Matrix metalloproteinase-10 effectively reduces infarct size in experimental stroke by enhancing fibrinolysis via a thrombin-activatable fibrinolysis inhibitor-mediated mechanism

BACKGROUND: The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate in thrombus dissolution and extracellular matrix proteolysis. The plasminogen/plasmin system activates MMPs, and some MMPs have been involved in the dissolution of fibrin by targeting fibrin(ogen) directly or by collaborating with plasmin. MMP-10 has been implicated in inflammatory/thrombotic processes and vascular integrity, but whether MMP-10 could have a profibrinolytic effect and represent a promising thrombolytic agent is unknown. METHODS AND RESULTS: The effect of MMP-10 on fibrinolysis was studied in vitro and in vivo, in MMP-10-null mice (Mmp10(-/-)), with the use of 2 different murine models of arterial thrombosis: laser-induced carotid injury and ischemic stroke. In vitro, we showed that MMP-10 was capable of enhancing tissue plasminogen activator-induced fibrinolysis via a thrombin-activatable fibrinolysis inhibitor inactivation-mediated mechanism. In vivo, delayed fibrinolysis observed after photochemical carotid injury in Mmp10(-/-) mice was reversed by active recombinant human MMP-10. In a thrombin-induced stroke model, the reperfusion and the infarct size in sham or tissue plasminogen activator-treated animals were severely impaired in Mmp10(-/-) mice. In this model, administration of active MMP-10 to wild-type animals significantly reduced blood reperfusion time and infarct size to the same extent as tissue plasminogen activator and was associated with shorter bleeding time and no intracranial hemorrhage. This effect was not observed in thrombin-activatable fibrinolysis inhibitor-deficient mice, suggesting thrombin-activatable fibrinolysis inhibitor inactivation as one of the mechanisms involved in the MMP-10 profibrinolytic effect. CONCLUSIONS: A novel profibrinolytic role for MMP-10 in experimental ischemic stroke is described, opening new pathways for innovative fibrinolytic strategies in arterial thrombosis

Spray-drying of the microalga dunaliella salina: Effects on B-Carotene content and isomer composition

The effects of spray-drying of the unicellular microalga Dunaliella salina on its B-carotene content and geometric isomer composition have been studied. The efficacy of a range of synthetic and natural antioxidants in preventing degradation of B-carotene has been determined. Losses of B-carotene and isomerization were minimal during processing for both the control (no exogenous antioxidants) and the samples containing butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ). However, the use of tocopherol-based antioxidants resulted in degradation of 52-72% of B-carotene during the drying process. All dried powders of Dunaliella proved to be unstable during storage in the presence of light and air, with B-carotene degraded according to a first-order kinetic model. Of the antioxidants studied, only TBHQ was successful in significantly minimizing degradation (degradation constants of 0.03 and 0.04 days-1, compared to 0.53 days-1 for the respective control). For control powders and those with BHT added to the feed, the degradation constants were reduced to values between 0.27 and 0.37 days-1 by restricting light and flushing with nitrogen; however, storage in the dark alone had no effect. For more slowly degrading powders having TBHQ added to the feed, it was clear that degradation of â-carotene was influenced by both light and oxygen. During storage the 9-cis isomer of B-carotene was significantly more unstable than the all-trans form. TBHQ was, however, successful in reducing relative losses of this isomer for samples stored in the dark. The results suggest a dominant photodegradative mechanism for the loss of the 9-cis isomer of B-carotene

Brain plasminogen system

Endothelial cells, glial and neuronal cells, representing the principal sources of plasminogen activator (tPA and uPA), are in close interaction in the neurovascular unit. Formation of plasmin at the surface of endothelial cells and neurons has important consequences on these cells (apoptosis of endothelial cells and detachment/aggregation of neurons). The plasminogen activation system is also known to participate in various inflammatory conditions of the central nervous system. In such pathologies, beyond circulating plasminogen, the origin of plasminogen is still a matter of debate. First, we have investigated the presence of plasminogen in human cerebro-spinal fluids. The presence of plasminogen and the activity of plasmin, tPA and uPA in inflammatory diseases (GBS, Guillain Barre Syndrome patients, n = 14; MS, Multiple sclerosis, n = 9) and also in non-inflammatory diseases (n = 13) were studied. Western blotting, zymography and chromogenic detection were used to evaluate antigens and activity of plasmin(ogen), uPA and tPA. In human, plasminogen was detectable in both inflammatory (66%) and non-inflammatory (65%) patients. Plasminogen was found in larger concentration in inflammatory diseases (4.6 nM in GBS, 6.5 nM in MS and 2.2 nM in non inflammatory diseases). Active plasmin was detected in GBS and MS patients (3.55 nM vs. 2.6 nM). uPA was detectable in a minority of patients (15% of GBS, 20% if MS and 7% of non-inflammatory diseases), and tPA was not detect. To further investigate the origin of plasminogen in the central nervous system, we are currently exploring the presence of plasminogen in mouse parenchyma in physiological and inflammatory conditions by immuno-histochemistry. In conclusion, we have shown that a plasminogen activation system is detectable in CSF of patient with inflammatory and non-inflammatory diseases. The role of these proteolytic messengers in diseases outcome remains to be determined

The brain-specific tissue-type plasminogen activator inhibitor, neuroserpin, protects neurons against excitotoxicity both in vitro and in vivo.

Considering its brain-specific expression, neuroserpin (NS), a potent inhibitor of tissue-type plasminogen activator (tPA), might be a good therapeutic target to limit the pro-excitotoxic effects of tPA within the cerebral parenchyma, without affecting the benefit from thrombolysis in stroke patients. Here, we aimed at determining the mechanisms of action responsible for the previously reported neuroprotective activity of NS in rodent experimental cerebral ischemia. First, we show in vivo that exogenous NS protects the cortex and the striatum against NMDA-induced injury. Then, the cellular mechanisms of this neuroprotection were investigated in primary cultures of cortical neurons. We show that NS fails to prevent serum deprivation-induced apoptotic neuronal death, while it selectively prevents NMDA- but not AMPA-induced excitotoxicity. This beneficial effect is associated to a decrease in NMDA receptor-mediated intracellular calcium influx. Altogether, these data suggest that an overexpression of neuroserpin in the brain parenchyma might limit the deleterious effect of tPA on NMDA receptor-mediated neuronal death, which occurs following experimental ischemia