Microglial cell activation is a source of metalloproteinase generation during hemorrhagic transformation - PubMed (original) (raw)

Microglial cell activation is a source of metalloproteinase generation during hemorrhagic transformation

Gregory J del Zoppo et al. J Cereb Blood Flow Metab. 2012 May.

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.

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Figures

Figure 1

Figure 1

Relative expressions of pro-MMP-9 in striatal tissue with detectable parenchymal hemorrhage versus autologous blood mixed with homogenized normal striatal tissue ex vivo, by zymography (integrated density (IDA), normalized to 10 _μ_g protein). pro-MMP-9 content of tissue with parenchymal hemorrhage (tissue, squares; _R_2=0.60, _2P_=0.015) was significantly greater than the equivalent amount of autologous blood added to homogenates of normal striatal tissue by hemoglobin content (control, triangles; _R_2=0.994, 2P<0.001). The difference between the two relationships is significant (_F_1,19=8.04, _2P_=0.01). See text. MMP, metalloproteinase.

Figure 2

Figure 2

Microglial cells within regions of hemorrhagic transformation of the ischemic core 24 hours after middle cerebral artery occlusion (MCAO) in paraformaldehyde-fixed sections (A). Cells expressing the surface markers CD11b and CD68/KP-1 (B, D, and F and C, E, and G, respectively), compatible with microglial cells or macrophages (arrows), are seen inside (D, E) and outside (F, G) the areas of hemoglobin deposition. Regions of interest outside and inside the areas of hemoglobin deposition are indicated in (B) (that also apply to C). The morphologic features of the identified cells (arrows) suggest activated microglia or macrophages (but not PMN leukocytes) between the two regions of interest in (D) and (E), compared with (F) and (G). Magnification bars: (A–C) 100 _μ_m; (D, E) 10 _μ_m; (F, G) 10 _μ_m.

Figure 3

Figure 3

Plasma matrix protein extravasation in a region of hemorrhagic transformation 2 hours after middle cerebral artery occlusion (MCAO) in paraformaldehyde-fixed sections (A). Hemoglobin deposition identified by gray-blue coloration within the region of injury. Extravasated albumin in tissue surrounding hemorrhage (B), coregisters with the deposition of fibronectin (C) and vitronectin (D) at the hemorrhage boundary. Magnification bar=200 _μ_m (see Supplementary Figure 1).

Figure 4

Figure 4

Gelatinase gene transcription responses of primary microglia and astrocytes to matrix exposure under oxygen-glucose deprivation (OGD) compared with normoxia. (A) Responses of microglia and astrocytes to poly-

D

-lysine (PDL), perlecan (HSPG), and laminin (LN). With OGD, microglia displayed no change in MMP-2 or MMP-9 gene expression, while astrocytes displayed increased MMP-2 transcription on LN (all changes not different from unity). _n_=5 to 8 experiments, each in duplicate. (B) Gelatinase gene transcription responses of primary microglia to fibrinogen (FN) and vitronectin (VN) exposed to OGD. VN stimulates a variable increase in MMP-9 gene expression (_2P_=0.15), and fibronectin a consistent decrease in MMP-2 gene expression (_2P_=0.03). _n_=7 experiments, each in duplicate. Bars=mean with standard deviation. MMP, metalloproteinase.

Figure 5

Figure 5

Microglial cell (left panel, _n_=8) and astrocyte (right panel, _n_=5) pro-MMP-2 and pro-MMP-9 secretion in response to matrix protein exposure, under normoxia with fetal bovine serum (FBS) depletion (by zymography). No active MMP-2 or MMP-9 was detected. Matrix substrates include poly-

D

-lysine (PDL), laminin (LN), fibronectin (FN), or vitronectin (VN). Microglial cells produced significant increases in pro-MMP-9 compared with baseline (2P<0.0005 each substrate). VN increased pro-MMP-9 production compared with PDL (_2P_=0.0002), whereas LN decreased pro-MMP-9 compared with PDL (_2P_=0.0148). Astrocytes produced a uniform increase in pro-MMP-2 generation compared with baseline (_2P_=0.0075 each substrate). Bars=mean with standard deviation. MMP, metalloproteinase.

Figure 6

Figure 6

Relative pro-MMP-2 and pro-MMP-9 secretion by primary microglia (left panel, _n_=8) and astrocytes (right panel, _n_=5) to matrix protein exposure, under oxygen-glucose deprivation (OGD) with fetal bovine serum (FBS) depletion (by zymography). Matrix substrates include poly-

D

-lysine (PDL), laminin (LN), fibronectin (FN), or vitronectin (VN). Note the additional increase in pro-MMP-9 production by microglial cells (2P<0.0004 each substrate), but no significant change in pro-MMP-2 production by astrocytes. For microglia, FN stimulated additional pro-MMP-9 production compared with PDL (_2P_=0.0006). The conditions did not appreciably change cell viability during each experiment (upper panels). Bars=mean with standard deviation. MMP, metalloproteinase.

Figure 7

Figure 7

pro-MMP-9 generation depends on relative microglial density and astrocyte exposure. Microglia enhance pro-MMP-9 generation in the presence of astrocytes (grown on collagen IV) under normoxia and oxygen-glucose deprivation (OGD), compared with microglia alone. At a microglia-to-astrocyte ratio of 3:1 pro-MMP-9 secretion significantly increased during OGD compared with normoxia (_F_1,8=12.34, _2P_=0.008), and pro-MMP-9 secretion significantly increased in microglia-to-astrocyte cocultures compared with microglia alone (_F_1,8=6.29, _2P_=0.036). Bars (triplicate, mean with standard deviation): black=normoxia; hatched=OGD. MMP, metalloproteinase.

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