Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

Effects of increased liver blood flow on the kinetics and dynamics of recombinant tissue-type plasminogen activator.

OBJECTIVE: To investigate the influence of increased liver blood flow on the pharmacokinetics and pharmacodynamics of recombinant tissue-type plasminogen activator (rt-PA) and to study the changes in endogenous urokinase-type plasminogen activator (u-PA). METHODS: This open, randomized, crossover trial was carried out in a clinical research unit. Eight healthy, nonsmoking volunteers received linear infusions of 24 mg rt-PA and 92 mg indocyanine green over 160 minutes. Sixty minutes after the infusions were started, the subjects consumed a standardized meal to increase liver blood flow on one occasion and abstained from taking food on the other occasion. Plasma concentrations of indocyanine green, tissue-type plasminogen activator (t-PA) antigen, t-PA activity, total u-PA antigen, plasmin-activatable single-chain u-PA (scu-PA), active two-chain u-PA (tcu-PA), fibrinogen, total fibrin, and fibrinogen/fibrin degradation products (TDP), and alpha 2-antiplasmin were measured. RESULTS: After the consumption of the meal, the area under the curve (AUC) was 35% (95% confidence interval [CI]: 25%, 43%) lower for indocyanine green, 15% (CI: 6%, 24%) lower for t-PA antigen, and 11% (CI: 2%, 19%) lower for t-PA activity compared to the AUC after subjects abstained from food. No changes were observed in fibrinogen, TDP, or alpha 2-antiplasmin concentrations that were attributable to the intake of food. The infusion of rt-PA caused a fivefold increase in the concentration of active tcu-PA and a concomitant decrease in scu-PA concentrations by more than 50%. CONCLUSIONS: Increased liver blood flow results in an increase in t-PA clearance. The conversion of the inactive zymogen scu-PA to the active tcu-PA is increased by an infusion of rt-PA, but total u-PA antigen concentrations remain unchanged.

Effects of liver blood flow on the pharmacokinetics of tissue-type plasminogen activator (alteplase) during thrombolysis in patients with acute myocardial infarction.

BACKGROUND: The removal of recombinant tissue-type plasminogen activator (rt-PA; alteplase) by the liver is so rapid that liver blood flow becomes rate determining for its clearance. In patients with myocardial infarction changes in liver blood flow may result from impaired cardiac performance or drug treatment. OBJECTIVE: To estimate the effect of variations in liver blood flow on t-PA plasma concentrations during thrombolytic therapy. METHODS: Fifteen patients with acute myocardial infarction were investigated in an open single-center study at the coronary care unit of University Hospital Leiden. Patients received thrombolytic treatment with 100 mg rt-PA over 3 hours. Liver blood flow was estimated by indocyanine green clearance and by Doppler echocardiography. Concentrations of t-PA antigen, t-PA activity, indocyanine green, alpha 2-antiplasmin, fibrinogen, and fibrin and fibrinogen degradation products were measured. RESULTS: Indocyanine green clearance and clearance of both t-PA antigen (r = 0.78; p < 0:01) and t-PA activity (r = 0.54; p < 0.05) were significantly related. Significant associations between t-PA antigen and fibrin and fibrinogen degradation products and between t-PA antigen and alpha 2-antiplasmin were also found. CONCLUSIONS: The liver blood flow of patients with myocardial infarction is inversely correlated with plasma concentrations of t-PA. In patients with severely impaired liver blood flow and heart failure, high t-PA plasma concentrations may occur if standard doses are given. This finding could contribute to optimization of the dosage of t-PA in certain patient groups.

Effects of changing liver blood flow by exercise and food on kinetics and dynamics of saruplase.

OBJECTIVE: To investigate the influence of changes in liver blood flow on the pharmacokinetics and pharmacodynamics of single-chain unglycosylated urokinase-type plasminogen activator. METHODS: This open, randomized, crossover trial was carried out in the clinical research unit. Infusions of 37.5 mg saruplase and 90 mg indocyanine green were administered over 150 minutes to 10 healthy male volunteers. After 60 minutes the subjects consumed a standardized meal to increase liver blood flow or performed an exercise test (20 minutes) to decrease liver blood flow. Indocyanine green concentrations, total urokinase-type plasminogen activator (u-PA) antigen, two-chain u-PA activity, fibrinogen, total degradation products, alpha 2-antiplasmin, and factor XII-dependent fibrinolytic activity were measured. Blood flow was measured after food intake in a portal vein branch with Doppler echography. RESULTS: The weighted average indocyanine green concentration after exercise was increased by 29% compared with baseline (steady-state concentration) values (95% confidence intervals [CI]: +6%, +56%). After food, the concentration was 27% lower compared with baseline values (95% CI: -35%, -19%), and portal vein flow was increased by a maximum of 103% (95% CI: +71%, +136%). Average maximal concentrations of u-PA antigen after exercise were increased by 130 ng/ml compared with baseline concentrations (95% CI: +65, +195 ng/ml) and, unexpectedly, 156 ng/ml higher after food (95% CI: +59, +253 ng/ml). Although not significant, an increase in average u-PA antigen concentration compared with baseline values was detected after both exercise (7%) and food (13%). This tendency toward a larger effect after food compared with the effect after exercise was reflected by minor changes in the pharmacodynamics. CONCLUSIONS: u-PA plasma concentrations were increased by reduced liver blood flow induced by exercise. Food intake produced an unexpected increase in u-PA concentrations despite increases in liver blood flow.

On the role of C1-inhibitor as inhibitor of tissue-type plasminogen activator in human plasma.

An enzyme immuno assay was developed to measure complexes of tissue-type plasminogen activator (t-PA) with C1-inhibitor in order to study the role of C1-inhibitor as an inhibitor of t-PA in plasma. In vitro experiments with melanoma and recombinant t-PA learned that purified C1-inhibitor reacts with both single chain t-PA and two chain t-PA. The rate constants ranged from 3.0 to 5.2 M-1s-1. In plasma, melanoma and recombinant two chain t-PA were hardly inhibited by C1-inhibitor, in contrast to melanoma and recombinant single chain t-PA which were inhibited to the same extent by endogenous C1-inhibitor as they were by purified C1-inhibitor. In vivo, t-PA/C1-inhibitor complex could be measured in plasma in a few cases in healthy volunteers (0.62 +/- 0.43 ng/ml t-PA equivalents), after exercise (0.84 +/- 0.25 ng/ml t-PA equivalents) and after a desmopression infusion (0.26 +/- 0.04 ng/ml t-PA equivalents). However, t-PA/C1-inhibitor complex was found in plasma in all cases after venous occlusion (1.7 +/- 0.5 ng/ml t-PA equivalents), in peritoneal fluid from patients suffering from peritoneal inflammatory disease (2.2 +/- 1.3 ng/ml t-PA equivalents) and in plasma from healthy volunteers during a t-PA infusion (27.7 +/- 18.5 ng/ml t-PA equivalents at peak level). In the last case, about 8% of the infused dose of recombinant t-PA (alteplase) was inhibited by C1-inhibitor at peak level.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviral gene transfer of a u-PA receptor-binding plasmin inhibitor and green fluorescent protein: inhibition of migration and visualization of expression.

Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

Effect of propranolol (long-acting) on the circadian fluctuation of tissue-plasminogen activator and plasminogen activator inhibitor-1.

The incidence of myocardial infarction and sudden cardiac death is highest in the morning. Inhibition of fibrinolytic activity in blood also peaks in the morning and this inhibition may favor the development of arterial thrombosis. It has been reported that patients treated with beta blockers do not show the typical circadian pattern of onset of myocardial infarction and sudden cardiac death. This study was undertaken to investigate whether beta blockade alters the circadian rhythm of 2 major fibrinolytic factors, tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Repeated blood samples were taken over a 24-hour period in 13 healthy volunteers: 7 taking 160 mg/day of long-acting propranolol orally for 14 days, and the other 6 taking no medications. Blood samples were analyzed for the plasma levels of t-PA activity, t-PA antigen, PAI activity and PAI-1 antigen. A significant circadian variation of all 4 parameters was present in both groups. No significant differences in peak and nadir values, 24-hour mean, amplitude of fluctuation, and time of peak and nadir were found between the treated and untreated subjects. The data therefore suggest that propranolol treatment does not affect the plasma concentrations at rest or the endogenous circadian rhythm of t-PA and PAI-1 in healthy volunteers. The reported alteration in the circadian pattern of onset of myocardial infarction and sudden cardiac death by beta blockers does not appear to be mediated by effects on the fibrinolytic system.

Improved adenovirus vectors for infection of cardiovascular tissues.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of a cell surface-targeted plasmin inhibitor.

OBJECTIVE: Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface-targeted plasmin inhibitor (ATF.BPTI). METHODS: Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast-dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. RESULTS: The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface-binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean +/-SEM cartilage degradation in vitro was reduced to 87.9+/-0.9% after LacZ gene transfer versus a reduction to 24.0+/-1.6% after ATF.BPTI gene transfer (P<0.0001). The mean +/- SEM in vivo cartilage invasion score was 3.1+/-0.4 in the control-transduced fibroblasts and 1.8+/-0.4 in the ATF.BPTI-transduced fibroblasts (P<0.05). CONCLUSION: These results indicate a role of the plasminogen activation system in synovial fibroblast-dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface-targeted plasmin inhibitor.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.