Summary:Matrix metalloproteinase-9 (MMP-9) activity increases in the brain during the first day after focal ischemia and might be involved in the pathogenesis of tissue damage. We previously showed MMP-9 in the extracellular space of brain parenchyma along with neutrophil recruitment after ischemia. In the present study, we tested whether neutrophils were a direct source of enhanced MMP-9 in the ischemic brain. Neutrophil infiltration was prevented either by injecting an antibody against ICAM-1, which abrogates neutrophil adhesion to the endothelial vessel wall, or by inducing neutropenia. Onehour intraluminal middle cerebral artery occlusion with reperfusion was induced, and studies were performed at 24 hours. Circulating neutrophils expressed 95-kDa MMP-9 and dimers, and infiltrated neutrophils stained positive for MMP-9. The expression of MMP-9 (mainly 95-kDa proform and dimers and, to a lesser extent, 88-kDa form) increased in brain after ischemia/reperfusion. Treatments preventing neutrophil infiltration failed to preclude the ischemia-induced increase in 88-kDa MMP-9 form and gelatinase activity in neurons and blood vessels. However, these treatments prevented the major increase in 95-kDa MMP-9 form and dimers. We conclude that neutrophil infiltration highly contributes to enhanced MMP-9 in the ischemic brain by releasing MMP-9 proform, which might participate in the tissular inflammatory reaction.

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The anticancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells-Manuscript Draft-Manuscript Number: KAUP-2019-0426R3 Full Title: The anticancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells

Matrix metalloproteinase-3 (MMP-3) degrades components of the extracellular matrix and may participate in the pathogenesis of stroke. Here we examine the expression, activation, and cellular location of MMP-3 and the cleavage of agrin, an MMP-3 substrate, following transient middle cerebral artery occlusion in the rat. MMP-3 was activated by ischemia/reperfusion, which was revealed by the appearance of a cleaved form and increased degradation of a substrate. MMP-3 was observed in ischemic neurons, oligodendrocytes, microvasculature, and reactive microglia/macrophages. In cell cultures, MMP-3 expression was observed in neurons and, to a lesser extent, in mature oligodendrocytes, but not in oligodendrocyte progenitors, astrocytes, or microglia. Casein zymography revealed MMP-3 in cultured neurons. Agrin was expressed in cultured neurons and cultured astrocytes. In brain tissue, agrin was detected in neurons, and following ischemia it was also detected in reactive astrocytes. Addition of MMP-3 to protein extracts from control brain caused neuronal agrin degradation. Following ischemia/reperfusion, agrin disappeared from the tissue membrane fraction and a cleaved agrin fragment was found in tissue protein extracts. The present results show MMP-3 activation and neuronal transmembrane agrin cleavage after ischemia/reperfusion. In addition, the finding that MMP-3 cleaves brain agrin strongly suggests that ischemia-induced MMP-3 activation causes agrin cleavage.

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and carry out key functions in cell development, cancer, injury, and regeneration. In addition to its well recognized extracellular action, functional intracellular MMP activity under certain conditions is supported by increasing evidence. In this study, we observed higher gelatinase activity by in situ zymography and increased MMP-9 immunoreactivity in human neuroblastoma cells and in bone marrow macrophages undergoing mitosis compared with resting cells. We studied the pattern of immunoreactivity at the different stages of cell division by confocal microscopy. Immunostaining with different monoclonal antibodies against MMP-9 revealed a precise, dynamic, and well orchestrated localization of MMP-9 at the different stages of cell division. The cellular distribution of MMP-9 staining was studied in relation to that of microtubules. The spatial pattern of MMP-9 immunoreactivity suggested some participation in both the reorganization of the nuclear content and the process of chromatid segmentation. We then used several MMP-9 inhibitors to find out whether MMP-9 might be involved in the cell cycle. These drugs impaired the entry of cells into mitosis, as revealed by flow cytometry, and reduced cell culture growth. In addition, the silencing of MMP-9 expression with small interfering RNA also reduced cell growth. Taken together, these results suggest that intracellular MMP-9 is involved in the process of cell division in neuroblastoma cells and in primary cultures of macrophages.

Matrix metalloproteinases (MMPs) are activated in focal cerebral ischemia. The activation of MMP-9 is involved in blood-brain barrier breakdown and tissue remodeling. The MMPs are released to the extracellular space, but the form and fate of secreted enzymes in brain are unknown. Using microdialysis in vivo, the authors studied whether ischemiainduced MMP-9 in brain tissue was related to free MMP-9 in the extracellular fluid. A microdialysis probe was placed into the right striatum and microdialysis was initiated 24 hours later in controls (n ‫ס‬ 7). One hour prior to microdialysis, a group of rats (n ‫ס‬ 7) was subjected to 1-hour occlusion of the right middle cerebral artery, followed by reperfusion. Dialysates were collected at discrete time points up to 24 hours, and subjected to zymography and Western blot analysis. The MMP-9 was released after ischemia and accumulated in the extracellular space at 24 hours (P < 0.05). Free MMP-9 forms include mainly the 95-kd proform, and, to a lesser extent, dimers and cleaved active forms (70 kd), but not the 88-kd form found in tissue. Probe implantation and microdialysis increased free MMP-9 in the dialysate. This increase was concomitant with neutrophil infiltration after the mechanical lesion, as myeloperoxidase was found by means of Western blot analysis in the brain hemisphere subjected to microdialysis (P < 0.005), and immunohistochemistry revealed the presence of myeloperoxidase stain surrounding the site of probe implantation. The results suggest that certain forms of MMP-9 are released and accumulate in the extracellular space after brain injury, and that vascular alterations and neutrophil recruitment elicit MMP-9 activation in the brain after focal ischemia and trauma.

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