Role of vascular endothelial growth factor in the pathophysiology of nonalcoholic steatohepatitis in two rodent models.

UNLABELLED: The pathophysiology of nonalcoholic steatohepatitis (NASH) should be approached as a multifactorial process. In several stages of NASH, a link between disease progression and hepatic microvasculature changes can be made. In this study we investigated the role of angiogenesis in two mouse models for NASH, and the effect of a preventive and therapeutic antiangiogenic treatment in a diet-induced mouse model for NASH. Protein and RNA levels of angiogenic and inflammatory factors were significantly up-regulated in the liver of C56BL/6 and db/db mice with NASH at different timepoints. To examine the effect of angiogenic factors on the disease progression of NASH, a prevention and treatment study was set up, blocking the placental growth factor (PlGF) or vascular endothelial growth factor receptor 2 (VEGFR2). Our study showed that treatment prevents the progression of NASH by attenuating steatosis and inflammation, both in a preventive and therapeutic setting, thereby confirming the hypothesis that angiogenic factors play an early role in the disease progression from steatosis to NASH. Anti-PlGF (αPlGF) did not significantly improve liver histology. Vascular corrosion casting showed a more disrupted liver vasculature in mice with NASH compared to controls. Treatment with αVEGFR2 showed an improvement of the liver vasculature. Moreover, fat-laden primary hepatocytes treated with αVEGFR2 stored significantly less lipids. CONCLUSION: Our results demonstrate that there is an increased expression of angiogenic factors in the liver in different mouse models for NASH. We found that VEGFR2 blockage attenuates steatosis and inflammation in a diet-induced mouse model for NASH in a preventive and therapeutic setting. Our findings warrant further investigation of the role of angiogenesis in the pathophysiology in NASH.

Inhibition of placental growth factor improves surgical outcome of glaucoma surgery.

Excessive post-operative wound healing with subsequent scarring frequently leads to surgical failure of glaucoma filtration surgery (trabeculectomy). We investigated the hypothesis that placental growth factor (PlGF) plays a role in post-operative scar formation, and that it therefore may be a target for improvement of filtration surgery outcome. ELISA experiments showed that PlGF levels were significantly increased in aqueous humour of glaucoma patients and after VEGF treatment, which may indicate an important contribution of this growth factor to wound healing after trabeculectomy. Using a mouse model of glaucoma filtration surgery, we were able to show that intracameral injection of a previously characterized anti-PlGF antibody (ThromboGenics NV) significantly improved surgical outcome by increasing bleb survival and bleb area. This was associated with a significant reduction in post-operative proliferation, inflammation and angiogenesis during the first post-operative days after surgery, and with a decrease in collagen deposition at later stages. Furthermore, inhibition of PlGF seemed to be more effective than anti-VEGF-R2 treatment in improving surgical outcome, possibly via its additional effect on inflammation. These results render PlGF an appealing target for ocular wound healing and point to potential therapeutic benefits of PlGF inhibition for the prevention of surgical failure.

The placental growth factor as a target against hepatocellular carcinoma in a diethylnitrosamine-induced mouse model.

BACKGROUND & AIMS: The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile. METHODS: We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PlGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies. RESULTS: We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC. CONCLUSIONS: Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC.

Serum protein N-glycan alterations of diethylnitrosamine-induced hepatocellular carcinoma mice and their evolution after inhibition of the placental growth factor.

Placental growth factor (PlGF) inhibition produced promising results in reducing tumor burden in a diethylnitrosamine (DEN)-induced mouse model for hepatocellular carcinoma (HCC). The aim of this study was to non-invasively assess the improved histology by performing a serum glycomic analysis. To elucidate the molecular mechanism underlying the observed glycomic effects, we investigated the transcription and expression of E26 transformation-specific sequence 1 (Ets-1), a transcription factor essential for the glycomic and angiogenic changes in malignant transformation, including its different phosphorylated forms that result from activation of the MAP kinase and a Ca(2+)-dependent pathway. In addition, three Ets-1-dependent glycosyltransferase genes, Mgat4a, Mgat4b, and Mgat5, were also evaluated. HCC was induced in mice by weekly injections with DEN for 16, 20, 25, and 30 w. In the treatment study, mice were injected with DEN for 25 w and subsequently treated with PlGF antibodies (5D11D4) for 5 w. Finally, PlGF-/- mice were injected with DEN for 20, 25, and 30 w. Serum N-glycans were analyzed with DNA sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology. Maximum altered N-glycan phenotype was reached after 20 w of DEN-injections, i.e., when the first neoplastic lesions started to appear. 5D11D4-treatment improved the glycomic phenotype in that 7 of the 11 altered glycans tended to normalize. The PlGF-/- mice also showed a normalization trend, although not to the same extent of the treatment group. Number of Ets1, Mgat4a, Mgat4b, and Mgat5 transcripts increased considerably in DEN-injected mice, however, a non-significant decrease was observed after 5D11D4-treatment. On the protein level, 5D11D4-treatment had a prominent effect on the MAP kinase pathway with a significant p38 activation, yet independent of Ets-1 function.

Placental growth factor contributes to bronchial neutrophilic inflammation and edema in allergic asthma.

Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.

Inhibition of placental growth factor activity reduces the severity of fibrosis, inflammation, and portal hypertension in cirrhotic mice.

UNLABELLED: 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 CCl(4) -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.

The role of different VEGF isoforms in scar formation after glaucoma filtration surgery.

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. Our previous studies showed a differential role of VEGF isoforms in retinal physiological angiogenesis. We also demonstrated that non-selective inhibition of VEGF by bevacizumab had a beneficial effect on surgical outcome after glaucoma filtration surgery by reducing angiogenesis as well as fibrosis. However, the function of the VEGF isoforms in pathological angiogenesis and wound healing in the eye still remains unidentified. This study was designed to elucidate the differential roles of VEGF isoforms in scar formation after trabeculectomy. Furthermore, we also investigated whether pegaptanib (Macugen™, Pfizer), an aptamer which specifically blocks VEGF(165), could improve surgical outcome by reducing postoperative scarring. VEGF-R2 and neuropilin-1 (NRP-1) expression was analyzed in vitro by RT-PCR, and were found to be expressed at higher levels in human umbilical vein endothelial cells (HUVEC) as compared to Tenon fibroblasts (TF). The effect of the different VEGF isoforms (VEGF(121), VEGF(165) and VEGF(189)) and pegaptanib on cell proliferation was determined via WST-1 assay. Endothelial cell proliferation was stimulated after addition of VEGF(121) and VEGF(165), whereas VEGF(121) and VEGF(189) increased fibroblast growth. These effects on proliferation were associated with an activation of the ERK pathway, as revealed using the TransAM c-Myc assay. Inhibition of the ERK pathway, by PD98059 administration, significantly reduced VEGF isoform induced cell growth. A dose-dependent reduction of endothelial cell proliferation was observed after pegaptanib administration, while only the highest dose was able to inhibit fibroblast growth. Next, the in vivo effect of pegaptanib was investigated in a rabbit model of trabeculectomy. The surgical outcome was evaluated by performing clinical investigations (IOP, bleb area, height and survival), as well as histomorphometric analyses of angiogenesis (CD31), inflammation (CD45) and fibrosis (Sirius Red). A single postoperative application of pegaptanib had a beneficial impact on surgical outcome, mainly by reducing angiogenesis, but not inflammation or collagen deposition. Repeated injections slightly improved surgical outcome, but again solely by reducing angiogenesis. In summary, our results revealed that the VEGF isoforms play a differential role in ocular wound healing: VEGF(165) and VEGF(121) predominantly affect blood vessel growth, whereas VEGF(189) is rather involved in fibrosis, an important process in wound healing.

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.

Thrombolytic efficacy of recombinant human microplasmin in a canine model of copper coil-induced coronary artery thrombosis.

We investigated the in vitro fibrinolytic properties of microplasmin, the isolated proteinase domain of plasmin, and its thrombolytic efficacy in a coronary artery thrombosis model in dogs. The amidolytic and fibrinolytic activity of recombinant microplasmin was compared with natural human plasmin. The thrombolytic efficacy of microplasmin was studied in a canine model of copper coil induced coronary artery thrombosis. Animals were randomly assigned to one of six treatment regimens, each with five animals per cohort. Four treatment groups received an intravenous bolus of microplasmin followed by an intravenous infusion of microplasmin for 1 h (1 mg/kg + 1.5 mg/kg/h with or without abciximab or 2 mg/kg + 3 mg/kg/h). In two treatment groups, microplasmin was administered intracoronary. Bolus administration was followed by a 1-h infusion if coronary flow was incompletely restored after the initial bolus administration (1 mg/kg + 1.5 mg/kg/h or 2 mg/kg + 3 mg/kg/h, respectively). The thrombolytic efficacy was documented by repeated angiographies and the coronary perfusion was assessed with the Thrombolysis in Myocardial Infarction (TIMI) grading. No significant differences between plasmin and microplasmin were observed with respect to the catalytic efficiencies towards the synthetic chromogenic substrates S-2403 or S-2444. The concentration required for 50% lysis of purified fibrin clots in 3 h, was approximately 100 nM for microplasmin compared to 20 nM for natural plasmin. Intravenous bolus administration of microplasmin restored TIMI 3 coronary flow in 0/5, 0/5, 1/5 and 2/5, respectively, whereas intracoronary bolus administration restored TIMI 3 coronary flow in 1/5 and 4/5 (1 mg/kg and 2 mg/kg, respectively) (ANOVA P < 0.05). TIMI 3 coronary flow was obtained in 0/5, 2/5, 2/5 and 3/5, respectively, during subsequent intravenous administration and in 5/5 and 4/5 in case of intracoronary administration (ANOVA P < 0.05). When compared to natural plasmin, the catalytic efficiency of microplasmin towards chromogenic substrates was similar, but the fibrinolytic potency of microplasmin towards fibrin clots was lower. Intracoronay administration of microplasmin effectively lysed coronary thrombosis.

Emerging treatments for thrombocytopenia: increasing platelet production.

Thrombocytopenia is a common medical problem. The first generation thrombopoietic agents, recombinant THPO and 'megakaryocyte growth and development factor' (PEG-rHuMGDF) entered clinical trials, but their development was discontinued owing to neutralizing auto-antibodies cross-reacting with endogenous THPO, causing thrombocytopenia in healthy volunteers. Although an approved drug for prevention of severe thrombocytopenia following myelosuppressive chemotherapy (human Interleukin-11) exists, the search for new thrombopoietic agents continued because its use is limited by side effects. Several second generation thrombopoietic factors have entered clinical trials and some new negative regulators of megakaryopoiesis have been found, such as platelet factor 4 (PF4) and the pituitary adenylate cyclase activating polypeptide (PACAP). Their inhibition may be useful in the treatment of thrombocytopenia. This article reviews second generation thrombopoietic factors and those recently discovered regulators of megakaryopoiesis.

In-vitro profile and ex-vivo anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate.

Dabigatran is a reversible and selective, direct thrombin inhibitor (DTI) undergoing advanced clinical development as its orally active prodrug, dabigatran etexilate. This study set out to determine the molecular potency and anticoagulant efficacy of dabigatran and its prodrug dabigatran etexilate. This was achieved through enzyme inhibition and selectivity analyses, surface plasmon resonance studies, platelet aggregation, thrombin generation and clotting assays in vitro and ex vivo. These studies demonstrated that dabigatran selectively and reversibly inhibited human thrombin (Ki: 4.5 nM) as well as thrombin-induced platelet aggregation (IC(50): 10 nM), while showing no inhibitory effect on other platelet-stimulating agents. Thrombin generation in platelet-poor plasma (PPP), measured as the endogenous thrombin potential (ETP) was inhibited concentration-dependently (IC(50): 0.56 microM). Dabigatran demonstrated concentration-dependent anticoagulant effects in various species in vitro, doubling the activated partial thromboplastin time (aPTT), prothrombin time (PT) and ecarin clotting time (ECT) in human PPP at concentrations of 0.23, 0.83 and 0.18 microM, respectively. In vivo, dabigatran prolonged the aPTT dose-dependently after intravenous administration in rats (0.3, 1 and 3 mg/kg) and rhesus monkeys (0.15, 0.3 and 0.6 mg/kg). Dose- and time-dependent anticoagulant effects were observed with dabigatran etexilate administered orally to conscious rats (10, 20 and 50 mg/kg) or rhesus monkeys (1, 2.5 or 5 mg/kg), with maximum effects observed between 30 and 120 min after administration, respectively. These data suggest that dabigatran is a potent, selective thrombin inhibitor and an orally active anticoagulant as the prodrug, dabigatran etexilate.

Effects of the direct thrombin inhibitor dabigatran and its orally active prodrug, dabigatran etexilate, on thrombus formation and bleeding time in rats.

Dabigatran is a reversible direct, selective thrombin inhibitor, undergoing clinical development as its orally active prodrug, dabigatran etexilate. The objective of this trial was to assess the antithrombotic and anticoagulant effects of dabigatran and dabigatran etexilate in a rat model of venous thrombosis. In order to do this a modified Wessler model was used to assess the antithrombotic and anticoagulant effects of intravenous (i.v.) dabigatran and oral dabigatran etexilate administration. In addition, a rat tail bleeding time model was used to investigate the antihemostatic effect of dabigatran. The study demonstrated that bolus administration of dabigatran (0.01-0.1 mg/kg) reduced thrombus formation dose-dependently, with an ED50 (50% of the effective dose) of 0.033 mg/kg and complete inhibition at 0.1 mg/kg. By comparison, ED50 values for heparin (0.03-0.3 mg/kg), hirudin (0.01-0.5 mg/kg) and melagatran (0.1-0.5 mg/kg) were 0.07, 0.15 and 0.12 mg/kg, respectively. Oral administration of dabigatran etexilate (5-30 mg/kg) inhibited thrombus formation in a dose- and time-dependent manner, with maximum inhibition within 30 min of pretreatment, suggesting a rapid onset of action. Following i.v. administration of dabigatran (0.1-1.0 mg/kg), a statistically significant prolongation of bleeding time was observed at doses at least 15- and 5-fold greater than ED50 and ED100 (100% of the effective dose) doses, respectively; there was no significant increase in bleeding tendency at the maximum therapeutically effective dose (0.1 mg/kg). It can be concluded that dabigatran and its oral prodrug, dabigatran etexilate, show promise in the management of thromboembolic disease.

Humanization by variable domain resurfacing and grafting on a human IgG4, using a new approach for determination of non-human like surface accessible framework residues based on homology modelling of variable domains.

Many antithrombotic agents have only a small therapeutic window, frequently leading to bleeding problems. However, interfering with platelet adhesion through the collagen-VWF-GPIbalpha axis is expected to cause less bleeding problems. Our group developed a monoclonal antibody, 82D6A3, directed against the von Willebrand factor (VWF) A3-domain, which inhibits the VWF-interaction to fibrillar collagen. 82D6A3 has antithrombotic effects in vivo without bleeding time prolongation. To further investigate the promising features of 82D6A3, we have humanized it by variable domain resurfacing and grafting on the constant regions of a human IgG4. First, the sequence of the variable domains was determined and the murine scFv was constructed. The expressed scFv had a comparable activity as the IgG of 82D6A3, and its DNA was thus used in subsequent humanization procedures. For this, a new approach was introduced to identify non-human like framework surface residues, since the general distribution of accessible residues described for human and murine heavy and light chain variable domains showed several discrepancies with the homology modelled Fv of 82D6A3. Identification of non-human like framework residues and evaluation of their surface accessibility within the context of the homology modelled Fv of 82D6A3, revealed 10 residues that need to be humanized without influencing the conformation of the CDR loops. Indeed, the humanized scFv of 82D6A3, obtained by mutating all 10 residues to their human counterpart, was still binding with high affinity to VWF and retained the inhibitory properties of the murine scFv. Next, in order to increase its half life and to decrease its immunogenicity, the humanized variable domains were grafted on the constant regions of a human IgG4, resulting in h82D6A3 with an in vitro activity comparable to that of the murine IgG.

Release of experimental retinal vein occlusions by direct intraluminal injection of ocriplasmin.

PURPOSE: Retinal vein occlusions (RVO) are a major cause of vision loss in people aged 50 years and older. Current therapeutic options limit the consequences of RVO but do not eliminate the cause. Cannulation of the involved vessel and removal of the clot may provide a more permanent solution with a less demanding follow-up. However, cannulation of smaller retinal veins remains challenging. This paper explores the use of ocriplasmin (recombinant plasmin without its kringles) to clear RVO, using a robotic micromanipulator. METHODS: Branch RVO were induced in a porcine model with rose bengal followed by 532 nm endolaser to the superior venous branch of the optic nerve. The vein was cannulated proximal to the occlusion or beyond the first branching vessel from the obstruction. The vein was infused with a physiologic citric acid buffer solution (CAM) or CAM/ocriplasmin. The time of cannulation, number of attempts, and the ability to release the thrombus were recorded. RESULTS: Cannulation and infusion was possible in all the cases. The use of a micromanipulator allowed for a consistent cannulation of the retinal vein and positional stability allowed the vein to remain cannulated for up to 20 min. In none of the attempts (5/5) with CAM did the thrombus dissolve, despite repeat infusion/relaxation cycles. In 7/7 injections of CAM/ocriplasmin near to the point of obstruction, the clot started to dissolve within a few minutes of injection. An infusion, attempted beyond the first venous branch point proximal to the clot, was unsuccessful in 2/3 attempts. CONCLUSIONS: Ocriplasmin is effective in resolving RVO if injected close to the site of occlusion with the use of a micromanipulator.

Robotic Assisted Cannulation of Occluded Retinal Veins.

PURPOSE: To develop a methodology for cannulating porcine retinal venules using a robotic assistive arm after inducing a retinal vein occlusion using the photosensitizer rose bengal. METHODOLOGY: Retinal vein occlusions proximal to the first vascular branch point were induced following intravenous injection of rose bengal by exposure to 532nm laser light delivered by slit-lamp or endolaser probe. Retinal veins were cannulated by positioning a glass catheter tip using a robotically controlled micromanipulator above venules with an outer diameter of 80µm or more and performing a preset piercing maneuver, controlled robotically. The ability of a balanced salt (BSS) solution to remove an occlusion by repeat distention of the retinal vein was also assessed. RESULTS: Cannulation using the preset piercing program was successful in 9 of 9 eyes. Piercing using the micromanipulator under manual control was successful in only 24 of 52 attempts, with several attempts leading to double piercing. The best location for cannulation was directly proximal to the occlusion. Infusion of BSS did not result in the resolution of the occlusion. CONCLUSION: Cannulation of venules using a robotic microassistive arm can be achieved with consistency, provided the piercing is robotically driven. The model appears robust enough to allow testing of therapeutic strategies aimed at eliminating a retinal vein thrombus and its evolution over time.

Inhibition of Rho-Associated Kinase Prevents Pathological Wound Healing and Neovascularization After Corneal Trauma.

PURPOSE: To investigate the effect of AMA0526, a specific inhibitor of rho-associated protein kinase (ROCK), on corneal neovascularization (NV) and scarring in different in vitro and in vivo experimental models. METHODS: The effect of AMA0526 on cell viability, proliferation, and migration of human umbilical vein endothelial cells was determined. Its in vivo topical effect on NV was investigated in the corneal micropocket mouse model (bevacizumab as a control). The vessel length, clock hours, and NV area were measured on photographs. The effect of AMA0526 on pathological wound healing was investigated in the alkali burn mouse model (dexamethasone as a control). Corneas were scored for corneal opacity (CO) and NV after burn injury. Immunohistochemistry was performed to study inflammation, blood vessel density, and collagen III deposition after 7 days. RESULTS: ROCK inhibition significantly inhibited vascular endothelial cell proliferation and migration in vitro in a dose-dependent manner. In the micropocket model, NV was significantly reduced by AMA0526 (37% reduction, P < 0.05) comparable to bevacizumab. CO and NV were reduced after AMA0526, compared with the vehicle (P < 0.05 at all time points from day 3) after chemical burn. AMA0526 resulted in decreased inflammatory cell infiltration (26% reduction, P < 0.01), angiogenesis (47% reduction, P < 0.01), and collagen III deposition (27% reduction, P = 0.009) in the alkali burn model. AMA0526 administration showed results similar to those of dexamethasone with an additional antifibrotic effect. CONCLUSIONS: The ROCK inhibitor, AMA0526, efficiently inhibited angiogenesis in vitro, reduced CO and NV, and controlled the complete process of wound healing in vivo. These results warrant further investigation of the therapeutic potential of AMA0526 for corneal NV and scarring.

Design, synthesis, and biological evaluation of novel, highly active soft ROCK inhibitors.

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed.

Structure-based design of novel potent nonpeptide thrombin inhibitors.

The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this enzyme. On the basis of the X-ray crystal structure of the peptide-like thrombin inhibitor NAPAP complexed with bovine thrombin, we have designed a new structural class of nonpeptidic inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin inhibition in the lower nanomolar range could be achieved although the binding energy mainly results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral activity in different animal species. On the basis of these results, 31 was chosen for clinical development.

Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative.

Interfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (K(A) = 4.8 +/- 0.2 x 10(7) M(-1) vs. 1.8 +/- 0.7 x 10(9) M(-1) for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (K(A) = 2.1 +/- 0.2 x 10(7) M(-1)). In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.

Importance of N-terminal residues in plasminogen activator inhibitor 1 on its antibody induced latency transition.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a well-known risk factor for thromboembolic and cardiovascular diseases. Many efforts have been made to reveal structure-function relationship in PAI-1, including the understanding of its unique latency transition. In this study, we describe the molecular characterization of PAI-1 neutralization by MA-159M12, a monoclonal antibody against rat PAI-1. Time-dependent inactivation of PAI-1, exposure of a trypsin cleavage site typically for the latent conformation and disappearance of elastase susceptibility revealed that MA-159M12 accelerated the active to latent, conformational transition (t1/2 120 +/- 12 min and 18 +/- 3.6 min in the absence and presence of MA-159M12, p < 0.0001). Cross-reactivity analysis of the antibody with various rat/human PAI-1 chimeras revealed that the epitope resides in alpha hA of rat PAI-1. Subsequent alanine-scanning mutagenesis and binding studies demonstrated that Pro2-Leu3-Pro4-Glu5 constitute the major residues of the epitope for MA-159M12. In conclusion, these findings demonstrate that, even though unexpected based on current knowledge on PAI-1 stability and function, interference with alpha hA results in a destabilisation of its active, inhibitory conformation. Therefore, alpha hA forms a putative target for the rational development of PAI-1 neutralizing components.