Biomedical subjects

D Collen

Publications and source records attributed to D Collen.

At least 73 records · Page 4Linked to original sources

PubMed2000

Blood coagulation factor X deficiency causes partial embryonic lethality and fatal neonatal bleeding in mice.

Mice with a total deficiency in blood coagulation Factor X (FX) were generated by targeted replacement of an 18-kb fragment of the FX gene, comprising all exons encoding the mature FX protein, with a neo(r) cassette. The genotype distribution among the offspring from heterozygous breeding pairs suggested that FX deficiency resulted in partial embryonic lethality, with approximately one-third of the FX-/- embryos dying around embryonic day (E) 11.5-12.5. Two of 44 non-resorbed FX-/- embryos analyzed at these stages showed signs of massive bleeding, one of which into the brain ventricles, but no histological defects in the vasculature of these embryos or their yolk sac were observed. The remainder of the FX-/- embryos appeared normal and survived to term, but the majority of neonates (90%) died within 5 days, most frequently from intraabdominal bleeding. The remaining FX-/- animals succumbed between postnatal day (P)5 and P20 with intraabdominal, subcutaneous, or intracranial bleeding or a combination thereof. The lethal phenotype of the FX-/- mice illustrates the importance of FX function in embryonic and postnatal survival and demonstrates that these mice serve as effective models of the bleeding disorders observed in severe FX deficiency in humans.

Animals↗

PubMed2000

Lack of tPA significantly affects antithrombotic therapy by a GPIIb/IIIa antagonist, but not by a thrombin inhibitor in mice.

The interaction of fibrinolytic components with platelets or coagulation factors after endothelial injury, was investigated in mouse deficient in tissue type plasminogen activator (tPA -/-), or urokinase (uPA -/-) and in their wild type control (tPA +/+, uPA +/+). A thrombus was induced in the murine carotid artery using the photochemical reaction. Blood flow was continuously monitored and the time needed before the vessel became completely obstructed was within 11 min in all types of mice. When GR144053, a platelet glycoprotein IIb/IIIa antagonist, or argatroban, a thrombin inhibitor, was applied, the time required to occlusion was prolonged in a dose-dependent manner in all types of mice. However, when GR144053 was injected in tPA -/- mice, the most significant changes were observed: that is the estimated ED50 was 14.8 times higher than the one in tPA +/+ mice. On the other hand, when argatroban was injected in tPA -/- mice, the estimated ED50 was not changed. Platelet aggregation, haemostasis tests and bleeding times were not significantly different among the different types of mice. In conclusion, the antithrombotic effect of platelet inhibition by a GPIIb/IIIa antagonist, is severely affected by the absence or presence of tPA-production. Thus, the lack of tPA significantly reduces the antithrombotic efficacy.

Animals↗

PubMed2000

Outcome and one year follow-up of intra-arterial staphylokinase in 191 patients with peripheral arterial occlusion.

Wild-type or equipotent variants of recombinant staphylokinase (rSak) were given intra-arterially (as a 2 mg bolus injection followed by an infusion of 1 mg/h or 0.5 mg/h overnight, with concomitant heparin [1000 IU/h]) to 191 patients of less than 80 years (62 +/- 1 years, mean +/- SEM), with a peripheral arterial occlusion (PAO) of less than 120 days (mean 14 +/- 1 days, median 11 days, 5 to 95 percentiles 3 to 30 days). Ninety nine patients presented with acute or subacute ischemia, 57 with severe claudication, 33 with chronic rest pain and 2 with gangrene. Occlusion occurred in 122 native arteries and in 69 grafts. Revascularization was complete in 83 percent (158/191), partial in 13 percent (24/191) and absent in 4 percent (7/191) after administration of 12 +/- 0.5 mg rSak over 14 +/- 0.7 h. Complete revascularization of acute occlusions of popliteal or more distal arteries was less frequent (60 percent, 15/25) than of acute occlusions of more proximal native arteries (95 percent, 37/39, p <0.001) or grafts (89 percent, 50/56, p = 0.005). Additional endovascular procedures were performed in 47 percent and subsequent elective bypass surgery in 23 percent of patients. Major bleeding occurred in 12 percent (23/191), one month mortality was 3.1 percent (6/191) and one year mortality was 6.9 percent (12/174). However, four patients (2.1 percent) had an intracranial bleeding following therapy: a 85 year old woman with severe diabetic arteriopathy, who was included in violation of the protocol, a 79 and a 74-year-old woman and a 74-year-old man, all with severe hypertension and limb threatening ischemia; these four patients died within two months after treatment. Amputations were performed within the first year in 16 of 162 surviving patients (9.8 percent): in 7 percent (7/96) with an occluded native artery and 14 percent (9/66) with an occluded graft (p = 0.19). No significant difference in lysis rate, one month mortality or one year amputation-free survival was observed in occlusions of recent onset (< or =14 days, n = 126) as compared to occlusions of longer duration (>14 days, n = 65). Treatment was interrupted prematurely in 4 patients because of a suspected allergic reaction. Fibrinogen levels remained unaffected during treatment (3.3 +/- 0.1 g/l before vs. 3.3 +/- 0.1 g/l after infusion, n = 167). In conclusion, rSak appears to be a highly effective thrombolytic agent in patients with PAO, resulting in a low one month mortality (3.1 percent) and a high one year amputation free survival (84 percent), with an acceptable incidence of major bleedings, but with occasional fatal intracranial hemorrhages.

Aged↗

PubMed2000

CD9 gene deficiency does not affect smooth muscle cell migration and neointima formation after vascular injury in mice.

The hypothesis that CD9, a member of the tetraspanin family, plays a role in smooth muscle cell (SMC) migration was tested with the use of a vascular injury model in wild-type (CD9+/+) and CD9-deficient (CD9-/-) mice. Neointima formation 3 weeks after electric injury of the femoral artery was not significantly different in CD9+/+ and CD9-/- mice (area of 0.019 +/- 0.0034 mm2 versus 0.013 +/- 0.0036 mm2; mean +/- SEM, n = 6). The medial areas were also comparable, resulting in intima/media ratio's of 1.3 +/- 0.15 and 0.90 +/- 0.22, respectively. Nuclear cell counts in cross-sectional areas of the injured region were comparable in media (33 +/- 5 versus 27 +/- 2) and neointima (135 +/- 16 versus 97 +/- 17) of CD9+/+ and CD9-/- arteries. Immunocytochemical analysis revealed expression of CD9 in the endothelium, by SMC in the media and by some fibroblasts in the adventitia of non-injured femoral arteries. Three weeks after injury, there appeared to be a gradient of increased CD9 expression from the adventitia to the neointima, in which SMC are abundantly present. Immunogold labeling and electron microscopy with non-injured femoral arteries of CD9+/+ mice confirmed the presence of CD9 at the surface of adventitial fibroblasts and in SMC or pericytes, as well as in the endothelium. Thus, in this model CD9 is highly expressed by migrating SMC, but deficiency of CD9 does not affect SMC migration or neointima formation after perivascular injury.

Animals↗

PubMed2000

Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha.

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.

Binding Sites↗

PubMed1999

1,25-Dihydroxyvitamin D(3) induction of the tissue-type plasminogen activator gene is mediated through its multihormone-responsive enhancer.

Tissue-type plasminogen activator (t-PA) is a positive modulator of the plasminogen-plasmin system, which is involved in bone remodeling. In the present study, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] was found to stimulate t-PA gene expression in ROS17/2.8 osteosarcoma cells. Transient transfection analysis and in vitro DNA binding studies identified two vitamin D-responsive elements (VDRE) in the human t-PA enhancer. The first VDRE (bp -7175 to -7146) comprised an inverted palindrome separated by 9 bp (IP9) overlapping a palindrome separated by 3 bp. The second VDRE (bp -7315 to -7302) is an IP2 element overlapping the previously identified retinoic acid-responsive element. 1,25(OH)(2)D(3) treatment of primary osteoblasts derived from t-PAlacZ transgenic mice containing 9 kb of 5' sequence of the human t-PA gene increased the number of lacZ-positive cells, fitting with the probability model of enhancer function.

Animals↗

PubMed1999

Circulating activated platelets assist THP-1 monocytoid/endothelial cell interaction under shear stress.

Circulating complexes of leukocytes and activated platelets are markers for atherosclerosis, but their interaction with the arterial endothelial lining has not been studied. Therefore, the effect of activated platelets on rolling and adhesion of labeled human THP-1 monocytoid cells to human umbilical vein endothelial cell (HUVEC) monolayers was studied by epifluorescence microscopy in a parallel plate flow chamber. In the absence of activated platelets, THP-1 rolling on resting HUVEC was negligible at shear rates greater than 300 s(-1). Activation of HUVEC with 100 nmol/L phorbol myristate acetate (PMA) increased THP-1 cell adhesion at shear rates less than 400 s(-1). Therefore, a shear rate of 400 s(-1) was identified as a threshold for THP-1 adhesion. THP-1 rolling on activated HUVEC was reduced by 64% after L-selectin inhibition but was not affected by P-selectin inhibition. The addition of 1 to 50 thrombin receptor-activating peptide (TRAP)-activated platelets per THP-1 cell enhanced interactions between THP-1 cells and HUVEC, resulting in a steep bell-shaped dose-response curve, with a peak of 10 +/- 3 rolling cells/50 seconds at 3 platelets per THP-1 cell (P <.01 v control) with a concomitant 2- to 3-fold increase of firmly adhering cells (P <.01 v control). In reconstituted blood, low numbers of activated platelets had the same effect on THP-1 rolling and adhesion. P-selectin inhibition reduced platelet/THP-1 cell interaction in suspension and deposition of the complexes on the endothelial monolayer. Inhibition of both P- and L-selectin reduced THP-1/HUVEC interactions to 14% (P <.01, n = 4). Sialidase digestion and removal of terminal sialic acid residues from HUVEC or THP-1 cells but not from platelets abolished the platelet mediated augmentation of THP-1 cell adhesion. Thus, THP-1 rolling on HUVEC is shear-dependent and largely mediated by L-selectin. P-selectin expressed on activated platelets increases monocytoid cell adhesion to endothelial cells at shear rates found in coronary arteries through interactions with both endothelial and monocytoid cells and may facilitate macrophage accumulation in the vessel wall.

Antibodies, Monoclonal↗

PubMed1999

Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice.

Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic disabilities. It is well suited for gene therapy because a moderate increase in plasma FVIII concentration has therapeutic effects. Improved retroviral vectors expressing high levels of human FVIII were pseudotyped with the vesicular stomatitis virus G glycoprotein, were concentrated to high-titers (10(9)-10(10) colony-forming units/ml), and were injected intravenously into newborn, FVIII-deficient mice. High-levels (>/=200 milliunits/ml) of functional human FVIII production could be detected in 6 of the 13 animals, 4 of which expressed physiologic or higher levels (500-12,500 milliunits/ml). Five of the six expressers produced FVIII and survived an otherwise lethal tail-clipping, demonstrating phenotypic correction of the bleeding disorder. FVIII expression was sustained for >14 months. Gene transfer occurred into liver, spleen, and lungs with predominant FVIII mRNA expression in the liver. Six of the seven animals with transient or no detectable human FVIII developed FVIII inhibitors (7-350 Bethesda units/ml). These findings indicate that a genetic disease can be corrected by in vivo gene therapy using retroviral vectors.

Animals↗

PubMed1999

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Amino Acid Sequence↗

PubMed1999

Involvement of Sp1 in basal and retinoic acid induced transcription of the human tissue-type plasminogen activator gene.

Transcription of the human tissue-type plasminogen activator (t-PA) gene is regulated by a multi-hormonal responsive enhancer at -7 kb. Transient co-transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t-PA reporter constructs showed that Sp1 and Sp3 activate the t-PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.

Animals↗

PubMed1999

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

Animals↗

PubMed1999

Guiding a docking mode by phage display: selection of correlated mutations at the staphylokinase-plasmin interface.

During co-evolution of interacting proteins, functionally disruptive mutations on one side of the interface may be compensated by local amino acid changes on the other to restore binding affinity. This information can be useful for geometry-based docking approaches by reducing the translational and rotational space available to the proteins. Here, we demonstrate that correlated mutations at a protein-protein interface can be rapidly identified by selecting a phage-displayed library of a randomly mutated component of the complex for complementation of mutations that decreased binding in the interacting partner. This approach was used to deduce the binding mode of staphylokinase (Sak), a 15.5 kDa "indirect" plasminogen activator on microplasmin (microPli), the 28 kDa serine protease domain of plasmin. Biopanning indicated that residues Arg94 and Gly174 in microPli are located in close proximity to Glu75 and the Glu88:Ile128 pair in Sak, respectively. The coupled mutations Glu94<-->Lys75 reversed and Gly174<-->Lys88:Val128 introduced a salt bridge, whereby the binding affinities (with coupling energies of 1.8 to 2.3 kcal mol-1, respectively) and the plasminogen activation ability of the mutated complexes were partially restored. These findings suggested a unique docking mode of Sak at the western rim of the active-site cleft of microPli, that is in agreement with the structure of the Sak-microPli complex as recently derived by other methods.

Amino Acid Substitution↗

PubMed1999

Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene.

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.

Binding Sites↗

PubMed1999

Structure-function analyses of thrombomodulin by gene-targeting in mice: the cytoplasmic domain is not required for normal fetal development.

Thrombomodulin (TM) is a widely expressed glycoprotein receptor that plays a physiologically important role in maintaining normal hemostatic balance postnatally. Inactivation of the TM gene in mice results in embryonic lethality without thrombosis, suggesting that structures of TM not recognized to be involved in coagulation might be critical for normal fetal development. Therefore, the in vivo role of the cytoplasmic domain of TM was studied by using homologous recombination in ES cells to create mice that lack this region of TM (TMcyt/cyt). Cross-breeding of F1 TMwt/cyt mice (1 wild-type and 1 mutant allele) resulted in more than 300 healthy offspring with a normal Mendelian inheritance pattern of 25.7% TMwt/wt, 46.6% TMwt/cyt, and 27.7% TMcyt/cyt mice, indicating that the tail of TM is not necessary for normal fetal development. Phenotypic analyses showed that the TMcyt/cyt mice responded identically to their wild-type littermates after procoagulant, proinflammatory, and skin wound challenges. Plasma levels of plasminogen, plasminogen activator inhibitor 1 (PAI-1), and alpha2-antiplasmin were unaltered, but plasmin:alpha2-antiplasmin (PAP) levels were significantly lower in TMcyt/cyt mice than in TMwt/wt mice (0.46 +/- 0.2 and 1.99 +/- 0.1 ng/mL, respectively; P <.001). Tissue levels of TM antigen were also unaffected. However, functional levels of plasma TM in the TMcyt/cyt mice, as measured by thrombin-dependent activation of protein C, were significantly increased (P <.001). This supported the hypothesis that suppression in PAP levels may be due to augmented activation of thrombin-activatable fibrinolysis inhibitor (TAFI), with resultant inhibition of plasmin generation. In conclusion, these studies exclude the cytoplasmic domain of TM from playing a role in the early embryonic lethality of TM-null mice and support its function in regulating plasmin generation in plasma.

Animals↗

PubMed1999

Nigral degeneration following striato-pallidal lesion in tissue type plasminogen activator deficient mice.

Tissue type plasminogen activator (tPA) has been suggested as a key factor in excitotoxic neuronal death in the hippocampus. Transneuronal degeneration of the substantia nigra pars reticulata (SNR) neurons after striato-pallidal lesions is attributable to excess excitatory glutamatergic inputs into the SNR following inhibitory GABAergic deafferentation and tPA may contribute to the mechanism of transneuronal degeneration of the SNR. To examine this possibility, we studied pathological changes in the SNR following striato-pallidal lesions produced by electrocoagulation in tPA-deficient mice. There was no difference in the degree of SNR degeneration, or in microglial activation and proliferation in the degenerating SNR of tPA-deficient and control mice. Our results indicate that tPA does not contribute to transneuronal degeneration in the SNR following striato-pallidal lesions in mice.

Animals↗

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↗