28,1% entre o pré-operatório e D365 (p < 0,01). Conclusão: A nefrectomia para doação de rim em 74 pacientes saudáveis elevou os valores de FGF23 durante todo o estudo juntamente com a FePO4. O fósforo, cálcio e PTH séricos apresentaram queda nos seus valores na primeira semana após a nefrectomia, e, com duas semanas após a cirurgia, retornaram aos valores basais mantendo-se estáveis até o final do estudo / Introduction: Living kidney donors (LKDs) experience an abrupt decline in glomerular filtration rate (GFR). Mineral metabolism adaptations in early CKD are still debated and not well studied in LKDs. We prospectively studied acute and long term mineral metabolism adaptation of LKDs. Materials and Methods: We measured renal function and mineral metabolites longitudinally for 1 year (days (D) 1, 2, 14, 180, & 365 post-operatively) in 74 healthy individuals who underwent kidney live donation. Results: eGFR (MDRD) decreased to 59% of its baseline on day 2 and started to increase at day 3, to its maximum at day 360 (75.3±15.6 ml/min/1.73m2, p < 0.01) wile FGF23 increased from 60.6 (25th-75th percentile 19-81 RU/mL) at baseline to 111.0±144.6 (p < 0.01) on day 1 and keep higher than baseline throwout the study. PTH rose maximally on day 1 (64.9 ± 30.3pg/ml) and returned to its base line on D2 and did not change after that. Total serum Calcium levels decreased from 9,40±0,48 mg/dL to a nadir of 7.99±0,51 mg/dL on day 1 (p < 0.001). Serum Phosphate levels reached their nadir on day 2 (2.61±0,52 mg/dL; p < 0.01). At D14 total calcium and phosphate levels had returned to baseline, but phosphate levels returned down on D360 (3.36±0,52 mg/dL; p < 0.001). Phosphate excretion fraction (FePO4) increased from base line (11.4±5.2%) up to 15.2±8.1% until D360 (p < 0.001). Conclusions: Abrupt reduction in eGFR induces physiological increases in FGF23 and PTH, and decreases in serum Ca and Pi in the first week. The changes in FGF23 and Pi urinary fractional excretion of Pi remain modestly yet significantly different from baseline throughout the first year after nephrectomy. Wile Ca, PTH and Pi serum levels are not significantly different from the baseline

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