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D Collen

Publications and source records attributed to D Collen.

At least 91 records · Page 5Linked to original sources
PubMed1999

Malondialdehyde-modified LDL as a marker of acute coronary syndromes.

CONTEXT: Release of circulating malondialdehyde (MDA)-modified low-density lipoprotein (LDL) may reflect endothelial injury or plaque instability. OBJECTIVE: To determine the usefulness of MDA-modified LDL for identifying patients with unstable angina and acute myocardial infarction (AMI). DESIGN: Blinded comparison of MDA-modified LDL, C-reactive protein, and troponin I followed by multiple receiver operating curve analysis. SETTING: University hospital. PARTICIPANTS: A total of 104 consecutive patients with acute coronary syndromes (42 with unstable angina and 62 with AMI), and 64 patients with stable coronary artery disease (CAD) without evidence of ischemia. MAIN OUTCOME MEASURES: Ability of MDA-modified LDL, C-reactive protein, and troponin I to discriminate patients with stable CAD, unstable angina, or AMI. RESULTS: Malondialdehyde-modified LDL (chi2 = 10.2; P = .001), but not troponin I or C-reactive protein, discriminated between stable CAD and unstable angina. Troponin I (chi2 = 14.5; P<.001), but not MDA-modified LDL or C-reactive protein, discriminated between unstable angina and AMI. Both MDA-modified LDL and troponin I (chi2 = 14.5; P<.001 and chi2 = 5.3; P = .02, respectively) but not C-reactive protein discriminated between stable CAD and AMI. The sensitivity of MDA-modified LDL was 95% for unstable angina and 95% for AMI, with a specificity of 95%. Values for troponin I were 38% and 90%, respectively, with a specificity of 95%. The combination of MDA-modified LDL and troponin I had a sensitivity of 98% for unstable angina and 100% for AMI, with a specificity of 99%. CONCLUSION: The combination of MDA-modified LDL, which may reflect endothelial injury or plaque instability, and troponin I, which reflects myocardial cell injury, allows better discrimination between stable CAD and acute coronary syndromes than troponin I alone.

Acute Disease↗
PubMed1999

Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice.

BACKGROUND: The role of plasminogen system components in focal cerebral ischemic infarction (FCI) was studied in mice deficient in plasminogen (Plg-/-), in tissue or urokinase plasminogen activator (tPA-/- or uPA-/-), or in plasminogen activator inhibitor-1 or alpha2-antiplasmin (PAI-1(-/-) or alpha2-AP-/-). METHODS AND RESULTS: FCI was produced by ligation of the left middle cerebral artery and measured after 24 hours by planimetry of stained brain slices. In control (wild-type) mice, infarct size was 7.6+/-1.1 mm3 (mean+/-SEM), uPA-/- mice had similar infarcts (7.8+/-1.0 mm3, P=NS), tPA-/- mice smaller (2.6+/-0.80 mm3, P<0.0001), PAI-1(-/-) mice larger (16+/-0.52 mm3, P<0.0001), and Plg-/- mice larger (12+/-1.2 mm3, P=0.037) infarcts. alpha2-AP-/- mice had smaller infarcts (2. 2+/-1.1 mm3, P<0.0001 versus wild-type), which increased to 13+/-2.5 mm3 (P<0.005 versus alpha2-AP-/-) after intravenous injection of human alpha2-AP. Injection into alpha2-AP-/- mice of Fab fragments of affinospecific rabbit IgG against human alpha2-AP, after injection of 200 microg human alpha2-AP, reduced FCI from 11+/-1.5 to 5.1+/-1.1 mm3 (P=0.004). CONCLUSIONS: Plg system components affect FCI at 2 different levels: (1) reduction of tPA activity (tPA gene inactivation) reduces whereas its augmentation (PAI-1 gene inactivation) increases infarct size, and (2) reduction of Plg activity (Plg gene inactivation or alpha2-AP injection) increases whereas its augmentation (alpha2-AP gene inactivation or alpha2-AP neutralization) reduces infarct size. Inhibition of alpha2-AP may constitute a potential avenue to treatment of FCI.

Adenoviridae↗
PubMed1999

Percutaneous adenoviral gene transfer into porcine coronary arteries: is catheter-based gene delivery adapted to coronary circulation?

Recombinant adenoviral (Ad) vectors represent an efficient gene transfer system for targeting the cardiovascular system. Phenotypic modulation of coronary vascular cells in vivo is, however, critically dependent on the efficacy of local delivery devices. Four local drug delivery catheters were tested for intracoronary gene transfer efficiency: the Infiltrator (INF, n = 10), the Crescendo (CRE, n = 10), the Infusasleeve (SLE, n = 8), and the Remedy balloon (channel balloon [CHA], n = 8). After balloon injury of the LAD, Ad vector containing the firefly luciferase cDNA (AdCMVluc, 1.5 x 10(10) plaque-forming units) was administered at the site of injury. On day 4, tissue samples from different regions in the heart and from the liver were assayed for luciferase activity to evaluate local and systemic gene transfer. INF, CRE, and SLE catheters showed higher transduction levels of the target LAD segment than did the CHA catheter (median luciferase activity = 4.2 x 10(6), 11 x 10(6), and 1.3 x 10(6) light units [LU]/vessel versus 0.09 x 10(6) LU/vessel, respectively, p < 0.05). Luciferase activity was occasionally observed in nontarget tissues (right and left ventricular free wall, distal LAD, and liver) and was not significantly different between groups. The viral circulatory half-life was similar for the four groups (<1 min). Gene transfer efficiency was positively correlated with the degree of injury for the intralumenal catheters (CRE, SLE, and CHA) but was independent of the vessel wall injury for the intramural INF. Local drug delivery catheters enable efficient vascular gene transfer in balloon-injured coronary arteries, a prerequisite for further development of intracoronary gene therapy for restenosis.

Adenoviridae↗
PubMed1999

Alpha2-antiplasmin gene deficiency in mice is associated with enhanced fibrinolytic potential without overt bleeding.

alpha2-antiplasmin (alpha2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine alpha2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with the neomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance of the inactivated alpha2-AP allele was observed, and homozygous deficient (alpha2-AP-/-) mice displayed normal fertility, viability, and development. Reverse transcription-polymerase chain reaction confirmed the absence of alpha2-AP mRNA in kidney and liver from alpha2-AP-/- mice, in contrast to wild-type (alpha2-AP+/+) mice. Immunologic and functional alpha2-AP levels were undetectable in plasma of alpha2-AP-/- mice, and were about half of wild-type in heterozygous littermates (alpha2-AP+/-). Other hemostasis parameters, including plasminogen activator inhibitor-1, plasminogen, fibrinogen, hemoglobin, hematocrit, and blood cell counts were comparable for alpha2-AP+/+, alpha2-AP+/-, and alpha2-AP-/- mice. After amputation of tail or toe tips, bleeding stopped spontaneously in alpha2-AP+/+, as well as in alpha2-AP+/- and alpha2-AP-/- mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin-labeled plasma clots was significantly higher in alpha2-AP-/- than in alpha2-AP+/+ mice when injecting clots prepared from alpha2-AP+/+ plasma (78% +/- 5% v 46% +/- 9%; mean +/- SEM, n = 6 to 7; P =.02) or from alpha2-AP-/- plasma (81% +/- 5% v 46% +/- 5%; mean +/- SEM, n = 5; P =.008). Four to 8 hours after endotoxin injection, fibrin deposition in the kidneys was significantly reduced in alpha2-AP-/- mice, as compared with alpha2-AP+/+ mice (P </=.005). Thus, alpha2-AP-/- mice develop and reproduce normally; they have an enhanced endogenous fibrinolytic capacity without overt bleeding.

Alleles↗
PubMed1999

Gene therapy in the cardiovascular system: an update.

This update reviews the remarkable progression in several cardiovascular gene transfer domains. The first chemical gene therapy protocols to stimulate angiogenesis in ischemic myocardium are discussed and both the great expectations as well as remaining hurdle are highlighted. In experimental models of restenosis and heart failure gene therapy shows promising results. Important question regarding vector-related limitations and suboptimal in vivo delivery systems will require expeditious attention for gene therapy to become a more widely applicable option in cardiovascular diseases.

Animals↗
PubMed1999

Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure.

Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Animals↗
PubMed1999

Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors.

Recombinant adenoviral vectors expressing u-PA, t-PA, PAI-1 and PAI-2 were employed to correlate the expression of components of the fibrinolytic system with the invasiveness of HT 1080 tumor cells. Migration through Transwell inserts in vitro in the presence of plasminogen was increased up to 22% by overexpression of u-PA, whereas t-PA had no effect. Gene transfer of PAI-1 or PAI-2 both reduced migration in a dose-dependent manner by up to 43% with PAI-1 and 29% with PAI-2. Two routes of gene transfer were used to alter metastasis of subcutaneously implanted HT 1080 cells expressing firefly luciferase in nude mice. Infection of cultured tumor cells with adenovirus expressing either PAI-1 or PAI-2 before implantation significantly reduced the incidence of lung metastasis by 60% compared with control virus. However, only PAI-2 reduced the incidence of lung and brain metastasis following liver gene transfer. Although PAI gene transfer by either route reduced primary tumor size, it had little effect on tumor vascularization or host survival. The migratory and metastatic phenotype of HT 1080 tumor cells is thus directly dependent on u-PA expression levels and can be altered by gene transfer of u-PA or plasminogen activator inhibitors.

Adenoviridae↗
PubMed1999

A fully functional deletion staphylokinase derivative crystallizes in two non-isomorphous monoclinic modifications.

Two non-isomorphous monoclinic types of diffraction-quality crystals of Delta10Sak, a fully functional staphylokinase derivative lacking the first ten amino acids, have been grown by the hanging-drop vapour-diffusion technique. Type I crystals grow from a solution containing Zn(OAc)2, Tris buffer pH 7.5 and PEG 8000, while type II crystals can be obtained from a solution containing MgCl2, Tris buffer pH 8.5 and PEG 4000. Both crystal types were suitable for data collection (to 2.4 and 2.6 A resolution, respectively) and further structural investigation.

Base Sequence↗
PubMed1999

Accelerated neointima formation after vascular injury in mice with stromelysin-3 (MMP-11) gene inactivation.

The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11(+/+)) and MMP-11-deficient (MMP-11(-/-)) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11(-/-) as compared with MMP-11(+/+) mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm(2) at 2 weeks, P<0.001) and those of a 50/50 mixed 129SV/BL6 background (0.030 versus 0.013 mm(2) at 3 weeks, P<0.05). The medial areas were comparable, resulting in intima/media ratios that were significantly increased in MMP-11(-/-) as compared with MMP-11(+/+) arteries, in mice of both the 129SV (1. 0 versus 0.18, P<0.001) and mixed (1.5 versus 0.70, P<0.05) backgrounds. Nuclear cell counts in cross-sectional areas of the intima of the injured region were higher in arteries from MMP-11(-/-) mice than in those from MMP-11(+/+) mice (210 versus 48, P<0.001, in pure 129SV mice and 290 versus 150, P<0.01, in mice of the mixed genetic background). Immunocytochemical analysis revealed that alpha-actin-positive and CD45-positive cells were more abundant in intimal sections of MMP-11(-/-) mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11(-/-) mice than in those of MMP-11(+/+) mice (39% versus 6.8% at 3 weeks, P<0. 005). The mechanisms by which MMP-11 could impair elastin degradation and cellular migration in this model remain, however, unknown.

Actins↗
PubMed1999

Factor VII deficiency rescues the intrauterine lethality in mice associated with a tissue factor pathway inhibitor deficit.

Mice doubly heterozygous for a modified tissue factor pathway inhibitor (TFPI) allele (tfpi delta) lacking its Kunitz-type domain-1 (TFPI+/delta) and for a deficiency of the factor VII gene (FVII+/-) were mated to generate 309 postnatal and 205 embryonic day 17.5 (E17. 5) offspring having all the predicted genotypic combinations. Progeny singly homozygous for the tfpidelta modification but with the wild-type fVII allele (FVII+/+/TFPIdelta/delta), and mice singly homozygous for the fVII deficiency and possessing the wild-type tfpi allele (FVII-/-/TFPI+/+), displayed previously detailed phenotypes (i.e., a high percentage of early embryonic lethality at E9.5 or normal development with severe perinatal bleeding, respectively). Surprisingly, mice of the combined FVII-/-/TFPIdelta/delta genotype were born at the expected mendelian frequency but suffered the fatal perinatal bleeding associated with the FVII-/- genotype. Mice carrying the FVII+/-/TFPIdelta/delta genotype were also rescued from the lethality associated with the FVII+/+/TFPIdelta/delta genotype but succumbed to perinatal consumptive coagulopathy. Thus, the rescue of TFPIdelta/delta embryos, either by an accompanying homozygous or heterozygous FVII deficiency, suggests that diminishment of FVII activity precludes the need for TFPI-mediated inhibition of the FVIIa/tissue factor coagulation pathway during embryogenesis. Furthermore, the phenotypes of these combined deficiency states suggest that embryonic FVII is produced in mice as early as E9.5 and that any level of maternal FVII in early-stage embryos is insufficient to cause a coagulopathy in TFPIdelta/delta mice.

Animals↗
PubMed1999

Structural and functional basis of plasminogen activation by staphylokinase.

Staphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of microplasmin (microPli), the serine protease domain of plasmin. Sak binding on microPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on microPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed beta-sheet. Several Sak residues located within the unique alpha-helix and the beta2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:microPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of microPli, protrudes into a broad groove near the catalytic triad of microPli, thereby generating a competent binding pocket for micro-plasminogen (microPlg), which buries approximately 2500 A of the Sak:microPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:microPli:microPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

Bacteriophages↗
PubMed1999

Differential role of components of the fibrinolytic system in the formation and removal of thrombus induced by endothelial injury.

The role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4+/-1.3, 9.8+/-1.1 or 9.7+/-1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (1 8.4+/-3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

Animals↗
PubMed1999

Temporal and topographic matrix metalloproteinase expression after vascular injury in mice.

Temporal and topographic expression of matrix metalloproteinases (MMPs) after perivascular electric injury was studied in wild-type (WT) and urokinase-deficient (u-PA-/-) mice. Neointima formation after injury of the femoral artery was significantly reduced in u-PA-/- mice as compared to WT mice (area of 0.002+/-0.0007 mm2 versus 0.008 + 0.002 mm2 at 3 weeks after injury; p <0.001), associated with impaired cellular migration (nuclear cell counts of 44+/-5 versus 82+/-9in cross-sectional areas; p <0.001). Zymographic and/or microscopic analysis indicated that MMP expression gradually increased to reach a maximum at 1 to 2 weeks after vascular injury. In general, MMP levels were lower in u-PA-/- than in WT mice. In non-injured arteries, MMP-2 (gelatinase A) and MMP-3 (stromelysin-1) were produced mainly by adventitial fibroblasts and/or non-contractile smooth muscle cells (SMC). One week after injury, MMP-2 and MMP-3 levels were enhanced due to an increased number and size of producing cells; 2 to 3 weeks after injury, MMP-2 and MMP-3 were produced also by some contractile SMC, which stained with alpha-actin antiserum. MMP-9 (gelatinase B), MMP-12 (metalloelastase) and MMP-13 (collagenase-3) were found in macrophages located mainly in the adventitia. Immunogold electron microscopic examination revealed that MMP-2 was located predominantly in association with the cell surface of fibroblasts or SMC, while MMP-9 and MMP- 12 were located in well defined storage granules within macrophages. MMP-2, MMP-3 and MMP-13, but not MMP-9 or MMP-12, were also found extracellularly, associated with elastin-containing structures (MMP-2), with the basement membrane and occasionally with collagen fibres (MMP-3), or with proteoglycans, collagen and elastin (MMP-13). The temporal and topographic expression pattern of MMPs after vascular injury, coinciding with smooth muscle cell migration and neointima formation, thus is compatible with a role in vascular remodeling.

Animals↗
PubMed1999

Plasmodium falciparum-infected erythrocytes: a mutational analysis of cytoadherence via murine thrombomodulin.

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Animals↗
PubMed1999

Modulation of cell-associated plasminogen activation by stromelysin-1 (MMP-3).

Stromelysin-1 (MMP-3) cleaves a 55 kDa kringle 1-4 fragment, containing the lysine-binding site(s) involved in cellular binding, from 92 kDa plasminogen and removes a 17 kDa NH2-terminal fragment, containing the cellular receptor-binding site, from 45 kDa urokinase (u-PA), but a potential role of MMP-3 in the regulation of cellular fibrinolytic activity by affecting binding and/or activation of plasminogen and/or single-chain u-PA has not been established. Human plasminogen (input concentration 100 nM for 4x10(6) cells per ml) was shown to bind specifically to human monocytoid THP-1 cells, to murine MMP-3 deficient smooth muscle cells (SMC) and fibroblasts (1.9, 0.92 and 1.0x10(6) molecules per cell, respectively). Treatment with MMP-3 (final concentration 0-50 nM) of cells saturated with bound plasminogen (about 25 nM), overnight at 37 degrees C, resulted in a dose-dependent reduction of the amount of u-PA activatable plasminogen (reduction to 25-40% of the value in the absence of MMP-3). Immunoblotting with specific monoclonal antibodies and autoradiography of eluates of the cells treated with MMP-3 revealed cleavage of plasminogen into the 55 kDa fragment and miniplasminogen (kringle 5 plus the proteinase domain). Binding of human single chain u-PA (scu-PA) to human THP-1 and HT 1080 cells amounted to 2.5x10(6) and 7.1x10(6) molecules per cell, respectively. Treatment with MMP-3 (final concentration 0-25 nM) of cell-bound u-PA (about 17 nM for THP-1 and 47 nM for HT1080 cells), overnight at 37 degrees C, did not alter cell-associated u-PA activity, measured in a direct chromogenic substrate assay or in a plasminogen-coupled chromogenic substrate assay (residual u-PA activity always > or =85% of that without MMP-3 treatment). Autoradiography of 125I-labeled u-PA moieties, removed from the cells by treatment with acid or with phosphatidylinositol phospholipase C, confirmed that u-PA remained essentially intact after MMP-3 treatment. These data indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity.

Binding Sites↗