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Biomedical subjects

B R Binder

Publications and source records attributed to B R Binder.

At least 19 recordsLinked to original sources
PubMed1995

High precision measurement of electrical resistance across endothelial cell monolayers.

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 omega cm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric field across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 omega resistance changes of the endothelium on top of a 21 omega series resistance of the support and bathing fluid layers. Histamine (10 microM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 microM) was less effective, PAF (380 nM) and LTC4 (1 microM) had no effect. TEER was also reduced by the calcium ionophore A-23187 (10 microM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.

Bradykinin↗
PubMed1995

Growth and fibrinolytic parameters of human umbilical vein endothelial cells seeded onto cardiovascular grafts.

It was the aim of this study to investigate possible effects of biomaterials used to produce vascular grafts on the fibrinolytic system of endothelial cells. Therefore growth conditions for human umbilical vein endothelial cells on polytetrafluoroethylene and on polyurethane were optimized. Tissue culture polystyrene was used as a control material. We could demonstrate that precoating of the materials with fibronectin significantly increased the growth rate of human umbilical vein endothelial cells on these materials. Furthermore, we showed that human umbilical vein endothelial cells grown on polytetrafluoroethylene or polyurethane released more plasminogen activator inhibitor-1 and tissue type-plasminogen activator into the conditioned media than did human umbilical vein endothelial cells grown on tissue culture polystyrene. Human umbilical vein endothelial cells cultured on polytetrafluoroethylene also deposited more plasminogen activator inhibitor-1 into the extracellular matrix than did control cells grown on tissue culture polystyrene. Our results give evidence that human umbilical vein endothelial cells grown on two biomaterials used to construct vascular grafts, namely polytetrafluoroethylene and polyurethane, produce tissue-type plasminogen activator as well as plasminogen activator inhibitor-1, two major components of the fibrinolytic system also expressed by endothelial cells in vivo. In conclusion, our data suggest that endothelial cells grown on vascular grafts show functional integrity concerning their fibrinolytic system, which in turn might contribute to reduce the thrombogenic properties of the graft material.

Biocompatible Materials↗
PubMed1995

Endothelial cell injury induced by plasmin in vitro.

We investigated the effect of plasmin on the integrity and function of endothelial cells to elucidate the mechanism by which bleeding or rethrombosis may be induced in thrombolytic therapy. When incubated with cultured human umbilical vein endothelial cells (HUVECs), plasmin increased the endothelial permeability to serum albumin 10 minutes after the incubation. Plasmin damaged the cell membranes 30 minutes after the incubation, detached the cells from the matrix 3 hours after the incubation, and finally induced cell lysis. Such damaging effects on HUVECs were not observed with plasminogen or plasmin pretreated with aprotinin and alpha 2-plasmin inhibitor, suggesting that the catalytic function of plasmin plays an important role in inducing this damage. Sulfur 35-labeled glycosaminoglycans (35S-GAGs) of HUVECs were decreased 1 hour after the incubation of plasmin with HUVECs, and the thrombomodulin (TM) activity of HUVECs measured by protein C activation capacity was decreased 6 hours after the incubation. Neither 35S-GAGs nor the TM activity of HUVECs was decreased after the incubation of plasmin pretreated with aprotinin and alpha 2-plasmin inhibitor. These findings suggest that the nonthrombogenic properties of endothelial cells can be damaged by the proteolytic action of plasmin. Our findings, taken together, suggest that plasmin damages the endothelial barrier function, endothelial cell integrity, and nonthrombogenic properties. These damaging effects of plasmin on endothelial cells may be related to the pathophysiology of bleeding or rethrombosis observed in patients undergoing high-dose thrombolytic therapy for thrombosis.

Cell Membrane Permeability↗
PubMed1994

Fibrinolytic system of cultured rabbit aortic endothelial cells.

The fibrinolytic system of rabbit aortic endothelial cells (RAECs) isolated from male New Zealand White rabbits was investigated. The components of the fibrinolytic system in the conditioned media (CM) were analysed by SDS-PAGE followed by fibrin autography and reverse fibrin autography. The major lytic zones appeared at 54 and 70 KD and minor lytic zones could be detected between 100 and 120 KD. A plasminogen activator inhibitor was localised at 50 KD. Addition of amiloride abolished the lytic zones at 54 KD and 100 KD. There was a time dependent increase in plasminogen activator and inhibitor activities as revealed by an assay involving plasminogen and chromogenic plasmin substrate. A plateau was reached after 16-20 hours. When mRNA obtained from RAECs cultured for 16 hours in serum free medium was analysed for the presence of specific messages of tissue type plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAI-1) using human probes, weak specific binding could be seen for both t-PA and mu-PA while the PAI-1 probe gave a strong specific signal at 3.4 Kb and a weak signal at 2.3 Kb. Both thrombin (1U/ml) and endotoxin (100 ng/ml) increased the release of PAI-1 activity into CM significantly, while there was no significant change in PAs. The increase of PAI-1 was reflected by increased PAI-1 mRNA levels. The data suggests that rabbit ECs can be used to investigate endothelial cell mediated fibrinolysis and supplement results obtained in investigations employing the rabbit as an in vivo animal model to study the fibrinolytic system.

Animals↗
PubMed1994

Hepatocyte growth factor stimulates expression of plasminogen activator inhibitor type 1 and tissue factor in HepG2 cells.

HGF is a powerful mitogen for both rat and human hepatocytes, epithelial cells and endothelial cells in vitro, and is angiogenic in vivo. It has considerable homology with plasminogen and has been shown to upregulate urokinase-type plasminogen activator (u-PA) in endothelial cells as well as u-PA and its receptor in kidney epithelial cells. In this study, we report that human recombinant HGF stimulates expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue factor (TF) in the human hepatoma cell line HepG2. PAI-1 antigen as determined by a specific enzyme-linked immunosorbent assay increased up to threefold in conditioned media of HepG2. This increase was dose dependent with maximum stimulation achieved with a concentration of 50 ng/mL of hepatocyte growth factor (HGF). PAI-1 antigen also increased up to fourfold in the extracellular matrix in HGF treated HepG2. The production of the PAI-1 binding protein vitronectin (Vn) was not affected by HGF. In contrast, TF activity in HepG2 treated with HGF increased up to twofold. As determined by Northern blotting, PAI-1 and TF-specific mRNA were increased significantly in the presence of HGF, whereas Vn mRNA was not affected. The increase in PAI-1 and TF mRNA was also seen when HepG2 were incubated with HGF in the presence of cycloheximide, thereby indicating that de novo protein synthesis is not required to mediate the effect. u-PA could be detected neither in unstimulated or HGF-stimulated HepG2 cells on the antigen level nor on the mRNA level. In conclusion, our data give evidence that HGF, in addition to its proliferative effect for different cell types, is also involved in the local regulation of fibrinolysis and coagulation. One could speculate that HGF might modulate processes requiring matrix degradation by increasing the expression of the protease u-PA in one cell type and by upregulating the expression of the serine protease inhibitor PAI-1 in a different cell type. Because u-PA has been shown to activate latent HGF to the active form, it could furthermore be speculated that by upregulating PAI-1, which in turn could inhibit u-PA, HGF might regulate its own activation.

Carcinoma, Hepatocellular↗
PubMed1994

Effect of type-1 plasminogen activator inhibitor on human leukocyte elastase.

When human leukocyte elastase (HLE) activity (1.0 microgram/ml) was analysed in the presence of PAI-1 (0.15.0 micrograms/ml), HLE activity, measured with the low molecular weight paranitroanilide substrate L-pyroglutamyl-prolyl-L-valine-p-nitroanilide was increased time and dose dependently (a plateau of stimulation was reached after 30 minutes) with a simultaneous decrease in PAI-1 inhibitory activity. This effect was neither influenced by the presence of vitronectin nor heparin. When PAI-1 was converted into its latent form by incubation for 48 hours at 37 degrees C or incubated with an excess of recombinant t-PA to convert free PAI-1 into t-PA-PAI-1 complexes, the stimulatory effect of both the latent and the complexed form of PAI-1 was significantly greater than that of the active form. Analysing HLE PAI-1 interaction on a molecular level using SDS-PAGE, no SDS stable complex formation between HLE and PAI-1 could be observed but lower molecular weight cleavage products of PAI-1 were generated. The stimulatory effect of PAI-1 on HLE activity was not restricted to the low molecular weight pNA-substrate but was also revealed using a natural substrate assay (bovine neck ligament elastin solubilization). Therefore interaction of HLE and PAI-1 seems not to be restricted just to decrease PAI-1 activity but would simultaneously also increase HLE activity, thereby supporting enzymatic activity necessary for migration of leukocytes, dissolution of blood clots and tissue remodelling.

Glycoproteins↗
PubMed1994

The somatomedin B domain of vitronectin. Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor.

We recently localized the high affinity binding site for activated type 1 plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e. amino acid (aa) 1-51) of vitronectin (Vn). In this study to further define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity. Vn polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding activity and stabilized PAI-1 to a similar extent as intact Vn, but polypeptides containing aa 1-30 did not bind to PAI-1 nor stabilize its activity. The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 binding was also determined. One mAb bound to Vn and blocked its ability to bind to PAI-1. It also dissociated pre-existing PAI-1.Vn complexes, and prevented the incorporation of PAI-1 into extracellular matrix of HT 1080 cells. This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30. A second randomly chosen mAb with similar affinity for Vn was inactive in these assays and bound to the region between aa 52 and 239. These results indicate that the high affinity binding site for active PAI-1 in Vn is between aa 1 and 40, and that this domain may also stabilize active PAI-1.

Amino Acid Sequence↗
PubMed1994

Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa.

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

Acrosin↗
PubMed1994

Effect of notoginsenoside R1 on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells.

Among other Chinese herb drugs, Panax notoginseng is used to treat cardiovascular diseases. To elucidate any possible effects of this drug on the hemostatic system in vitro, we analyzed the influence of one of its major active constituents on fibrinolytic parameters of cultured human umbilical vein endothelial cells (HUVECs). When confluent cultures of HUVECs (passages 2 to 3) were conditioned with purified notoginsenoside R1 (NR1), a dose- (0.01 to 100 micrograms NR1/mL) and time-dependent increase in tissue-type plasminogen activator (TPA) synthesis was observed, which was significant from 0.1 microgram NR1/mL and from 6 hours of incubation with 100 micrograms NR1/mL on. TPA antigen increased from 3.9 +/- 0.2 ng per 10(5) cells per 24 hours to 8.0 +/- 0.5 ng per 10(5) cells per 24 hours on addition of 100 micrograms NR1/mL. In contrast, no change in urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) antigen synthesis was seen. There was also no effect of NR1 on PAI-1 deposition in the extracellular matrix. As judged from fibrin autography and reverse fibrin autography, TPA activity and TPA-PAI-1 complexes reached a maximal stimulation of more than threefold and twofold, respectively, at a concentration of 100 micrograms NR1/mL in conditioned media. On the contrary, NR1 induced a more than fivefold decrease in PAI-1 activity at the same concentration of NR1 in conditioned media. On Northern blot analysis of RNA obtained from NR1-stimulated and control HUVECs, NR1 induced a significant increase in TPA mRNA (192% of control value at 100 micrograms NR1/mL) while PAI-1 mRNA remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents, Phytogenic↗
PubMed1994

Fibrinogen, t-PA, and PAI-1 plasma levels in patients with pulmonary hypertension.

We measured fibrinogen levels as well as the fibrinolytic parameters tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) in plasma samples obtained at basal conditions and after stimulating the fibrinolytic system by venous occlusion (VO). Samples were taken from patients with primary pulmonary hypertension (PPH), with secondary thromboembolic pulmonary hypertension (SPHTH), with secondary pulmonary hypertension due to congenital heart disease with Eisenmenger's reaction (SPHCD), and from healthy control individuals (CON). Fibrinogen levels were not significantly different between the groups with PPH and SPHTH or between SPHCD and CON. The latter groups, however, exhibited significantly lower fibrinogen plasma levels compared with PPH or SPHTH (p < 0.01). Basal plasma levels of t-PA antigen, t-PA activity, and PAI-1 activity, respectively, did not differ significantly between the study groups. After VO, mean t-PA activity levels increased to a higher extent in control subjects compared with patients with PPH, or SPHTH, or SPHCD, with significant differences only between CON and SPHTH or CON and PPH (p < 0.03). Patients with PPH and SPHTH exhibit both increased fibrinogen plasma levels and a diminished fibrinolytic response compared with healthy subjects. Moreover, the fibrinogen plasma levels in patients with SPHCD are in normal range, and the fibrinolytic response is similar to CON compared with PPH and SPHTH, thus indicating the existence of a comparable prothrombotic situation in patients with PPH and SPHTH.

Adult↗
PubMed1994

Secretory response of the vessel wall to DDAVP and venous occlusion in von Willebrand's disease.

The aim of this study was to compare the secretory response of the vascular wall in vivo to DDAVP (i.v. 0.3 microgram/kg, 30 min) and to venous occlusion (VO, 20 min) in control healthy subjects, patients with von Willebrand's disease type I (vWd I) and patients with von Willebrand's disease type III (vWd III). In controls (n = 10) and vWd I (n = 12), DDAVP induced a 2 to 3-fold rise in plasma von Willebrand factor antigen (vWf: Ag), factor VIII coagulant activity (VIII: C) and tissue--type plasminogen activator antigen (t-PA:Ag). VO was less effective in increasing vWf: Ag and VIII:C but produced a greater rise in t-PA:Ag. Large increments (over 10-fold) were observed in plasmin-alpha 2-antiplasmin complexes following both stimuli. In vWd III (n = 10), DDAVP and VO failed to increase vWf:Ag, VIII:C and t-PA:Ag. No significant changes in plasmin-alpha 2-antiplasmin complexes were observed in this group. Moreover, the baseline t-PA:Ag values were significantly lower in vWd III (2.17 +/- 1.13 ng/ml) than in controls (4.84 +/- 1.97 ng/ml, p < 0.001). A significant increase in urokinase--type plasminogen activator antigen (u-PA:Ag) was found only in controls after VO. Neither controls nor patients with vWd showed any changes in plasma fibronectin levels following DDAVP. The low t-PA:Ag results and the abnormal fibrinolytic response to DDAVP and VO in patients with severe (type III) vWd indicate that their endothelial cell abnormality is more extensive than the defect in the synthesis or release of vWf.

Adult↗
PubMed1993

Organization of the gene encoding mouse vitronectin.

The gene (Vn) encoding the mouse vitronectin was isolated and its nucleotide sequence determined. The gene covers approximately 3 kb of genomic DNA. Alignment of the genomic sequence with that of the cDNA revealed that Vn consists of eight exons, interrupted by seven introns ranging in size from 78 to 723 bp.

Amino Acid Sequence↗
PubMed1992

Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor.

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.

Amides↗
PubMed1992

Polar secretion of endothelin-1 by cultured endothelial cells.

The aim of this study was to determine the permeability of endothelial monolayers for endothelin-1 and a possible directionality of the endothelin-1 secretion process. Human umbilical vein endothelial cells were cultured on acellular amniotic membranes, dividing the tissue culture wells into an apical (luminal) and a basolateral (abluminal) compartment. Whereas in the absence of endothelial monolayers 44.9 +/- 2.3 and 43.5 +/- 2.0% of the unilaterally added endothelin-1 permeated from the apical to the basolateral side and from the basolateral to the apical side, respectively, only 6.5 +/- 0.6 and 6.6 +/- 0.4% diffused in the presence of endothelial cells. Analyzing endothelin-1 secretion, approximately 80% of the total amount of synthesized endothelin-1 was found in the basolateral compartment; thrombin (10 units/ml) stimulated the production of endothelin-1 approximately 2-fold, but did not change the relative distribution of endothelin-1 between the apical and basolateral compartments. In the presence of dexamethasone (10(-7) M), a decrease in the level of endothelin-1 was found in the apical compartment, whereas the total amount of endothelin-1 produced was not affected. Dexamethasone did not influence the permeability of human umbilical vein endothelial cell monolayers for endothelin-1. These results strongly support the hypothesis that endothelin-1 is a local paracrine regulator of vasotone.

Amnion↗
PubMed1992

Direct evidence for systemic fibrinogenolysis in a patient with metastatic prostatic cancer.

Although the possible occurrence of systemic fibrinogenolysis has been suggested in patients with metastasising prostatic cancer (MPC), direct evidence is lacking. We report on a patient with MPC whose laboratory data were consistent with hyperfibrinolysis: marked decrease of alpha 2-antiplasmin (AP) level (less than 50% of normal), increase of plasmin-alpha 2-antiplasmin complex, D-fragment of fibrin and fibrinogen degradation products [FDP(D)] and cross-linked fibrin degradation products (XDP). The patient neither showed laboratory nor clinical evidence for consumption coagulopathy except for a slight increase in thrombin-antithrombin III complex level. Immunoblotting of the patient's serum using an anti-fibrinogen antibody revealed the presence of a 250 kDa protein in addition to DD fragments. Following reduction of this protein by 2-mercaptoethanol after extraction from SDS-PAGE gel, gamma-chain of fibrinogen (47 kDa) was found by immunoblotting using a monoclonal antibody recognising a 86-302 residue of the gamma-remnant of fibrinogen. Moreover, the 250 kDa protein did not bind to Sepharose 4B to which a monoclonal antibody recognising the N-terminus of fragment D was conjugated. These findings indicated that this protein was not fragment DY, but rather fibrinogen fragment X. With the retraction of the prostatic tumour by an effective therapy, the patient's AP level increased gradually. When the plasma AP level rose to 60% of normal, the fragment X was no longer detectable. These findings suggested that systemic fibrinogenolysis occurred in the patient with MPC only when AP levels were markedly decreased.

Aged↗
PubMed1992

Modulation of heparin cofactor II activity by glycosaminoglycans and adhesive glycoproteins.

Heparin cofactor II (HCII) is a specific thrombin inhibitor; its inhibitory activity is stimulated by heparin (Hep) and dermatan sulfate (DS). Vitronectin (VN), a heparin binding adhesive glycoprotein present in plasma and extracellular matrix, has been shown to decrease the stimulatory effect of Hep but not of DS on thrombin inhibition by HCII (Preissner and Sié, 1988). We analyzed the effect of glycosaminoglycans (GAGs) and GAG-binding proteins on the HCII/thrombin interaction in more detail. HCII was purified from the supernatant of barium citrate adsorbed normal human plasma by polyethylene glycol precipitation followed by affinity chromatography on heparin-Sepharose CL-6B and ion-exchange chromatography on a QAE-Sephadex A-50. Inhibition of thrombin by HCII was studied in the absence and presence of GAGs (hep 0.03-30 micrograms/ml, DS 0.05-50 micrograms/ml, heparan sulfate (HS) 0.05-50 micrograms/ml) in a chromogenic substrate assay using S-2366 as a thrombin substrate. The effects of VN and fibronectin (FN) on HCII stimulation by GAGs were determined. In addition to Hep and DS the inhibitory effect of HCII was stimulated by HS, however, to a lesser extent. Using 0.03U/ml thrombin and 1nM HCII the stimulatory effect of GAGs was completely inhibited when Hep (less than or equal to 0.3 micrograms/ml) was preincubated with VN (60 micrograms/ml) and decreased to less than 50% when HS (50 micrograms/ml) was preincubated with VN (60 micrograms/ml). VN had no effect on DS as already described previously.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗