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.

In situ hybridization of human erythropoietin in pre- and postnatal kidneys.

Due to the transient transcription of the gene of erythropoietin (EPO) in response to hypoxic stimuli, localization within the cell of origin in humans has not been possible. Under acutely induced transcription of the EPO gene, through severe bleeding (hematocrit < 10%), EPO transcripts were detected within renal interstitial cells in rodents. However, by immunohistochemistry or tissue culture, human tubular epithelial cells or glomerular mesangium have been shown to produce EPO, suggesting a species difference. Aiming to investigate the precise location of EPO in intact human kidneys, 38 fetal, infant, and adult kidneys were examined by in situ hybridization. Kidneys from patients with conditions predisposing to severe hypoxia and/or induced polycythemia were utilized, because under these conditions active EPO mRNA synthesis is expected. We found specific EPO mRNA transcripts within a small population of cortical interstitial cells of near-term or postnatal kidneys.

Human eosinophils stimulate DNA synthesis and matrix production in dermal fibroblasts.

Fibrosis is frequently found in diseases exhibiting tissue eosinophilia, such as some parasitic worm infections, eosinophilia-myalgia syndrome, and eosinophilic fasciitis. Previously, eosinophil extracts have been shown to induce proliferation in neonatal foreskin fibroblasts in vitro. To determine if living eosinophils can induce synthesis of DNA and components of the extracellular matrix in dermal fibroblasts, we cultured purified human eosinophils for 2 or 7 days in the presence of the eosinophil-active cytokines granulocyte/macrophage colony stimulating factor, interleukin-3, or interleukin-5, and added eosinophil-conditioned medium to cultures of dermal fibroblasts. Using flow cytometry, we found that eosinophil-conditioned medium increased by two-fold the percentage of fibroblasts in S-phase. This stimulation of fibroblast DNA synthesis was corroborated using a standard tritiated thymidine assay and the two methods were shown to correlate well with each other. Eosinophil-conditioned medium stimulation of DNA synthesis was dose dependent and conditioned medium from eosinophils treated with any one of the three cytokines induced increased DNA synthesis. Treatment of fibroblasts with cytokines alone did not induce enhanced DNA synthesis. Eosinophil-conditioned medium also affected fibroblast matrix production. Eosinophil-conditioned medium induced a two-fold increase in soluble and cell-associated fibroblast glycosaminoglycan production and a 76% increase in collagen production. These observations support the concept that eosinophils may be active contributors to the pathophysiology of eosinophil-associated fibrotic disease.

Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense.

In our studies of host defense against the intracellular parasite Leishmania major, we obtained evidence for a novel mechanism of macrophage activation for antimicrobial defense that involves direct cell contact between CD4+ T lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive as it does not involve secretion of lymphokines but is apparently mediated by the membrane-anchored form of tumor necrosis factor (mTNF; approximately 50-60 kd) present on the surface of the effector T lymphocytes. Furthermore, it is not cytotoxic to the host cell and its expression is antigen specific and genetically restricted. We prepared a Leishmania-specific cloned T-T cell hybridoma line 1B6 (CD4+, TH1) that expresses membrane-bound TNF but does not secrete TNF or other macrophage activators. We now report that 1B6 cells can activate antileishmanial defense in inflammatory macrophages, whereas soluble recombinant murine TNF (sTNF) alone is unable to do so. On the other hand, both 1B6 cells and sTNF can act synergistically with recombinant murine interferon-gamma (IFN-gamma, a known soluble macrophage-activating factor) in activating antimicrobial defense and NO2- release. The effects of 1B6 alone and the synergistic effects of 1B6 and IFN-gamma or sTNF and IFN-gamma are arginine dependent. These results suggest that mTNF may be more efficient than sTNF in macrophage activation and that contact with effector CD4+ lymphocytes that express mTNF may be an important mechanism of host defense.