Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation.

The molecular mechanisms predisposing to atherosclerotic aneurysm formation remain undefined. Nevertheless, rupture of aortic aneurysms is a major cause of death in Western societies, with few available treatments and poor long-term prognosis. Indirect evidence suggests that matrix metalloproteinases (MMPs) and plasminogen activators (PAs) are involved in its pathogenesis. MMPs are secreted as inactive zymogens (pro-MMPs), requiring activation in the extracellular compartment. Plasmin, generated from the zymogen plasminogen by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA; refs 14,15), has been proposed as a possible activator in vitro, but evidence for such a role in vivo is lacking. Analysis of atherosclerotic aorta in mice with a deficiency of apoliprotein E (Apoe-/-; ref. 18), singly or combined with a deficiency of t-PA (Apoe-/-:Plat-/-) or of u-PA (Apoe-/-:Plau-/-; ref. 19), indicated that deficiency of u-PA protected against media destruction and aneurysm formation, probably by means of reduced plasmin-dependent activation of pro-MMPs. This genetic evidence suggests that plasmin is a pathophysiologically significant activator of pro-MMPs in vivo and may have implications for the design of therapeutic strategies to prevent aortic-wall destruction by controlling Plau gene function.

A mouse model for Zellweger syndrome.

The cerebro-hepato-renal syndrome of Zellweger is a fatal inherited disease caused by deficient import of peroxisomal matrix proteins. The pathogenic mechanisms leading to extreme hypotonia, severe mental retardation and early death are unknown. We generated a Zellweger animal model through inactivation of the murine Pxr1 gene (formally known as Pex5) that encodes the import receptor for most peroxisomal matrix proteins. Pxr1-/- mice lacked morphologically identifiable peroxisomes and exhibited the typical biochemical abnormalities of Zellweger patients. They displayed intrauterine growth retardation, were severely hypotonic at birth and died within 72 hours. Analysis of the neocortex revealed impaired neuronal migration and maturation and extensive apoptotic death of neurons.

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization.

Acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression. High levels of components of the plasminogen activation system, including urokinase, but paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI1), have been correlated with a poor prognosis for some cancers. We report here that deficient PAI1 expression in host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes. When this PAI1 deficiency was circumvented by intravenous injection of a replication-defective adenoviral vector expressing human PAI1, invasion and associated angiogenesis were restored. This experimental evidence demonstrates that host-produced PAI is essential for cancer cell invasion and angiogenesis.

Abrupt rate accelerations or premature beats cause life-threatening arrhythmias in mice with long-QT3 syndrome.

Deletion of amino-acid residues 1505-1507 (KPQ) in the cardiac SCN5A Na(+) channel causes autosomal dominant prolongation of the electrocardiographic QT interval (long-QT syndrome type 3 or LQT3). Excessive prolongation of the action potential at low heart rates predisposes individuals with LQT3 to fatal arrhythmias, typically at rest or during sleep. Here we report that mice heterozygous for a knock-in KPQ-deletion (SCN5A(Delta/+)) show the essential LQT3 features and spontaneously develop life-threatening polymorphous ventricular arrhythmias. Unexpectedly, sudden accelerations in heart rate or premature beats caused lengthening of the action potential with early afterdepolarization and triggered arrhythmias in Scn5a(Delta/+) mice. Adrenergic agonists normalized the response to rate acceleration in vitro and suppressed arrhythmias upon premature stimulation in vivo. These results show the possible risk of sudden heart-rate accelerations. The Scn5a(Delta/+) mouse with its predisposition for pacing-induced arrhythmia might be useful for the development of new treatments for the LQT3 syndrome.

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.

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.

Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulonephritis.

The plasminogen/plasmin system has the potential to affect the outcome of inflammatory diseases by regulating accumulation of fibrin and other matrix proteins. In human and experimental crescentic glomerulonephritis (GN), fibrin is an important mediator of glomerular injury and renal impairment. Glomerular deposition of matrix proteins is a feature of progressive disease. To study the role of plasminogen and plasminogen activators in the development of inflammatory glomerular injury, GN was induced in mice in which the genes for these proteins had been disrupted by homologous recombination. Deficiency of plasminogen or combined deficiency of tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA) was associated with severe functional and histological exacerbation of glomerular injury. Deficiency of tPA, the predominant plasminogen activator expressed in glomeruli, also exacerbated disease. uPA deficiency reduced glomerular macrophage infiltration and did not significantly exacerbate disease. uPA receptor deficiency did not effect the expression of GN. These studies demonstrate that plasminogen plays an important role in protecting the glomerulus from acute inflammatory injury and that tPA is the major protective plasminogen activator.

The tissue-type plasminogen activator story.

Milestones in the development of tissue-type plasminogen activator (t-PA) as a fibrin-specific thrombolytic agent include: purification of human t-PA from the culture fluid of the Bowes melanoma cell line, elucidation of the molecular basis of fibrin-specific plasminogen activation, first experimental animal models of thrombosis, first patient (renal allograft) treated with melanoma t-PA, pilot studies in patients with acute myocardial infarction, cloning and expression of recombinant t-PA providing sufficient amounts for large scale clinical use, and demonstration of its therapeutic benefit in large multicenter clinical trials.

Receptor-independent role of urokinase-type plasminogen activator in pericellular plasmin and matrix metalloproteinase proteolysis during vascular wound healing in mice.

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR-/-). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR-/- smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR-/- and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR-/- cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA-mediated plasmin proteolysis to allow cellular migration into a vascular wound.

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Alpha 2-antiplasmin Enschede: alanine insertion and abolition of plasmin inhibitory activity.

An abnormal alpha 2-antiplasmin that is associated with a serious bleeding tendency has been found in a Dutch family and is referred to as alpha 2-antiplasmin Enschede. This abnormal alpha 2-antiplasmin is converted from an inhibitor of plasmin to a substrate. The molecular defect of alpha 2-antiplasmin Enschede, as revealed by sequencing of cloned genomic DNA fragments, consists of an alanine insertion near the active site region of the molecule. Substitution of this fragment into complementary DNA for a wild-type alpha 2-antiplasmin yields a translation product with physical and functional properties typical of the abnormal alpha 2-antiplasmin Enschede. The naturally occurring mutant may serve as a model for investigating the structures that determine the properties of an inhibitor versus those of a substrate in serine protease inhibitors.

Clot-selective coronary thrombolysis with tissue-type plasminogen activator.

Coronary thrombolysis, an intervention that can abort the sequelae of acute myocardial infarction, was accomplished within 10 minutes in dogs by intravenous administration of clot-selective, tissue-type plasminogen activator. In addition to inducing clot lysis, this promising fibrinolytic agent restored intermediary metabolism and nutritional myocardial blood flow, detectable noninvasively with positron tomography, without inducing a systemic fibrinolytic state.

Inhibition of vascular endothelial growth factor receptor tyrosine kinases impairs adipose tissue development in mouse models of obesity.

We have studied the effect of PTK787 (Vatalanib), an inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, on adipose tissue development. Oral administration of PTK787 for 4 weeks (2 mg/g high fat diet, HFD) to C57Bl/6 mice resulted in a significant reduction in total body weight and of subcutaneous (SC) and gonadal (GON) adipose tissue mass, as compared to control animals fed HFD only (all p<0.0005). In the GON adipose tissue adipocytes were hypertrophic after PTK787 treatment. Blood vessel size and density were not significantly affected by PTK787 treatment. Expression of Flk-1 (VEGFR-2) mRNA was significantly reduced in SC and GON adipose tissues of PTK787 treated mice. De novo fat pad formation following injection of preadipocytes in NUDE mice was significantly (p<0.005) impaired by PTK787 administration (2 mg/g HFD for 4 weeks), without associated effect on blood vessel size or density. Thus, in nutritionally induced murine obesity models, oral administration of the VEGFR tyrosine kinases inhibitor PTK787 resulted in reduced adipose tissue development.

Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors.

The angiogenic mechanism and therapeutic potential of PDGF-CC, a recently discovered member of the VEGF/PDGF superfamily, remain incompletely characterized. Here we report that PDGF-CC mobilized endothelial progenitor cells in ischemic conditions; induced differentiation of bone marrow cells into ECs; and stimulated migration of ECs. Furthermore, PDGF-CC induced the differentiation of bone marrow cells into smooth muscle cells and stimulated their growth during vessel sprouting. Moreover, delivery of PDGF-CC enhanced postischemic revascularization of the heart and limb. Modulating the activity of PDGF-CC may provide novel opportunities for treating ischemic diseases.

Metabolism of fibrinogen in cirrhosis of the liver.

The metabolism of human fibrinogen labeled with radioactive iodine was studied in 50 patients with documented cirrhosis of the liver and in 35 healthy control subjects. Results in cirrhotic subjects were the following: plasma volume 47 +/- 10 ml/kg; plasma fibrinogen concentration 250 +/- 102 mg/100 ml; total plasma fibrinogen pool 118 +/- 59 mg/kg, representing 0.73 +/- 0.10 of the total body pool; fibrinogen half-life 2.99 +/- 0.59 days; fractional catabolic rate 0.34 +/- 0.09 of the plasma pool per day; absolute catabolic rate 39 +/- 20 mg/kg per day; fractional transcapillary efflux rate 0.82 +/- 0.30 of the plasma pool per day. Results in the control subjects were the following: plasma volume 42 +/- 7 ml/kg; plasma fibrinogen concentration 284 +/- 71 mg/100 ml; total plasma fibrinogen pool 119 +/- 40 mg/kg, representing 0.72 +/- 0.07 of the total body pool; fibrinogen half-life 4.14 +/- 0.56 days; fractional catabolic rate 0.24 +/- 0.04 of the plasma pool per day; absolute catabolic rate 28 +/- 9 mg/kg per day; fractional transcapillary efflux rate 0.60 +/- 0.26 of the plasma pool per day.A significant difference between cirrhotics and controls was observed for plasma volume, fibrinogen half-life, fractional and total catabolic rates, and transcapillary efflux rate. During heparinization of 10 cirrhotic patients the fibrinogen half-life was prolonged from 3.15 +/- 0.69 to 4.59 +/- 0.79 days. This was associated with a rise in plasma fibrinogen in six out of eight patients. Heparinization did not influence the fibrinogen half-life in five control subjects. Inhibition of the fibrinolytic system in 17 patients resulted in prolongation of the plasma radioactivity half-life of more than 1 day in only three patients, an incidence comparable with that in five control subjects. These results strongly support the concept of accelerated fibrinogen consumption by a process of disseminated intravascular coagulation in cirrhosis of the liver.

Influence of protein C activation on blood coagulation and fibrinolysis in squirrel monkeys.

Protein C is a circulating proenzyme which, upon activation, exerts a potent anticoagulant activity. Infusion of activated bovine protein C into dogs is accompanied by an increase of circulating tissue plasminogen activator (PA) activity. However, the evidence that human protein C shares a similar profibrinolytic capacity is still lacking. Therefore, we investigated the profibrinolytic properties of human protein C in squirrel monkeys (Samiri sciureus). Injection of activated human protein C resulted in prolongation of the activated partial thromboplastin time but was not associated with increased fibrinolytic activity of blood. Similarly, activation of endogenous protein C (up to 20-30%) by infusion of thrombin-thrombomodulin complex markedly reduced blood coagulability without being accompanied by an increase of circulating PA activity. The in vivo-generated anticoagulant activity was identified as activated protein C by the following observations. It was neutralized by rabbit anti-human protein C-IgG, was slowly inhibited by plasma but not by anti-thrombin III, was adsorbable on barium citrate, and expressed amidolytic activity. Activation of protein C appeared to be selective since other parameters such as thrombin time, platelet count, fibrinogen, and factor V levels were unaffected by thrombin-thrombomodulin infusion. Infusion of human plasma derived from whole blood incubated in vitro with human activated protein C also did not induce a fibrinolytic response, suggesting that no second messengers with PA-releasing activity were being generated in blood. It is concluded that in a primate, neither the administration of activated human protein C nor the activation of endogenous protein C are associated with an increase of fibrinolytic activity. These findings question the role of this enzyme in the regulation of PA release in man.

Generation in plasma of a fast-acting inhibitor of plasminogen activator in response to endotoxin stimulation.

Endotoxin producing bacteria cause disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in man is still unclear. Impairment of the fibrinolytic system has been suggested as a contributing mechanism. A single injection of Escherichia coli lipopolysaccharide in rabbits resulted in a marked and prolonged increase of the levels of a fast-acting inhibitor of plasminogen activator (PA-inhibitor) in plasma (from 3.9 +/- 0.7 to 41 +/- 13.2 U/ml after 3 h). Gel filtration studies indicated that inhibition of human tissue-type plasminogen activator (t-PA) by rabbit plasma is accompanied by a change in the elution profile of the activator compatible with the formation of an enzyme-inhibitor complex with an apparent molecular weight of 100,000. Injection of human t-PA (1,500 IU/kg body wt) in endotoxin treated animals resulted in very fast inhibition of t-PA and formation of a similar complex. The half-life of circulating PA-inhibitor activity in rabbits was about 7 min as estimated by donor receiver plasma transfusion experiments. Stimulation of cultured human endothelial cells with endotoxin resulted in enhanced rate of accumulation of PA-inhibitor activity in the culture medium (two- to sevenfold increase). In five patients with septicemia, markedly increased levels of PA-inhibitor (14.3 +/- 15.5 U/ml) as compared with control subjects (1.3 +/- 0.7 U/ml) were observed in plasma. A very strong correlation (r = 0.98) was found between inhibition of t-PA and of urokinase in all conditions, suggesting that this fast-acting inhibitor reacts with both plasminogen activators. These data suggest that the appearance of this fast-acting PA-inhibitor is very sensitive to endotoxin stimulation. The marked increase in the level of PA-inhibitor in blood may contribute to the pathogenesis of DIC in septicemia.

Thrombolysis with human extrinsic (tissue-type) plasminogen activator in rabbits with experimental jugular vein thrombosis. Effect of molecular form and dose of activator, age of the thrombus, and route of administration.

A simple venous thrombosis model in rabbits was used for the quantitative evaluation of the thrombolytic effect of human extrinsic (tissue-type) plasminogen activator as compared with urokinase.A thrombus was formed in an isolated segment of the jugular vein from a mixture of (125)I-labeled fibrinogen, whole rabbit blood, and thrombin. In order to immobilize the thrombus during lysis, it was formed around a woolen thread introduced longitudinally in the lumen of the vein. Thrombotic extension of the clot was prevented by subcutaneous injection of heparin. The extent of thrombolysis was measured as the difference between the radioactivity introduced in the clot and that recovered in the vein segment at the end of the experiment. In control animals the extent of thrombolysis was 5.6+/-1.4% (n = 5) after 6 h, 14.5+/-1.7% (n = 10) after 30 h, 16.0+/-1.5% (n = 11) after 78 h, and 48.1+/-2.7% (n = 10) after 174 h (mean+/-SEM). Extrinsic (tissue-type) plasminogen activator, highly purified from the culture fluid of a human melanoma cell line, was administered systemically or locally over a time period of 4 h and the percent thrombolysis measured 2 h after the end of the infusion. One- and two-chain extrinsic plasminogen activator had very similar thrombolytic potency. Systemic infusion resulted in a dose-dependent degree of thrombolysis. The activator-induced thrombolysis, after infusion of 100,000 IU ( congruent with1 mg protein), was approximately 75% for fresh clots, 35% for 1-d-old clots, 30% for 3-d-old clots, and 50% for 7-d-old clots. The thrombolytic activity of urokinase was more than five times lower than that of extrinsic plasminogen activator: Infusion of 500,000 IU resulted in approximately 40% lysis of fresh clots and 25% of 1-3-d-old clots, while 7-d-old clots appeared to have become resistent to urokinase. Local infusion resulted in a 5-10 times higher thrombolytic effect of both extrinsic plasminogen activator and urokinase. Thrombolysis with extrinsic plasminogen activator was not associated with systemic activation of the fibrinolytic system as evidenced by unaltered plasma levels of fibrinogen, plasminogen, and alpha(2)-antiplasmin. Systemic infusion of urokinase resulted in significant thrombolysis only at doses that were associated with disseminated plasminogen activation. Local infusion of urokinase required a 5-10-fold higher dose than extrinsic plasminogen activator to obtain a similar degree of thrombolysis, which also occurred in the absence of systemic activation of the fibrinolytic system. It is concluded that the extent of thrombolysis by extrinsic plasminogen activator is mainly determined by the dose of activator and its delivery in the vicinity of the thrombus and much less by the age of the thrombus or the molecular form of the activator. Extrinsic plasminogen activator appears to be superior to urokinase because of its higher (5-10-fold) specific thrombolytic activity and the absence of systemic activation of the fibrinolytic system, which results in defibrinogenation and a bleeding tendency.