Microglial Cell Activation is a Source of Metalloproteinase Generation during Hemorrhagic Transformation

Abstract

Hemorrhage and edema accompany evolving brain tissue injury after ischemic stroke. In patients, these events have been associated with metalloproteinase (MMP)-9 in plasma. Both the causes and cellular sources of MMP-9 generation in this setting have not been defined. MMP-2 and MMP-9 in nonhuman primate tissue in regions of plasma leakage, and primary murine microglia and astrocytes, were assayed by immunocytochemistry, zymography, and real-time RT-PCR. Ischemia-related hemorrhage was associated with microglial activation in vivo, and with the leakage of plasma fibronectin and vitronectin into the surrounding tissue. In strict serum-depleted primary cultures, by zymography, pro-MMP-9 was generated by primary murine microglia when exposed to vitronectin and fibronectin. Protease secretion was enhanced by experimental ischemia (oxygen-glucose deprivation, OGD). Primary astrocytes, on each matrix, generated only pro-MMP-2, which decreased during OGD. Microglia—astrocyte contact enhanced pro-MMP-9 generation in a cell density-dependent manner under normoxia and OGD. Compatible with observations in a high quality model of focal cerebral ischemia, microglia, but not astrocytes, respond to vitronectin and fibronectin, found when plasma extravasates into the injured region. Astrocytes alone do not generate pro-MMP-9. These events explain the appearance of MMP-9 antigen in association with ischemia-induced cerebral hemorrhage and edema.

Hemorrhagic Transformation after Ischemic Stroke in Animals and Humans

“…92,93 Delayed Hemorrhagic Transformation: Role of Brain-Derived Metalloproteinases At 18 to 24 hours after stroke, brain cells become an increasingly more important source of MMP-9 and MMP-3. 81,86 Indeed, when neutrophils are depleted in a rat stroke model brain levels of MMP-9 or MMP-2 measured at 24 hours remain relatively unchanged compared with controls. 86 This suggests that the major source of MMP-9 and MMP-2 in the brain at 24 hours is not neutrophils, but brain.…”

“…In addition, other proteases derived from brain cells also contribute to delayed HT, including MMP-10, MMP-13, MMP-14, TNF-a converting enzyme, plasmins (tPA and urokinase), and cathepsins. 32,34,38,41,56,81 Several brain cells including astrocytes, neurons, microglia, and endothelium can express MMP-9 and have been found to be MMP-9 immunoreactive after stroke. The expression of MMP-9 may vary by severity of ischemia and time from stroke onset.…”

Cathepsin L Acutely Alters Microvessel Integrity within the Neurovascular Unit during Focal Cerebral Ischemia

“…Murine astrocytes and microglial cells were obtained from 1-3-day-old post-natal C57Bl/6 mouse pups (Charles River Laboratories, Hollister, CA, USA) by the procedures previously described. 15,20 Cortices were cleaned of meninges and external blood vessels, chopped finely, then dissociated in a solution containing 30 U/mL papain (Worthington) and 40 μg/mL DNase I (Sigma) in 1 mL MEM-HEPES. After trituration, the cell suspensions, in DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, penicillin, and streptomycin (all Sigma), were plated onto PDL-coated T75 flasks (Corning, Corning, NY, USA).…”

“…15,20 Before induction of OGD, serum was completely removed from the cell cultures. 15 The cells were then cultured in strictly serum-free high glucose medium (4.5 g/L, DMEM containing 4 mM L-glutamine, penicillin, and streptomycin, supplemented with N1 medium) for normoxia or in low-glucose medium (1.0 g/L, supplemented DMEM) for OGD. Cultures containing low-glucose medium were placed in a hypoxia chamber (Billups-Rothenburg, Del Mar, CA, USA), which was flushed through with a mixture of 95% N 2 /5% CO 2 for 1 hour, and then closed for the duration of the experiment.…”

“…15 (pro-)MMP-9 release is stimulated by the exposure of resting microglia, but not astrocytes, to the circulating plasma matrix proteins FN and vitronectin (VN) that extravasate with edema. 15 This suggests a precise non-neuronal cellular origin of select matrix proteases during focal cerebral ischemia. Often forgotten in tissue disruption is the role of the pH changes caused by focal cerebral ischemia.…”