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1.
Allergy ; 69(2): 237-45, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24127643

ABSTRACT

BACKGROUND: Edema represents a key feature of nasal polyp (NP) disease. Members of the vascular endothelial growth factor (VEGF) family may be involved, but the precise role of VEGF-A, VEGF-B, placental growth factor (PlGF), and their receptors VEGFR1 and VEGFR2 in NP edema formation remains elusive. OBJECTIVE: Exploring the expression of VEGF family members and their receptors and their correlation with clinical, radiological, and edema markers in NP. METHODS: The expression of VEGF-A, VEGF-B, PlGF, VEGFR1, and VEGFR2 was measured in NP (n = 23) and control tissue (n = 22) at mRNA and protein level. Edema was evaluated by measuring albumin levels and wet/dry ratios. Computed tomography (CT) scans were scored using the Lund-Mackay scoring system. IL-5 mRNA expression was determined by real-time RT-PCR. Cell suspensions from NP (n = 10) and control tissue (n = 12) were stimulated in vitro with IL-1ß or TNFα. RESULTS: mRNA expression of VEGFR1 and VEGF-B was significantly higher in NP compared with control tissue. Expression levels of VEGF-B and VEGFR1 significantly correlated with NP albumin content (VEGF-B: P = 0.0208; VEGFR1: P = 0.0293), CT scan scores (VEGF-B: P = 0.0075; VEGFR1: P = 0.0068), and IL-5 mRNA (VEGF-B: P = 0.0027; VEGFR1: P = 0.0001). In vitro stimulation of control and NP tissue cell suspensions with IL-1ß or TNFα significantly reduced the expression of VEGFR2 in control tissue, without altering VEGFR1 and VEGF-B expression. hVEGF-B induced nitric oxide production in NP macrophages (P < 0.05). CONCLUSION: Expression levels of VEGFR1 and VEGF-B correlate with edema and clinical markers of NP disease and therefore represent potential therapeutic targets.


Subject(s)
Nasal Polyps/metabolism , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Female , Humans , Immunohistochemistry , Male , Membrane Proteins/analysis , Membrane Proteins/biosynthesis , Nasal Polyps/pathology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor B/analysis , Vascular Endothelial Growth Factor B/biosynthesis , Vascular Endothelial Growth Factor Receptor-1/analysis , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor Receptor-2/biosynthesis
2.
Br J Cancer ; 106(4): 678-84, 2012 Feb 14.
Article in English | MEDLINE | ID: mdl-22333707

ABSTRACT

BACKGROUND: TB-403 (RO 5323441), a humanised monoclonal antibody, is a novel antiangiogenesis agent directed against placental growth factor. The safety, pharmacokinetics (PK), and antitumour activity of TB-403 were assessed in a phase I, dose-escalation study in patients with advanced solid tumours. METHODS: Patients in sequential dose groups received either weekly doses of 1.25, 5.0, or 10 mg kg(-1) or doses of 20 or 30 mg kg(-1) every third week. RESULTS: Twenty-three patients were enrolled and received TB-403. The most common adverse events (AEs) were fatigue, constipation, pyrexia, dyspnoea, and nausea. One serious AE, a lung embolus in a patient with non-small cell lung cancer treated with 10 mg kg(-1) weekly, was deemed possibly related to TB-403. No dose-limiting toxicities were observed, and a maximum-tolerated dose was not reached. The PK parameters were dose linear and the terminal half-life values ranged from 9 to 14 days. Six patients exhibited stable disease for at least 8 weeks. Two patients, (oesophageal squamous cell carcinoma and pancreatic adenocarcinoma) both treated with 5 mg kg(-1) weekly, remained stable for 12 months. CONCLUSION: TB-403 treatment in this patient population is well tolerated, with a safety profile distinct from that of vascular endothelial growth factor-axis inhibitors.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Pregnancy Proteins/immunology , Adult , Aged , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/adverse effects , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal, Humanized/administration & dosage , Drug Administration Schedule , Female , Humans , Infusions, Intravenous , Male , Maximum Tolerated Dose , Middle Aged , Placenta Growth Factor
3.
J Thromb Haemost ; 7(3): 429-37, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19207364

ABSTRACT

BACKGROUND: The inhibitory activity of an anti-factor VIII (FVIII) antibody can be modulated through glycosylation of the antigen binding site, as has recently been described. This offers the opportunity to develop an optimized anticoagulant agent targeting partial FVIII inhibition. OBJECTIVES: We investigated in non-human primates the antithrombotic activity, pharmacokinetics,and pharmacodynamics of a human monoclonal antibody, Mab-LE2E9Q, inhibiting FVIII activity partially. METHODS: The ability of Mab-LE2E9Q to prevent thrombosis was evaluated in baboons after administration of 1.25 and 5 mg kg(-1) antibody or saline as a single intravenous (i.v.) bolus. Thrombus development was recorded in expansion ('venous') and in Dacron ('arterial') thrombosis chambers incorporated in an extracorporeal arteriovenous shunt implanted between the femoral vessels 1 h, 24 h and 7 days after the administration of Mab-LE2E9Q. RESULTS: Mab-LE2E9Q reduced thrombus growth to a similar extend 1 h, 1 day and 1 week after administration of the antibody. Ex vivo pharmacodynamic analysis indicated that the evaluation of the residual FVIII activity was strongly dependent on the type of FVIII assay and on the phospholipid concentration in the assay. No significant difference in bleedings was observed between animals treated with Mab-LE2E9Q or with saline. CONCLUSIONS: Understanding the role of glycosylation in FVIII inhibition by a human monoclonal antibody allowed selection of an antibody inhibiting only moderately FVIII activity while significantly reducing thrombus development in a baboon extracorporeal model. As that antibody did not increase the bleeding tendency, it may represent a novel type of a long-acting antithrombotic agent with an optimal safety/efficacy profile.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Factor VIII/immunology , Thrombosis/prevention & control , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/therapeutic use , Drug Evaluation, Preclinical , Drug-Related Side Effects and Adverse Reactions , Glycosylation , Hemorrhage/chemically induced , Humans , Papio , Time Factors
4.
J Thromb Haemost ; 5(6): 1237-42, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17362226

ABSTRACT

BACKGROUND: Oral anticoagulant therapies targeted at thrombin are being developed to overcome limitations associated with current standard therapies. OBJECTIVES: This study was undertaken to assess and compare the antithrombotic and anticoagulant effects of the novel, selective and reversible, direct thrombin inhibitor (DTI), dabigatran, and its oral prodrug dabigatran etexilate, to that of unfractionated heparin (UFH), hirudin and melagatran using a rabbit model of venous thrombosis. METHODS: A rabbit model of venous thrombosis consisting of endothelial damage with blood flow reduction was used with minor modifications. RESULTS: All compounds demonstrated a dose-dependent reduction in thrombus formation following i.v. administration with complete or almost complete inhibition at the highest doses. Dabigatran (in the dose range 0.03-0.5 mg kg(-1)) had a 50% effective dose of 0.066 mg kg(-1). By comparison, UFH (5-50 U kg(-1)), hirudin (0.01-0.05 mg kg(-1)) and melagatran (0.01-0.3 mg kg(-1)) had a 50% effective dose of 9.8 U kg(-1), 0.016 mg kg(-1) and 0.058 mg kg(-1), respectively. Similarly, oral dabigatran etexilate (1-20 mg kg(-1)) inhibited thrombus formation in a dose-dependent manner. Maximum inhibition was achieved within 1 h of administration, suggesting a rapid onset of action. For both routes of administration, inhibition of thrombus formation directly correlated with prolongation of the activated partial thromboplastin time. CONCLUSIONS: These findings demonstrate the potent anticoagulant and antithrombotic activity of dabigatran as a selective thrombin inhibitor in a rabbit model of venous thrombosis. Notably, dose-dependent and long-lasting antithrombotic efficacy was observed after application of its oral form dabigatran etexilate, which is currently undergoing phase III clinical development.


Subject(s)
Anticoagulants/therapeutic use , Benzimidazoles/therapeutic use , Fibrinolytic Agents/therapeutic use , Prodrugs/therapeutic use , Pyridines/therapeutic use , Venous Thrombosis/drug therapy , Administration, Oral , Animals , Anticoagulants/administration & dosage , Azetidines/administration & dosage , Azetidines/therapeutic use , Benzimidazoles/administration & dosage , Benzylamines/administration & dosage , Benzylamines/therapeutic use , Dabigatran , Disease Models, Animal , Dose-Response Relationship, Drug , Fibrinolytic Agents/administration & dosage , Heparin/therapeutic use , Hirudin Therapy , Injections, Intravenous , Male , Partial Thromboplastin Time , Prodrugs/administration & dosage , Pyridines/administration & dosage , Rabbits , Venous Thrombosis/blood
5.
Curr Med Chem ; 11(17): 2245-60, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15379710

ABSTRACT

The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis also termed primary, secondary and tertiary hemostasis. From the platelet transcriptome 6000 mRNA species and represent receptors, ion channels, signalling molecules, kinases, phosphatases, and structural, metabolic and regulatory proteins. This abundance of regulatory proteins points towards the importance of signal transduction in platelet function. First platelets adhere to collagen, this induces activation signals such as TXA(2) that induces further Ca(2+) increase. Consecutively, fibrinogen binds to the integrin alpha(IIb)beta(3) resulting in aggregation.This self-amplifying process is controlled by signals, from endothelial cells, to restrict the platelet plug to the site of vessel injury. Secondary hemostasis (coagulation) consists of an extrinsic and intrinsic pathway. Thrombin is generated via Factor Xa resulting from the extrinsic tenase reaction that is turned of by tissue factor pathway inhibitor. While thrombin generation is maintained via positive feedback mechanisms activating factors V, VIII and XI. Excess thrombin is inhibited by antithrombin or by autodownregulation via activation of protein C. Since minor injuries are common, platelets and plasma clotting factors constantly produce clots to stop bleeding. If clots remained after the tissue healing, the vascular bed would become obstructed with clots therefore this is regulated by fibrinolysis, tertiary hemostasis. Tissue-type plasminogen activator synthesised by the endothelium, converts plasminogen to plasmin, the clot lysis enzyme. Plasmin clears the blood vessels by degrading fibrin. Fibrinolysis is controlled by plasminogen activators inhibitor (PAI-1), alpha2-antiplasmin and alpha2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI).


Subject(s)
Fibrin/physiology , Hemostasis/physiology , Antifibrinolytic Agents/pharmacology , Blood Coagulation Factors/metabolism , Blood Platelets/drug effects , Blood Platelets/physiology , Fibrinolysis/physiology , Hemostasis/drug effects , Humans , Platelet Adhesiveness/physiology , Platelet Aggregation/physiology , Signal Transduction/physiology , Thrombosis/etiology
6.
J Biol Chem ; 276(48): 44912-8, 2001 Nov 30.
Article in English | MEDLINE | ID: mdl-11559698

ABSTRACT

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.


Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Plasminogen Activator Inhibitor 1/metabolism , Alanine/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Arginine/chemistry , Aspartic Acid/chemistry , Binding Sites , DNA/metabolism , Epitopes , Glutamic Acid/chemistry , Histidine/chemistry , Humans , Kinetics , Lysine/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Rats , Sequence Homology, Amino Acid , Surface Plasmon Resonance , Swine , Time Factors
7.
Structure ; 9(1): 29-37, 2001 Jan 10.
Article in English | MEDLINE | ID: mdl-11342132

ABSTRACT

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.


Subject(s)
Blood Coagulation Factor Inhibitors/pharmacology , Blood Coagulation/drug effects , Factor Xa Inhibitors , Factor Xa/chemistry , Thrombin/antagonists & inhibitors , Thrombin/chemistry , Thrombin/metabolism , Animals , Cattle , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Thrombosis/prevention & control , Trypsin/metabolism
8.
Thromb Haemost ; 85(5): 866-74, 2001 May.
Article in English | MEDLINE | ID: mdl-11372681

ABSTRACT

Increased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore. eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising alpha-helices hD, hE and hF, beta-strands s4C, s3A, s2A and s1A and the loops connecting these elements). (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data. together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.


Subject(s)
Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Plasminogen Activator Inhibitor 1/immunology , Serine Proteinase Inhibitors/immunology , Animals , Antibodies, Monoclonal/metabolism , Dose-Response Relationship, Drug , Epitope Mapping , Epitopes/metabolism , Genetic Variation , Humans , Models, Molecular , Mutagenesis , Plasminogen Activator Inhibitor 1/pharmacology , Rats , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Serine Proteinase Inhibitors/pharmacology , Tissue Plasminogen Activator/antagonists & inhibitors
9.
J Mol Biol ; 297(3): 683-95, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10731421

ABSTRACT

The crystal structure of a constitutively active multiple site mutant of plasminogen activator inhibitor 1 (PAI-1) was determined and refined at a resolution of 2.7 A. The present structure comprises a dimer of two crystallographically independent PAI-1 molecules that pack by association of the residues P6 to P3 of the reactive centre loop of one molecule (A) with the edge of the main beta-sheet A of the other molecule (B).Thus, the reactive centre loop is ordered for molecule A by crystal packing forces, while for molecule B it is unconstrained by crystal packing contacts and is disordered. The overall structure of active PAI-1 is similar to the structures of other active inhibitory serpins exhibiting as the major secondary structural feature a five-stranded beta-sheet A and an intact proteinase-binding loop protruding from the one end of the elongated molecule. No preinsertion of the reactive centre loop is observed in this structure.A comparison of the present structure with the previously determined crystal structures of PAI-1 in its alternative conformations reveals that, upon cleavage of an intact form of PAI-1 or formation of latent PAI-1, the well-characterised rearrangements of the serpin secondary structural elements are accompanied by dramatic and partly unexpected conformational changes of helical and loop structures proximal to beta-sheet A. The present structure explains the stabilising effects of the mutated residues, reveals the structural cause for the observed spectroscopic differences between active and latent PAI-1, and provides new insights into possible mechanisms of stabilisation by its natural binding partner, vitronectin.


Subject(s)
Plasminogen Activator Inhibitor 1/chemistry , Plasminogen Activator Inhibitor 1/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Biopolymers/chemistry , Biopolymers/metabolism , Crystallization , Crystallography, X-Ray , Dimerization , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Plasminogen Activator Inhibitor 1/genetics , Protein Structure, Secondary , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Thermodynamics , Vitronectin/metabolism
10.
J Biol Chem ; 275(9): 6375-80, 2000 Mar 03.
Article in English | MEDLINE | ID: mdl-10692438

ABSTRACT

The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu(128)-Val(129)-Glu(130)-Arg(131) and Lys(154) (at the hinge region between alpha-helix F and the main part of the PAI-1-molecule) might form the major site of interaction. Therefore a variety of alanine mutants were generated and evaluated for their affinity toward both monoclonal antibodies. The affinity constants of MA-55F4C12 and MA-33H1F7 for PAI-1 were 2.7 +/- 1.6 x 10(9) M(-1) and 5.4 +/- 1.7 x 10(9) M(-1), respectively, but decreased between 13- and 270-fold upon mutation of Lys(154) to Ala(154) or Glu(128)-Val(129)-Glu(130)-Arg(131) to Ala-Ala-Ala-Ala. The combined mutations (PAI-1-EVER/K), however, resulted in an absence of binding to either of the antibodies. Both antibodies bound to PAI-1-wt/t-PA complexes with a similar affinity as to PAI-1-wt (K(A) = 4-5 x 10(9) M(-1)). The epitope localization reveals the molecular basis for the neutralizing properties of both monoclonal antibodies. In addition, it provides new insights into the validity of various models that have been proposed for the serpin/proteinase complex, excluding full insertion of the reactive-site loop.


Subject(s)
Antibodies, Monoclonal/immunology , Epitopes/immunology , Plasminogen Activator Inhibitor 1/chemistry , Serine Proteinase Inhibitors/chemistry , Amino Acid Sequence , Animals , Binding Sites , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Plasminogen Activator Inhibitor 1/immunology , Protein Structure, Secondary , Sequence Alignment , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/immunology
11.
Basic Res Cardiol ; 95(6): 449-56, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11192365

ABSTRACT

Na+/H+ exchange (NHE) plays an important role in the regulation of the intracellular pH (pHi) and in cardiac cell injury induced by ischemia and reperfusion. In the present study, we investigated the effects of BIIB513, a selective NHE-1 inhibitor on myocardial ischemia induced arrhythmias and myocardial infarction, provoked by 30 minutes of left main coronary artery occlusion followed by 2 hours of reperfusion in an anesthetized rat model. Intravenous administration of BIIB513 (0.01-3.0 mg/kg) did not induce changes in blood pressure or heart rate. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced ventricular premature beats, ventricular tachycardia, and a complete suppression of ventricular fibrillation down to the dose of 0.1 mg/kg. BIIB513 (0.01, 0.1, 0.3, 1.0, 3.0 mg/kg) given prior to the coronary artery occlusion dose-dependently reduced the infarct size with an ED50 value of 0.16 mg/kg. BIIB513 (1.0 mg/kg) given prior to reperfusion also reduced infarct size by 47.3 +/- 13.1%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase (CPK). In conclusion, the present study demonstrates the cardioprotective ability of NHE-1 inhibition during myocardial ischemia and reperfusion by reducing serious ventricular arrhythmias and myocardial infarct size in anesthetized rats.


Subject(s)
Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/etiology , Mesylates/therapeutic use , Myocardial Infarction/drug therapy , Myocardial Infarction/etiology , Myocardial Ischemia/complications , Animals , Arrhythmias, Cardiac/physiopathology , Coronary Disease/drug therapy , Coronary Disease/physiopathology , Creatine Kinase/blood , Epinephrine/blood , Hemodynamics/drug effects , Male , Myocardial Reperfusion Injury/drug therapy , Myocardial Reperfusion Injury/physiopathology , Norepinephrine/blood , Rats , Rats, Wistar , Reference Values , Severity of Illness Index
12.
Thromb Haemost ; 79(1): 202-10, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9459348

ABSTRACT

von Willebrand factor (vWF) is instrumental in arterial but has also been implicated in venous thrombogenesis. To address its role in venous thrombosis, experimental thrombosis was induced in the carotid artery and the femoral vein of hamsters, following which thrombus prevention by two different antagonists of vWF was studied. The first antagonist was the anti-human vWF monoclonal antibody AJvW-2, which inhibits the botrocetin and ristocetin induced aggregation of human blood platelets. AJvW-2 reacts with an epitope present in the A1 domain of vWF in very different species (human, pig, rabbit, dog, Guinea pig and rat). This epitope was found to be conformational and overlapping with vWF binding sites for aurin tricarboxylic acid (ATA), but not for botrocetin and heparin. AJvW-2 has affinities for vWF in the absence (Kd = 0.5 +/- 0.03 nmol/l in solution) and in the presence of shear stress (Kd = 3.3 +/- 0.6 nmol/l during perfusion at 1,300 s over subendothelial matrix associated vWF) sufficiently elevated to neutralize vWF. During perfusion of subendothelial matrix with anticoagulated human blood, the surface covered by adhering platelets was reduced by AJvW-2, with IC50s equal to 6.6 +/- 0.34 microg/ml at 1,300 s(-1) and to 1 +/- 0.01 microg/ml at 2,700 s(-1). As a second antagonist, molecular size gel filtered ATA was selected. Fractionated ATA inhibited platelet adhesion to matrix with IC50s equal to 0.27 +/- 0.09 mmol/l at 1,300 s(-1) and 0.16 +/- 0.008 mmol/l at 2,700 s(-1). When administered to hamsters, AJvW-2 prevented thrombosis in the injured carotid artery dose-dependently (ED50 = 0.15 +/- 0.01 mg/kg). Thrombosis in the similarly injured femoral vein was however also inhibited (ED50 = 0.37 +/- 0.06 mg/kg). Likewise, fractionated ATA completely inhibited carotid artery thrombosis (ED50 = 0.42 +/- 0.13 mg/kg), but also interfered with femoral vein thrombosis (apparent ED50 between 2 and 3 mg/kg). We conclude that antagonizing the vWF A1 domain by AJvW-2 and to a lesser extent also by fractionated ATA, inhibits thrombosis not only in the arterial but also in the venous circulation. Since venous thrombi were prevented at only 3-5-fold higher doses of antagonist, vWF participates in injury induced venous thrombosis.


Subject(s)
Carotid Artery Diseases/drug therapy , Femoral Vein/injuries , Thrombophlebitis/drug therapy , Thrombosis/drug therapy , von Willebrand Factor/antagonists & inhibitors , Animals , Antibodies, Monoclonal , Antigen-Antibody Reactions , Cricetinae , Disease Models, Animal , Endothelium, Vascular/cytology , Epitopes , Protein Conformation
13.
Thromb Haemost ; 79(1): 222-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9459351

ABSTRACT

Intravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 +/- 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 +/- 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests. In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50's of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 +/- 0.3 mM. A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced "spontaneous" platelet aggregation in human whole blood. It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.


Subject(s)
Fibrinolytic Agents/therapeutic use , Piracetam/therapeutic use , Platelet Aggregation Inhibitors/therapeutic use , Thrombosis/drug therapy , Animals , Bleeding Time , Cricetinae , Disease Models, Animal , Fibrinogen/metabolism , Humans , In Vitro Techniques , Male , Platelet Count , Protein Binding , Rats , Thrombosis/metabolism
14.
Arterioscler Thromb Vasc Biol ; 18(1): 100-7, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9445262

ABSTRACT

The monoclonal antibody 4E6-based ELISA was used to quantify levels of oxidized LDL in plasma of 65 control subjects, 47 patients transplanted for dilated cardiomyopathy (DCM), and 60 patients transplanted for coronary artery disease (CAD). Levels of oxidized LDL were 0.68+/-0.039 mg/dL (mean+/-SEM), 1.27+/-0.14 mg/dL (P<.001 versus control), and 1.73+/-0.13 mg/dL (P<.001 versus control and <0.01 versus DCM), respectively. Levels of oxidized LDL were significantly lower in transplanted patients with angiographically normal coronary arteries (grade 0, 1.16+/-0.053 mg/dL; n=79) than in patients with mild (grade 1, 2.13+/-0.30 mg/dL; n=18; P<.001 versus grade 0) or severe (grade 2, 3.18+/-0.45 mg/dL; n=10; P<.001 versus grade 0 and P<.05 versus grade 1) coronary artery stenosis. Logistic regression analysis identified three parameters that were significantly and independently correlated with posttransplant CAD: plasma levels of oxidized LDL (P=.0001), length of follow-up (P=.0008), and donor age (P=.047). Thus, the present study demonstrates that plasma levels of oxidized LDL correlate with the extent of CAD in heart transplant patients and suggests that elevated levels of oxidized LDL may be a marker for CAD.


Subject(s)
Coronary Disease/etiology , Heart Transplantation/adverse effects , Lipoproteins, LDL/blood , Coronary Disease/blood , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lipoproteins, HDL/blood , Male , Middle Aged , Oxidation-Reduction , Triglycerides/blood
16.
Blood ; 90(4): 1527-34, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9269770

ABSTRACT

Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1-resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA(-/-)) and PAI-1-overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin-labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA-expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA-deficient mice) or augment (in PAI-1-overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.


Subject(s)
Fibrinolysis , Genetic Therapy , Plasminogen Activator Inhibitor 1/metabolism , Thrombosis/therapy , Tissue Plasminogen Activator/metabolism , Adenoviridae , Alleles , Animals , Cells, Cultured , Fibrinolysis/drug effects , Gene Transfer Techniques , Genetic Vectors , Humans , Mice , Mice, Transgenic , Plasminogen Activator Inhibitor 1/genetics , Recombinant Proteins/pharmacology , Tissue Plasminogen Activator/deficiency , Tissue Plasminogen Activator/genetics
17.
Am J Pathol ; 150(2): 761-76, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9033288

ABSTRACT

Vascular interventions for atherothrombotic disease frequently induce neointima formation, which can contribute to restenosis of blood vessels. As the molecular mechanisms of this process remain largely unknown, quantitative models of arterial injury in transgenic animals may be useful to study this process at the genetic level. Here, an injury model is proposed in which surgically exposed femoral arteries in mice were injured perivascularly via a single delivery of an electric current. Transmission electron microscopy, light microscopy, and immunohistochemistry revealed that electric injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and transiently induced platelet-rich mural thrombosis. A vascular wound-healing response resulted that was characterized by degradation of the mural thrombus, transient infiltration of the vessel wall by inflammatory cells, and progressive removal of the necrotic debris. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the uninjured borders and progressing into the necrotic center. Within 3 weeks after injury, a neointima of 0.026 +/- 0.003 mm2 (n = 7 arteries) was formed that contained a maximum of 12 +/- 1 layers of smooth muscle alpha-actin-immunoreactive cells. Evans blue staining in five electrically injured arteries revealed a denuded distance of 2.8 +/- 0.2 mm immediately after injury, which became progressively re-endothelialized from the uninjured borders to 2.2 +/- 0.08 mm (P = 0.013 vs freshly injured by analysis of variance), 0.8 +/- 0.22 mm (P < 0.001), and 0.005 +/- 0.003 mm (P < 0.001) within 2, 7, and 14 days after injury, respectively. Analysis of 5'-bromo-2'-deoxyuridine incorporation revealed that a maximum of 35 +/- 10% endothelial cells proliferated within 2 days after injury and that in the media and neointima, a maximum of, respectively, 12 +/- 2% and 18 +/- 3% smooth muscle cells proliferated within 2 weeks after injury. Thus, electric injury of arteries provides a model of vascular wound healing with arterial neointima formation and re-endothelialization that may be useful for the genetic analysis of its molecular mechanisms in transgenic mice.


Subject(s)
Electric Injuries/physiopathology , Femoral Artery/injuries , Femoral Artery/physiopathology , Tunica Intima/physiopathology , Wound Healing , Animals , Electric Injuries/complications , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Female , Humans , Immunohistochemistry , Leukocytes/physiology , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Thrombosis/etiology , Thrombosis/pathology
18.
Ann Plast Surg ; 38(1): 56-62, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9015541

ABSTRACT

Numerous clinically relevant animal models exist for thrombosis studies. Few of these are suitable for both arteries and veins. In this investigation, an established venous thrombosis model was adapted through minimal technical adjustments to allow also the study of arterial thrombosis. A standardized subintimal crush injury was performed to carotid arteries or femoral veins of hamsters. Thrombus volumes were then quantified by direct morphometric measurements from serial microscopic sections or by on-line image analysis of light intensity changes from transilluminated vessels. The platelet-rich mural thrombus, which was established within minutes of the trauma, disintegrated during the observation period. The life cycle of the thrombus was different in arteries and veins, but significant linear correlation (p < 0.01) was found in both types of vessel between thrombus volumes measured by the two techniques. The model can consequently be used for comparative in vivo thrombosis studies in small (approximately 1-mm) arteries and veins.


Subject(s)
Carotid Artery Thrombosis/pathology , Femoral Vein , Thrombosis/pathology , Animals , Carotid Arteries/pathology , Carotid Artery Thrombosis/etiology , Cricetinae , Disease Models, Animal , Femoral Vein/pathology , Male , Microscopy, Electron , Microscopy, Electron, Scanning , Thrombosis/etiology , Transillumination
19.
Ann Plast Surg ; 38(1): 63-8, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9015542

ABSTRACT

After a standardized trauma to carotid arteries or femoral veins of hamsters, the antithrombotic effects of two antiplatelet agents (aspirin and the glycoprotein IIb/IIIa antagonist G4120) and two anticoagulants (heparin and the direct thrombin inhibitor r-hirudin) were studied in vivo. The thrombus area volume was assessed by image analysis of the transilluminated experimental vessels. Heparin, r-hirudin, and G-4120 demonstrated a dose-dependent complete inhibition of arterial and venous thrombosis. In contrast, the antithrombotic effect of aspirin was only partial in both vessel types. A significant correlation between activated partial thromboplastin time (aPTT) at the end of the experiments and the antithrombotic effect was observed with the anticoagulant agents. However, only r-hirudin inhibited thrombus formation at a therapeutical prolongation of aPTT, while heparin required supratherapeutical amounts to achieve the same inhibition. The data confirm that the inhibition of aspirin, heparin, r-hirudin, and G-4120 on the formation of platelet-rich thrombi is independent of the blood flow rate.


Subject(s)
Anticoagulants/therapeutic use , Aspirin/therapeutic use , Carotid Artery Thrombosis/prevention & control , Femoral Vein , Heparin/therapeutic use , Hirudin Therapy , Peptides, Cyclic/therapeutic use , Platelet Aggregation Inhibitors/therapeutic use , Sulfoxides/therapeutic use , Thrombosis/prevention & control , Animals , Carotid Artery Thrombosis/etiology , Cricetinae , Disease Models, Animal , Dose-Response Relationship, Drug , Linear Models , Male , Partial Thromboplastin Time , Thrombosis/etiology
20.
Circulation ; 94(8): 1941-7, 1996 Oct 15.
Article in English | MEDLINE | ID: mdl-8873672

ABSTRACT

BACKGROUND: Angiotensin II may contribute to hypoxic pulmonary hypertension via its vasoconstrictor and growth-stimulatory effects on vascular smooth muscle cells (VSMCs). Therefore, the use of ACE inhibitors might reduce hypoxic pulmonary hypertension by decreasing pulmonary vasomotor tone or vascular remodeling. METHODS AND RESULTS: Pulmonary hemodynamics and vascular remodeling were compared in chronically hypoxic (FIO2 = 0.10) rats treated with 0, 1, and 10 mg.kg-1.d-1 quinapril, a potent tissue ACE inhibitor, both during and after the development of pulmonary hypertension. Quinapril reduced the development of pulmonary hypertension after 12 days of hypoxia from 26 +/- 1 to 19 +/- 1 mm Hg (P < .05). When started in established pulmonary hypertension, quinapril reduced pulmonary artery pressure and total pulmonary resistance index from 29 +/- 1 to 25 +/- 1 mm Hg and from 0.136 +/- 0.01 to 0.101 +/- 0.005 mm Hg .mL-1.min-1 per kg, respectively (P < .05). Chronically hypoxic rats showed a small pulmonary vasoconstrictor response that was not affected by quinapril. In contrast, percent medial thickness in alveolar duct blood vessels was reduced by quinapril treatment both in developing and in established pulmonary hypertension (10.0 +/- 0.2% versus 8.9 +/- 0.1% [P < .05] and 11.2 +/- 0.2% versus 9.1 +/- 0.2% [P < .05], respectively). 5'-Bromo-deoxyuridine-positive VSMCs were detected in 56 +/- 3% of hypoxic control pulmonary resistance vessels versus 41 +/- 3% of vessels after quinapril treatment (P < .05). CONCLUSIONS: Pulmonary ACE and angiotensin II contribute to the development and maintenance of hypoxic pulmonary hypertension in rats. ACE inhibition with quinapril reduces the development of hypoxic pulmonary hypertension and in part reverses established pulmonary hypertension, most likely via inhibition of pulmonary VSMC proliferation and/or growth.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Antihypertensive Agents/therapeutic use , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , Hypoxia/complications , Isoquinolines/therapeutic use , Pulmonary Circulation , Tetrahydroisoquinolines , Animals , Blood Vessels/metabolism , Blood Vessels/pathology , Blood Vessels/physiopathology , Bromodeoxyuridine/pharmacokinetics , Hypertension, Pulmonary/physiopathology , Immunohistochemistry , Male , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Quinapril , Rats , Rats, Wistar , Vasoconstriction/drug effects
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