A new pathobiologic hypothesis has recently emerged that the alveolar structural destruction and loss in emphysema are caused by the deficiency of vascular endothelial growth factor (VEGF). Therefore, this project hypothesized that such pathobiologic VEGF deficiency of emphysematous lungs can be recovered with a natural caffeic acid tetramer, salvianolic acid B (SalB), through activation of signal transducer and activator of transcription 3 (STAT3), so that emphysema can be reversed as a result of inhibition of induced cell death, stimulation of cell proliferation and migration, and promotion of stem cell recruitment to the lungs. SalB was first shown to be potently anti-oxidative (IC50 = 3.7 μM), but devoid of anti-elastase activity. SalB was then administered to the lungs of healthy rats at 0.2 mg/kg for two weeks, verifying ~1.7-fold increased lung tissue expressions of phosphorylated STAT3 (pSTAT3; an activated form of STAT3) and VEGF. Subsequently, SalB was examined in the anti-cell death assay, cell proliferation and migration assays, and trans- endothelial stem cell recruitment assay in the in vitro lung epithelial (A549) and endothelial (HMVEC-L) cell systems. SalB at 25 μM exerted significant 48-88 % inhibitory activities against cell death induced with oxidative stress and VEGF receptor blockade (with SU5416) in both cell systems, measured by the trypan blue exclusion and propidium iodide-based flow cytometry assays. SalB at 25 μM also stimulated A549 and HMVEC-L cell proliferation by ~1.4-fold and promoted cell migration by ~1.6-fold, while recovering stem cell recruitment impaired with SU5416 by 60 %. The anti-cell death, and proliferation and migration stimulatory activities of SalB were significantly opposed by pharmacological inhibitors of JAK2 (Janus kinase 2; an upper signal of STAT3), STAT3 and VEGF. SalB was then examined for its in vivo reversal activities in emphysema induced with porcine pancreatic elastase (PPE) and cigarette smoke extract (CSE) in rats. Upon establishment of emphysema on day 21, SalB was administered to the lungs three times weekly over three weeks. SalB at 0.2 mg/kg significantly recovered ~85 %-impaired treadmill exercise endurance by 57-82 %; and reduced abnormal airspace enlargement by 59-75 %. In the PPE-induced emphysematous rats, SalB also reduced the 4-fold greater alveolar destruction index by 61 %. The lung tissue protein expression by Western blot analysis found that cleaved caspase 3 (cell apoptotic marker) was induced by 13-fold, and VEGF was reduced by 60 % in the PPE -induced emphysematous rats. However, pulmonary treatment with SalB at 0.2 mg/kg normalized these proteins, and also significantly increased the expression of a cell proliferation marker, proliferative cell nuclear antigen (PCNA) by 2.6-fold. Note however that SalB treatment did not reduce the neutrophilic myeloperoxidase activity in the lungs induced in the PPE-induced rats. Taken all together, this study has demonstrated that SalB potently inhibited lung cell death, stimulates lung cell proliferation and migration, and restores stem cell migration with its mechanism of STAT3 activation and VEGF elevation and reversed established emphysema in rat models.

La protéine d’échafaudage Gab1 amplifie la signalisation de plusieurs récepteurs à fonction tyrosine kinase (RTK). Entre autres, elle promeut la signalisation du VEGFR2, un RTK essentiel à la médiation de l’angiogenèse via le VEGF dans les cellules endothéliales. En réponse au VEGF, Gab1 est phosphorylé sur tyrosine, ce qui résulte en la formation d’un complexe de protéines de signalisation impliqué dans le remodelage du cytosquelette d’actine et la migration des cellules endothéliales. Gab1 est un modulateur essentiel de l’angiogenèse in vitro et in vivo. Toutefois, malgré l’importance de Gab1 dans les cellules endothéliales, les mécanismes moléculaires impliqués dans la médiation de ses fonctions, demeurent mal définis et la participation du second membre de la famille, Gab2, reste inconnue. Dans un premier temps, nous avons démontré que tout comme Gab1, Gab2 est phosphorylé sur tyrosine, qu’il s’associe de façon similaire avec des protéines de signalisation et qu’il médie la migration des cellules endothéliales en réponse au VEGF. Cependant, contrairement à Gab1, Gab2 n’interagit pas avec le VEGFR2 et n’est pas essentiel pour l’activation d’Akt et la promotion de la survie cellulaire. En fait, nous avons constaté que l’expression de Gab2 atténue l’expression de Gab1 et l’activation de la signalisation médiée par le VEGF. Ainsi, Gab2 semble agir plutôt comme un régulateur négatif des signaux pro-angiogéniques induits par Gab1. La migration cellulaire est une des étapes cruciales de l’angiogenèse. Nous avons démontré que Gab1 médie l’activation de la GTPase Rac1 via la formation et la localisation d’un complexe protéique incluant la GEF VAV2, la p120Caténine et la Cortactine aux lamellipodes des cellules endothéliales en réponse au VEGF. De plus, nous montrons que l’assemblage de ce complexe corrèle avec la capacité du VEGF à induire l’invasion des cellules endothéliales et le bourgeonnement de capillaires, deux phénomènes essentiels au processus angiogénique. La régulation des RhoGTPases est également régulée par des inactivateurs spécifiques les « Rho GTPases activating proteins », ou GAPs. Nous décrivons ici pour la première fois le rôle de la GAP CdGAP dans les cellules endothéliales et démontrons son importance dans la médiation de la signalisation du VEGF via la phosphorylation sur tyrosine de Gab1 et l’activation des RhoGTPases Rac1 et Cdc42. Ainsi, dù à son importance sur l’activation de voies de signalisation du VEGF, CdGAP représente un régulateur crucial de la promotion de diverses activités biologiques essentielles à l’angiogenèse telles que la migration cellulaire, et le bourgeonnement de capillaires in vitro et d’aortes de souris ex vivo. De plus, les embryons de souris CdGAP KO présentent des hémorragies et de l’œdème, et ces défauts vasculaires pourraient être responsables de la mortalité de 44% des souris CdGAP knock-out attendues. Nos études amènent donc une meilleure compréhension des mécanismes moléculaires induits par le VEGF et démontrent l’implication centrale de Gab1 et des régulateurs des RhoGTPases dans la promotion de l’angiogenèse. Cette meilleure compréhension pourrait mener à l’identification de nouvelles cibles ou approches thérapeutiques afin d’améliorer le traitement des patients souffrant de maladies associées à une néovascularisation incontrôlée telles que le cancer. / The Gab1 scaffolding protein allows signaling of multiple Receptors Tyrosine Kinase (RTKs). Among other things, it allows VEGFR2 signaling, an essential RTK to mediate angiogenesis via VEGF in endothelial cells. In response to VEGF, Gab1 is tyrosine phosphorylated, resulting in the formation of a signaling protein complex involved in the remodeling of the actin cytoskeleton and the migration of endothelial cells. Gab1 is a key modulator of angiogenesis in vitro and in vivo. However, despite the importance of Gab1 in endothelial cells, the molecular mechanisms involved in mediating its functions remain poorly defined and the participation of the second family member, Gab2, remains unknown. Initially, we demonstrated that as with Gab1, Gab2 is tyrosine phosphorylated, it associates with similar signaling proteins and induces cell migration in response to VEGF in endothelial cells. However, Gab2 does not interact with VEGFR2 and is not essential for the activation of Akt and the promotion of cell survival. In fact, we found that the expression of Gab2 attenuates the expression of Gab1 and activation of VEGF-mediated signaling. In light of these results, we propose that in endothelial cells stimulated with VEGF, Gab2 acts as a negative regulator of pro-angiogenic signals induced by Gab1. Cell migration is a crucial step in angiogenesis, though, few studies have investigated the involvement of Gab1 in regulating different molecular mechanisms for actin remodeling leading to endothelial cell migration. We demonstrated that Gab1 mediates activation of Rac1 GTPase via the formation and localization of a protein complex including the GEF VAV2, p120 Catenin and Cortactin to lamellipodia of endothelial cells in response to VEGF. Furthermore, we show that the assembly of this complex correlates with the ability of VEGF to induce endothelial cell invasion and capillary sprouting, phenomena essential to the angiogenic process. RhoGTPases are also regulated by specific inactivators, "Rho GTPase activating proteins" or GAPs. The involvement of GAPs in promoting angiogenesis is relatively poorly described. Here we describe for the first time the role of the GAP CdGAP in endothelial cells and demonstrate its importance in mediating VEGF signaling via tyrosine phosphorylation of Gab1 and activation of Rac1 and Cdc42 RhoGTPases. Due to its importance in the activation of signaling pathways critical in VEGF signaling, CdGAP is thus an important protein for the regulation of various essential biological activities such as cell migration, sprouting and therefore in vitro and ex vivo angiogenesis. In addition, embryos of CdGAP knock-out mice exhibit vascular defects, excessive branching vessels, haemorrhages and edema which may be responsible for the 44% mortality seen in CdGAP knock-out mice expected. Our studies contribute to a better understanding of the molecular mechanisms induced by VEGF and demonstrate the central involvement of Gab1 and regulators of RhoGTPases in promoting angiogenesis. This understanding could lead to the identification of new targets and therapeutic approaches to improve the treatment of patients with uncontrolled neovascularization associated with diseases such as cancer.

Le VEGF-A est impliqué dans plusieurs pathologies, dont les maladies cardiovasculaires et plusieurs types de cancer. L'existence de voies de signalisation communes entre le VEGF-A, les molécules d'adhésion cellulaire et des molécules de l'inflammation pourrait permettre d'expliquer la large gamme de fonctions du VEGF-A dans les différentes situations pathologiques. Dans le cadre de cette thèse, nous avons développé une approche intégrative pour l’étude du VEGF-A et de son positionnement au sein de plusieurs voies métaboliques. Cette approche associe les études d’identification des variants génétiques associés au VEGF-A et leur fonctionnalité biologique par une approche transcriptomique. Ainsi, le but général de cette thèse est d’investiguer les relations complexes des polymorphismes liés au VEGF-A, des taux plasmatiques et de l’expression du VEGF-A avec les molécules d'adhésion cellulaire et les molécules de l’inflammation, les lipides plasmatiques, les gènes candidats NOS3, CD14, MMP3 et IL4, et avec les facteurs de risque cardiovasculaires (obésité, pression artérielle) chez des individus en bonne santé. Pour la réalisation de ces études, nous avons utilisé des sous-groupes de populations de la cohorte STANISLAS et autres populations du Centre de Ressources Biologiques IGE-PCV. L’expérimentation transcriptomique est réalisée avec des cellules mononucléaires du sang périphérique.Parmi les résultats obtenus nous avons montré : • Une association entre l'isoforme VEGF-A145 et l'ARNm d'ICAM-1, de sélectine L et de TNF-α. • Une association entre les taux du VEGF-A et les taux d’ICAM-1 et de la sélectine E. • Des interactions épistatiques entre les variants du VEGF-A pour les taux de la sélectine E, du TNF-α, de l'ICAM-1 et de l'IL-6. • Une association significative entre rs4416670 et les niveaux de l'ARNm de la sélectine-L • Une association entre le variant rs6921438 et les niveaux de HDL-C et LDL-C. • Une interaction entre rs4416670 et hypertension pour la variation interindividuelle de l'apolipoprotéine E. • Des associations significatives entre l’expression de l’isoforme VEGF-A145 et les polymorphismes de NOS3, CD14, MMP3, IL4R, et IL4. • Des interactions épistatiques significatives entre les variants génétiques de NOS3, CD14, MMP3, IL4R, et IL4 et les quatre polymorphismes liés au VEGF-A sur les taux plasmatiques de VEGF-A. • Des interactions significatives entre le rs1800779 de NOS3 et HDL-C, les triglycérides et l’obésité ainsi que l’interaction de rs6921438 avec l’hypertension pour les niveaux plasmatique de VEGF-A • Des associations significatives et interactions gène × lipides du sang entre tous les variants génétiques de VEGF-A et les phénotypes d’obésité. • Une association significative entre le rs4416670 et la pression pulsée. • Une interaction épistatique entre le rs6921438 et le rs10738760 pour la pression pulsée. • Des associations significatives entre le variant rs10738760 de VEGF-A et le risque de syndrome métabolique. Les résultats de cette thèse montrent le rôle central du VEGF-A dans la régulation des différents processus physiologiques et permettant de proposer le VEGF-A comme un nouveau biomarqueur potentiel des maladies cardiovasculaires à évaluer cliniquement / VEGF-A is involved in several diseases, including cardiovascular disease and several types of cancer. The existence of common signaling between the VEGF-A, cell adhesion molecules and inflammatory molecules may help to explain the wide range of functions of VEGF-A in different pathological situations. As part of this thesis, we have developed an integrative approach to study of VEGF-A and its position in several metabolic pathways. This approach involves the identification of genetic variants associated with VEGF-A and their biological function by a transcriptomic approach. Thus, the general aim of this thesis is to investigate the complex relationships between four polymorphisms associated with VEGF-A, its plasma levels and its expression with cell adhesion molecules, inflammatory molecules, plasma lipids, candidate genes (NOS3, CD14, MMP3 and IL-4) and with cardiovascular risk factors (obesity and blood pressure) in healthy individuals. For our studies, we used a subgroup of the STANISLAS Family Study and other populations available in the Biological Resources Center IGE-PCV. Our transcriptomics experiments have been performed with peripheral blood mononuclear cells. The results showed: • An association between VEGF-A145 isoform with the levels of ICAM-1 mRNA, L-selectin mRNA and TNF-α mRNA. • An association between the levels of VEGF-A and the levels of ICAM-1 and E selectin. • An epistatic interactions between the VEGF-A related variants for the levels of E selectin, TNF-α], ICAM-1 and IL-6. • An association of rs4416670 with levels of mRNA of L selectin. • An association between rs6921438 and levels of HDL-C and LDL-C. • An interaction between rs4416670 and hypertension for the interindividual variation of apolipoprotein E. • Significant associations between the expression of VEGF-A with NOS3, CD14, MMP3, IL4R and IL-4 polymorphisms. • Significant epistatic interactions between genetic variants of NOS3, CD14, MMP3, IL4R, and IL4 and the four polymorphisms related to VEGF-A on the plasma levels of VEGF-A. • Significant interactions between rs1800779 in NOS3 and HDL-C, triglycerides, and obesity, as well as interactions of rs6921438 with hypertension on plasma levels of VEGF-A. • Significant associations and gene × blood lipids interactions between all genetic variants of VEGF-A with obesity traits. • A significant association between rs4416670 and pulse pressure. • An epistatic interaction between rs6921438 and rs10738760 on pulse pressure. • Significant associations between the rs10738760 variant of VEGF-A and the risk of metabolic syndrome. The results of this thesis indicate the central role of VEGF-A in the regulation of various physiological processes and offer VEGF-A as a potential novel biomarker for cardiovascular disease to be further evaluated clinically

Le cancer de vessie représente la sixième cause de mortalité par cancer en France. Son incidence a augmenté ces 20 dernières années, mais les taux de survie restent inchangés. La carcinogénèse vésicale fait intervenir différents mécanismes moléculaires qui agissent en réseau comme c’est le cas dans de nombreux cancers. Le développement récent de nouveaux traitements prenant spécifiquement pour cible certaines voies de signalisation apportent de nouveaux espoirs thérapeutiques. Nous nous sommes intéressés dans ce travail à trois axes de recherche pour tenter d’identifier, dans les carcinomes urothéliaux, de nouveaux marqueurs pronostiques moléculaires et de nouvelles cibles thérapeutiques potentielles: l’angiogénèse, la voie de signalisation Hedgehog et les microARNs. Nous avons choisi la RT-PCR quantitative en temps réel à grande échelle permettant d’évaluer le niveau d’expression de nombreux gènes, avec une quantification précise et reproductible des transcrits. L’expression de ces gènes a été corrélée aux données de suivi clinique afin d’identifier de nouveaux biomarqueurs moléculaires prédictifs de l’évolution des tumeurs de vessie.Nous avons ainsi pu démontrer que les niveaux d’expression de certains de ces gènes variaient de façon significative dans les tumeurs de vessie, confirmant le rôle de l’angiogénèse dans la carcinogénèse urothéliale, et plus particulièrement de la voie du VEGF, et suggérant une implication majeure de la voie de signalisation Hedgehog et des microARNs. Par ailleurs, nous avons également pu identifier plusieurs biomarqueurs ayant une valeur pronostique en terme de survie globale dans les tumeurs infiltrantes. C’est le cas du VEGF, qui semble être un biomarqueur moléculaire particulièrement intéressant puisqu’il existe des thérapies ciblées spécifiquement dirigées contre ce ligand ou ses récepteurs avec plusieurs essais cliniques actuellement en cours dans le cancer de vessie. C’est également le cas d’une signature moléculaire associant 3 miARNs (miR-9, miR-182 et miR-200b) ayant une valeur péjorative dans les tumeurs infiltrantes, ouvrant la voie vers de nouvelles stratégies thérapeutiques.L’ensemble de ces études confirment l’intérêt majeur d’une meilleure compréhension des bases moléculaires de la carcinogénèse urothéliale vésicale débouchant sur l’utilisation rationnelle de nouvelles thérapies ciblées dans le cancer de vessie, avec l’espoir d’en améliorer la prise en charge et l’évolution. / Bladder cancer is the sixth cause of cancer mortality in France and its incidence is increasing since the last 20 years, with no improvement in survival outcomes. Bladder carcinogenesis involves different molecular mechanisms such as in many cancers. The recent development of new targeted therapies targeting signaling pathways provides new therapeutic hopes.In this work, we choose to study three molecular pathways in order to identify new prognostic markers and new therapeutic targets in urothelial carcinoma: angiogenesis, Hedgehog signaling pathway, and microRNAs. Real-time quantitative RT-PCR was performed to measure simultaneously expression levels of several genes with precise and reproductible RNA quantification. Our results were correlated with clinical outcomes to identify new molecular markers associated with bladder cancer evolution and to guide the potential use of targeted therapies.We were able to demonstrate that expression levels of several transcripts differ significantly in bladder tumors as compared to normal bladder and that some of them may have a prognostic implication. This is the case of VEGF, which appears to be an interesting molecular marker since there are targeted therapies specifically targeting the pathway and several ongoing trials in bladder cancer. The Hedgehog pathway also appears to be altered in bladder tumors, with a ligand-dependent activation. Then, we were able to identify several deregulated microRNAs and describe a molecular 3 miRNA-signature (miR-9, miR-182 and miR-200b) having a prognostic value in muscle-invasive bladder tumors. All these studies confirm the major interest of molecular biology and new targeted therapies in the treatment of bladder cancer, with the hope of improving management and evolution.

Introdução: A hipertensão arterial sistêmica (HAS) é uma doença que pode determinar lesões em diversos órgãos inclusive nos olhos. As doenças vasculares oculares constituem a grande maioria das causas de cegueira na atualidade e a HAS tem contribuição importante nesta estatística. A variabilidade da pressão arterial tem sido implicada na gênese de uma série de lesões de órgãos-alvo. Na tentativa de compreender melhor a patogênese das doenças vasculares oculares testamos a hipótese de que não apenas os efeitos da HAS, mas também a variabilidade da pressão arterial (PA) poderia determinar lesão de órgão-alvo (ocular). Materiais e Métodos: A desnervação sino-aórtica (DSA), um modelo experimental de aumento da variabilidade da pressão arterial foi utilizado nos experimentos. Foram obtidas medidas da pressão intraocular e a partir destas medidas, a pressão de perfusão ocular. Foram analisados marcadores de estresse oxidativo (8-OHdG e nitrotirosina),VEGF e receptores AT1 na retina de animais normotensos e hipertensos com e sem DSA aguda (12 e 24 horas) e crônica (10 semanas). Resultados: Os animais desnervados apresentaram aumento da variabilidade da PA sem modificar a PA basal e redução da sensibilidade do barorreflexo. Houve aumento da modulação simpática vascular e da pressão de perfusão ocular (PPO), nos animais hipertensos, com aumento adicional da PPO nos hipertensos e desnervados crônicos.Observou-seestresse oxidativo retiniano nos animais desnervados agudos e noshipertensos desnervados crônicos, além do aumento da expressão de receptores AT1 da Angiotensina II nos animais hipertensos. Os níveis de VEGF retinianos dos animais desnervados crônicos, apresentaram comportamento inverso aos níveis de Caspase-3. Conclusão: Tais resultados indicam que só a HAS, mas também a variabilidade da PA podem determinar variações na pressão de perfusão ocular, assim como também podem induzir dano oxidativo às células retinianas. Além disso, pode-se sugerir efeito neuroprotetor retiniano do VEGF / Introduction: High blood pressure (HBP) is a disease that can determine lesions in many organs including the eyes. The ocular vascular diseases constitute the vast majority of causes of blindness and hypertension has important contribution in this statistic. Blood pressure variability has been implicated in the genesis of a series of end-organ damage. In an attempt to better understand the pathogenesis of ocular vascular diseases, we hypothesized that not only the effects of hypertension, but also the variability of blood pressure (BPV) could determine target end-organ damage (ocular). Materials and Methods: Sino-aortic denervation (SAD), an experimental model of increased blood pressure variability was used in the experiments. The intraocular pressure measurements were performed and from these measurements the ocular perfusion pressure was estimated. Oxidative stress markers (8-OHdG and nitrotyrosine), VEGF and AT1 receptor in rat retinas were analyzed inacute and chronic hypertensive and normotensiveSAD rats and in controls. Results: Denervated animals showed increased BP variability without altering the basal BP, while presenting reduced baroreflex sensitivity.There was an increase in sympatheticvascular modulation and in OPP,in hypertensive animals, that was additionally in chronic denervated hypertensive animals.Acute denervated and chronic hypertensive denervated animals showed retinal oxidative stress as well as hypertensive animals presented increased expression of AT1 receptors of angiotensin II. The levels of retinal VEGF of chronically denervated animals showed inverse behavior of levels of Caspase-3 Conclusion: These results suggest that, apart from the arterial hypertension, BP variability not only determines changes in ocular perfusion pressure, but also induces oxidative damage to retinal cells. Furthermore, one can suggest retinal neuroprotective effect of VEGF

<p>Colorectal cancer is one of the most common malignant diseases in Sweden – more than 5000 new cases are diagnosed each year. The overall five-year survival is about 60% and in cases of recurrence the prognosis is poor.</p><p>In a phase III study in advanced colorectal cancer the response rate was doubled when 5-FU was given as a bolus injection versus as a short infusion. The toxicity was similar and time to progression was longer in the injection group. However, overall survival was not significantly different. Dose-effect relationships of 5-FU were studied in another phase III study recruiting 312 patients. A decrease from 500 mg/m² to 400 mg/m² worsened the treatment results. A low incidence of severe toxicity was seen in both groups. An increase to 600 mg/m² worsened the toxicity without any improvement of the results.</p><p>A cytotoxic drug sensitivity test in different tumour types, mainly gastrointestinal cancer, poorly predicted treatment outcome in a phase II study.</p><p>The conventional Nordic Flv regimen was split in a phase I/II trial. An escalation of dose was possible and the response rate was 20%.</p><p>Thymidylate synthase (TS) and the gene expression of p53 were investigated by immunohistochemical technique in the primary tumours of 132 patients. None of the markers predicted the later palliative chemotherapy result. However, TS significantly predicted time to recurrence.</p><p>Serum markers were analysed before and during FLv treatment to early predict outcomes among 87 patients. TPS is promising, both as a predictive marker before start of treatment and after a short period of treatment. In the same setting, CEA had lower predictive value. S-VEGF and S-bFGF did not yield any prognostic information of later outcome. In all studies B-haemoglobin values, performance status and subjective response were strong markers, both for prediction of objective response and for survival.</p>

<p>Formation of blood vessels occurs through two processes: vasculogenesis and angiogenesis, which are regulated by various growth factors such as vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor. The present study was carried out in order to investigate the roles of the Shb and Cbl proteins in growth factor-mediated signal transduction and blood vessel formation. Shb was found to be involved in NGF-stimulated Rap1 signaling in PC12 cells by forming a complex with CrkII and a 130-135 kDa protein. The Rap1 signaling pathway contributed to NGF-dependent neurite outgrowth. In immortomouse brain endothelial (IBE) cells, Shb increased cell spreading, migration and cytoskeletal rearrangements. Such effects may partly be due to altered Rap1 activation in Shb overexpressing IBE cells. Shb was required for tubular morphogenesis in collagen gels in the presence of FGF-2. In embryoid bodies (EBs) derived from murine embryonic stem cells, Shb up-regulated both VEGFR2 and Tal1 expression at early stages of EB development and thus promoted blood vessel formation both in the absence and in the presence of growth factors. In IBE cells, Cbl positively regulated FGF-2 signaling and increased cell proliferation. Mutation of RING finger alone did not affect blood vessel formation in EBs. However, EBs overexpressing the oncogenic form Cbl 70Z, which had a deletion of the linker region and the first cysteine of the RING finger, exhibited intense CD31 positive sheet-like staining and blood vessel. The results suggested that Cbl had dual roles in endothelial cells: it promoted FGF-2-induced proliferation whereas down-regulated proliferation of endothelial progenitor cells.</p><p>The present work suggests that Shb and Cbl play a crucial role in cell differentiation and blood vessel formation.</p>

<p>Neuroblastoma is a childhood cancer that originates from neuroblasts in the peripheral nervous system. Neuroblastoma show considerable heterogeneity with respect to location, responsiveness to treatment and prognosis. Since current therapy involves drugs with risk of serious side effects in the growing child, there is a clinical need for more effective and less toxic treatment strategies.</p><p>Angiogenesis, the formation of new blood vessels, is critical for tumor progression. Specific inhibition of tumor-induced angiogenesis should restrict growth of most solid tumors and thereby provide a new treatment strategy. The aim of this study was to investigate the effects of angiogenic inhibition in experimental neuroblastoma in mice.</p><p>We found that experimental neuroblastomas expressed the perhaps most potent angiogenic growth factor, VEGF-A, and that plasma VEGF-A levels correlated with tumor size. SU5416, a novel antagonist of VEGFR-1 and 2, reduced angiogenesis and tumor growth in our model. We also investigated the properties of SU11657, a new, orally available, synthetic small molecule multi-targeted tyrosine kinase inhibitor. SU11657, at a well-tolerated dose, was more potent than SU5416 in reducing tumor growth rate and angiogenesis, even in MYCN-amplified tumors. Chemotherapeutics can also inhibit angiogenesis, when administrated daily in a non-toxic dose. CHS 828, a new chemotherapeutic, given orally, alone induced complete neuroblastoma regression in 44 % of the animals. Furthermore, the bisphosphonate zoledronic acid, developed to reduce bone resorption, showed anti-tumor activity in our model. Zoledronic acid was more potent than the angiogenic inhibitor TNP-470. Thus bisphosphonates may have other beneficial properties in patients with cancer apart from preventing bone resorption.</p><p>In conclusion, SU5416, SU11657, CHS 828, and zoledronic acid represent new drugs with potent anti-tumor effects. Angiogenic inhibition as single therapy or in combination with chemotherapeutics may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood such as neuroblastoma.</p>

<p>Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods.</p><p>Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. </p><p>In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF. </p>

<p>Angiogenesis, de novo synthesis of blood vessels from the pre-existing vasculature, is required both during embryonic development and in pathophysiological conditions. In particular, tumor growth needs new capillary vessels in order to both deliver oxygen and nutrients and to remove toxin and metabolites. Growth of most solid tumors would be restricted to a microscopic size in the absence of neovascularization. Angiogenesis ensues as a result of a shift in the balance between pro- and anti-angiogenic molecules.</p><p>Histidine-rich glycoprotein (HRGP) is a heparin-binding plasma protein. We showed that HRGP inhibits endothelial cell migration and adhesion to vitronectin. As a consequence, HRGP attenuates growth and vascularization of mouse model tumors. The anti-angiogenic effect of HRGP is mediated by the central histidine/proline (His/Pro)-rich domain, which must be released from the parent molecule to exert its effect. A 35-amino acid residue peptide denoted HRGP330, derived from the His/Pro-rich domain, was identified as a minimal active anti-angiogenic domain of HRGP. HRGP330 induces disruption of molecular interactions required for cell motility, such as the integrin-linked kinase/paxillin complex. Moreover, HRGP330 inhibits VEGF-induced tyrosine phosphorylation of α-actinin, a focal adhesion kinase (FAK) substrate. Consequently, the motility of endothelial cells is arrested. By use of a signal transduction antibody array, we identified FAK, paxillin and growth factor receptor-bound 2 (Grb2) as tyrosine phosphorylated in HRGP330-treated cells. We confirmed that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures. A critical role of FAK in HRGP-inhibition of angiogenesis was validated using a FAK inhibitor, geldanamycin, which allowed rescue of endothelial cell actin rearrangement.</p><p>We identified another potential mechanism in the HRGP/HRGP330 anti-angiogenic effects, exerted through regulation of tumor-associated macrophages (TAMs). HRGP/HRGP330 treatment led to reduced TAM infiltration, which in turn caused a marked decrease in VEGF and MMP-9 levels in the tumor. </p><p>Taken together, our present studies show that HRGP/HRGP330 target endothelial cell adhesion, migration, focal adhesions, and furthermore, that HRGP is involved in regulation of macrophage infiltration.</p>