Background: Traumatic spinal cord injury (SCI) results in vascular disruption which appears to contribute to the pathobiology of SCI. Vascular endothelial growth factor (VEGF) is known for vascular development and repair, and more recently for its neuroprotective properties. Given this, I investigated the temporal-spatial changes to the spinal vasculature, as well as examined the role of VEGF as a therapeutic approach for SCI. Hypothesis: It is hypothesized that clip-compression injury will result in significant vascular changes, and that ZFP-VEGF gene therapy will enhance molecular and functional recovery following spinal cord injury. Methods: Briefly, female Wistar rats received a two-level laminectomy and a 35g clip-compression injury at T6-T7 for 1 minute. Control animals received a laminectomy only. AdV-ZFP-VEGF or AdV-eGFP was administered 24 hour post-injury by intraspinal injection. For molecular and vascular analysis, tissues were extracted at various time points between 1 hour and 14 days post-SCI. For behavioural experiments animals were studied for 8 consecutive weeks. Results: I have shown that vasculature undergoes structural and functional changes, which occur as early as 1 hour following SCI. Although endogenous improvement is observed, SCI results in permanent vascular damage. Animals receiving AdV-ZFP-VEGF treatment had increased levels of VEGF mRNA and protein. AdV-ZFP-VEGF resulted in neuroprotection, as observed by increased NF200 protein and NeuN counts, and decreased TUNEL staining. Animals treated with AdV-ZFP-VEGF also showed an increased number of newly formed vessels (angiogenesis), as well as an increase in total number of vessels. Moreover, animals treated with AdV-ZFP-VEGF showed significant increases in hindlimb weight support and reduction neuropathic pain. Conclusions: I have characterized the dramatic temporal-spatial changes which occur in the spinal vasculature following SCI. Additionally, I have demonstrated that AdV-ZFP-VEGF administration results in beneficial molecular and functional outcomes. Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and neurobehavioural benefits, by acting through angiogenic and neuroprotective mechanisms.

Background: Traumatic spinal cord injury (SCI) results in vascular disruption which appears to contribute to the pathobiology of SCI. Vascular endothelial growth factor (VEGF) is known for vascular development and repair, and more recently for its neuroprotective properties. Given this, I investigated the temporal-spatial changes to the spinal vasculature, as well as examined the role of VEGF as a therapeutic approach for SCI. Hypothesis: It is hypothesized that clip-compression injury will result in significant vascular changes, and that ZFP-VEGF gene therapy will enhance molecular and functional recovery following spinal cord injury. Methods: Briefly, female Wistar rats received a two-level laminectomy and a 35g clip-compression injury at T6-T7 for 1 minute. Control animals received a laminectomy only. AdV-ZFP-VEGF or AdV-eGFP was administered 24 hour post-injury by intraspinal injection. For molecular and vascular analysis, tissues were extracted at various time points between 1 hour and 14 days post-SCI. For behavioural experiments animals were studied for 8 consecutive weeks. Results: I have shown that vasculature undergoes structural and functional changes, which occur as early as 1 hour following SCI. Although endogenous improvement is observed, SCI results in permanent vascular damage. Animals receiving AdV-ZFP-VEGF treatment had increased levels of VEGF mRNA and protein. AdV-ZFP-VEGF resulted in neuroprotection, as observed by increased NF200 protein and NeuN counts, and decreased TUNEL staining. Animals treated with AdV-ZFP-VEGF also showed an increased number of newly formed vessels (angiogenesis), as well as an increase in total number of vessels. Moreover, animals treated with AdV-ZFP-VEGF showed significant increases in hindlimb weight support and reduction neuropathic pain. Conclusions: I have characterized the dramatic temporal-spatial changes which occur in the spinal vasculature following SCI. Additionally, I have demonstrated that AdV-ZFP-VEGF administration results in beneficial molecular and functional outcomes. Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and neurobehavioural benefits, by acting through angiogenic and neuroprotective mechanisms.

Vascular endothelial growth factor-A (VEGF) is required for endothelial cell differentiation and survival. To investigate the renoprotective properties of VEGF in diabetes an inducible Cre-loxP gene targeting system was used to excise VEGF from podocytes of adult mice (VEGFKO). Diabetes was induced by streptozotocin (STZ) at 2.5 weeks of age and VEGFKO was induced by doxycycline (dox) at 3-4 weeks of age. Blood and urine were collected weekly to monitor for hyperglycaemia and proteinuria, respectively. Mice were dissected 8 weeks after diabetes induction or earlier if morbidly ill; twenty percent of the mice in the DM+VEGFKO group died before the surrogate endpoint. Glomerular VEGF mRNA expression was decreased in VEGFKO mice compared to controls. However, DM+VEGFKO mice had significantly greater proteinuria, degrees of glomerular sclerosis, and glomerular cell apoptosis. These results confirm that VEGF is normally upregulated in diabetes but reducing VEGF expression in diabetes causes severe kidney injury.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 176-207).

Dissertation (M.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 71-84).

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 190-220.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 161-178).

A bioengenharia tecidual tem sido aplicada na Endodontia na busca de tratamentos mais biológicos. A revascularização pulpar tem sido observada em dentes com ápice aberto, porém há poucos estudos direcionados aos dentes com ápice formado. A angiogênese é primordial para a nutrição celular durante a regeneração tecidual. O objetivo deste estudo foi analisar o efeito do fator de crescimento endotelial (VEGF) na angiogênese após pulpectomia de canais radiculares de rato com apicigênese completa. A câmara pulpar dos primeiros molares superiores de 12 ratos machos Wistar com 13 semanas foi acessada. A polpa radicular do canal mésio vestibular foi removida e o canal instrumentado até a lima K25. Com uma lima K20, além do limite foraminal, foi induzido o sangramento periapical para o canal radicular. Os dentes do lado direito da arcada foram preenchidos com coágulo sanguíneo (GS), e nos dentes do lado esquerdo foi utilizado VEGF165 recombinante (Prospec, Israel) adicionado ao coágulo (GV). Os dentes foram selados com ionômero de vidro fotopolimerizável, e 60 dias depois os animais foram sacrificados. A maxila foi dissecada, fixada, descalcificada e incluída em parafina. Cortes transversais seriados foram feitos e corados com hematoxilina-eosina para avaliação morfológica ou incubados com anticorpo de coelho anti-fator VIII (Bioss, EUA) para análise da angiogênese. A análise histológica demonstrou canais preenchidos por tecido conjuntivo sem presença de odontoblastos nos dois grupos. A imuno-histoquímica demonstrou positividade para o anticorpo no tecido neoformado em ambos os grupos. No grupo 1 foi predominante a marcação difusa, e no grupo 2 foi a marcação definida. Pode-se concluir que o uso de VEGF acelera a angiogênese que ocorre em canais pulpectomizados de molares com apicigênese completa. / The tissue engineering has been applied in Endodontics in search of more biological treatments. The pulp revascularization has been observed in immature teeth, but there are few studies directed to the closed-apex teeth. Angiogenesis is essential for cellular nutrition during tissue regeneration. The aim of this study was to analyze the effect of vascular endothelial growth factor (VEGF) in angiogenesis after pulp removal in rats with mature teeth. The pulp chamber of upper first molars of 12 male Wistar rats with 13-weeks-old was accessed. The pulp of the mesiobuccal root canal was removed, and the root canal was shaped up to 25K-file. Then, a 20K-file was introduced beyond the apex to induce bleeding. The teeth on the right side of the arch are filled using blood clot alone (GS) and the teeth on the left side was filled using blood clot + VEGF165 recombinant (Prospec, Israel) (GV). The teeth were coronally sealed using light-curing glass ionomer, and the animals were sacrificed after 60 days. The samples were dissected, fixed, decalcified and included in paraffin. Transverse serial sections were made, and stained using hematoxylin-eosin to morphological evaluation or expressed by immunostaining using anti-Factor VIII rabbit antibody (BIOSs, USA) to angiogenesis analysis. Morphological analysis showed root canals filled by connective tissue without the presence of odontoblasts in both groups. Immunohistochemistry showed positivity for the antibody in the new tissue in both groups, with marking defined in group 2. Diffuse marking was predominant in the group 1, and the well-maked type was predominant in the group 2. It can be concluded that the use of VEGF accelerates angiogenesis that occurs in root canal of closed-apex molars after pulpectomy.

A formação de novos vasos sanguíneos é fundamental para o crescimento tumoral e a disseminação metastática, sendo o fator de crescimento endotelial vascular (VEGF) uma das chaves reguladoras deste processo. O objetivo do presente estudo foi avaliar a expressão imunohistoquímica de VEGF nos hemangiossarcomas e hemangiomas esplênicos, e rever a prevalência das demais afecções esplênicas através da análise retrospectiva do diagnóstico histopatológico de cães submetidos à esplenectomia. Os resultados foram confrontados com os exames laboratoriais, as manifestações clínicas, a presença de arritmias cardíacas e de hemoperitôneo. Participaram do estudo retrospectivo 109 cães atendidos no Serviço de Cirurgia de Pequenos Animais do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, entre os anos de 2002 e 2009. A média de idade foi de 10 anos (± 3), não foi observado predileção sexual. Cães sem raça definida foram os mais acometidos, com peso médio de 22 kg (± 13). Cinqüenta e dois por cento (57/109) dos animais foram esplenectomizados devido a afecções não neoplásicas, enquanto que 48% (52/109), por neoplasias esplênicas. Dentre estes, o diagnóstico mais freqüente foi o hemangiossarcoma, acometendo 28 (54%) animais. Os sintomas mais freqüentes foram disorexia, apatia e emese. Cães com neoplasias malignas apresentaram níveis de hematócrito e hemácias significativamente menores que os acometidos por massas benignas. Do mesmo modo, a presença de hemoperitôneo, secundário à ruptura esplênica, correlacionou-se significativamente com a presença de neoplasia maligna. Arritmias cardíacas não foram fatores preditivos para a diferenciação da esplenomegalia. A avaliação imunohistoquímica da expressão tecidual de VEGF foi realizada em 23 hemangiossarcomas e 7 hemangiomas, revelando-se significativamente maior nas neoplasias malignas. Tal resultado sugere que a expressão deste fator pode estar relacionada à proliferação maligna observada nos hemangiossarcomas. / New blood vessel formation is a fundamental event in the process of tumor growth and metastatic dissemination, being the vascular endothelial growth factor (VEGF) one of the key regulators of this process. The aim of this study was evaluate the VEGF immunohistochemical expression in splenic hemangiosarcomas and hemangiomas, and review the prevalence of canine splenic disorders through retrospective analysis of histological diagnosis after splenectomy. The results were confronted with laboratory findings, clinical signs and presence of cardiac arrhythmia and hemoperitoneum. A hundred nine dogs were included in the retrospective study at Veterinary Hospital of School of Veterinary Medicine, University of Sao Paulo, between 2002 and 2009. The average age was 10 year ± 3, without sexual predilection. Mix breeds were the most frequent, and average weigh was 22kg ± 13. Overall, 52% (57/109) of dogs were splenectomized for nonneoplastic disease, although 48% (52/109) were splenectomized for neoplasia. Among these dogs the most common diagnosis was hemangiossarcoma (28 dogs, 54%). Frequently clinical signs included anorexia, lethargy and vomiting. Dogs with malignant neoplasia had significantly lower red blood cells counts and packed cell volume compared with values for dogs with benign masses. Similarly, hemoperitoneum secondary to splenic rupture had a significant correlation with malignant tumor. Cardiac arrhythmia was not useful in differentiating dogs with splenomegaly. Expression of vascular endothelial growth factor was made by immunohistochemical analyses in 23 hemangiosarcomas and 7 hemangiomas being significantly higher in malignant tumor. These data suggest that VEGF expression may contribute to malignant proliferation of hemangiossarcoma.