Lee Anna Cunningham

Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures

Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinases (MMPs) are associated with the opening of the BBB, but their cellular localization and activation mechanisms are uncertain. We used immunohistochemistry to determine the cellular localization of the MMPs in reperfused rat brain, and cell cultures to study their activation. Spontaneously hypertensive rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunostained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostaining in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunoreactivity for MMP-3 and MMP-9 appeared. MMP-3 co-localized with activated microglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days intense MMP-2 staining was seen in reactive astrocytes around the ischemic core. Studies of activation of the MMP were done in lipopolysaccharide (LPS)-stimulated astrocyte and microglia cultures. Stimulated astrocytes produced an activated form of MMP-2. When microglia were stimulated, they activated MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cells. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is normally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP-9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the proteolytic disruption of the BBB.

Multiple roles for MMPs and TIMPs in cerebral ischemia

Matrix metalloproteinases (MMPs) are matrix‐degrading enzymes involved in diverse homeostatic and pathological processes. Several MMPs are expressed within the CNS and serve important normal and pathological functions during development and adulthood. An early and major pathological effect of MMP activity after cerebral ischemia is opening of the blood‐brain barrier (BBB). More recent work demonstrates emerging roles for MMPs and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in the regulation of neuronal cell death. In addition, MMPs and TIMPs are likely to play important roles during the repair phases of cerebral ischemia, particularly during angiogenesis and reestablishment of cerebral blood flow. This review attempts to elucidate how MMPs and TIMPs may provide detrimental or beneficial actions during the injury and repair processes after cerebral ischemia. These processes will have important implications for therapies using MMP inhibitors in stroke.

Endogenous Matrix Metalloproteinase (MMP)-3 and MMP-9 Promote the Differentiation and Migration of Adult Neural Progenitor Cells in Response to Chemokines

Adult neurogenesis is regulated by both intrinsic programs and extrinsic stimuli. The enhanced proliferation of adult neural stem/progenitor cells (aNPCs) in the subventricular zone and the migration of neuroblasts toward the ischemic region in adult brains present a unique challenge as well as an opportunity to understand the molecular mechanisms underlying the extrinsic cue‐induced neurogenic responses. Matrix metalloproteinases (MMPs) are a family of proteinases known to play a role in extracellular matrix remodeling and cell migration. However, their presence in aNPCs and their potential function in injury‐induced aNPC migration remain largely unexplored. Here we demonstrate that in response to two injury‐induced chemokines, stromal cell‐derived factor 1 (SDF‐1) and vascular endothelial growth factor, aNPCs differentiated into migratory cells that expressed increased levels of MMP‐3 and MMP‐9. Whereas differentiated neuroblasts and a subpopulation of astrocytes migrated toward the chemokines, undifferentiated progenitors did not migrate. Blocking the expression of MMP‐3 or MMP‐9 in aNPCs interfered with both the differentiation of aNPCs and chemokine‐induced cell migration. Thus, endogenous MMPs expressed by aNPCs are important for mediating their neurogenic response to extrinsic signals.

Angiogenic recruitment of pericytes from bone marrow after stroke.

Bone marrow-derived cells (BMDCs) contribute to revascularization after ischemia. However, the mechanisms by which BMDCs support vessel remodeling after cerebral ischemia are not clear. Using mouse chimeras that express enhanced green fluorescent protein in reconstituted bone marrow, we investigated the role of BMDCs in revascularization and brain repair after middle cerebral artery occlusion of murine brain. After ischemia, two populations of BMDCs were observed, one in the brain parenchyma and another associated with the vasculature. The number of BMDCs that infiltrated the brain parenchyma peaked at 7 days and persisted through 14 days, the last time point observed. The majority of BMDCs were characterized as microglia, based on cell-type-specific marker expression. We observed a robust angiogenic response after cerebral ischemia. Bone marrow-derived cells associated with remodeling blood vessels were negative for endothelial markers, but were surrounded by basal lamina and expressed desmin and vimentin, identifying these cells as pericytes. Quantification of BMDCs that expressed desmin revealed increasing desmin expression with time. Perivascular associated BMDCs that expressed desmin were immunoreactive for the angiogenic factors vascular endothelial growth factor and transforming growth factor-β. These findings suggest that pericytes are recruited from the periphery and are involved in blood vessel stabilization during ischemiainduced angiogenesis.

Astrocyte delivery of glial cell line-derived neurotrophic factor in a mouse model of Parkinson's disease.

Primary astrocytes were genetically modified ex vivo to express recombinant glial cell line-derived neurotrophic factor (GDNF) and subsequently were tested for their ability to provide neuroprotection to dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinsons disease. A replication-defective retrovirus was constructed, which contained the rat GDNF sequence and a sequence encoding a beta-galactosidase (beta-gal)/neomycin phosphotransferase fusion protein, linked via an internal ribosomal entry site. Murine astrocytes transduced with this vector secreted GDNF into the culture media at the rate of 115 +/- 34 pg/24 h/10(5) cells and expressed cytoplasmic beta-gal, whereas control nontransduced astrocytes were negative for GDNF production and cytoplasmic beta-gal expression. Mice that received implants of GDNF-producing astrocytes into the striatum or nigra displayed elevated levels of GDNF compared to mice that received control nontransduced astrocytes. In addition, tissue content of GDNF was increased bilaterally and in brain regions both proximal and distal to the graft, even though astrocyte migration away from the graft site did not occur. Importantly, GDNF-producing astrocytes provided marked neuroprotection of nigral dopaminergic perikarya, and partial protection of striatal dopaminergic fibers, when implanted into the midbrain 6 days prior to a retrograde 6-OHDA lesion, as assessed by tyrosine hydroxylase immunohistochemistry. Similarly, GDNF-producing astrocytes prevented the acquisition of amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented dopamine depletion within the substantia nigra, as assessed by high-performance liquid chromatography. These results indicate that continuous exposure to low levels of GDNF provided by transgenic astrocytes provides marked neuroprotection of nigral dopaminergic neurons. (c)2002 Elsevier Science (USA).

Focal cerebral ischemia induces a multilineage cytogenic response from adult subventricular zone that is predominantly gliogenic

The purpose of this study was to ascertain the relative contribution of neural stem/progenitor cells (NSPCs) of the subventricular zone (SVZ) to lineages that repopulate the injured striatum following focal ischemia. We utilized a tamoxifen‐inducible Cre/loxP system under control of the nestin promoter, which provides permanent YFP labeling of multipotent nestin+ SVZ‐NSPCs prior to ischemic injury and continued YFP expression in all subsequent progeny following stroke. YFP reporter expression was induced in adult male nestin‐CreERT2:R26R‐YFP mice by tamoxifen administration (180 mg kg−1, daily for 5 days). Fourteen days later, mice were subjected to 60‐min transient middle cerebral artery occlusion (MCAO) and sacrificed at 2 days, 2 weeks, or 6 weeks post‐MCAO for phenotypic fate mapping of YFP+ cells using lineage‐specific markers. Migration of YFP+ cells from SVZ into the injured striatal parenchyma was apparent at 2 and 6 weeks, but not 2 days, post‐MCAO. At 2 weeks post‐MCAO, the average percent distribution of YFP+ cells within the injured striatal parenchyma was as follows: 10% Dcx+ neuroblasts, 15–20% oligodendrocyte progenitors, 59% GFAP+ astrocytes, and only rare NeuN+ postmitotic neurons. A similar phenotypic distribution was observed at 6 weeks, except for an increased average percentage of YFP+ cells that expressed Dcx+ (20%) or NeuN (5%). YFP+ cells did not express endothelial markers, but displayed unique anatomical relationships with striatal vasculature. These results indicate that nestin+ NSPCs within the SVZ mount a multilineage response to stroke that includes a gliogenic component more predominant than previously appreciated.

Bone marrow-derived microglia contribute to the neuroinflammatory response and express iNOS in the MPTP mouse model of Parkinson's disease

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication in mice results in dopamine neuron degeneration that is alleviated by prevention of microglia cell activation and blockade of iNOS production. However, the role of peripherally derived microglia in this response has not been well characterized. In the present study, we investigated the time course of infiltration and phenotypic differentiation of bone marrow-derived cells (BMDCs) following MPTP treatment in mice, using green fluorescent protein (GFP) bone marrow chimeras. BMDCs were found in the meninges, choroid plexus, blood vessels, and brain parenchyma in both saline and MPTP-treated mice. MPTP stimulated a transient, two-fold increase in the rate of BMDC infiltration into the brain, concomitant with the onset of microglia activation. The majority of BMDCs were microglial in phenotype, as assessed by morphology and expression of the pan-hematopoietic marker CD45 and the microglia marker CD11b. We did not observe BMDCs that expressed neuronal or astroglial markers. Over 90% of bone marrow-derived microglia expressed the inducible form of nitric oxide synthase (iNOS), suggesting that peripherally derived microglia may play a deleterious role in MPTP-induced degeneration.

Neural Stem/Progenitor Cells Promote Endothelial Cell Morphogenesis and Protect Endothelial Cells against Ischemia via HIF-1α-Regulated VEGF Signaling:

Vascular cells provide a neural stem/progenitor cell (NSPC) niche that regulates expansion and differentiation of NSPCs within the germinal zones of the embryonic and adult brain under both physiologic and pathologic conditions. Here, we examined the NSPC—endothelial cell (NSPC/EC) interaction under conditions of ischemia, both in vitro and after intracerebral transplantation. In culture, embryonic mouse NSPCs supported capillary morphogenesis and protected ECs from cell death induced by serum starvation or by transient oxygen and glucose deprivation (OGD). Neural stem/progenitor cells constitutively expressed hypoxia-inducible factor 1α (HIF-1α) transcription factor and vascular endothelial growth factor (VEGF), both of which were increased approximately twofold after the exposure of NSPCs to OGD. The protective effects of NSPCs on ECs under conditions of serum starvation and hypoxia were blocked by pharmacological inhibitors of VEGF signaling, SU1498 and Flt-1-Fc. After intracerebral transplantation, NSPCs continued to express HIF-1α and VEGF, and promoted microvascular density after focal ischemia. These studies support a role for NSPCs in stabilization of vasculature during ischemia, mediated via HIF-1α-VEGF signaling pathways, and suggest therapeutic application of NSPCs to promote revascularization and repair after brain injury.

Murine Neural Stem/Progenitor Cells Protect Neurons against Ischemia by HIF-1α–Regulated VEGF Signaling

Focal cerebral ischemia following middle cerebral artery occlusion (MCAO) stimulates a robust cytogenic response from the adult subventricular zone (SVZ) that includes massive proliferation of neural stem/progenitor cells (NSPCs) and cellular migration into the injury area. To begin to explore beneficial roles of NSPCs in this response, we investigated the ability of embryonic and postnatal NSPCs to promote neuronal survival under conditions of in vivo and in vitro ischemia. Intracerebral transplantation of NSPCs attenuated neuronal apoptosis in response to focal ischemia induced by transient MCAO, and prevented neuronal cell death of cortical neurons in response to oxygen-glucose deprivation (OGD) in culture. NSPC-mediated neuroprotection was blocked by the pharmacological inhibitors of vascular endothelial growth factor (VEGF), SU1498 and Flt-1Fc. Embryonic and postnatal NSPCs were both intrinsically resistant to brief OGD exposure, and constitutively expressed both hypoxia-inducible factor 1α (HIF-1α) transcription factor and its downstream target, VEGF. Genomic deletion of HIF-1α by Cre-mediated excision of exon 2 in NSPC cultures resulted in >50% reduction of VEGF production and ablation of NSPC-mediated neuroprotection. These findings indicate that NSPCs promote neuronal survival under ischemic conditions via HIF-1α-VEGF signaling pathways and support a role for NSPCs in promotion of neuronal survival following stroke.

Tissue inhibitor of metalloproteinases-3 facilitates Fas-mediated neuronal cell death following mild ischemia

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a natural inhibitor of metalloproteinases involved in matrix degradation and ectodomain shedding of many cell-surface proteins, including death receptors and/or their ligands. In the present study, we examined the role of TIMP-3 in Fas-mediated neuronal cell death following cerebral ischemia, using both gene deletion and pharmacological approaches. In culture, exposure of primary cortical neurons to 2 h of oxygen–glucose deprivation (OGD) resulted in delayed neuronal cell death that was dependent on activation of the death receptor, Fas. Cortical cultures derived from timp-3−/− mice displayed partial resistance against OGD-induced neuronal cell death and also displayed increased shedding of Fas ligand (FasL) into the culture media, compared to wild-type control cultures. Both the increased neuroprotection and increased FasL shedding in timp-3−/− cultures were reversed by addition of exogenous metalloproteinase inhibitors, recombinant TIMP-3 or GM6001. In vivo, timp-3−/− mice showed marked resistance to a brief (30 min) middle cerebral artery occlusion (MCAO), but were not protected against more severe lesions induced by 90 min of MCAO. These studies demonstrate that TIMP-3 facilitates Fas-mediated neuronal cell death following OGD and plays a pro-apoptotic role in mild cerebral ischemia.