Jean Marie Stassen

Inhibition of Placental Growth Factor Activity Reduces the Severity of Fibrosis, Inflammation, and Portal Hypertension in Cirrhotic Mice

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;)

Role of Placental Growth Factor in Mesenteric Neoangiogenesis in a Mouse Model of Portal Hypertension

BACKGROUND & AIMS Portal hypertension is responsible for the major complications associated with cirrhosis. Angiogenesis has been associated with the pathophysiology of portal hypertension. We investigated the role of placental growth factor (PlGF) and tested the effects of monoclonal antibodies against PlGF (alphaPlGF) in a mouse model of portal hypertension. METHODS Using a mouse model of prehepatic portal hypertension, we measured PlGF levels in the mesenteric tissue at different time points. We used knockout mice and alphaPlGF to determine the role of PlGF in the splanchnic hyperdynamic system and portosystemic collateral formation, examining its effects before and after portal hypertension was induced. RESULTS PlGF was significantly up-regulated in the mesenteric tissue of mice with portal hypertension. Compared with wild-type animals, the vascular density in the mesentery was reduced in PlGF knockout hypertensive mice, preventing collateral formation and attenuation of mesenteric artery flow without affecting portal pressure. In the prevention study, alphaPlGF showed similar findings as in the knockout study. In mice with portal hypertension, administration of alphaPlGF resulted in a 32% decrease in portal pressure, compared with mice given immunoglobulin G(1) (control). CONCLUSIONS Pathologic angiogenesis in the mesenteric tissues of mice with portal hypertension is mediated by PlGF. Blocking PlGF could be an effective strategy for reducing collateral formation and lowering portal pressure; further research into the effects in cirrhosis is warranted.

Anti–Placental Growth Factor Reduces Bone Metastasis by Blocking Tumor Cell Engraftment and Osteoclast Differentiation

Treatment of bone metastases is largely symptomatic and is still an unmet medical need. Current therapies mainly target the late phase of tumor-induced osteoclast activation and hereby inhibit further metastatic growth. This treatment method is, however, less effective in preventing initial tumor engraftment, a process that is supposed to depend on the bone microenvironment. We explored whether bone-derived placental growth factor (PlGF), a homologue of vascular endothelial growth factor-A, regulates osteolytic metastasis. Osteogenic cells secrete PlGF, the expression of which is enhanced by bone-metastasizing breast tumor cells. Selective neutralization of host-derived PlGF by anti-mouse PlGF (alphaPlGF) reduced the incidence, number, and size of bone metastases, and preserved bone mass. alphaPlGF did not affect metastatic tumor angiogenesis but inhibited osteoclast formation by preventing the upregulation of the osteoclastogenic cytokine receptor activator of NF-kappaB ligand in osteogenic cells, as well as by blocking the autocrine osteoclastogenic activity of PlGF. alphaPlGF also reduced the engraftment of tumor cells in the bone and inhibited their interaction with matrix components in the metastatic niche. alphaPlGF therefore inhibits not only the progression of metastasis but also the settlement of tumor in the bone. These findings identify novel properties of PlGF and suggest that alphaPlGF might offer opportunities for adjuvant therapy of bone metastasis.

Thrombolytic profiles of clot-targeted plasminogen activators. Parameters determining potency and initial and maximal rates.

BackgroundTargeting of plasminogen activators to the thrombus by means of fibrin-specific monoclonal antibodies may enhance their thrombolytic potency. The kinetics of clot binding of two humanfibrin-specific monoclonal antibodies (MA-12B3 and MA-15C5) and of clot lysiswith their chemical 1:1 stoichiometric complexes with recombinant single-chain urokinase-type plasminogen activator (rscu-PA)(rscu-PA/MA-12B3 and rscu-PA/MA-15C5) were determined in hamsters and rabbits. Thrombolyticpotencies, maximal rates of clot lysis, and the duration of the lag phases before clot lysis of theantibody/rscu-PA conjugates were compared with those of rscu-PA and tissue-type plasminogen activator (rt-PA). Methods and ResultsBolus injection of 7.5 μg of 125I-labeled antibody in rabbits with an extracorporeal arteriovenous loopcontaining a 0.3-mL human plasma clot produced clot-to-blood ratios of 6.6±1.0 (mean±SEM) for MA-12B3 and 1.1±0.15 for MA-15C5 (p<0.001 versus MA-12B3) within 6 hours. Progressive digestion of the clot did not alter the binding of MA-12B3 but resulted in as much as a 10-fold increase of the binding of MA-15C5. The conjugates infused intravenously over 90 minutes in hamsters with a human plasma clot in thepulmonary artery produced dose-related in vivo clot lysis. Thrombolytic potencies (maximal slope of the percent lysis versus dose in milligrams of u-PA equivalent per kilogram body weight) were 2,500±440 for rscu-PA/MA-12B3, 3,600±640 for rscu-PA/MA-15C5 (p=NS vs. rscu-PA/MA-12B3), 60±8 for rscu-PA (p<0.001 versus both conjugates), and 380+66 for rt-PA (p<0.001 versus both conjugates). The plasma clearances of the conjugates were fourfold to sixfold slower than those of rscu-PA and rt-PA. Maximal rates of clot lysis, determined by continuous external radioisotope scanning over the thorax, were 0.90±0.13%, 0.91+0.17%, 0.84±0.12%, and 1.1+0.16% lysis per minute for rscu-PA/MA-12B3, rscu-PA/MA-1SC5, rscu-PA, and rt-PA, respectively; these maximal rates were obtained with 0.016,0.016, 1.0, and 0.25 mg/kg, respectively, and were associated with minimal lag phases of 18±3.2, 28±4.9, 34+3.7, and 25±3.9 minutes, respectively. ConclusionThe thrombolytic potency of the rscu-PA/antifibrin conjugates is determined by their clearance, as well as by rate and extent of initial binding to clots and by changes in binding during clotlysis. Clot targeting of rscu-PA with fibrin-specific antibodies increases its thrombolytic potency but does not alter the maximal rate or the minimal lag phase of clot lysis. These parameters appear to be independent of the nature of the plasminogen activator and of targeting.

Pharmacokinetics of Ocriplasmin in Vitreous

PURPOSE Ocriplasmin contains the active moiety of plasmin enzyme. At a physiologic pH, ocriplasmin is highly proteolytic and autolytic, limiting its duration of activity. Specific inhibitors of plasmin are present in the vitreous under normal and disease conditions and could affect its activity. Each may contribute to its mode of action. METHODS Degradation characteristics were determined in porcine, human vitreous, and PBS under reducing conditions with different incubation periods between 0 and 24 hours on SDS-PAGE Tris-glycine gels. Residual activity was determined by spectrophotometry of p-nitroaniline release through hydrolysis of L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroaniline hydrochloride. The presence of endogenous inactivators of ocriplasmin in human vitreous was determined in a series of vitreous samples using an ELISA specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin. RESULTS Degradation productions from autolysis are similar between vitreous and PBS with a significant prolongation of the effect in vitreous. Both follow a nonlinear pattern over time. The degradation corresponds best to a second-order kinetic process. The resulting rate constants were 207 ± 60 M(-1) s(-1) in PBS, 81 ± 15 M(-1) s(-1) in porcine vitreous, and 195 M(-1) s(-1) in human vitreous natural inhibitors were identified in samples of donor vitreous. Amounts differed significantly between samples, which may help explain the observed variability in human subjects. CONCLUSIONS Ocriplasmin is autolytic in vitreous. Biologic activity extends to several days following injection. The exact duration will vary based on the presence and concentration of serine protease inhibitors.

Inhibition of human pulmonary artery smooth muscle cell proliferation and migration by sabiporide, a new specific NHE-1 inhibitor.

Abstract: Abnormal growth of vascular smooth muscle cells is seen in various pathological conditions such as hypertension, atherosclerosis, and restenosis. Na+/H+ exchanger (NHE) activation appears to play a permissive role in vascular smooth muscle cell proliferation and vascular remodeling. The present study investigated the effect of a new specific NHE-1 inhibitor, sabiporide, on human pulmonary artery smooth muscle cell proliferation and migration. Concentrations of sabiporide as low as 20 μmol/L in the culture medium containing growth factors inhibited cell proliferation, as measured by cell counting, and also inhibited the rate of DNA synthesis, as examined by measuring BrdU incorporation into DNA. Cell growth inhibition was not caused by cell death, as demonstrated by the measurement of intracellular lactate dehydrogenase release and by the reversibility of inhibition upon washing. By fluorescent-activated cell sorting analysis, we are the first to demonstrate that NHE-1 inhibition arrests the cell cycle progression at G0/G1 phase, suggesting that NHE activation plays a permissive role in entrance of cells into the cell cycle. Sabiporide also concentration-dependently inhibited human pulmonary artery smooth muscle cell migration. The present study showed that sabiporide inhibits vascular smooth muscle cell proliferation and migration by blocking the cell cycle progression at G0/G1 phase.

Intraoperative thrombolytic treatment of microarterial occlusion by selective rt-PA infusion

The development of microvascular thrombosis during replantation surgery or free-flap transfer is generally best treated by identification of the problem, vascular revision, and reanastomosis. It is not unique, however, that surgical measures alone are insufficient or undesirable. Pharmacologic adjuncts are widely used for the prevention and sometimes treatment of microvascular thrombosis in surgical practice, but the benefit of thrombolytic agents, effective in dissolving an already established thrombus, is usually considered counterlevered by the fear of uncontrollable bleeding. However, selective infusion of the drug reduces the risk for systemic complications considerably and may therefore be considered in peripheral microvascular reconstruction. The technique was used successfully in a case of digital revascularization, where an arterial thrombosis was dissolved with the use of recombinant tissue plasminogen activator (rt-PA).

Contribution of platelets and the vessel wall to the antithrombotic effects of a single bolus injection of Fab fragments of the antiplatelet GPIIb/IIIa antibody 7E3 in a canine arterial eversion graft preparation.

The contribution of platelets and the vessel wall to the antithrombotic effects of a single intravenous bolus injection of 0.8 mg/kg Fab fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody 7E3 (7E3-Fab), combined with continuous heparin anticoagulation (100 U/kg bolus and 50 U/kg per hour), was studied in a canine preparation consisting of an everted (inside out) carotid arterial segment that had been inserted into a transected femoral artery. In all 6 control dogs without antibody, persistent or transient eversion graft occlusion occurred during an initial 2-hour observation period, and 5 of the 6 grafts were occluded at 24 hours. In 6 dogs given 7E3-Fab 24 hours before receiving an everted carotid artery segment from a donor dog, cyclic occlusion and reflow occurred in all dogs, whereas the grafts were patent at the end of a 2-hour observation period in 5 of the 6 dogs (P = .056 versus control). When transferred back to the donor dogs, the patient eversion segments showed brief periods of cyclic occlusion and reflow within 2 hours in 3 of 5 dogs (P = .034 versus control), whereas all of the 5 eversion segments were patent at 24 hours (P < .005 versus control).(ABSTRACT TRUNCATED AT 250 WORDS)

A technique to investigate microvascular mural thrombus formation in arteries and veins: II. Effects of aspirin, heparin, r-hirudin, and G-4120.

After a standardized trauma to carotid arteries or femoral veins of hamsters, the antithrombotic effects of two antiplatelet agents (aspirin and the glycoprotein llb/llla antagonist G4120) and two anticoagulants (heparin and the direct thrombin inhibitor rhirudin) were studied in vivo. The thrombus area volume was assessed by image analysis of the transilluminated experimental vessels. Heparin, r-hirudin, and G-4120 demonstrated a dosedependent complete inhibition of arterial and venous thrombosis. In contrast, the antithrombotic effect of aspirin was only partial in both vessel types. A significant correlation between activated partial thromboplastin time (aPTT) at the end of the experiments and the antithrombotic effect was observed with the anticoagulant agents. However, only r-hirudin inhibited thrombus formation at a therapeutical prolongation of aPTT, while heparin required supratherapeutical amounts to achieve the same inhibition. The data confirm that the inhibition of aspirin, heparin, r-hirudin, and G-4120 on the formation of platelet-rich thrombi is independent of the blood flow rate. Stockmans F, Stassen JM, Vermylen J, Hoylaerts MF, Nystrom A. A technique to investigate microvascular mural thrombus formation in arteries and veins. II. Effects of aspirin, heparin, r-hirudin, and G-4120. Ann Plast Surg 1997;38:63-68

Comparative antigenicity of recombinant wild-type staphylokinase (SakSTAR) and a selected mutant (SakSTAR.M38) in a baboon thrombolysis model.

Staphylokinase, a bacterial plasminogen activator, is a potent, highly fibrin-specific but antigenic thrombolytic agent in humans. In an effort to attenuate the antigenicity of wild-type staphylokinase (SakSTAR variant), 2 of its 3 immunodominant epitopes were altered by substituting clusters of 2 or 3 charged amino acids with alanine, yielding the mutant SakSTAR.M38 (K35A, E38A, K74A, E75A, R77A), which was less antigenic in inbred New Zealand White rabbits. In the present study, groups of 6 baboons (Papio hamadryas) were randomized to SakSTAR (group 1) or SakSTAR.M38 (group 2). The thrombolytic potencies of 50 micrograms/kg compound at baseline, assessed in an extracorporeal thrombosis model, were similar: 77 +/- 2.9% (mean +/- SEM) clot lysis in group 1 and 83 +/- 3.6% in group 2. Groups 1 and 2 were immunized subcutaneously at 2, 3, and 5 weeks with 500 micrograms SakSTAR or SakSTAR.M38, respectively. From 6 weeks, group 1 developed significantly more antibody-related neutralizing activity than group 2 (maximal titer at 8 weeks of 100 +/- 23 micrograms SakSTAR and of 22 +/- 7.1 micrograms SakSTAR.M38 neutralized per milliliter of plasma, respectively). Neutralizing activities subsequently decreased gradually to 10-20% of peak values at 18 weeks. At 6 weeks, both groups were resistant to thrombolysis with 50 micrograms/kg of either compound. Rechallenge at 18 weeks with 250 micrograms/kg of the immunizing compound showed a significantly better recovery of the thrombolytic potency of SakSTAR.M38 (68 +/- 4.5% clot lysis) than of SakSTAR (39 +/- 5.3% clot lysis). Neither agent degraded fibrinogen or depleted alpha 2-antiplasmin. Therefore, SakSTAR.M38 is comparably active and fibrin-specific but less antigenic than wild-type SakSTAR. These findings in outbred primates confirm and extend earlier observations in inbred rabbits and provide a basis for the further development of staphylokinase variants with reduced antigenicity in humans.