Effective lysis of model thrombi by a t-PA mutant (A473S) that is resistant to alpha2-antiplasmin.

This study used two mutants of tissue-type plasminogen activator (t-PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)-1 and alpha2-antiplasmin (alpha2-AP) to the control of fibrin lysis. Wild-type t-PA was compared with KHRR296-299AAAA, which is resistant to PAI-1, and with A473S, which is resistant to alpha2-AP. We examined these forms of t-PA in model systems that are physiologically relevant. Neutralization of alpha2-AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild-type t-PA or KHRR296-299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of alpha2-AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild-type t-PA; the rate and extent of lysis were enhanced by the addition of antibodies to alpha2-AP or PAI-1. A473S was more effective than wild-type t-PA without the addition of antibodies by virtue of its resistance to alpha2-AP. This resistance was remarkable, in that no complex formed between A473S t-PA and alpha2-AP, even after extended incubation, when 50% of wild-type t-PA could be converted to complex. Comparison of A473S and KHRR296-299AAAA mutants showed their similar effectiveness in lysis of platelet-rich model thrombi. Thus, PAI-1 and alpha2-AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of alpha2-AP as a direct inhibitor of t-PA and further explains the basis of the accepted role of alpha2-AP as a regulator of fibrin persistence and thrombus resistance to lysis.

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator.

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.

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.

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 pM rhVEGF165 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 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 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 microM 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.

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

An 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 microg/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 microg/ml of blood. However, TNK-tPA, at 1 microg/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 microg/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 microg/ml revealed slight fibrinogenolysis that became extensive at 10 microg/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.

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.

Substantially attenuated hemodynamic responses to Escherichia coli-derived vascular endothelial growth factor given by intravenous infusion compared with bolus injection.

Vascular endothelial growth factor (VEGF) produces beneficial angiogenesis in animal models of coronary and peripheral ischemia. However, intravenous bolus injection of Chinese hamster ovary cell (CHO)-derived VEGF produces adverse effects on hemodynamics. The present study examined pharmacokinetic and hemodynamic responses to Escherichia coli-derived VEGF, which will be used in clinical patients, compared with responses to CHO-derived VEGF, and tested whether intravenous infusion of E. coli-derived VEGF attenuates the hemodynamic responses compared with the responses observed with intravenous bolus injection. Hemodynamic parameters were measured before and after administration of VEGF in conscious, instrumented rats. Intravenous injection of both CHO- and E. coli-derived VEGF produced a similar maximal reduction in arterial pressure, although E. coli-derived VEGF exhibited less of a depressor effect in the initial phase after injection. Either infusion or injection of E. coli-derived VEGF caused hypotension, tachycardia and reduced cardiac output and stroke volume, which were significantly attenuated when given by infusion compared with injection. The maximal hypotensive and tachycardiac responses to infusion were decreased by 50 to 60% compared with those responses observed after injection. Cardiac output was maximally reduced by 34% after injection, but only 18% after infusion. A sustained elevation in systemic vascular resistance observed after injection was avoided after infusion. Thus, the hemodynamic side effects of VEGF administration can be substantially attenuated by controlling the rate of VEGF infusion. The data indicate that infusion, instead of bolus injection, is a more appropriate regimen for VEGF administration.

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.

1H, 13C, and 15N backbone assignment and secondary structure of the receptor-binding domain of vascular endothelial growth factor.

Nearly complete sequence-specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor-binding domain of vascular endothelial growth factor (VEGF), a 23-kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple-resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA-(CO)NH] and a 3D 15N-TOCSY-HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45 degrees C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1H alpha, 13C alpha, 13C beta, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N-NOESY-HSQC spectrum. Two helices and a significant amount of beta-sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192-7197].

Disulfide structure of the heparin binding domain in vascular endothelial growth factor: characterization of posttranslational modifications in VEGF.

Preparations of recombinant human vascular endothelial growth factor (VEGF165) expressed in Chinese hamster ovary (CHO) cells and Escherichia coli were compared using a variety of analytical methods. Amino terminal sequence analyses of both the CHO- and E. coli-derived VEGF165 confirmed the predicted amino terminal sequence for VEGF165, although the CHO VEGF165 exhibited a heterogeneous amino terminus with sequences beginning at Ala-1 (76%), Pro-2 (4%), Ala-4 (13%), and Glu-5 (7%). Tryptic digests of reduced and carboxymethylated CHO- and E. coli-derived VEGF165 were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF165. The N-linked carbohydrate in the CHO-derived VEGF165 was determined to be a complex fucosylated biantennary structure. The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF165 confirmed 98% of the primary structure. Disulfide linkages for the eight cysteine residues in the carboxyl terminal heparin binding domain were assigned by amino terminal sequencing of fragments produced by tryptic digests of each native molecule. The following disulfides have been identified for both CHO- and E. coli-derived VEGF165: Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160. Plasmin cleavage of VEGF165 yields an N-terminal homodimeric VEGF110 and a 55-amino-acid carboxyl terminal domain. VEGF110 was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated. The 55-amino-acid carboxyl terminal region of VEGF165 appears to be a unique heparin binding domain with no known protein homology.

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 production 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 P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, 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.

Limiting systemic plasminogenolysis reduces the bleeding potential for tissue-type plasminogen activators but not for streptokinase.

Clinical experience suggests that thrombolytic-induced bleeding is associated with systemic activation of the thrombolytic system. Using fibrin specific variants of tissue-type plasminogen activator (t-PA) and making use of the apparent fibrin specificity of streptokinase (SK) in the rabbit we tested the hypothesis that minimizing systemic plasmin production and fibrinogenolysis will decrease hemorrhages in models of peripheral bleeding and embolic stroke. t-PA consumed 51% of the available fibrinogen; caused cerebral bleeds and increased peripheral bleeding time. Fibrin-specific variants of t-PA depleted less than 20% of the fibrinogen and did not cause peripheral or cerebral bleeding. However, an equipotent dose of SK converted only 12% of the available fibrinogen but increased bleeding time and caused hemorrhagic conversion in 75% of embolic stroke model animals treated. The data suggest that bleeding associated with tissue-type plasminogen activators is linked to systemic plasmin generation and subsequent fibrinogenolysis. This hypothesis does not explain the mechanism(s) of SK-induced bleeding.

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. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, 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.

Locating the unpaired cysteine of tissue-type plasminogen activator.

Variants of tissue-type plasminogen activator (t-PA) were constructed with selected cysteines replaced by alanine to evaluate the role of an unpaired cysteine, which has been presumed to be in the growth factor module. C75A, C83A, C84A and CC83-84AA variants of t-PA were expressed transiently in human embryonic kidney cells. The biochemical properties of these variants provided experimental evidence to identify the unpaired cysteine in t-PA. Assays of amidolytic activity, plasminogen activation (in the presence or absence of fibrinogen or fibrin), plasma clot lysis, fibrin binding, clearance in mice, and interaction with a panel of monoclonal antibodies were performed as the basis for comparing these variants with wild-type t-PA. In all assays, C83A t-PA was biochemically equivalent to wild-type t-PA. C75A t-PA, C84A t-PA and CC83-84AA t-PA variants exhibited reduced activities in a variety of functional assays. These variants displayed two-to threefold lower activity in fibrinogen or fibrin stimulated plasminogen activation, and fivefold reduced plasma clot lysis activity compared with that of wild-type t-PA. The affinity of C75A t-PA and C84A t-PA for fibrin was decreased more than two orders of magnitude compared with C83A t-PA or wild-type t-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced 2-fold in mice. The C75A, C84A and CC83-84AA variants displayed significantly decreased reactivity with anti-tPA monoclonal antibodies specific for finger/growth factor domain epitopes. These data are consistent with a disulfide linkage of Cys75 with Cys84 and that Cys83 exists as an unpaired sulfhydryl. The significance of the unpaired cysteine is as yet undetermined since C83A t-PA and wild-type t-PA are functionally equivalent.

The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

Hypoxic regulation of vascular endothelial growth factor in retinal cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an angiogenic protein and vasopermeability factor whose intraocular concentrations are closely correlated with active neovascularization in patients with diabetes mellitus, central retinal vein occlusion, retinopathy of prematurity, and rubeosis iridis. OBJECTIVE: To determine whether hypoxia could induce expression of VEGF in retinal cells, which then promotes retinal endothelial cell proliferation. METHODS: Retinal pigment epithelial cells, pericytes, and microvascular endothelial cells were exposed to hypoxic conditions in vitro, and RNA expression of VEGF was evaluated by Northern blot analysis. The VEGF-specific proliferative potential of the medium was measured by means of retinal endothelial cell growth assays and VEGF-neutralizing VEGF receptor IgG chimeric protein. RESULTS: The VEGF RNA levels increased within 4 hours and reached elevations of threefold to 30-fold after 18 hours of hypoxia (0% to 5% oxygen, 5% carbon dioxide, 90% to 95% nitrogen) in all cell types (.01 < P < .03). Stimulation was dependent on oxygen concentration. The VEGF RNA levels were normalized by reinstitution of normoxia for 24 hours (P < .004). Medium conditioned by hypoxic retinal pericytes and retinal pigment epithelial cells stimulated retinal endothelial cell growth by 20% (P = .04), and this stimulation was entirely inhibited by VEGF-neutralizing receptor chimeric protein (P = .02). CONCLUSION: Hypoxia increases VEGF expression in retinal cells, which promotes retinal endothelial cell proliferation, suggesting that VEGF plays a major role in mediating intraocular neovascularization resulting from ischemic retinal diseases.

New variant of human tissue plasminogen activator (TPA) with enhanced efficacy and lower incidence of bleeding compared with recombinant human TPA.

BACKGROUND: The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model. METHODS AND RESULTS: After 60 minutes of arterial occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV infusion for 90 minutes; n = 22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as IV bolus; n = 16) was administered. Blood flow through the artery was monitored for an additional 120 minutes. Bleeding was assessed by weighing the amount of blood absorbed in a gauze pad placed in a subcutaneous muscular incision. Recanalization rates and duration of recanalization were dose dependent. The doses that produced > 80% recanalization rates with the longest duration of recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg for TNK-TPA. At these doses, time to reperfusion (mean +/- SEM) was significantly faster (11 +/- 2 versus 23 +/- 7 minutes) and duration of recanalization longer (77 +/- 9 versus 51 +/- 18 minutes) for TNK-TPA compared with TPA (P < .025). Weights of the residual thrombi of the TPA group were greater than those of the TNK-TPA group (P = .004). Concentrations of fibrinogen, plasminogen, and alpha 2-antiplasmin at 120 minutes were significantly higher for TNK-TPA-treated animals compared with TPA-treated animals (P < .001). ANOVA of the blood loss data determined that there were significant differences between thrombolytic agents but not between doses. After correction for saline controls, total blood loss for pooled doses of TPA and TNK-TPA was 82 +/- 6 mg and 40 +/- 4 mg, respectively (P < .01). CONCLUSIONS: From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.

Visualization of thrombi in pulmonary arteries with radiolabeled, enzymatically inactivated tissue-type plasminogen activator.

BACKGROUND: Despite the high frequency of pulmonary thromboembolism and its significant morbidity and mortality, diagnosis remains suboptimal. We have been developing a method for prompt detection with the use of radiolabeled, inactivated tissue-type plasminogen activator (TPA) and performed the present study to determine whether its use permits rapid scintigraphic visualization of pulmonary thrombi in vivo. METHODS AND RESULTS: The thrombolytic, but not fibrin-binding, property of TPA was inactivated with a tripeptide chloromethyl ketone (YPACK) that had already been iodinated with 123I to radiolabel the TPA. Pulmonary arterial thrombosis was induced in nine dogs with the use of guide wires modified to provide thrombogenic tips. 123I-YPACK-TPA (1.1 to 7.8 mCi, 0.5 to 7.8 mg) was infused for 5 minutes into either the systemic or the pulmonary circulation. Clearance of radioactivity from the blood was rapid and indistinguishable from that of unlabeled, thrombolytically active TPA, with only 6.7 +/- 1.0% (mean +/- SEM) of peak radioactivity remaining after 60 minutes and minimal release of labeled fragments from the liver during this interval. Thrombi were visualized with single photon emission computed tomography and/or planar imaging 40 to 120 minutes after infusion of tracer in all seven animals given at least 3.7 mCi of 123I-YPACK-TPA. Ratios of radioactivity in thrombus (wet mass, 610 +/- 64 mg) to blood were high (14 +/- 3:1). CONCLUSIONS: The use of radiolabeled TPA in which thrombolytic activity is inactivated permits prompt scintigraphic detection of thrombi in pulmonary arteries in vivo.

Scintigraphic visualization of pulmonary thrombi with 123I-YPACK-TNK-tPA.

BACKGROUND: We have previously demonstrated that radiolabeled tissue-type plasminogen activation (tPA) in which the plasminogen-activating catalytic site has been inactivated binds avidly to thrombi and can be used for scintigraphic detection of pulmonary thrombi in vivo. The present study was performed to overcome identified limitations of the initially developed approach and to determine whether a tracer made with a molecular variant of tPA, TNK-tPA, would provide superior images of pulmonary thrombi and hence facilitate differential diagnosis of pulmonary embolism from acute myocardial infarction. It was thought that TNK-tPA may be superior in view of its longer biological half-life and less avid uptake by macrophages that would otherwise contribute to high background because of non-clot-selective uptake of the tracer. METHODS: 123I-tyrosylprolylarginyl chloromethyl ketone (123I-YPACK-TNK-tPA) was infused into the systemic circulation of dogs with either pulmonary or right ventricular thrombi induced with thrombogenic tips of modified guide wires. Planar and single-photon emission computed tomography (SPECT) scintigraphic data were obtained, and blood and tissue samples were acquired for analysis of the distribution of the radiotracer over time. RESULTS: Tracer cleared from blood with an alpha phase half-life of 10 +/- 1 min, paralleling the clearance of unlabeled TNK-tPA. Only minimal release of labeled fragments from liver into blood occurred during the entire time course of the imaging studies. Pulmonary thrombi were visualized with SPECT within 30-120 min in all dogs. Images were superior to those obtained after infusion of labeled wild-type tPA, primarily because of diminished background radioactivity and consequently increased scintigraphic contrast. In one dog which had a right ventricular thrombus, the thrombus was readily detectable in both planar and SPECT images. CONCLUSIONS: Radiolabeled TNK-tPA in which plasminogen-activating activity has been inhibited biochemically is an excellent radiopharmaceutical for prompt scintigraphic detection of pulmonary and ventricular thrombi in vivo, and an attractive candidate for rapid, sensitive and non-invasive diagnosis of pulmonary thromboembolic disease in patients.

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.