INTRODUCCIÓ L’angiogènesi és essencial per al creixement dels tumors sòlids i participa en el procés metastàtic. El VEGF-C, un potent factor angiogènic, es troba elevat en pacients amb càncer colorectal (CCR). Hi ha evidències que suggereixen que la COX-2 és un factor important en el procés de carcinogènesi colorectal. L’objectiu principal de l’estudi és investigar l’efecte que un inhibidor selectiu de la ciclooxigenasa-2, pugui tenir a un model de carcinogènesi colònica experimental sobre la superfície tumoral del còlon, i la seva traducció als nivells sèrics de VEGF-C i a l’expressió de ciclooxigenasa-2 (COX-2). MATERIAL I MÈTODES Es van utilitzar 100 rates Sprague-Dawley no consanguínies distribuïdes en cinc grups: grup control, grup amb CCR induït amb DMH, grup de rates tractades amb celecoxib, grup de rates tractades amb àcid acetilsalicílic i grup de rates tractades amb indometacina. Després del sacrifici, es van prendre mostres de sang per determinar el VEGF-C sèric mitjançant ELISA i mostres dels segments del còlon i d’aquells suggestius de contenir neoplàsia per a estudiar l’expressió de COX-2 als tumors i a la mucosa normal del còlon descendent mitjançant immunohistoquímica. Es va utilitzar la prova estadística més idònia a cada supòsit d’estudi i es va considerar significatiu quan la p£0,05. RESULTATS Vam trobar expressió positiva de COX-2 en el 57,14-66,67% dels tumors de còlon. El 72% de tumors carcinomes mucinosos van presentar un score de tinció de COX-2 positiu, vs. un 48,46% dels adenocarcinomes (p<0,05). Es van trobar diferències significatives entre els grups que van rebre fàrmacs quan es va comparar el nivell sèric de VEGF-C (p=0,008). El grup que va prendre àcid acetilsalicílic presentà uns nivells sèrics menors als del grup que va rebre celecoxib (63,67 vs. 92,58 pg/ml) (p=0,003). No es van apreciar diferències significatives entre el grup que va rebre celecoxib i el grup control amb inducció tumoral (92,58 vs. 89,89 pg/ml; p=0,698). No es van trobar diferències estadísticament significatives entre els valors de VEGF-C sèric i l’expressió tumoral de COX-2 a cap dels grups d’estudi (p>0,05). Es van trobar diferències significatives entre el valor sèric mig de VEGF-C i l’extensió de l’expressió de COX-2 en el tumor (p=0,035). També es va trobar correlació estadísticament significativa entre el VEGF-C sèric i la suma de superfície tumoral (p=0,045). Es van trobar diferències estadísticament significatives en el VEGF-C sèric segons els animals presentessin o no metàstasis a distància o hematògenes (p=0,015). CONCLUSIONS La major part de tumors de còlon van expressar COX-2. L’expressió de COX-2 es va associar al tipus de tumor, i s’incrementa d’acord amb l’augment de la malignitat de la histologia del tumor. El VEGF-C sèric es relaciona amb la progressió de la malaltia. Valors elevats es van correlacionar amb superfícies tumorals altes de manera estadísticament significativa. Celecoxib no va reduir de manera significativa l’expressió de COX-2 als tumors respecte dels grups tractats amb fàrmacs no selectius, ni va disminuir de manera significativa els valors sèrics de VEGF-C, ni la suma de superfície tumoral en el còlon. L’àcid acetilsalicílic indueix una disminució en els nivells sèrics de VEGF-C, propers als nivells control, però no van existir canvis en l’expressió tumoral de COX-2, ni en la suma de superfície tumoral en comparar aquest grup amb els altres. / INTRODUCCIÓN La angiogénesis es esencial para el crecimiento de los tumores sólidos y participa en el proceso metastático. El VEGF-C, un potente factor angiogénico, se encuentra elevado en pacientes con cáncer colorrectal (CCR).Hay muchas evidencias que sugieren que la COX-2 es un factor importante en el proceso de carcinogénesis colorrectal. El objetivo principal del estudio es investigar el efecto que un inhibidor selectivo de la ciclooxigenasa-2, comparándolo con otros no selectivos, pueda tener, en un modelo de carcinógesis colónica experimental, sobre la superficie tumoral del colon y su traducción en los niveles séricos de VEGF-C y en la expresión de ciclooxigenasa-2 (COX-2). MATERIAL Y MÉTODOS Se utilizaron 100 ratas Sprague-Dawley no consanguíneas distribuidas en cinco grupos: grupo control, grupo con CCR inducido con DMH, grupo de ratas tratadas con celecoxib, grupo de ratas tratadas con ácido acetilsalicílico y grupo de ratas tratadas con indometacina. Tras el sacrificio, se tomaron muestras de sangre para determinar el VEGF-C sérico mediante ELISA y muestras de los segmentos del colon y de aquellos sugestivos de contener neoplasia para estudiar la expresión de COX-2 en los tumores y en la mucosa normal del colon descendente mediante inmunohistoquímica. Se utilizó la prueba estadística más idónea en cada supuesto de estudio y se consideró significativo cuando la p£ 0,05. RESULTADOS Encontramos expresión positiva de COX-2 en el 57,14 - 66,67% de los tumores de colon. El 72% de tumores carcinomas mucinosos presentaron un score de tinción de COX-2 positivo, vs. un 48,46% de los adenocarcinomas (p<0,05). Se encontraron diferencias significativas entre los grupos que recibieron fármacos cuando se comparó el valor sérico de VEGF-C (p=0,008). El grupo que tomó ácido acetilsalicílico presentó unos niveles séricos menores a los del grupo que recibió celecoxib (63,67 vs. 92,58 pg/ml) (p=0,003). No se apreciaron diferencias significativas entre el grupo que recibió celecoxib y el grupo control con inducción tumoral (92,58 vs. 89,89 pg/ml.) (p=0,698). No se encontraron diferencias estadísticamente significativas entre los valores de VEGF-C sérico y la expresión tumoral de COX-2 en ninguno de los grupos de estudio (p>0,05). Se encontraron diferencias significativas entre el valor sérico medio de VEGF-C y la extensión de la expresión de COX-2 en el tumor (p=0,035). También se halló correlación estadísticamente significativa entre el valor circulante de VEGF-C sérico y la suma de superficie tumoral (p=0,045). Se encontraron diferencias estadísticamente significativas en el valor sérico de VEGF-C según los animales presentasen o no metástasis a distancia o hematógenas (p=0,015). CONCLUSIONES La mayoría de tumores de colon inducidos experimentalmente en la rata expresaron COX-2. La expresión de COX-2 en el CCR se asocia de forma significativa al tipo de tumor, incrementándose conforme aumentaba la malignidad de la histología del tumor. El VEGF-C sérico se relaciona con la progresión de la enfermedad. Valores elevados se correlacionaron con sumas de superficies tumorales elevadas de un modo estadísticamente significativo. Celecoxib no redujo de un modo significativo la expresión de COX-2 en los tumores respecto de los grupos tratados con fármacos no selectivos, ni disminuyó de forma significativa los valores séricos de VEGF-C, ni la suma de superficie tumoral en el colon. El ácido acetilsalicílico induce una disminución en los niveles séricos circulantes de VEGF-C cercanos a los niveles control. Sin embargo, no existieron cambios en la expresión de COX-2 en los tumores, ni en la suma de superficie tumoral al comparar este grupo con los demás. / INTRODUCTION Angiogenesis is essential for the growth of solid tumours and takes part in the metastatic process. VEGF-C, a strong angiogenic factor, is increased in patients with colorectal cancer (CRC). There are evidences suggesting that COX-2 is an important factor in the colorectal carcinogenesis process. The main aim of the study is to investigate the effect that a selective COX-2 inhibitor, comparing it with non-selective ones, could have on experimental colon carcinogenesis, on the colorectal tumour area, and its results in the serum VEGF-C levels and in the cyclooxigenase-2 (COX-2) expression. MATERIAL AND METHODS 100 Sprague-Dawley rats without consanguinity were used. They were divided into five groups: control group, group with DMH induced CRC, group that was treated with celecoxib, group that was treated with aspirin and group that was treated with indomethacin. Blood samples were taken after slaughter of rats, in order to determine serum VEGF-C with ELISA technique. Colon segments samples and specimens from the areas that were suggested to contain neoplasms were taken too, in order to study COX-2 expression in colon tumors and in the normal mucosa with immunohistochemistry technique. The most appropriate statistical test was used in each case of the study and it was considered significant when p£ 0.05. RESULTS We found positive COX-2 expression in 57.14-66.67% of colon tumours. 72% of mucinous carcinomas presented a positive score for COX-2 expression, vs. 48.46% of adenocarcinomas (p<0.05). We found significant differences between groups that received drugs when we compared the serum VEGF-C level (p=0.008). The AAS group presented lower serum VEGF-C levels than the celecoxib group (63.67 vs. 92.58 pg/ml) (p=0.003). We didn’t appreciate significant differences between the celecoxib group and the control group with tumor induction (92.58 vs. 89.89 pg/ml) (p=0.698). There were no statistically significant differences between the serum VEGF-C levels and tumor expression of COX-2 in any of the groups of the study (p>0.05). We found significant differences between the serum VEGF-C level and the extension of COX-2 expression in the tumor (p=0.035). We also found statistically significant correlation between the value of serum VEGF-C and the sum of tumor area (p = 0.045). Statistically significant differences were found in the serum VEGF-C level between animals having or not hematogenous metastasis (p=0.015). CONCLUSIONS Most experimentally induced colon tumors in rats expressed COX-2. The expression of COX-2 in CRC is significantly associated with the type of tumor, and increases according to the increases of malignancy of the tumor histology. Serum VEGF-C is related to disease progression. High values were correlated with high sum of tumor surfaces in a statistically significant manner. Celecoxib didn’t reduce significantly the expression of COX-2 in tumors regarding groups treated with non-selective drugs, nor significantly decreased serum VEGF-C, or the sum of tumor area in the colon. Aspirin decreases serum VEGF-C levels which were similar to control levels. However, there were no changes in the expression of COX-2 in tumors, or the sum of tumor area by comparing this group with other treated groups.

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.

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).