Bruce A. Keyt

Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders

BACKGROUND Retinal ischemia induces intraocular neovascularization, which often leads to glaucoma, vitreous hemorrhage, and retinal detachment, presumably by stimulating the release of angiogenic molecules. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific angiogenic factor whose production is increased by hypoxia. METHODS We measured the concentration of VEGF in 210 specimens of ocular fluid obtained from 164 patients undergoing intraocular surgery, using both radioimmuno-assays and radioreceptor assays. Vitreous proliferative potential was measured with in vitro assays of the growth of retinal endothelial cells and with VEGF-neutralizing antibody. RESULTS VEGF was detected in 69 of 136 ocular-fluid samples from patients with diabetic retinopathy, 29 of 38 samples from patients with neovascularization of the iris, and 3 of 4 samples from patients with ischemic occlusion of the central retinal vein, as compared with 2 of 31 samples from patients with no neovascular disorders (P < 0.001, P < 0.001, and P = 0.006, respectively). The mean (+/- SD) VEGF concentration in 70 samples of ocular fluid from patients with active proliferative diabetic retinopathy (3.6 +/- 6.3 ng per milliliter) was higher than that in 25 samples from patients with nonproliferative diabetic retinopathy (0.1 +/- 0.1 ng per milliliter, P = 0.008), 41 samples from patients with quiescent proliferative diabetic retinopathy (0.2 +/- 0.6 ng per milliliter, P < 0.001), or 31 samples from nondiabetic patients (0.1 +/- 0.2 ng per milliliter, P = 0.003). Concentrations of VEGF in vitreous fluid (8.8 +/- 9.9 ng per milliliter) were higher than those in aqueous fluid (5.6 +/- 8.6 ng per milliliter, P = 0.033) in all 10 pairs of samples obtained simultaneously from the same patient; VEGF concentrations in vitreous fluid declined after successful laser photocoagulation. VEGF stimulated the growth of retinal endothelial cells in vitro, as did vitreous fluid containing measurable VEGF. Stimulation was inhibited by VEGF-neutralizing antibodies. CONCLUSIONS Our data suggest that VEGF plays a major part in mediating active intraocular neovascularization in patients with ischemic retinal diseases, such as diabetic retinopathy and retinal-vein occlusion.

Vascular Endothelial Growth Factor Regulates Endothelial Cell Survival through the Phosphatidylinositol 3′-Kinase/Akt Signal Transduction Pathway REQUIREMENT FOR Flk-1/KDR ACTIVATION

Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3′-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.

Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis.

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg, Lys and His, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp, Glu, and Glu, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.

HOMOLOGOUS UP-REGULATION OF KDR/FLK-1 RECEPTOR EXPRESSION BY VASCULAR ENDOTHELIAL GROWTH FACTOR IN VITRO

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pmrhVEGF165 for 0–7 days. Western blot analysis showed a 3–5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2–3-fold increase in high affinity receptor number (5.0 × 104/cellversus 2.4 × 104/cell) without significantly affecting receptor binding affinity (K d 76 pm versus 72 pm). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 μm sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Solution structure of the heparin-binding domain of vascular endothelial growth factor

BACKGROUND Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and is a potent angiogenic and vascular permeabilizing factor. VEGF is also an important mediator of pathological angiogenesis associated with cancer, rheumatoid arthritis and proliferative retinopathy. The binding of VEGF to its two known receptors, KDR and Flt-1, is modulated by cell-surface-associated heparin-like glycosaminoglycans and exogenous heparin or heparan sulfate. Heparin binding to VEGF165, the most abundantly expressed isoform of VEGF, has been localized to the carboxy-terminal 55 residues; plasmin cleavage of VEGF165 yields a homodimeric 110-residue amino-terminal receptor-binding domain (VEGF110) and two 55-residue carboxy-terminal heparin-binding fragments. The endothelial cell mitogenic potency of VEGF110 is decreased significantly relative to VEGF165, indicating that the heparin-binding domains are critical for stimulating endothelial cell proliferation. RESULTS The solution structure of the 55-residue heparin-binding domain of VEGF165 has been solved using data from two-dimensional homonuclear and three-dimensional heteronuclear NMR spectroscopy. The structure has two subdomains, each containing two disulfide bridges and a short two-stranded antiparallel beta sheet; the carboxy-terminal subdomain also contains a short alpha helix. Hydrophobic interactions are limited to sidechains packing against the disulfide bridges. CONCLUSIONS The heparin-binding domain of VEGF has no significant sequence or structural similarity to any known proteins and thus represents a novel heparin-binding domain. Most of the positively charged amino acid sidechains are localized on one side of the carboxy-terminal subdomain or on an adjacent disordered loop in the amino-terminal subdomain. The observed distribution of surface charges suggests that these residues constitute a heparin interaction site.

A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF165) in human plasma. The ELISA can be performed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF (rhVEGF) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF165 isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the intact VEGF165 homodimer, however, the homodimers VEGF121, VEGF110 and the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy individuals range from 20 to 141 pg/ml, with a mean of 42 +/- 22 pg/ml. There were no significant differences in VEGF levels between males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml.

Crystallization of the receptor binding domain of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the product on of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet‐derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P21, with cell parameters a = 55.6 Å, b = 60.4 Å, c = 77.7 Å, β = 90.0°, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild‐type VEGF.

Antibody Discovery via Multiplexed Single Cell Characterization

The secreted immunoglobulin footprint of single hybridoma cells, containing ~10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cells product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at <1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.

Real-time measurement of lysis of mural platelet deposits by fibrinolytic agents under arterial flow

AbstractAn in vitro whole blood reperfusion model was employed to quantify: (a) initial rates of lysis of mural platelet deposits from flowing blood onto fibrin-coated surfaces and (b) plasmin-mediated consumption of plasma plasminogen and fibrinogen, by recombinant tissue-type plasminogen activator (rt-PA) and two t-PA variants, KHRR 296–299 AAAA (K-tPA) and T103N, N117Q, KHRR 296–299 AAAA (TNK-tPA), at wall shear rates of either 500 or 1000 s−1. K- and TNK-tPA are more fibrin-specific than rt-PA, and are also resistant to inactivation by plasminogen activator inhibitor−1 (PAI−1). At 500 s−1, no agent showed significant lysis of mural platelet deposits on fibrin, even at concentrations as high as 10 μ g/ml of blood. At 1000 s−1, each agent demonstrated a dose-dependent lysis of mural platelet deposits, due to plasmin-mediated lysis of the fibrin substrate (fibrinolysis). The local concentration of thrombolytic agents close to the fibrin-coated surface is probably higher than the concentration of released PAI−1 from the adherent and activated platelets. Hence, the initial rates of lysis achieved by K- and TNK-tPA were not significantly different from that by rt-PA, when each agent was tested at either 1 or 10 μ g/ml of blood. However, TNK-tPA, at 1 μ g/ml,> caused the most extensive lysis at the end of the 50 min reperfusion period (50% vs 29% and 17% by rt-PA and K-tPA, respectively). K- and TNK-tPA, at concentrations as high as 10 μ g/ml of blood, caused plasminogen activation that was controlled by the natural plasmin inhibitors, and, thus, no proteolytic degradation of plasma fibrinogen (fibrinogenolysis). On the contrary, rt-PA at 1 μ g/ml revealed slight fibrinogenolysis that became extensive at 10 μ g/ml. This study demonstrates the potential use of an in vitro model, that mimics the in vivo hemodynamic environment, in evaluating the performance of thrombolytic agents. The data suggest that: (a) adequate flow must accompany fibrinolysis for successful embolization, and (b) the TNK variant may lyse annular thrombi after recanalization, at least as efficiently as rt-PA does, while causing lesser defect of systemic hemostasis.