Clinical manipulation of angiogenesis (the formation of new blood vessels from pre-existing vasculature) is of interest to treat diseases such as cancer and ischemic tissue where it is not properly regulated. Several treatments targeting vascular endothelial growth factor (VEGF) and its receptors - which are abundant at sites of angiogenesis - are currently in use to treat various types of cancer, however they have severe vascular side effects. Conversely, VEGF has been used clinically to promote angiogenesis to treat ischemic tissue. However, despite encouraging data from pre-clinical models, trials in humans have been disappointing. For further therapies to be developed, more information on how VEGF interacts with its receptors is required. Heparan sulfate (HS) is a ubiquitous glycosaminoglycan involved in a number of physiological processes including angiogenesis. HS facilitates the interaction of VEGF with its receptors, which is crucial for angiogenesis. Modification of this interaction via synthetic mimetics of HS may allow clinical intervention of angiogenesis. The current investigation aims first, to clarify the requirement for the interaction between VEGF and HS in angiogenesis; second to characterise the structure of HS that binds to VEGF so that mimetics can be developed; and third, to determine the effect of HS mimetics on angiogenesis in vivo. To determine the requirement for VEGF/HS interaction in angiogenesis, several mutants of VEGF165 that had lower affinities for HS were assayed for their ability to induce ectopic angiogenesis in the subintestinal baskets of zebrafish embryos. Wild type VEGF165 induced a 200-250% increase in ectopic vessels, which was matched only by a control mutant. Other mutants did not induce ectopic vessels, suggesting that this interaction is required for angiogenesis. To characterise the structure of HS that binds to VEGF, various HS mimetics were assayed against heparin in a VEGF competition assay using Biacore. Of these, the strongest inhibition (IC¬50 =~16nM) was with 2O10, an oligosaccharide that consisted of two highly sulfated octasaccharide domains (NS domains) that flanked an unsulfated dodecasaccharide region. To determine the type of sulfation required for this interaction, HS fragments were assayed for interaction with VEGF165 using the filter binding assay, and analysed by HPLC which indicated 6-O sulfation may be preferential for VEGF binding to HS.To investigate the ability of HS to affect angiogenesis, the effects of HS mimetics on zebrafish embryo subintestinal baskets were measured. The most interesting of these was with 2O10, which had a biphasic response whereby low doses (3ng) increased basket vasculature by 30% and high doses (30ng) decreased the endogenous vessels by 20%. As 2O10 had a high affinity for VEGF, its effects on the vasculature may be due to interaction with endogenous VEGF, which would indicate that HS mimetics can be used to control angiogenesis by modification of growth factor signalling. The investigation concludes that the interaction between VEGF and HS is critical for angiogenesis, and that this can be modulated by the application of HS mimetics that bind strongly to VEGF.

No description available.

Die Tumorangiogenese ist ein Prozess, der zur Ausbildung eines tumoreigenen Gefäßnetzwerks führt und kritisch ist für die Progression des Tumorwachstums, sowie für dessen Malignisierung und Metastasierung. Zytokine wie VEGF und PDGF steuern angiogene Prozesse. Die resultierende Tumorvaskulatur ist jedoch dysfunktional und unterscheidet sich in Struktur und Funktion stark von normalen Gefäßen. Die antiangiogene Therapie richtet sich gegen die Tumorvaskulatur indem Angiogenese-induzierende Signalwege inhibiert werden. Es existieren zahlreiche therapeutische Ansätze, zu denen u.a. Anti-VEGF- Antikörper und Rezeptortyrosinkinaseinhibitoren zählen. Ziel der antiangiogenen Therapie ist es, die Ausbildung neuer Blutgefäße im Tumor zu stoppen sowie existierende unreife Blutgefäße zu zerstören. Das Konzept der Gefäßnormalisierung beschreibt im Rahmen der antiangiogenen Therapie Prozesse, die zu einer transienten Verbesserung dieser defekten Tumorvaskulatur und zu ihrer tendenziellen Angleichung an Struktur und Funktion von normalen Gefäßen führen sollen. In dieser Studie wurden Veränderungen von Gefäßparametern in murinen AT3- Mammakarzinomen und murinen Lewis-lung-Karzinomen miteinander verglichen, die entweder (a) mit mG6-31, einem monoklonalen Anti-VEGF- Antikörper, (b) mit Axitinib, einem niedermolekularen VEGF-R-/PDGF-R- Tyrosinkinaseinhibitor antiangiogen behandelt oder (c) nicht behandelt wurden. Ziel war es dabei, Aussagen über die antiangiogene Wirksamkeit sowie die Gefäß- normalisierende Effektivität der o.g. Antiangiogenetika zu treffen. In einer bereits abgeschlossenen Forschungsarbeit von Ascheid (vgl. Absatz 7.2) wurden mit dem gleichen Experimentalaufbau wie zuvor beschrieben ebenfalls murine Tumoren hinsichtlich makroskopischer Gefäßstruktur und -organisation untersucht. Dabei wurde aufgezeigt, dass Gefäß-normalisierende Prozesse durch o.g. Angiogenetika in geringem Umfang stattfanden. Die durchgeführte Studie zielte darauf ab, die bereits erfassten Resultate zu komplettieren und somit eine abschließende Aussage über das Auftreten von Gefäßnormalisierung zu ermöglichen. 88 In den mG6-31-/Axitinib-/unbehandelten AT3-/LLC-Tumorschnitten wurden die Parameter Gefäßdichte, Apoptoserate, Proliferationsrate, Perizytenbesatz, Intaktheit der vaskulären Basalmembran und endotheliale Expression von TRPC6-Kanälen immunhistochemisch bzw. mittels Immunfluoreszenz detektiert, mikroskopisch aufgenommen und quantifiziert. Diese Arbeit zeigt, dass Axitinib deutliche antiangiogene Effekte in der Tumorvaskulatur hervorruft, mG6-31 hingegen wirkt schwächer antiangiogen. Im Unterschied zu den Ergebnissen aus Ascheids Arbeit (Ascheid, 2018) konnten- Effekte auf der Ebene der individuellen Blutgefäße nachgewiesen werden, die in der Literatur als Anzeichen für eine Gefäßnormalisierung beschrieben werden. Wiederum waren diese Effekte unter Axitinib stärker ausgeprägt als unter mG6- 31-Behandlung. Die Resultate beider Forschungsarbeiten zusammengefasst betrachtet, kann man feststellen, dass die Zusammenfassung der gefäßverändernden Effekte, die antiangiogene Wirkstoffe hervorrufen, unter dem Begriff „Normalisierung“ in Frage gestellt werden sollte. / Tumor angiogenesis is a process which leads to the formation of a tumor specific capillary system. It is a critical step towards tumor growth, malignancy and metastasis. Cytokins like VEGF and PDGF regulate angiogenic processes. The resulting tumor vasculature, however, is dysfunctional and strongly differs from structure and function in normal blood vessels. Antiangiogenic therapy is targeted against tumor vessels by inhibition of angiogenesis inducing signaling pathways. Numerous therapeutic approaches exist, for instance anti-VEGF antibodies and receptor tyrosine kinase inhibitors. Antiangiogenic therapy aims to stop the formation of new blood vessels and to prune the existing immature blood vessels. The concept of vessel normalization as part of antiangiogenic therapy describes a transient optimization of the defective tumor vasculature by acquisition of a phenotype more similar to the one of normal vessels in healthy tissue. In this study differences of vessel parameters in murine AT3 breast cancer and murine Lewis lung carcinoma were compared to ultimately evaluate the antiangiogenic and vessel normalizing effectiveness of the studied agents. Tumors were either (a) treated with mG6-31, a monoclonal anti-VEGF antibody or (b) treated with Axitinib, a VEGF-R-/PDGF-R-tyrosine kinase inhibitor or (c) untreated. In a previous study by Ascheid the same tumor models were examined with the same experimental design but with regard to macroscopic vessel structure and organization. It has been demonstrated that under the mentioned antiangiogenic agents vessel normalizing effects appeared only slightly. The current study aims to complete these results and to, in consideration of both, allow a statement concerning the appearance of vessel normalization. In the mG6-31/Axitinib/untreated AT3/LLC tumors the parameters vessel density, apoptosis rate, proliferation rate, pericyte covering, integrity of the vascular basement membrane and endothelial expression of TRPC6 channels were detected via immunohistochemistry or immunofluorescence, microscopically captured and quantified. This study demonstrates that Axitinib causes distinct antiangiogenic effects in tumor vasculature, whereas mG6-31 shows only light antiangiogenic action. In contrast to the results obtained by Ascheid vessel normalization did occur in this 90 study – more frequently under Axitinib than under mG6-31. Reflecting on the combined out-comes of the complementary study it has to be stated that the concept of a general normalization of tumor vasculature is highly questionable and subsequently has to be reconsidered.

Hintergrund: Kürzlich veröffentlichte Berichte deuten an, dass eine Metformineinnahme mit einem positiven Outcome hinsichtlich klarzelligen Nierenzellkarzinomen (clear cell renal cell carcinoma, ccRCC) in Verbindung gebracht werden kann, wenn diese Patienten mit Sunitinib oder Axitinib behandelt werden. Daher wurde ein potenzieller Synergismus zwischen Metformin (MF) und den Tyrosinkinaseinhibitoren (TKI) Sunitinib (SUT) und Axitinib (AX) in ccRCC-Zellen und seine Interaktion mit dem Tumorsuppressor miR-205 untersucht. Methoden: Es wurden qRT-PCR, Zellviabilitäts-Assays und Western Blots durchgeführt, um die Effekte von MF und TKI auf ccRCC-Zellen zu untersuchen. Ein Modell der Tetracyclin-Induzierbarkeit wurde genutzt um die Überexpression von miR-205 und die Regulation seines vorhergesagten Zielgens VEGF-A in RCC4- und 786-O-Zellen darzustellen. Zudem wurde die Expression von miR-205 und VEGF-A in der TCGA-ccRCC-Kohorte und einer hauseigenen RCC-Kohorte analysiert. Mithilfe von HUVEC-Wachstums-Assays mit einem Überstand von ccRCC-Zellen wurde der durch miR-205 vermittelte Effekt auf die Initiierung der Angiogenese und das Gefäßwachstum beobachtet. Um eine mögliche Rolle des Von-Hippel-Lindau-Tumorsuppressors (VHL) zu ermitteln, wurden RCC4- und 786-O-Zellen, beide ursprünglich VHLmut, mit RCC4- und 786-O-Zellen verglichen, die konstitutiv VHLwt exprimieren. Ergebnisse: Die Zellviabilitäts-Assays bestätigten einen dosisabhängigen Effekt von MF gegenüber SUT und AX in RCC4 und 786-O-Zellen. Zudem führte die miR-205 Überexpression im Tetrazyklin-Induzierbarkeitsmodell zu einer deutlichen Verringerung der VEGF-A Expression in beiden untersuchten Zellreihen, was ebenfalls durch eine alleinige Hochdosis-MF-Beimpfung erreicht werden konnte. Die weiteren Expressionsanalysen von miR-205 und VEGF-A in der TCGA-Kohorte und der hauseigenen Datenbank offenbarten eine Herabregulierung von miR-205 mit gleichzeitiger Hochregulierung von VEGF-A im Tumorgewebe. Dementsprechend führte auch eine miR-205 Induktion unserer Tet-On Modelle zu einer deutlich geringeren und kürzeren Gefäßneubildung in HUVEC-Wachstums-Assays. Zudem konnten in VHL-Wildtyp exprimierenden RCC4- und 786-O-Zellen signifikant höhere Mengen an miR-205 und deutlich geringere Mengen an VEGF-A im Vergleich zu den Kontroll-Zelltypen nachgewiesen werden. Fazit: Die Ergebnisse unterstützen die protektive Rolle von MF und erlauben Einsicht in den klinischen Synergismus von MF und Angiogenese-Inhibitoren. Des Weiteren können die erhobenen Daten eine Basis für klinische Studien mit MF als ein zusätzliches Adjuvanz in ccRCC Standardtherapien darstellen / Background: Recently published reports suggest that metformin use may be associated with a favorable outcome in clear cell renal cell carcinoma (ccRCC) when these patients are treated with sunitinib or axitinib. Therefore, a potential synergism between metformin (MF) and the tyrosine kinase inhibitors (TKIs) sunitinib (SUT) and axitinib (AX) in ccRCC cells and its interaction with the tumor suppressor miR-205 was investigated. Methods: qRT-PCR, cell viability assays and Western blots were performed to investigate the effects of MF and TKI on ccRCC cells. A tetracycline inducibility model was used to visualize the overexpression of miR-205 and the regulation of its predicted target gene VEGF-A in RCC4 and 786-O cells. In addition, the expression of miR-205 and VEGF-A was analyzed in the TCGA-ccRCC cohort and an in-house RCC cohort. Using HUVEC growth assays with a supernatant of ccRCC cells, the effect mediated by miR-205 on the initiation of angiogenesis and vessel growth was observed. To determine a possible role of the Von Hippel-Lindau (VHL) tumor suppressor, RCC4 and 786-O cells, both originally VHLmut, were compared with RCC4 and 786-O cells constitutively expressing VHLwt. Results: Cell viability assays confirmed a dose-dependent effect of MF versus SUT and AX in RCC4 and 786-O cells. In addition, miR-205 overexpression in the tetracycline inducibility model led to a significant reduction of VEGF-A expression in both cell lines studied, which could also be achieved by high-dose MF inoculation alone. The further expression analyses of miR-205 and VEGF-A in the TCGA cohort and the in-house database revealed a downregulation of miR-205 with simultaneous upregulation of VEGF-A in the tumor tissue. Accordingly, miR-205 induction of our Tet-On models also led to significantly less and shorter vessel formation in HUVEC growth assays. In addition, significantly higher levels of miR-205 and significantly lower levels of VEGF-A were detected in VHL wild-type expressing RCC4 and 786-O cells compared to control cell types. Conclusion: The results support the protective role of MF and provide insight into the clinical synergism of MF and angiogenesis inhibitors. Furthermore, the collected data may provide a basis for clinical trials with MF as an additional adjuvant in ccRCC standard therapies

The neuropilin (Nrp) family consists of multifunctional cell surface receptors with critical roles in a number of different cell and tissue types. A core aspect of Nrp function is ligand-dependent cellular adhesion and migration, where it controls the multistep process of cellular motility through integration of ligand binding, receptor coupling and signaling via the coordinated action of its extracellular and intracellular domains. While Nrp regulates cellular adhesion and motility in the cardiovascular and nervous systems under physiological conditions, the emerging pathological role of Nrp in tumor cell migration and metastasis has been identified and provides motivation for continued efforts toward developing Nrp inhibitors. At the molecular level, the role of Nrp in adhesion and migration is intimately connected to the control of adhesive interactions and cytoskeletal reorganization. The adhesive “interactome” for Nrp draws much attention because of its lack of enzymatic activity and inability to transduce signals on its own. It is an active area of research and is still expanding dramatically. Nrp has been well defined as a co-receptor for vascular endothelial growth factor receptor (VEGFR)/vascular endothelial growth factor (VEGF) signaling through enhancing receptor-ligand interaction in angiogenesis. Here, we contribute to this concept through characterization in more biochemical detail about Nrp-1/VEGF physical interactions. VEGF has been shown to compete with Sema3 for binding to Nrp-1 b1 ligand binding pocket. This competition fine-tunes VEGF-induced angiogenesis. Our data provides a molecular mechanism for high affinity Sema3F binding to Nrp-1 in the b1 domain. As to the VEGFR-independent function, Nrp/integrin association has been demonstrated. The functional integration has been shown for Nrp/integrin in angiogenic sprouting. Both proteins are highly expressed in endothelial tip cells to mediate endothelial cell migration during angiogenesis and knockdown of either one in mice leads to embryonic lethality due to similar defects in vascular development. To identify the structure and function correlation, we characterized in more detail about Nrp-1/integrin physical interactions with biochemical and cell-based assays. Through an integrated approach of biochemical, molecular and cellular methods, we defined the direct physical interactions between Nrp-1 and integrins. We have also extended this work to demonstrate the functional importance and contribution of the interactions in integrin-mediated cell adhesion on extracellular matrix (ECM) in angiogenesis and platelet function during wound healing and provide a molecular basis for the integration of Nrps/integrins in cell migration, adhesion to ECM, breast cancer initiation and breast cancer stem cell fate determination.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 85-102).

Vascular endothelial growth factor (VEGF-A) was first described in 1989 for its angiogenic and mitogenic properties. Early studies indicated that VEGF-A acts primarily in a paracrine pathway which is limited to vascular endothelium. Further investigation showed that VEGF-A and VEGF receptor-2 (VEGFR-2) are expressed by many solid tumors and improve cell growth and survival. Therefore, VEGF-A may act via an autocrine pathway that effects tumor cellular proliferation by binding VEGFR-2 at the cell surface. This study utilizes small interfering RNA (siRNA) technology to investigate the presence of an autocrine loop in human RKO colorectal cancer cells. RT-PCR demonstrated the expression of VEGF-A, VEGF-B, VEGF-D, placental growth factor (PlGF), VEGFR-2, neuropilin-1 (NP-1) and neuropilin-2 (NP-2) in vitro by RKO cells. Transfection with siRNA against VEGF-A resulted in a 94% knockdown of VEGF-A expression by ELISA. Northern blot, quantitative real time PCR and semiquantitative RT-PCR confirmed the knockdown data. In addition, transfected RKO cells showed a 67% decrease in cellular proliferation by WST-1 assay. This data correlated to the ELISA results. In summary, the presence of VEGF-A and VEGFR-2 argues in favor of an autocrine loop in human colorectal cancer cells. siRNA targeting of VEGF-A remains a promising anti-tumor therapeutic strategy.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.