VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.

VEGF is a potent angiogenic and endothelial cell growth factor that is key for the development of the glomerulus, the main filtration unit of the kidney. It is continued to be expressed in the mature glomerulus, with podocytes being the major site of production. VEGF binds to two receptors, VEGFR-1 and VEGFR-2, which are expressed by the adjacent endothelial cells (ECs). VEGFR-2 is the primary mediator of VEGF signaling while VEGFR-1is thought to function as a ‘decoy’ receptor, sequestering VEGF away from VEGFR-2. Gene targeting studies in mice show that VEGF loss from the podocyte results in profound defects of the ECs, consistent with a paracrine signaling loop. However, the identification of VEGF receptors on podocytes in vitro suggests an additional autocrine signaling pathway for VEGF may exist. To further study the role of VEGF in the glomerulus and to address whether a VEGF autocrine loop is functional in vivo, we generated a transgenic mouse model with inducible VEGF upregulation in the podocyte and genetically deleted VEGFR-2 and VEGFR-1 from the podocyte using the Cre-loxP system. Increased VEGF production from the podocyte leads to increased glomerular permeability and ultrastructural changes in the glomerular filtration barrier depending on the time and length of induction. Podocyte-selective deletion of VEGFR-2 did not cause glomerular disease. In contrast, VEGFR-1 loss from the podocyte led to proteinuria and glomerular defects at 6 weeks of age with extensive podocyte foot process effacement. In keeping with the model that VEGFR-1 functions as a VEGF trap, similarities were observed between the glomerular lesions of VEGFR-1 mutant mice and transgenic mice that overexpress VEGF within podocytes. Strikingly, in vitro studies also revealed an increase in podocyte cell adhesion to sVEGFR-1, suggesting additional roles for sVEGFR-1. Together, these data suggest that a tight regulation of VEGF must be maintained in the adult glomerulus. Furthermore, these findings provide the first genetic evidence that VEGF autocrine signaling loop through VEGFR-2 is dispensable in normal glomeruli. In addition, podocytes express sVEGFR-1 and is required in podocytes in vivo to maintain glomerular integrity by regulating VEGF availability and podocyte cell adhesive properties.

La polyarthrite rhumatoïde (PR) est le rhumatisme inflammatoire le plus fréquent. Cette maladie s’accompagne d’une hyperplasie de la membrane synoviale qui entoure les articulations. La formation du pannus synovial est sous la dépendance de cytokines pro-inflammatoires et pro-angiogéniques. Les immunothérapies anti-TNF utilisées dans le traitement de la PR présentent des inconvénients (perte d’efficacité, risque infectieux), ce qui laisse la place pour le développement d’une stratégie vaccinale anti-TNF. Dans la première partie de mes travaux, nous avons développé des vaccins anti-VEGF afin d’étudier les liens entre angiogenèse et inflammation dans l’arthrite expérimentale au collagène (AEC). Dans la deuxième partie, nous avons développé un vaccin anti-TNF de souris afin d’évaluer les conséquences de la neutralisation du TNF-α par la vaccination dans des modèles infectieux. Le ciblage du VEGF avec un vaccin constitué de VEGF entier ou de peptides du VEGF couplés à la KLH, a permis une protection clinique et histologique dans l’AEC. Dans notre deuxième axe de recherche, nous avons développé un vaccin anti-TNF de souris (TNF-KLH). Ce vaccin est aussi efficace que l’etanercept dans l’AEC, mais n’augmente pas le risque infectieux dans un modèle d’infection à Mycobacterium tuberculosis. Dans le modèle d’infection à Listeria monocytogenes, TNF-KLH n’augmente pas la charge bactérienne et n’induit pas de mortalité, contrairement à l’etanercept. Mes travaux de thèse montrent que la stratégie vaccinale anti-cytokine est efficace dans l’arthrite, et que le ciblage du TNF par une telle stratégie ne semble pas altérer la réponse anti-infectieuse dans nos modèles. / Rheumatoid arthritis (RA) is the most frequent inflammatory rheumatism. This disease is accompanied by hyperplasia of the synovial membrane surrounding the joint. Pannus formation is controlled by pro-inflammatory and pro-angiogenic cytokines. Anti-TNF immunotherapies used in the treatment of RA presents many drawbacks (loss of efficacy, infections), which leaves some place for the development of an anti-TNF immunization strategy. In the first part of my work, we developed an anti-VEGF vaccine to study the links between angiogenesis and inflammation in collagen-induced arthritis (CIA) model. In the second part, we developed a mouse anti-TNF vaccine to assess the consequences of the neutralization of TNF-α by vaccination in infectious models. Inhibition of VEGF with a vaccine consisting of whole VEGF or VEGF peptide coupled to KLH, showed a clinical and histological protection in the CIA model. In the second part of my work, we developed a mouse anti-TNF vaccine (TNF-KLH). This vaccine is as effective as etanercept in CIA, but does not increase the risk of infection in Mycobacterium tuberculosis model. In Listeria monocytogenes model, unlike etanercept, immunization with TNF-KLH does not increase the bacterial burden and mortality. My work contributed to the development of active anti-VEGF vaccine and our results show a partial protection with this strategy. Also, we demonstrate that targeting TNF by active immunotherapy does not alter the immune response in our models of infections.

O objetivo deste trabalho foi avaliar a expressão de fatores de crescimento responsáveis pelo processo vasculogênico durante o desenvolvimento e involução do timo em cães. Para este propósito, timos de fetos de cães (30, 40 50 e 60 dias de desenvolvimento), cães jovens (6 meses de idade) e cães adultos (1 ano de idade) foram coletados. Os tecidos foram analisados estereológicamente e submetidos à imunohistoquímica, PCR tempo real e western blot para os componentes do sistema VEGF (VEGF-A, VEGF-C, Flt-1, KDR e Flt-4) e endocrine gland-derived (EG)-VEGF. Pela estereologia, observamos um aumento no número dos vasos durante o desenvolvimento do órgão e uma diminuição durante a involução (p<0.05). Todos os componentes do sistema VEGF e do EG-VEGF foram detectados em todos os estágios durante o desenvolvimento e involução. As proteínas correspondentes foram localizadas nas células endoteliais e células epiteliais da região cortical e medular, sendo que o EG-VEGF foi observado apenas nas células epiteliais da região medular. A expressão do RNAm do sistema VEGF e do EG-VEGF apresentou um perfil tempo-dependente durante o desenvolvimento e involução. O sistema VEGF apresentou uma expressão constante durante o desenvolvimento e um aumento progressivo durante a involução, com exceção do KDR que se mostrou crescente durante todo o período de desenvolvimento e involução e do VEGF-C que apresentou alta expressão durante a involução. A expressão do RNAm do EG-VEGF foi crescente durante as fases estudadas, já a expressão da sua proteína foi alta no início do desenvolvimento do timo e diminui na involução, onde permanece praticamente constante durante todo o período. Nossos resultados sugerem que o sistema VEGF e o EG-VEGF possuem atividades biológicas similares nas glândulas endócrinas e diferentes papéis durante o desenvolvimento e involução, o qual pode ser especificado em estudos futuros. Possíveis funções incluem um efeito modulatório na vasculogênese tímica e microambiente, influenciando na diferenciação e proliferação de timócitos, maturação de células T, interação célula-célula e secreção de hormônios tímicos. / The aim this study was therefore to evaluate the expression of growth factors responsible for the vasculogenic process throughout development and initial involution of the canine thymus. For that purpose, thymuses from fetuses (30, 40, 50 and 60 days), juvenile (6 months) and adult (1 year) dogs were collected. The tissues were examined stereologically and subjected to immunohistochemistry, Real Time PCR and western blot for components of the VEGF-system (VEGF-A, VEGF-C, Flt-1, KDR and Flt-4) and endocrine gland-derived (EG)-VEGF. By means of stereology, the total number of blood vessels increased during development and decreased during involution. These changes were statistically significant (p<0.05). All components of the VEGF-system and EG-VEGF were detected in all stages during development and involution. The corresponding proteins were localized in endothelial and epithelial cells of cortical and medullar regions, and EG-VEGF was detected only in epithelial cells of medular region. The VEGF-system and EG-VEGF protein and mRNA expression showed a specific time-dependent profile during development and involution. The VEGF system showed a constant expression during development and a progressive increase during involution, except KDR that showed increased during all development and involution and VEGF-C that showed high expression during involution. The EG-VEGF RNAm expression was increased during development and involution, but protein expression was high in early thymus development and decreased in involution, and remains constant during all period. Our results suggest that VEGF-system and EG-VEGF have similar biological activities in endocrine glands and different roles during thymus development and involution which have to be specified in further studies. Possible functions include a modulatory effect on thymic vasculogenesis and microenvironment, influencing thymocyte proliferation and differentiation, T-cell maturation, cell-cell interaction and thymic hormonal secretion.

archives@tulane.edu / Vascular endothelial growth factor A (VEGFA) is a key contributor to the formation of new blood vessels and angiogenesis is commonly seen in wound healing, cancer, and inflammatory diseases. However, whether interleukin-17 (IL-17) can induce the expression of VEGFA in prostate cancer cells remains unknown. In this study, Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis validated that expression of VEGFA in human prostate cancer LNCaP cells under IL-17 treatment was significantly higher than the untreated control group. The conditioned culture medium (CM) of LNCaP cells treated with IL-17 increased tube number, tube nodes, and tube length formed by human umbilical vein endothelial cells (HUVEC) in tube formation assays, compared with the control CM without IL-17 treatment. Collectively, these findings reveal that the expression of VEGFA is induced by IL-17 in LNCaP prostate cancer cells, which leads to increased angiogenesis of HUVEC cells. This study suggests that expression of VEGFA may be up-regulated by IL-17 in prostate cancer to enhance tumor angiogenesis. / 1 / Benyu Li

Stroke is the leading cause of long term disability and the third leading cause of death in the United States. Perlecan plays a significant role in brain development by sequestering and delivering growth factors to developing neuronal precursor cells in a neurovascular niche. Previous results demonstrated that perlecan proteolysis results in the cleavage of perlecan’s most C-terminal domain five (DV) in the post-ischemic brain. As post-stroke angiogenesis is an important step in post-stroke brain repair, I focused on the mechanism of DV’s role in brain angiogenesis in vitro. I first demonstrated that DV significantly increased brain endothelial (BE) cell migration, proliferation and tube-like formation suggesting DV is a pro-angiogenic factor for BE cells. I next investigated VEGF secretion from BE cells in the presence of DV. DV significantly increased VEGF secretion into the cell media, which was both dose and time dependent. Using quantitative real-time PCR, DV induced a maximal nine-fold increase in VEGF expression, compared to control, indicating DV is an upstream regulator of VEGF transcription. DV treated cells show an increase in phosphorylation of ERK-(1/2) that could be blocked by the pharmacological inhibitor U0126. This inhibitor could also block DV’s effect on VEGF mRNA expression and secretion indicating ERK is involved with DV’s effect on VEGF regulation. Optical sensor binding assays confirmed that DV binds to the α5β1 integrin with a Kd of 160nM, and cells treated with DV showed a visual representation of integrin α5β1-DV colocalization. Furthermore, shRNA-mediated knockdown of integrin α5 blocked DV’s effect on VEGF mRNA expression, indicating integrin α5 is involved with DV’s regulation of VEGF expression. In conclusion, these results demonstrate that DV has an unexpected proangiogenic effect in brain angiogenesis. This occurs via a previously unreported interaction between DV and the α5β1 integrin, resulting in the activation of the ERK, eIF4A and HIF1α signaling pathway and an ultimate increase in VEGF mRNA expression and VEGF secretion. As DV is generated post-stroke, these results suggest a novel mechanism by which brain tissue recovery following ischemia is influenced by processed fragments from the extracellular matrix.

Whether vascular endothelial growth factor A (VEGF) plays an augmented role during pregnancy is unknown. In this thesis expression of VEGF in the placenta, maternal circulation and organs in mice was examined using qRT-PCR, LacZ expression, and/or ELISAs. Normal pregnancies and pregnancies with transgenic conceptuses that over-express VEGF in the placenta were examined. In normal pregnancies, VEGF120/164 levels in the ovary increased in parallel with that of the maternal circulation. In pregnancies where the placenta over-expressed VEGF, maternal circulating VEGF120/164 levels decreased and so did levels in the maternal ovary. Surprisingly, VEGF protein levels (per mg of total protein) decreased in the growing, highly vascular placenta during pregnancy. In conclusion this thesis provides evidence for an important ovarian source of maternal circulating VEGF120/164 during pregnancy.

Whether vascular endothelial growth factor A (VEGF) plays an augmented role during pregnancy is unknown. In this thesis expression of VEGF in the placenta, maternal circulation and organs in mice was examined using qRT-PCR, LacZ expression, and/or ELISAs. Normal pregnancies and pregnancies with transgenic conceptuses that over-express VEGF in the placenta were examined. In normal pregnancies, VEGF120/164 levels in the ovary increased in parallel with that of the maternal circulation. In pregnancies where the placenta over-expressed VEGF, maternal circulating VEGF120/164 levels decreased and so did levels in the maternal ovary. Surprisingly, VEGF protein levels (per mg of total protein) decreased in the growing, highly vascular placenta during pregnancy. In conclusion this thesis provides evidence for an important ovarian source of maternal circulating VEGF120/164 during pregnancy.

Acute lymphoblastic leukaemia (ALL) is the most common cause from death of disease in children. Whilst cure rates over the last 30 years have drastically improved, the children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments. The role that vascular endothelial growth factor (VEGF), an integral component of both neovascularisation and normal haematopoiesis, plays in the progression and invasiveness of solid tumours is well established. However, its function in haematological malignancies has been a more recent and thus less considered observation. Human leukaemia cells secrete VEGF, which may act in a paracrine manner with the bone marrow microenvironment to promote the survival and proliferation of leukaemia cells. In addition to VEGF being produced by leukaemias, it also increases vascularity in the bone marrow and lymph nodes of patients. Our previous work has established a panel of 10 childhood acute lymphoblastic leukaemia xenografts from patient biopsies in NOD/SCID mice. Several of these secrete VEGF, and express the FMS-like tyrosine kinase-3 (FLT-3). FLT 3, a receptor tyrosine kinase (RTK), and its ligand, play an essential role in regulating normal haematopoiesis. This thesis builds on the previous work by examining the relationship between VEGF and FLT 3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients. The results show that the high expression and activation of FLT 3, significantly increases the secretion VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of a class III receptor tyrosine kinase (RTK) inhibitor (SU11657), humanised anti-FLT 3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT 3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous gene expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FL on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells. Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional chemotherapy are warranted.

O objetivo deste trabalho foi avaliar a expressão de fatores de crescimento responsáveis pelo processo vasculogênico durante o desenvolvimento e involução do timo em cães. Para este propósito, timos de fetos de cães (30, 40 50 e 60 dias de desenvolvimento), cães jovens (6 meses de idade) e cães adultos (1 ano de idade) foram coletados. Os tecidos foram analisados estereológicamente e submetidos à imunohistoquímica, PCR tempo real e western blot para os componentes do sistema VEGF (VEGF-A, VEGF-C, Flt-1, KDR e Flt-4) e endocrine gland-derived (EG)-VEGF. Pela estereologia, observamos um aumento no número dos vasos durante o desenvolvimento do órgão e uma diminuição durante a involução (p<0.05). Todos os componentes do sistema VEGF e do EG-VEGF foram detectados em todos os estágios durante o desenvolvimento e involução. As proteínas correspondentes foram localizadas nas células endoteliais e células epiteliais da região cortical e medular, sendo que o EG-VEGF foi observado apenas nas células epiteliais da região medular. A expressão do RNAm do sistema VEGF e do EG-VEGF apresentou um perfil tempo-dependente durante o desenvolvimento e involução. O sistema VEGF apresentou uma expressão constante durante o desenvolvimento e um aumento progressivo durante a involução, com exceção do KDR que se mostrou crescente durante todo o período de desenvolvimento e involução e do VEGF-C que apresentou alta expressão durante a involução. A expressão do RNAm do EG-VEGF foi crescente durante as fases estudadas, já a expressão da sua proteína foi alta no início do desenvolvimento do timo e diminui na involução, onde permanece praticamente constante durante todo o período. Nossos resultados sugerem que o sistema VEGF e o EG-VEGF possuem atividades biológicas similares nas glândulas endócrinas e diferentes papéis durante o desenvolvimento e involução, o qual pode ser especificado em estudos futuros. Possíveis funções incluem um efeito modulatório na vasculogênese tímica e microambiente, influenciando na diferenciação e proliferação de timócitos, maturação de células T, interação célula-célula e secreção de hormônios tímicos. / The aim this study was therefore to evaluate the expression of growth factors responsible for the vasculogenic process throughout development and initial involution of the canine thymus. For that purpose, thymuses from fetuses (30, 40, 50 and 60 days), juvenile (6 months) and adult (1 year) dogs were collected. The tissues were examined stereologically and subjected to immunohistochemistry, Real Time PCR and western blot for components of the VEGF-system (VEGF-A, VEGF-C, Flt-1, KDR and Flt-4) and endocrine gland-derived (EG)-VEGF. By means of stereology, the total number of blood vessels increased during development and decreased during involution. These changes were statistically significant (p<0.05). All components of the VEGF-system and EG-VEGF were detected in all stages during development and involution. The corresponding proteins were localized in endothelial and epithelial cells of cortical and medullar regions, and EG-VEGF was detected only in epithelial cells of medular region. The VEGF-system and EG-VEGF protein and mRNA expression showed a specific time-dependent profile during development and involution. The VEGF system showed a constant expression during development and a progressive increase during involution, except KDR that showed increased during all development and involution and VEGF-C that showed high expression during involution. The EG-VEGF RNAm expression was increased during development and involution, but protein expression was high in early thymus development and decreased in involution, and remains constant during all period. Our results suggest that VEGF-system and EG-VEGF have similar biological activities in endocrine glands and different roles during thymus development and involution which have to be specified in further studies. Possible functions include a modulatory effect on thymic vasculogenesis and microenvironment, influencing thymocyte proliferation and differentiation, T-cell maturation, cell-cell interaction and thymic hormonal secretion.