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Kyu Suk Cho,
So Hyun Joo,
Chang Soon Choi,
Ki Chan Kim,
Hyun Myung Ko,
Jin Hee Park,
Pitna Kim,
Jun Hur, Sung Hoon Lee,
Geon Ho Bahn,
Jong Hoon Ryu,
Jongmin Lee,
Seol-Heui Han,
Kyoung Ja Kwon,
Chan Young Shin
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ABSTRACT: AIMS: Tissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS. MAIN METHODS: Rat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting. KEY FINDINGS: Glucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3β, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes. SIGNIFICANCE: Rapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis.
Life sciences 04/2013; · 2.56 Impact Factor
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Kyu Suk Cho,
Kyoung Ja Kwon,
Chang Soon Choi,
Se Jin Jeon,
Ki Chan Kim,
Jin Hee Park,
Hyun Myung Ko, Sung Hoon Lee,
Jae Hoon Cheong,
Jong Hoon Ryu,
Seol Heui Han,
Chan Young Shin
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ABSTRACT: Tissue plasminogen activator (tPA) is expressed in several regions of brain and plays regulatory roles such as neurite outgrowth, synaptic plasticity and long term potentiation. The activity of tPA is regulated by an endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1), which is expressed mainly in astrocytes. Valproic acid (VPA), a histone deacetylase inhibitor that is used for the treatment of epilepsy and bipolar disorders, promotes neurite extension, neuronal growth and has neuroprotective effect in neurodegenerative diseases. In this study, we examined whether the neurite extension effects of VPA is mediated by modulating tPA/PAI-1 system. VPA dose-dependently increased tPA activity and decreased PAI-1 activity in rat primary astrocytes but not in neurons. PAI-1 protein level secreted into the culture medium but not tPA per se was decreased by VPA. In co-culture system or in neuronal culture stimulated with astrocyte conditioned media but not in pure neuronal cell culture, VPA induced neurite outgrowth via increased tPA activity due to the decreased PAI-1 activity in astrocytes. The decrease in PAI-1 activity and increased neurite extension was regulated via JNK mediated post-transcriptional pathway. The essential role of tPA/PAI-1 system in the regulation of VPA-mediated neurite extension was further demonstrated by experiments using astrocyte conditioned media obtained from tPA or PAI-1 knockout mice. Regulation of PAI-1 activity in astrocyte by VPA may affect both physiological and pathological processes in brain by upregulating tPA activity. GLIA 2013.
Glia 02/2013; · 4.82 Impact Factor
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Se Jin Jeon,
So Young Rhee,
Jung Eun Seo,
Hae Rang Bak, Sung Hoon Lee,
Jong Hoon Ryu,
Jae Hoon Cheong,
Chan Young Shin,
Gun-Hee Kim,
Yong Soo Lee,
Kwang Ho Ko
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ABSTRACT: Oroxylin A (5,7-dihydroxy-6-methoxyfavone) is a flavonoid compound originated from the root of Scutellaria baicalensis Georgi. Our previous reports suggested that oroxylin A improves memory function in rat, at least in part, by its antagonistic effects on GABA(A) receptor. In addition, oroxylin A protects neurons from ischemic damage by mechanisms currently not clear. In this study we determined whether oroxylin A modulates the level of brain derived neurotrophic factor (BDNF) in primary rat cortical neuronal culture, which is well known for its role on neuronal survival, neurogenesis, differentiation of neurons and synapses and learning and memory. Treatment of oroxylin A for 3-48h increased BDNF expression which was analyzed by ELISA assay and Western blot analysis. Oroxylin A induced slow but sustained increases in intracellular calcium level and activated ERK1/2 mitogen activated protein kinase (MAPK). In addition, oroxylin A phosphorylated cyclic AMP response element binding protein (CREB) at Ser 133 in concentration and time dependent manner. Pretreatment with the MAPK inhibitor PD98059 (10μM) attenuated phosphorylation of ERK1/2 and CREB as well as BDNF production, which suggests that oroxylin A regulates BDNF production by activating MAPK-CREB pathway. GABA(A) antagonist bicuculline mimicked the effects of oroxylin A on BDNF production as well as MAPK-CREB pathway. Increase in intracellular Ca(2+) concentration, phosphorylation of ERK1/2 and CREB, and BDNF expression by oroxylin A was blocked by NMDA receptor inhibitor MK-801 (10μM) as well as tetrodotoxin (TTX, 0.5 and 1μM). The results from the present study suggest that the calcium and p-CREB dependent induction of BDNF expression, possibly via activation of synaptic NMDA receptor through the blockade of GABA(A) activity in cortical neuronal circuitry, might be responsible for the neuroprotective or memory enhancing effects of oroxylin A.
Neuroscience Research 03/2011; 69(3):214-22. · 2.25 Impact Factor
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ABSTRACT: Although originally known as a plasma serine protease involved in clot dissolution, tPA and its primary inhibitor, PAI-1, play crucial roles in synaptic reorganization and plasticity in the central nervous system. In contrast to the wide array of work conducted in neural cells, relatively little is known about the regulatory mechanism governing tPA/PAI-1 expression in astrocytes. Glucocorticoids (GCs) such as hydrocortisone regulate the expression of tPA/PAI-1 in various biological systems in a tissue-specific manner. However, little is known about GC-mediated regulation of tPA/PAI-1 system in CNS. The aims of the present study were to investigate whether tPA/PAI-1 expression is regulated by hydrocortisone in rat primary astrocytes. Enzyme activity of tPA was decreased in a concentration-dependent manner by hydrocortisone treatment, and the activity of PAI-1 was increased by hydrocortisone. Hydrocortisone did not affect the level of tPA mRNA, which suggests that transcriptional down-regulation of tPA mRNA is not involved in the down-regulation of tPA enzyme activity in astrocytes. However, the level of PAI-1 mRNA and protein was increased. Both hydrocortisone and a tPA-Stop treatment prevented glutamate-induced neurotoxicity in rat cortical primary mixed astrocyte-neuron culture, which suggests a neurotoxic role for tPA in our culture system. Interestingly, hydrocortisone further increased LPS-induced up-regulation of PAI-1 while inhibiting the up-regulation of iNOS and COX-2 expression. Our data show that hydrocortisone up-regulated PAI-1 expression along with down-regulation of tPA activity in both normal and inflammatory conditions.
Journal of Neuroscience Research 03/2011; 89(7):1059-69. · 2.74 Impact Factor
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Ji Woon Kim, Sung Hoon Lee,
Hyun Myung Ko,
Kyoung Ja Kwon,
Kyu Suk Cho,
Chang Soon Choi,
Jin-Hee Park,
Hahn Young Kim,
Jongmin Lee,
Seol-Heui Han,
Louis J Ignarro,
Jae Hoon Cheong,
Won-Ki Kim,
Chan Young Shin
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ABSTRACT: In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.
Neurochemistry International 02/2011; 58(3):423-33. · 2.86 Impact Factor
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ABSTRACT: In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.
Neurochemical Research 02/2010; 35(7):976-85. · 2.24 Impact Factor
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ABSTRACT: Neural progenitor cells (NPC) are self-renewing multipotent cells that generate neurons and glial cells in the brain. NPCs generate neurons and glia not only during development but also after neural injury. Recent studies have shown that endogenous NPCs are activated after brain injury and migrate toward damaged areas where astrocyte activation occurs. Considering the massive proliferation of astrocytes as well as the production of several kinds of cytoactive molecules after brain injury, such as NO, growth factors and cytokines, it is tempting to think that cytoactive molecules released by activated glial cells regulate neural progenitor differentiation and proliferation through inflammatory mediators. To test this hypothesis, we stimulated rat primary astrocytes with lipopolysaccharide (LPS) to induce the activation of astrocytes. After addition of the conditioned media from LPS-stimulated astrocytes to NPC culture, proliferation was examined by MTT assay and bromodeoxyuridine (BrdU) incorporation. The differentiation of NPC into neurons and astrocytes was examined by Western blot, ELISA and immunocytochemical staining with cell-type-specific markers. Conditioned media from LPS-stimulated astrocytes increased NPC proliferation as well as gliogenesis as compared with control conditioned media from astrocytes without LPS stimulation. In contrast, neurogenesis was decreased by LPS-conditioned media. To investigate the molecular mechanism mediating glial differentiation and proliferation of NPC by reactive astrocytes, we added inhibitors of the Erk and JNK pathways during LPS stimulation. These inhibitors - except for a p38 inhibitor - decreased NPC proliferation and glial differentiation. These results suggest that LPS stimulated astrocytes generate factors regulating NPC proliferation and gliogenesis via the Erk and JNK pathways.
NeuroImmunoModulation 08/2009; 16(6):365-76. · 2.38 Impact Factor
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ABSTRACT: Buerger's disease, also known as thromboangiitis obliterans, is a nonatherosclerotic, inflammatory, vasoocclusive disease. It is characterized pathologically as a panangiitis of medium and small blood vessels, including both arteries and adjacent veins, especially the distal extremities (the feet and the hands). There is no curative medication or surgery for this disease. In the present study, we transplanted human leukocyte antigen-matched human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) into four men with Buerger's disease who had already received medical treatment and surgical therapies. After the stem cell transplantation, ischemic rest pain suddenly disappeared from their affected extremities. The necrotic skin lesions were healed within 4 weeks. In the follow-up angiography, digital capillaries were increased in number and size. In addition, vascular resistance in the affected extremities, compared with the preoperative examination, was markedly decreased due to improvement of the peripheral circulation. Because an animal model of Buerger's disease is absent and also to understand human results, we transplanted human UCB-derived MSCs to athymic nude mice with hind limb ischemia by femoral artery ligation. Up to 60% of the hind limbs were salvaged in the femoral artery-ligated animals. By in situ hybridization, the human UCB-derived MSCs were detected in the arterial walls of the ischemic hind limb in the treated group. Therefore, it is suggested that human UCB-derived MSC transplantation may be a new and useful therapeutic armament for Buerger's disease and similar ischemic diseases.
Stem Cells 07/2006; 24(6):1620-6. · 7.78 Impact Factor
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ABSTRACT: Buerger's disease, also known as thromboangiitis obliterans, is a nonatherosclerotic, inflammatory, vasoocclusive disease. It is characterized pathologically as a panangiitis of medium and small blood vessels, including both arteries and adjacent veins, especially the distal extremities (the feet and the hands). There is no curative medication or surgery for this disease. In the present study, we transplanted human leukocyte antigen-matched human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) into four men with Buerger's disease who had already received medical treatment and surgical therapies. After the stem cell transplantation, ischemic rest pain suddenly disappeared from their affected extremities. The necrotic skin lesions were healed within 4 weeks. In the follow-up angiography, digital capillaries were increased in number and size. In addition, vascular resistance in the affected extremities, compared with the preoperative examination, was markedly decreased due to improvement of the peripheral circulation. Because an animal model of Buerger's disease is absent and also to understand human results, we transplanted human UCB-derived MSCs to athymic nude mice with hind limb ischemia by femoral artery ligation. Up to 60% of the hind limbs were salvaged in the femoral artery-ligated animals. By in situ hybridization, the human UCB-derived MSCs were detected in the arterial walls of the ischemic hind limb in the treated group. Therefore, it is suggested that human UCB-derived MSC transplantation may be a new and useful therapeutic armament for Buerger's disease and similar ischemic diseases.
Stem Cells 05/2006; 24(6):1620 - 1626. · 7.78 Impact Factor
