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International audience; Little information exists regarding what occurs in the affected artery in the days after acute ischemic stroke and its impact in the outcome. We sought to determine the hemodynamic evolution and correlated this evoution with clinical outcome in stroke patients treated with intravenous thrombolysis. Using serial transcranial Doppler ultrasound (TCD) on days 1 (TCD1), 3 to 6 (TCD2), and 7 to 10 (TCD3) after stroke, we determined the hemodynamics in the affected artery by means of the thrombolysis in brain ischemia (TIBI) score and compared this with clinical outcome (National Institutes of Health Stroke Scale [NIHSS] score) and functional outcome (modified Rankin Scale score) at discharge and at 3 months. Thirty-four patients were studied. There were 24 men with a mean (± SD) age of 72.9 ± 16.2 years. The mean time from stroke onset to the administration of intravenous tissue plasminogen activator was 181 ± 54.4 minutes, and the mean NIHSS score at admission was 16.9 ± 9. Hemodynamic changes were observed in 23 (68%) patients, including improvement in 17 (50%) patients and worsening in 6 (18%) patients within the first 10 days poststroke. Clinical deterioration (NIHSS ≥4 points) was timely associated with hemodynamic deterioration in 3 cases. Patients achieving full recanalization at TCD3 had better mRS scores at 3 months (4 v 3; P = .02). Hemodynamic changes in the affected artery occurred in about two-thirds of patients within the first 10 days after receiving intravenous thrombolysis; 18% had hemodynamic deterioration, which was associated with clinical worsening in half of these cases.

International audience; AIM:Practitioners are faced with a substantial challenge when considering recombinant tissue plasminogen activator (rt-PA) therapy for older patients with ischemic stroke. Patients aged over 80 years suffer from the most severe cerebral infarcts. The benefit of rt-PA treatment compared with single standard care only in stroke units remains to be clearly assessed.METHODS:We collected data from 321 patients aged over 80 years admitted for acute cerebral infarction to the stroke unit of Nancy University Hospital in France between 1 January 2009 and 31 December 2012. Patients were stratified into two groups: treated or not with rt-PA. Baseline characteristics and outcome were collected and compared between both groups. Good outcome at 3 months was defined as modified Rankin Scale score ≤2.RESULTS:The 55 patients treated with rt-PA had a higher National Institute of Health Stroke Scale score on admission than those without (15 vs 5; P 2 (57 vs 54 %), but without reaching significance (P > 0.05).CONCLUSIONS:Rt-PA therapy would appear to improve prognosis in the elderly with ischemic stroke. This suggests that age alone should no longer be a barrier to rt-PA therapy.

International audience; An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

International audience; Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

International audience; Fine-tuning of NMDA glutamatergic receptor signalling strategically controls crucial brain functions. This process depends on several ligands and modulators, one of which unexpectedly includes the serine protease tissue-type plasminogen activator (tPA). In vitro, tPA increases NMDA-receptor-mediated calcium influx by interacting with, and then cleaving, the NR1 subunit within its N-terminal domain. Owing to lack of in vivo evidence of the relevance and contribution of this mechanism in physiological and pathological brain processes, active immunisation was developed here in mice, to allow transient and specific prevention of the interaction of tPA with the NR1 subunit. Immunisation significantly reduced the severity of ischemic and excitotoxic insults in the mouse brain. Cognitive function was altered in some, but not all behavioural tasks affected in tPA-deficient mice. Our data demonstrate that in vivo, tPA controls neurotoxicity and the encoding of novel spatial experiences by binding to and cleaving the NMDA receptor NR1 subunit. Interesting therapeutic possibilities for several brain pathologies that involve excitotoxicity may now be envisaged.

International audience; Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems.

International audience; Activation of glucocorticoid receptors (GR) by glucocorticoid hormones (GC) enhances contextual fear memories through the activation of the Erk1/2(MAPK) signaling pathway. However, the molecular mechanism mediating this effect of GC remains unknown. Here we used complementary molecular and behavioral approaches in mice and rats and in genetically modified mice in which the GR was conditionally deleted (GR(NesCre)). We identified the tPA-BDNF-TrkB signaling pathway as the upstream molecular effectors of GR-mediated phosphorylation of Erk1/2(MAPK) responsible for the enhancement of contextual fear memory. These findings complete our knowledge of the molecular cascade through which GC enhance contextual fear memory and highlight the role of tPA-BDNF-TrkB-Erk1/2(MAPK) signaling pathways as one of the core effectors of stress-related effects of GC.

International audience; BACKGROUND: The role of fibrinolytic therapy in patients with intermediate-risk pulmonary embolism is controversial. METHODS: In a randomized, double-blind trial, we compared tenecteplase plus heparin with placebo plus heparin in normotensive patients with intermediate-risk pulmonary embolism. Eligible patients had right ventricular dysfunction on echocardiography or computed tomography, as well as myocardial injury as indicated by a positive test for cardiac troponin I or troponin T. The primary outcome was death or hemodynamic decompensation (or collapse) within 7 days after randomization. The main safety outcomes were major extracranial bleeding and ischemic or hemorrhagic stroke within 7 days after randomization. RESULTS: Of 1006 patients who underwent randomization, 1005 were included in the intention-to-treat analysis. Death or hemodynamic decompensation occurred in 13 of 506 patients (2.6%) in the tenecteplase group as compared with 28 of 499 (5.6%) in the placebo group (odds ratio, 0.44; 95% confidence interval, 0.23 to 0.87; P=0.02). Between randomization and day 7, a total of 6 patients (1.2%) in the tenecteplase group and 9 (1.8%) in the placebo group died (P=0.42). Extracranial bleeding occurred in 32 patients (6.3%) in the tenecteplase group and 6 patients (1.2%) in the placebo group (P

International audience; During early post-natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue-type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix. Crucial role of tissue plasminogen activator (tPA) in interneuron migration. Interneuron migration is a critical step for normal establishment of neuronal network. This study indicates that, in the post-natal cerebellum, tPA facilitates the opposite migration of immature excitatory granule neurons (GN) and immature inhibitory basket/stellate cells (B/SC) along the same migratory route. These data show that tPA exerts a pivotal role in neurodevelopment.

International audience; The estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined.

International audience; Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.

International audience; Owing to its ability to generate the clot-dissolving protease plasmin, tissue plasminogen activator (tPA) is the only approved drug for the acute treatment of ischemic stroke. However, tPA also promotes hemorrhagic transformation and excitotoxic events. High mobility group box-1 protein (HMGB-1) is a non-histone transcription factor and a pro-inflammatory cytokine, which has also been shown to bind to both tPA and plasminogen. We thus investigated the cellular and molecular effects through which HMGB-1 could influence the vascular and parenchymal effects of tPA during ischemia. We demonstrate that HMGB-1 not only increases clot lysis by tPA, but also reduces the passage of vascular tPA across the blood-brain barrier, as well as tPA-driven leakage of the blood-brain barrier. In addition, HMGB-1 prevents the pro-neurotoxic effect of tPA, by blocking its interaction with N-methyl-D-aspartate (NMDA) receptors and the attendant potentiation of NMDA-induced neuronal Ca²⁺ influx. In conclusion, we show in vitro that HMGB-1 can promote the beneficial effects of tPA while counteracting its deleterious properties. We suggest that derivatives of HMGB-1, devoid of pro-inflammatory properties, could be used as adjunctive therapies to improve the overall benefit of tPA-mediated thrombolysis following stroke.

International audience; Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.

International audience; OBJECTIVE: To evaluate whether very early neurologic improvement (VENI) after intravenous (i.v.) recombinant tissue plasminogen activator (rt-PA) perfusion in patients with acute ischemic stroke (AIS) predicts favorable outcome at 3 months. DESIGN: Retrospective analysis of prospective data. SETTING: Stroke registry at the Stroke Unit, Tenon University Hospital. PATIENTS: We analyzed consecutive patients with AIS treated with i.v. rt-PA between November 11, 2002, and December 24, 2007. MAIN OUTCOME MEASURES: VENI at 1 hour was defined as a National Institute of Health Stroke Scale score of 0 at the end of rt-PA perfusion or an improvement of 5 or more points compared with baseline. Favorable outcome was defined as a modified Rankin Scale score of 1 or less at 3 months. RESULTS: Of 120 patients with AIS treated with i.v. rt-PA, 22 (18.3%) had VENI after i.v. rt-PA perfusion. Favorable outcome was observed in 15 patients with VENI (68.2%) and in 29 patients without VENI (29.6%) (P < .001). No symptomatic intracerebral hemorrhage occurred in patients with VENI. Mortality rates were 0% in the patients with VENI and 17.3% in patients without VENI. Baseline scores for VENI (adjusted odds ratio, 6.23; 95% confidence interval, 2.03-19.13; P = .001) and the National Institute of Health Stroke Scale (0.83; 0.76-0.91; P < .001) were the only 2 factors associated with favorable outcome (modified Rankin Scale score of ≤1). CONCLUSIONS: VENI at the end of i.v. rt-PA perfusion in patients with AIS independently predicts favorable outcome at 3 months.

International audience; Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.

International audience; Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.

International audience; Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

PPACK-Desmodus rotundus salivary plasminogen activator (cDSPAα1) prevents the passage of tissuetype plasminogen activator (rt-PA) across the blood-brain barrier and neurotoxicity

International audience; Recombinant tissue-type plasminogen activator (tPA) is the fibrinolytic drug of choice to treat stroke patients. However, a growing body of evidence indicates that besides its beneficial thrombolytic role, tPA can also have a deleterious effect on the ischaemic brain. Although ageing influences stroke incidence, complications and outcome, age-dependent relationships between endogenous tPA and stroke injuries have not been investigated yet. Here, we report that ageing is associated with a selective lowering of brain tPA expression in the murine brain. Moreover, our results show that albumin D site-binding protein (DBP) as a key age-associated regulator of the neuronal transcription of tPA. Additionally, inhibition of DBP-mediated tPA expression confers in vitro neuroprotection. Accordingly, reduced levels of tPA in old mice are associated with smaller excitotoxic/ischaemic injuries and protection of the permeability of the neurovascular unit during cerebral ischaemia. Likewise, we provide neuroradiological evidence indicating the existence of an inverse relationship between age and the volume of the ischaemic lesion in patients with acute ischaemic stroke. Together, these results indicate that the relationship among DBP, tPA and ageing play an important role in the outcome of cerebral ischaemia.

International audience; We examined the capacity of delayed inhibition of plasminogen activator inhibitor-1 (PAI-1) to reduce tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO) in mice. Small peptides mimicking parts of urokinase (uPA) and tissular plasminogen activator (tPA) and serving as decoy molecules for PAI-1 were administered daily during the late stages (3 to 8 days) of UUO. Treatment with PAI-1 decoys reduced interstitial deposition of fibronectin, collagen III and collagen IV without changes in macrophage and myofibroblast infiltration. Interestingly, while PAI-1 activity was reduced and the combined uPA and tPA activity was increased, the antifibrotic effect was obtained without modification of plasmin activity but with increased of hepatocyte growth factor (HGF) expression. We show for the first time that treatment with small PAI-1 decoy peptides reduces established tubulointerstitial fibrosis. This protective effect probably resulted from increased degradation of the extracellular matrix by an HGF dependent mechanism.

International audience; Tissue-type plasminogen activator (tPA) plays a major role in the fibrinolytic system. According to several reports, tPA may also have antiangiogenic properties, especially in combination with a free sulfhydryl donor (FSD). In the rat C6 glioma model, in vitro and in vivo tPA synthesis by glioma cells is enhanced by differentiation therapy. To address the antiangiogenic potential of tPA in this model, tPA was overexpressed in glioma tumors by ex vivo transduction of C6 cells with a lentiviral vector encoding tPA. The transduced cells were subcutaneously implanted into nude mice. Gene transfer allowed for efficient synthesis of tPA by the C6 tumors. Although the treatment of tPA+ tumor-bearing animals with the FSD captopril generated angiostatin in situ and reduced endothelial vascularization of the tumors, it had no effect on tumor growth. Alternative mechanisms could account for this lack of effect and consequently have important implications for vascular the treatment of glioblastoma.

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (KD=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment. © 2008 ISCBFM All rights reserved., This work was supported by grants from INSERM, the French Ministry of Research and Technology, the Regional Council of Lower Normandy, the Regional Platform for Cell Imaging of Upper Normandy, the Medical Research Foundation, the University of Caen, Fonds Europeéns de Développement Régional, the Paul Hamel Foundation, the Diagnosis in Molecular Imaging Program (FP6-project DiMI-LSHB-CT-2005-512146-), and PAION Deutschland GmbH.

International audience; In the present study, we have analyzed four highly polymorphic regions (STRs) chosen from four candidate genes involved in: (1) Platelet aggregation: alpha subunit of the platelet GpIIb/GpIIIa integrin complex (GpIIIa (CT)n; 17q21.31), (2) Coagulation fibrinolysis: Plasminogen Activator Tissue (PLAT5 (TG)14(TA)12; 8p12-q11.2) and Plasminogen Activator Inhibitor-1 (PAI-1 (CA)n; 7q21.3-q22), (3) Oxidative metabolism: the inducible nitric oxide (NO) synthase (iNOS) gene (NOS2A (CCTTT)n; 17cen-q11.2). Allele frequencies for these four STR loci were investigated in several Mediterranean populations. The population data deviate from the Hardy-Weinberg equilibrium in all populations for GpIIIa (CT)n polymorphism.

We previously demonstrated that human gingival fibroblasts (HGF), but not their dermal counterparts, when seeded in retracting fibrin lattices induced intense fibrinolysis that was observed at the earliest stages of contraction and led to complete matrix degradation by day 7 of culture. Our aim was to examine the influence of mechanical forces in such fibrinolytic processes. HGF were seeded in retracting (R) e.g. free floating or non retracting (NR) e.g. anchored fibrin lattices (FL). Cultures were analysed from day 1-12 by phase contrast microscopy and scanning electron microscopy (s.e.m.). Levels of fibrin degradation products (FDP) and tissue plasminogen activator (tPA) accumulating in culture media were quantified by ELISA. Urokinase (uPA) and gelatinase A (MMP2) were identified by zymographic techniques. At the s.e.m. level, vacuolization around some HGF was noticed at the earliest stages of culture for RFL and complete degradation of lattices occurred at day 7. Formation of lysed matrix cavity was far less intense in NRFL even after 12 days of culture. FDP amounts at day 4 of culture were equal to 79 +/- 14 and 8.5 +/- 0.6 micrograms/10(5) cells for RFL and NRFL, respectively; tPA levels were equal to 5.8 +/- 0.6 (RFL) and 2.1 +/- 0.3 ng/10(5) cells (NRFL) and differences were still evident at day 7. The kinetics of tPA production were identical in either retracting fibrin or collagen lattices. On the contrary, uPA and proMMP2 productions were similar in RFL and NRFL. Isometric forces, but not the matrix support, were responsible for accelerated tPA production and fibrinolysis in HGF populated lattices.

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen of fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by epsilon-amino-caproic acid (epsilon ACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva.

International audience; Purpose: Intravitreal recombinant tissue plasminogen activator (rtPA) is used off-label for the surgical management of submacular hemorrhage, a severe complication of neovascular age-related macular degeneration. rtPA is approved for coronary and cerebral thrombolysis. However, in ischemic stroke rtPA is known to increase excitotoxic neural cell death by interacting with the N-methyl-D-aspartate (NMDA) receptor. We therefore investigated the retinal toxicity of rtPA in healthy rats and in a model of NMDA-induced retinal excitotoxicity.Methods: First, rtPA at three different doses (2.16 µg/5 µl, 0.54 µg/5 µl, and 0.27 µg/5 µl) or vehicle (NaCl 0.9%) was injected intravitreally in healthy rat eyes. Electroretinograms (ERGs) were performed at 24 h or 7 days. Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptotic retinal ganglion cells (RGCs) were counted on flatmounted retinas at 24 h or 7 days. Next, NMDA + vehicle or NMDA + rtPA (0.27 µg/5 µl) was injected intravitreally to generate excitotoxic conditions. Apoptotic annexin V-FITC-labeled RGCs and surviving Brn3a-labeled RGCs were quantified on flatmounted retinas and radial sections, 18 h after treatment.Results: In healthy rat eyes, the number of apoptotic RGCs was statistically significantly increased 24 h after the administration of rtPA at the highest dose (2.16 µg/5 µl; p = 0.0250) but not at the lower doses of 0.54 and 0.27 µg/5 µl (p = 0.36 and p = 0.20), compared to vehicle. At day 7, there was no difference in the apoptotic RGC count between the rtPA- and vehicle-injected eyes (p = 0.70, p = 0.52, p = 0.11). ERG amplitudes and implicit times were not modified at 24 h or 7 days after injection of any tested rtPA doses, compared to the baseline. Intravitreal administration of NMDA induced RGC death, but under these excitotoxic conditions, coadministration of rtPA did not increase the number of dead RGCs (p = 0.70). Similarly, the number of surviving RGCs on the flatmounted retinas and retinal sections did not differ between the eyes injected with NMDA + vehicle and NMDA + rtPA (p = 0.59 and p = 0.67).Conclusions: At low clinical equivalent doses corresponding to 25 µg/0.1 ml in humans, intravitreal rtPA is not toxic for healthy rat retinas and does not enhance NMDA-induced excitotoxicity. Vitreal equivalent doses ≥200 µg/0.1 ml should be avoided in patients, due to potential RGC toxicity.