Structural and functional analysis of the heparin-binding domain of VEGF164.

Several of the multitude of functions attributed to Vascular Endothelial Growth Factor-A (VEGF-A) are coordinated by its various isoforms, which are generated as a result of alternative splicing from a single gene. Despite the fact that the VEGF isoforms exhibit distinct biochemical properties, little has been done to clarify their functions and their contributions to physiological and pathological processes. In this thesis I describe the biochemical and biological characterization of the heparin-binding domain of mouse VEGF 164 through structure-function analysis. To investigate the functional significance of heparin binding, mutations were introduced into exon 7 of VEGF 164 to identify essential residues for heparin binding. Three key amino acids involved in this interaction were identified. Mutants with alterations in these amino acids were unable to bind heparin and were compromised in their ability to bind to cell-surface heparan sulfate. These mutants, however, retained wild-type like potency in inducing tissue factor expression in vitro and microvessel growth ex vivo, and maintained the capacity to bind to the receptors neuropilin-1, VEGFR-1, and VEGFR-2. A second goal of this work was to better define the role of VEGF 164 in mediating inflammatory processes during pathological vascularization of the retina. Analysis of VEGF 164-deficient (VEGF1ZU/ iao) mice subjected to neovascularization-inducing conditions and rats injected intravitreally with recombinant VEGF variants demonstrated that endogenous and exogenous VEGF 164 increases leukocyte adhesiveness to retinal vessels compared to the non-heparin-binding isoform, VEGF 120. Interestingly, the three basic residues that confer heparin binding of VEGF 164, appear to be critical for its pro inflammatory activity, but not for its angiogenic activity. In addition, both mutants (and VEGF 120) exhibited a reduced affinity for VEGFR-1, a leukocyte-expressed receptor that mediates VEGF-induced migration. Results of in vivo experiments using P1GF, VEGF-E, as well as a VEGFR-1 neutralizing antibody, further demonstrate a role for VEGFR-1 in retinal leukostasis

Glycosaminoglycans and Sialylated Glycans Sequentially Facilitate Merkel Cell Polyomavirus Infectious Entry

Merkel cell polyomavirus (MCV or MCPyV) appears to be a causal factor in the development of Merkel cell carcinoma, a rare but highly lethal form of skin cancer. Although recent reports indicate that MCV virions are commonly shed from apparently healthy human skin, the precise cellular tropism of the virus in healthy subjects remains unclear. To begin to explore this question, we set out to identify the cellular receptors or co-receptors required for the infectious entry of MCV. Although several previously studied polyomavirus species have been shown to bind to cell surface sialic acid residues associated with glycolipids or glycoproteins, we found that sialylated glycans are not required for initial attachment of MCV virions to cultured human cell lines. Instead, glycosaminoglycans (GAGs), such as heparan sulfate (HS) and chondroitin sulfate (CS), serve as initial attachment receptors during the MCV infectious entry process. Using cell lines deficient in GAG biosynthesis, we found that N-sulfated and/or 6-O-sulfated forms of HS mediate infectious entry of MCV reporter vectors, while CS appears to be dispensable. Intriguingly, although cell lines deficient in sialylated glycans readily bind MCV capsids, the cells are highly resistant to MCV reporter vector-mediated gene transduction. This suggests that sialylated glycans play a post-attachment role in the infectious entry process. Results observed using MCV reporter vectors were confirmed using a novel system for infectious propagation of native MCV virions. Taken together, the findings suggest a model in which MCV infectious entry occurs via initial cell binding mediated primarily by HS, followed by secondary interactions with a sialylated entry co-factor. The study should facilitate the development of inhibitors of MCV infection and help shed light on the infectious entry pathways and cellular tropism of the virus

Comparison of the ligand‐binding properties of fluorescent VEGF‐A isoforms to VEGF receptor 2 in living cells and membrane preparations using NanoBRET

Background and Purpose: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to NanoLuciferase-tagged VEGF Receptor 2 (VEGFR2) in living cells is that the bioluminescence resonance energy transfer (BRET) signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations.Experimental Approach: Ligand binding kinetics were monitored in both intact HEK293T cells and membranes (expressing NanoLuciferase tagged VEGFR2) using BRET between the tagged receptor and fluorescent analogues of VEGF165a, VEGF165b and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging.Key Results: Quantitiative analysis of the effect of fluorescent VEGF-A isoforms onVEGFR2 endocytosis in cells demonstrated that they produced a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand binding association curves were maintained for the duration of the 90 minute experiment. Measurement of koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4.Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to intracellular endosomes

A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF(165)

Aptamers recognize their targets with extraordinary affinity and specificity. The aptamer-based therapeutic, Macugen, is derived from a modified 2′fluoro pyrimidine RNA inhibitor to vascular endothelial growth factor (VEGF) and is now being used to treat the wet form of age-related macular degeneration. This VEGF(165) aptamer binds specifically to the VEGF(165) isoform, a dimeric protein with a receptor-binding domain and a heparin-binding domain (HBD). To understand the molecular recognition between VEGF and this aptamer, binding experiments were used to show that the HBD contributes the majority of binding energy in the VEGF(165)–aptamer complex. A tissue culture-based competition assay demonstrated that the HBD effectively competes with VEGF(165) for aptamer binding in vivo. Comparison of NMR spectra revealed that structural features of the smaller HBD–aptamer complex are present in the full-length VEGF(164)–aptamer complex. These data show that the HBD provides the binding site for the aptamer and is the primary determinant for the affinity and specificity in the VEGF(165)–aptamer complex

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the Kd of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar Kd values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications

Multivalent ligands control stem cell behaviour in vitro and in vivo

There is broad interest in designing nanostructured materials that can interact with cells and regulate key downstream functions. In particular, materials with nanoscale features may enable control over multivalent interactions, which involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another and are ubiquitous throughout biology. Cellular signal transduction of growth factor and morphogen cues (which have critical roles in regulating cell function and fate) often begins with such multivalent binding of ligands, either secreted or cell-surface-tethered to target cell receptors, leading to receptor clustering. Cellular mechanisms that orchestrate ligand-receptor oligomerization are complex, however, so the capacity to control multivalent interactions and thereby modulate key signalling events within living systems is currently very limited. Here, we demonstrate the design of potent multivalent conjugates that can organize stem cell receptors into nanoscale clusters and control stem cell behaviour in vitro and in vivo. The ectodomain of ephrin-B2, normally an integral membrane protein ligand, was conjugated to a soluble biopolymer to yield multivalent nanoscale conjugates that potently induce signalling in neural stem cells and promote their neuronal differentiation both in culture and within the brain. Super-resolution microscopy analysis yielded insights into the organization of the receptor-ligand clusters at the nanoscale. We also found that synthetic multivalent conjugates of ephrin-B1 strongly enhance human embryonic and induced pluripotent stem cell differentiation into functional dopaminergic neurons. Multivalent bioconjugates are therefore powerful tools and potential nanoscale therapeutics for controlling the behaviour of target stem cells in vitro and in vivo

Glycosaminoglycan-based hydrogels to modulate heterocellular communication in in vitro angiogenesis models

Angiogenesis, the outgrowth of blood vessels, is crucial in development, disease and regeneration. Studying angiogenesis in vitro remains challenging because the capillary morphogenesis of endothelial cells (ECs) is controlled by multiple exogenous signals. Therefore, a set of in situ-forming starPEG-heparin hydrogels was used to identify matrix parameters and cellular interactions that best support EC morphogenesis. We showed that a particular type of soft, matrix metalloproteinase-degradable hydrogel containing covalently bound integrin ligands and reversibly conjugated pro-angiogenic growth factors could boost the development of highly branched, interconnected, and lumenized endothelial capillary networks. Using these effective matrix conditions, 3D heterocellular interactions of ECs with different mural cells were demonstrated that enabled EC network modulation and maintenance of stable vascular capillaries over periods of about one month in vitro. The approach was also shown to permit in vitro tumor vascularization experiments with unprecedented levels of control over both ECs and tumor cells. In total, the introduced 3D hydrogel co-culture system could offer unique options for dissecting and adjusting biochemical, biophysical, and cell-cell triggers in tissue-related vascularization models