Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells.

Vascular Endothelial Growth Factor, from Basic Research to Clinical Applications.

Judah Folkman's landmark discovery in the 1970s showing that tumors, growing beyond a few millimeters in diameter, depend on de novo vascularization triggered by specific growth factors released by tumor cells encountering hypoxia [...

ScFvs as Allosteric Inhibitors of VEGFR-2: Novel Tools to Harness VEGF Signaling.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity.

VEGFR2 promotes central endothelial activation and the spread of pain in inflammatory arthritis.

Chronic pain can develop in response to conditions such as inflammatory arthritis. The central mechanisms underlying the development and maintenance of chronic pain in humans are not well elucidated although there is evidence for a role of microglia and astrocytes. However in pre-clinical models of pain, including models of inflammatory arthritis, there is a wealth of evidence indicating roles for pathological glial reactivity within the CNS.

Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions.

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution.

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist.

Angiogenesis plays a key role in various physiological and pathological conditions, including inflammation and tumor growth. The bone morphogenetic protein (BMP) antagonist gremlin has been identified as a novel pro-angiogenic factor. Gremlin promotes neovascular responses via a BMP-independent activation of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2).

Highly efficient baculovirus-mediated multigene delivery in primary cells.

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters.

VEGFR-2 conformational switch in response to ligand binding.

VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated.

VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread.

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation.

Subcellular object quantification with Squassh3C and SquasshAnalyst.

Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst.

Vascular endothelial growth factor-A165b prevents diabetic neuropathic pain and sensory neuronal degeneration.

Diabetic peripheral neuropathy affects up to half of diabetic patients. This neuronal damage leads to sensory disturbances, including allodynia and hyperalgesia. Many growth factors have been suggested as useful treatments for prevention of neurodegeneration, including the vascular endothelial growth factor (VEGF) family.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy.

Cell lines expressing recombinant transmembrane domain-activated receptor kinases as tools for drug discovery.

Many receptor tyrosine kinases (RTKs) represent bona fide drug targets in oncology. Effective compounds are available, but treatment invariably leads to resistance, often due to RTK mutations. The discovery of second-generation inhibitors requires cellular models of resistant RTKs.

Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation.

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. VEGF receptor 2 (VEGFR-2) is the major receptor involved in vasculogenesis and angiogenesis and regulates endothelial cell survival, migration, and mitogenesis. Ligand-mediated receptor dimerization instigates transmembrane signaling, thereby promoting activation of the intracellular kinase domain.

Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.

Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation.

High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis.

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin.

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation.

Targeting extracellular domains D4 and D7 of vascular endothelial growth factor receptor 2 reveals allosteric receptor regulatory sites.

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains).

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization.

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligand-binding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation.

Novel functional germline variants in the VEGF receptor 2 gene and their effect on gene expression and microvessel density in lung cancer.

Purpose: VEGF receptor 2 (VEGFR-2) plays a crucial role in mediating angiogenic endothelial cell responses via the VEGF pathway, and angiogenesis inhibitors targeting VEGFR-2 are in clinical use. As angiogenesis is a host-driven process, functional heritable variation in KDR, the gene encoding VEGFR-2, may affect VEGFR-2 function and, ultimately, the extent of tumor angiogenesis.

Experimental Design: We resequenced KDR using 24 DNAs each from healthy Caucasian, African American, and Asian groups.

Structural analysis of vascular endothelial growth factor receptor-2/ligand complexes by small-angle X-ray solution scattering.

Receptor tyrosine kinases play essential roles in tissue development and homeostasis, and aberrant signaling by these molecules is the basis of many diseases. Understanding the activation mechanism of these receptors is thus of high clinical relevance. We investigated vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which regulate blood and lymph vessel formation.

Neuropilin-1 promotes VEGFR-2 trafficking through Rab11 vesicles thereby specifying signal output.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development by activating 3 receptor tyrosine kinases (RTKs), VEGFR-1, -2, and -3, and by binding to coreceptors such as neuropilin-1 (NRP-1). We investigated how different VEGF-A isoforms, in particular VEGF-A(165)a and VEGF-A(165)b, control the balance between VEGFR-2 recycling, degradation, and signaling. Stimulation of cells with the NRP-1-binding VEGF-A(165)a led to sequential NRP-1-mediated VEGFR-2 recycling through Rab5, Rab4, and Rab11 vesicles.

Structural determinants of vascular endothelial growth factor-D receptor binding and specificity.

Vascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus.

A plasmid-based multigene expression system for mammalian cells.

The introduction of heterologous genetic information, particularly of multiple genes, into mammalian cells is a key technology in contemporary experimental biological research. The coexpression of fluorescently tagged sensors is required to simultaneously analyse multiple parameters in living cells and the coexpression of several proteins is necessary to manipulate cell fate in stem cell biology. Current technologies for multigene expression in mammalian cells are inefficient, inflexible and time-consuming.