Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl4-induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF−/− mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. Conclusion: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile. (Hepatology 2011;)

A B S T R A C T 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 125I-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 (n1 mg protein), was -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 -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 a2-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. 368J. Clin. Invest.

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

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

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