A angiogênese é um processo fundamental e fisiológico de organismos vertebrados, sendo responsável pela formação de novos vasos sanguíneos a partir dos já existentes. Entretanto, a angiogênese pode ocorrer também em condições patológicas, como causa ou consequência de doenças. Um exemplo disso está nos tumores, que para crescer além de alguns milímetros cúbicos, necessitam de um suprimento adequado de oxigênio e nutrientes, e, portanto, dependem da angiogênese. Por isso, compostos que inibem a angiogênese já estão em uso na clínica, não só para o tratamento de tumores, mas também de outras doenças dependentes da angiogênese, as retinopatias. Neste projeto, daremos continuidade à linha de pesquisa do nosso grupo, que procura identificar e validar peptídeos com potencial translacional (pré-fármacos), por apresentarem atividade anti-angiogênica. Utilizando a metodologia do Phage Display, nosso grupo identificou e caracterizou um hexapeptídeo, que foi selecionado por interagir com os receptores do principal fator iniciador da angiogênese, o VEGF (fator de crescimento endotelial vascular). Os receptores de VEGF (ou VEGFR) são proteínas do tipo receptor tirosina quinase, expressos em células endoteliais e essenciais para a iniciação e progressão da neovascularização. O hexapeptídeo identificado em nosso laboratório liga-se ao VEGFRs e inibe a formação de vasos sanguíneos in vivo em modelos animais de angiogênese. Neste trabalho, procuramos estender os estudos com este hexapeptídeo para identificar o sítio de ligação do mesmo no VEGFR e avançar em modelos que permitam a determinação dos requisitos estruturais de interação peptídeoreceptor. Com estes conhecimentos, poderemos num futuro próximo, caminhar para o desenvolvimento racional de moléculas peptideomiméticas com propriedades anti-angiogênicas. / Angiogenesis is a fundamental and physiological process for vertebrate organisms, being responsible for the formation of new blood vessels, sprouting from the existent ones. However, angiogenesis may occur in pathological conditions, being cause or consequence of diseases. One example is tumor development. To grow beyond a few cubic millimeters, tumors need a suitable supply of oxygen and nutrients, and, therefore, they are dependent of angiogenesis. In fact, anti-angiogenic compounds are already in therapeutic use, targeting not only tumors but other angiogenesis dependent diseases, like retinopathies. In this project, we expand research from our own group to identify and develop anti-angiogenic peptides with translational potential (pre-drugs). Using Phage Display methodology, our group identified and characterized a hexapeptide, that was selected based on its capacity to interact with the receptors for the main initiator factor of angiogenesis, the VEGF (Vascular Endothelial Growth Factor). VEGF receptors (VEGFR) are tyrosine kinase proteins, expressed by endothelial cells and essential for neovascularization initiation and progress. The hexapeptide identified in our lab binds to VEGFRs and inhibit blood vessel formation in vivo when tested in an angiogenesis animal model. In this study, we seek to further understand the interaction of this hexapeptide with its receptor by identifying its binding domain on VEGFR and develop models that will allow the determination of the structural requirements for interaction of this receptor ligand pair. With this knowledge, we can in a near future progress to a rational development of novel peptidemimetic molecules with angiogenic properties similar to this hexapeptide.

A angiogênese é um processo fundamental e fisiológico de organismos vertebrados, sendo responsável pela formação de novos vasos sanguíneos a partir dos já existentes. Entretanto, a angiogênese pode ocorrer também em condições patológicas, como causa ou consequência de doenças. Um exemplo disso está nos tumores, que para crescer além de alguns milímetros cúbicos, necessitam de um suprimento adequado de oxigênio e nutrientes, e, portanto, dependem da angiogênese. Por isso, compostos que inibem a angiogênese já estão em uso na clínica, não só para o tratamento de tumores, mas também de outras doenças dependentes da angiogênese, as retinopatias. Neste projeto, daremos continuidade à linha de pesquisa do nosso grupo, que procura identificar e validar peptídeos com potencial translacional (pré-fármacos), por apresentarem atividade anti-angiogênica. Utilizando a metodologia do Phage Display, nosso grupo identificou e caracterizou um hexapeptídeo, que foi selecionado por interagir com os receptores do principal fator iniciador da angiogênese, o VEGF (fator de crescimento endotelial vascular). Os receptores de VEGF (ou VEGFR) são proteínas do tipo receptor tirosina quinase, expressos em células endoteliais e essenciais para a iniciação e progressão da neovascularização. O hexapeptídeo identificado em nosso laboratório liga-se ao VEGFRs e inibe a formação de vasos sanguíneos in vivo em modelos animais de angiogênese. Neste trabalho, procuramos estender os estudos com este hexapeptídeo para identificar o sítio de ligação do mesmo no VEGFR e avançar em modelos que permitam a determinação dos requisitos estruturais de interação peptídeoreceptor. Com estes conhecimentos, poderemos num futuro próximo, caminhar para o desenvolvimento racional de moléculas peptideomiméticas com propriedades anti-angiogênicas. / Angiogenesis is a fundamental and physiological process for vertebrate organisms, being responsible for the formation of new blood vessels, sprouting from the existent ones. However, angiogenesis may occur in pathological conditions, being cause or consequence of diseases. One example is tumor development. To grow beyond a few cubic millimeters, tumors need a suitable supply of oxygen and nutrients, and, therefore, they are dependent of angiogenesis. In fact, anti-angiogenic compounds are already in therapeutic use, targeting not only tumors but other angiogenesis dependent diseases, like retinopathies. In this project, we expand research from our own group to identify and develop anti-angiogenic peptides with translational potential (pre-drugs). Using Phage Display methodology, our group identified and characterized a hexapeptide, that was selected based on its capacity to interact with the receptors for the main initiator factor of angiogenesis, the VEGF (Vascular Endothelial Growth Factor). VEGF receptors (VEGFR) are tyrosine kinase proteins, expressed by endothelial cells and essential for neovascularization initiation and progress. The hexapeptide identified in our lab binds to VEGFRs and inhibit blood vessel formation in vivo when tested in an angiogenesis animal model. In this study, we seek to further understand the interaction of this hexapeptide with its receptor by identifying its binding domain on VEGFR and develop models that will allow the determination of the structural requirements for interaction of this receptor ligand pair. With this knowledge, we can in a near future progress to a rational development of novel peptidemimetic molecules with angiogenic properties similar to this hexapeptide.

Em roedores, o aumento da permeabilidade vascular, a transformação decidual, e angiogênese são eventos cruciais para o sucesso da gestação. O fator endotelial vascular (VEGF) é um mitogênico para células endoteliais e um indutor de angiogênese. O VEGF age via dois receptores da família das tirosina quinases: VEGFR1 e VEGFR2. O objetivo deste estudo foi investigar usando o método imunohistoquímico, a expressão espacial e temporal do VEGF e os receptores VEGFR1 e VEGFR2 em células endometriais de camundongo entre o 4º e 8º dias de gestação. No 4º dia de gestação, VEGF, VEGFR1 e VEGFR2 foram expressos pelo epitélio luminal e glandular e fracamente pelo estroma endometrial. Do 5º ao 8º dias de gestação, o VEGF foi expresso nas células deciduais mesometriais. VEGFR1 e VEGFR2 foram expresso pelas células do epitélio luminal e glandular e mostraram uma marcação diferencial na decídua mesometrial e antimesometrial. Os receptores VEGFR1 e VEGFR2 foram intensamente expressos pelas células endoteliais dos capilares sinusóides mesometriais e pelas células Nk uterinas. / In rodents, increase of vascular permeability, decidual cell transformation, and uterine angiogenesis are crucial events for the success of pregnancy. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and an inducer of angiogenesis. VEGF acts via two tyrosine kinase family receptors: VEGFR1 and VEGFR2. The aim of this study was to investigate using the immunohistochemical method, the spatiotemporal expression of VEGF and its receptors VEGFR1 e VEGFR2 by mouse endometrial cells on days 4 to 8 of pregnancy. On day 4, VEGF, VEGFR1 and VEGFR2 were expressed mostly by the luminal and glandular epithelium. Stromal cells showed a very weak labeling. On days 5-8, VEGF and its receptors showed an increased labeling throughout the mesometrial decidua. The expression of VEGF, VEGFR1, and VEGFR2 were differentially expressed in the mesometrial cells and in the predecidual cells of the antimesometrial decidua. VEGFR1 and VEGF R2 were highly expressed by endothelial cells of the mesometrial sinusoids, and Nk uterine cells.

Em roedores, o aumento da permeabilidade vascular, a transformação decidual, e angiogênese são eventos cruciais para o sucesso da gestação. O fator endotelial vascular (VEGF) é um mitogênico para células endoteliais e um indutor de angiogênese. O VEGF age via dois receptores da família das tirosina quinases: VEGFR1 e VEGFR2. O objetivo deste estudo foi investigar usando o método imunohistoquímico, a expressão espacial e temporal do VEGF e os receptores VEGFR1 e VEGFR2 em células endometriais de camundongo entre o 4º e 8º dias de gestação. No 4º dia de gestação, VEGF, VEGFR1 e VEGFR2 foram expressos pelo epitélio luminal e glandular e fracamente pelo estroma endometrial. Do 5º ao 8º dias de gestação, o VEGF foi expresso nas células deciduais mesometriais. VEGFR1 e VEGFR2 foram expresso pelas células do epitélio luminal e glandular e mostraram uma marcação diferencial na decídua mesometrial e antimesometrial. Os receptores VEGFR1 e VEGFR2 foram intensamente expressos pelas células endoteliais dos capilares sinusóides mesometriais e pelas células Nk uterinas. / In rodents, increase of vascular permeability, decidual cell transformation, and uterine angiogenesis are crucial events for the success of pregnancy. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and an inducer of angiogenesis. VEGF acts via two tyrosine kinase family receptors: VEGFR1 and VEGFR2. The aim of this study was to investigate using the immunohistochemical method, the spatiotemporal expression of VEGF and its receptors VEGFR1 e VEGFR2 by mouse endometrial cells on days 4 to 8 of pregnancy. On day 4, VEGF, VEGFR1 and VEGFR2 were expressed mostly by the luminal and glandular epithelium. Stromal cells showed a very weak labeling. On days 5-8, VEGF and its receptors showed an increased labeling throughout the mesometrial decidua. The expression of VEGF, VEGFR1, and VEGFR2 were differentially expressed in the mesometrial cells and in the predecidual cells of the antimesometrial decidua. VEGFR1 and VEGF R2 were highly expressed by endothelial cells of the mesometrial sinusoids, and Nk uterine cells.

Head and neck cancer ranks high among the most common cancers world wide. In addition, there is a high recurrence rate, as well as a high prevalence of loco-regional tumor spread. Among many factors contributing to metastasis is vascular endothelial cell growth factor C. VEGF-C is primarily an inducer of new lymph vessel formation, typically during embryogenesis; however, some advanced cancers show a significant increase in VEGF-C expression, suggesting a role in metastasis. In the current study, the effects of VEGF-C expression were tested in HN12 cells, which are highly metastatic and known to express high levels of the chemokine CXCL5. A connection between VEGF-C and CXCL5 expression was made in previous studies. VEGF-C expression was downregulated or upregulated in appropriate target cells, in order to test its effect on proliferation and migration. Downregulation of VEGF-C in HN12 cells resulted in a decrease in proliferation, migration and motility. Conversely, upregulation of VEGF-C in HN4 cells led to an increase in cell proliferation. In addition, downregulation of VEGF-C significantly lowered tumorigenicity in athymic mice. All results suggest VEGF-C is contributing to an increase in proliferation, migration and motility in this HNSCC model system.

L’angiogenèse tumorale réfère à la capacité d’une tumeur à stimuler la formation de nouveaux vaisseaux sanguins. L’induction de l’angiogenèse dépend notamment de la présence de certains récepteurs exprimés à la surface de cellules endothéliales et tumorales. Ces récepteurs sont impliqués dans la formation de nouveaux vaisseaux sanguins mais aussi dans la progression des tumeurs, l’invasion locale des tissus avoisinants et la formation de métastases. Nous nous intéressons ici essentiellement aux récepteurs de type intégrines (et surtout l’intégrine αvß3) ou neuropiline-1 (NRP1).Les intégrines sont des récepteurs transmembranaires décrits initialement parce qu’ils permettent aux cellules d’adhérer et de se déplacer sur la matrice extracellulaire (ECM) en particulier parce qu’elles se lient à la séquence tri-peptidique RGD, mais elles interviennent aussi directement et indirectement dans les échanges biochimiques entre les cellules et leur micro-environnement. NRP1 est un corécepteur du VEGF (vascular endothelial growth factor). Pour cela, NRP1 s’associe au récepteur principal VEGFR2, surexprimé dans les tumeurs et dont l’expression a été corrélée avec l’angiogenèse. Il est très important de noter que l’intégrine αvß3 et le récepteur NRP1 peuvent interagir physiquement et fonctionnellement. Notre hypothèse de travail est alors qu’en bloquant la fonction de ces 2 récepteurs nous pourrons augmenter l’efficacité des thérapies anti-angiogèniques anti-tumorales.Nous avons généré des nanoparticules de silices bifonctionnelles car elles présentent à leur surface à la fois des peptides cycliques cRGD ciblant l’intégrine αvß3 et ATWLPPR qui cible NRP1. Nous avons testé des ratio différents de peptides cRGD et ATWLPPR (100/0, 25/75, 50,75/50/25 et 0/100), et nous avons aussi optimisé le nombre total de ces ligands/NP. Nous avons analysé l’affinité des différentes molécules, leur sélectivité et activité biologique ainsi que leurs propriétés anti-angiogéniques et anti-tumorale en particulier sur des cellules endothéliales humaines (ECs) et sur des lignées de cellules tumorales.Notre étude suggère que ces nanoparticules bifonctionnelles présentent un grand potentiel si leur composition est soigneusement définie. En particulier, elles peuvent présenter des activités extrêmement variables voir opposées suivant la nature et composition de leur surface et de la concentration à laquelle les NPs sont utilisées. En effet, à « haute concentration » en NP, ce qui correspond en fait à une faible concentration en peptides, nous montrons qu’il est possible d’obtenir un effet « pro-angiogénique » lié au recrutement d’autres récepteurs de facteurs de croissance (IGF1-R/IR) qui a priori ne devaient pas intervenir dans notre système, mais semblent pouvoir être fonctionnellement liés aux intégrines et/ou NRP1 en réponse aux particules présentant les 2 peptides cRGD et ATWLPPR. Ces résultats contribuent à expliquer certains échecs thérapeutiques des agents anti-angiogéniques mais nous permettent aussi de proposer des solutions attractives pour la définitions nouveaux agents thérapeutiques. / Tumor angiogenesis refers to the ability of a tumor to stimulate new blood vessels formation. Angiogenesis strongly depends on cell surface receptors and integrin activation to promote tumor progression, local invasion and dissemination. Integrins (especially integrin αvß3) and Neuropilin-1 (NRP1), a co-receptor of VEGFR2, are over-expressed in the tumor vasculature and by tumor cells, and their expression has been correlated with tumor progression. Importantly, integrin αvß3 and NRP1 can physically and functionally interact.The use of dual targeted drugs that block the integrin αvß3 and the NRP1 receptor simultaneously is thus expected to augment the anti-angiogenic and anti-tumor activities, as compared to each “mono-therapy” separately. During my PhD studies, in collaboration with the group of chemists leaded by Pr G. Subra, we generated different batches of bifunctional cRGD/ATWLPPR peptides coated nanoparticles (NPs) targeting integrin αvß3 and NRP1 simultaneously. We introduced different ratio of cRGD and ATWLPPR peptides (100/0, 25/75, 50/50, 75/25 and 0/100), and we also increased the amount of total ligands on the surface of the silica NPs. Systematic studies including molecules' affinity, selectivity, and biological activity as well as anti-angiogenic and anti-tumoral effects were performed on primary endothelial cells (ECs), immortalized ECs and several tumor cells. NPs properties were also evaluated in vivo in a mouse tumor model. We report here that these NPs present highly variable biological activities in ECs and tumor cells depending on the peptides ratio, surface coating of the NPs and on their concentration. In particular, “elevated” concentrations of NPs, which actually correspond to usual concentrations of peptides, can activate an unexpected IGF1-R/IR-AKT signaling pathway that could lead to a counter-productive pro-angiogenic activity (agonist instead of antagonist). This could mimic the conflicting results obtained in clinical trials using Cilengitide, an RGD-presenting peptide, and thus provide new areas of investigations and new possibilities to design active nano-drugs.This work can thus participate to the general effort of our research community to design efficient targeted anti-angiogenic therapies that could be applied in particular for cancer treatment.

Les thiénopyrimidinones et les thiénofuranes sont considérés comme des structures prometteuses dans le design de molécules bioactives, notamment des composés anti-angiogéniques. L'angiogenèse pathologique est l'un des évènements majeurs dans le développement tumoral. Une des stratégies thérapeutiques étudiée repose donc sur la conception de petites molécules organiques susceptibles d'inhiber l'activité tyrosine kinase du récepteur VEGFR-2, un des acteurs clé du processus angiogénique. Dans ce contexte, nos travaux ont essentiellement porté sur la synthèse d'une nouvelle série de thiénopyrimidinones et thiénofuranes fonctionnalisés à potentiel inhibiteur du VEGFR-2. Pour chacune des nouvelles structures, nous avons étudié, par 3D-QSAR, l'influence sur le potentiel biologique de différentes modulations structurelles (fonctionnalisations et isomérie du noyau central, et structure d’une chaîne latérale). Puis, plusieurs stratégies de synthèse ont été élaborées et discutées afin de pouvoir préparer les molécules cibles. Pour accéder sélectivement à l'un des isomères du thiénofurane, nous avons mis au point une méthode de synthèse chimiosélective à partir de thiophènes 3-O-alkylés judicieusement fonctionnalisés. Ces composés peuvent ensuite être fonctionnalisés pour accéder aux molécules cibles. Parallèlement, la synthèse des thiéno[2,3-d]pyrimidinones a été abordée selon deux stratégies complémentaires : une voie de synthèse convergente permettant d'articuler autour du noyau central les modulations envisagées pour les cibles en série azotée (hétéroatome de jonction), et une voie de synthèse linéaire plus adéquate à la modulation des cibles en séries oxygénée et soufrée / As bioisosteres of quinazolinones and benzofuranes respectively, thienopyrimidinones and thienofuranes can be envisioned as promising precursors for the design of potential bioactive compounds, especially anti-angiogenic molecules. Tumoral angiogenesis plays an important role in the tumor growth and metastasis of cancer. Therefore, one of the most studied anti-cancer strategies implies the design of small kinase inhibitors, targeting more specifically VEGFR-2 (Vascular Endothelial Growth Factor Receptor 2), which is considered as key component in the angiogenic process. In this context, our work has essentially focused on the synthesis of new series of thienopyrimidinone and thienofuran derivatives with potential inhibitory activity of VEGFR-2. For each of the designed structures, we have first studied, by 3D-QSAR, the influence of various modulations of the heterocyclic core (including substitution and isomery of the core, nature of the extending chain) on the predicted activity. Then, we have elaborated several synthetic pathways allowing access to the targeted compounds. Selective synthesis of a choosen thienofuran isomer has been performed through a chemoselective methodology starting from wisely functionalized 3-O-alkylated thiophenes. These molecules could be then further functionalized to access to final compounds. Simultaneously, synthesis of thieno[2,3-d]pyrimidinone derivatives has been carried out according to two strategies : a convergent pathway allowed a direct introduction of some of the envisioned modulations on the central core in nitrogen series (linking heteroatom), while a linear pathway has ensured the desired modulations on oxygen and sulfur series

This work explored features of canine mast cell tumours (MCT) to improve prognosis and to discover potential therapeutic targets. Subcutaneous MCT - a subset of these tumours arising in the subcutis - are usually grouped with cutaneous MCT, but there is evidence that they may be clinically different. The first objective was to develop a grading scheme for subcutaneous MCT. Over 300 canine subcutaneous MCT were evaluated retrospectively and parameters were correlated with clinical outcomes, making this the largest retrospective survival study of these tumours to date. The results of the study showed that the majority of subcutaneous MCT had excellent outcomes and key prognostic markers were identified (mitotic index, surgical margins and degree of infiltration). A subset of the subcutaneous MCT from the retrospective study was further evaluated to assess the cellular localization of KIT - a receptor tyrosine kinase (RTK) which is dysregulated and constitutively activated in some cutaneous MCT - as well as Ki67, a proliferation marker. In addition, evaluation of mutations of c-KIT, the gene for KIT, was determined for each MCT. Cytoplasmic KIT localization and high Ki67 values were predictive of decreased survival time and time to local reoccurrence, but no c-KIT mutations were detected. The majority of canine MCT do not appear to depend solely upon KIT for tumour progression and few other RTK targets have been studied in canine MCT. Based on evidence that vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) - may play a role in the progression of canine MCT; the expression and distribution of these RTK were evaluated. The results showed that canine MCT have unique expression profiles and activity of KIT, VEGFR2 and PDGFR. Two novel mast cell tumour cell lines were generated and used to assess signalling of KIT and VEGFR2 in vitro. Stimulatory and inhibitory responses were assessed and found to be different in both cell lines. Both had autophosphorylated VEGFR2 and an autocrine VEGF/VEGFR2 signalling pathway existed for both cell lines. These findings are unique and the first that identify autocrine VEGF signalling as a possible survival mechanism for canine MCT. / Pet Trust Foundation, Ontario Veterinary College

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases.

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-κB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-κB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-κB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-κB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-κB p50 and p65 transcription factors. This also revealed that Prox1 and NF-κB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter. Characterization of p50 knockout mice revealed significantly decreased lymphatic vessel density in several organs that corresponded to reduced VEGFR-3 and Prox1 expression. Activation of NF-κB by inflammatory stimuli also elevated expression of NF-κB, Prox1 and VEGFR-3 in cultured lymphatic endothelial cells, which enhanced proliferation and migration in response to the VEGFR-3-specific ligand, VEGF-C152S. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.