edoc

Generation and functional characterization of specific receptor inhibitors based on an analysis of the VEGFR-2 activation mechanism

Giese, Alexandra. Generation and functional characterization of specific receptor inhibitors based on an analysis of the VEGFR-2 activation mechanism. 2012, PhD Thesis, University of Basel, Faculty of Science.

[img]
Preview
PDF
24Mb

Official URL: http://edoc.unibas.ch/diss/DissB_9938

Downloads: Statistics Overview

Abstract

Angiogenesis, the formation of new blood vessels from preexisting vasculature, mainly occurs during embryonic development. In the adult it takes place in the female reproductive system and during wound healing. Unregulated angiogenesis is associated with various diseases such as atherosclerosis, retinopathies, lymphoproliferative or rheumatoid disease, and cancer. Tumor neovascularization enables cancer cells to enter the blood circulation and to metastasize to other organs and is a hallmark of many types of cancer.
Vascular Endothelial Growth Factors (VEGFs) constitute a family of proteins that play an important role in blood and lymphatic vessel development. VEGFs interact with three type V receptor tyrosine kinases, VEGFR�1, �2, and �3 promoting endothelial cell survival, migration, proliferation, and differentiation. VEGFR-2 is the major mediator of angiogenic signaling in endothelial cells and its activity is regulated at multiple levels. Ligand binding to the extracellular domain (ECD) of VEGFR-2 leads to receptor dimerization followed by activation of the intracellular kinase domain and downstream signaling. However, at present the specific structural changes in the ECD and the exact molecular mechanisms underlying kinase activation are only partially understood. In this study, we investigated the role of ECD Immunoglobulin (Ig)�homology domains D4 and D7 in receptor dimerization and activation. We expressed a series of receptor ECD mutants in tissue culture cells and determined receptor activation. Mutation or deletion of D4 or D7 drastically reduced receptor activation. We interpret these data as the demonstration that Ig�homology domains 4 and 7 are required for correctly aligning receptor monomers in active dimers and are thus indispensable for kinase activation.
Based on our insights into the activation mechanism of VEGFR�2, we generated two types of ECD binders, single chain Fvs (scFvs) and Designed Ankyrin Repeat Proteins (DARPins), specifically interacting with single Ig-homology domains. We tested these reagents for inhibition of ligand-stimulated receptor activation. We identified several DARPins interacting with D2-3 and thereby blocking ligand binding and receptor activation. Most interestingly, DARPins binding to D4 inhibited receptor activation without interfering with receptor dimerization and therefore behave as allosteric regulators of VEGFR-2. Furthermore, scFvs specifically binding to D7 were identified. These new reagents will be useful for in vivo studies aiming at vessel imaging or for inhibiting VEGFR�2.
We furthermore characterized variations in the VEGFR-2 gene which might contribute to the phenotypic variability in tumor endothelial function and, consequently, may affect cancer progression and the susceptibility of tumors to VEGFR-2 inhibitors. VEGFR-2 genomic sequencing in three different ethnic groups led to the discovery of 120 genetic variants, single nucleotide polymorphisms (SNPs), of which 25 had not been previously reported. The functionality of the genetic variants was assessed by phosphorylation assays, mRNA and protein expression arrays, as well as by the measurement of microvessel density in non-small cell lung cancer (NSCLC) tumor samples. The correlations found may have important implications for understanding the molecular basis of genetic associations between VEGFR-2 variation and clinical phenotypes related to VEGFR-2 function.
Finally, we were interested in the influence of extracellular matrix components on VEGF�induced angiogenesis. In vivo, endothelial cells are exposed to concentration gradients of soluble growth factors and matrix immobilized guidance cues. To better mimic the complexity of angiogenic tissues in vitro, we generated micropatterned coverslips derivatized with VEGF for the cultivation of endothelial cells and the monitoring of their migration. Immobilized VEGF was shown to be biologically active and endothelial cells migrated and adhered to the patterned surfaces. The micropatterned coverslips present a robust and reproducible platform for the characterization of complex cellular behaviors generated by multiple VEGF isoforms.
Advisors:Ballmer-Hofer, Kurt
Committee Members:Resink, Thérèse
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Further Research Groups at DBM > Signal Transduction (Resink/Erne)
Item Type:Thesis
Thesis no:9938
Bibsysno:Link to catalogue
Number of Pages:159 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:49
Deposited On:24 Jul 2012 13:15

Repository Staff Only: item control page