Jeong-Sun Choi

Upregulation of gp130 and STAT3 activation in the rat hippocampus following transient forebrain ischemia

To determine whether the pathophysiological processes after transient forebrain ischemia are mediated via a signal pathway involving gp130 (a signal transducer for the interleukin‐6 family), we analyzed changes in the expression of gp130 and its downstream transcription factor, signal transducer and activator of transcription factor 3 (STAT3), in the rat hippocampus of a four‐vessel occlusive ischemia model. Expression of gp130 mRNA was restricted to neurons of the pyramidal cell and granule cell layers in control animals. Four hours after ischemic injury, astrocytes expressed gp130 mRNA. Expression of gp130 increased preferentially in the CA1 and dentate hilar regions, and was maintained for at least 2 weeks. Increase in gp130 expression was accompanied by the activation of STAT3 following ischemic injury. Four hours after injury, STAT3 and phosphorylated STAT3 (pSTAT3) were observed in the nuclei of the dentate hilar region, and sequentially in the CA1 region at day 1. By day 3, STAT3 immunoreactivity markedly increased in these areas, where small cells with the morphology of astrocytes showed nuclear and cytoplasmic STAT3 and nuclear pSTAT3 immunoreactivities. These patterns were especially maintained in the CA1 area until 14 days of reperfusion. Double‐labeling experiments revealed that the cells expressing STAT3 and pSTAT3 were glial fibrillary acidic protein‐expressing reactive astrocytes. These results show a coordinated and long‐lasting upregulation of gp130 mRNA and STAT3 activation in reactive astrocytes of the postischemic hippocampus, indicating that they may be involved in the astrocytic response to an ischemic insult. GLIA 41:237–246, 2003.

Auranofin blocks interleukin‐6 signalling by inhibiting phosphorylation of JAK1 and STAT3

Auranofin (AF) is a sulphur‐containing gold compound. Because of its anti‐inflammatory and immunosuppressive activities, AF has been widely used for the therapeutic treatment of rheumatoid arthritis. However, little is known about its mechanism of action. To elucidate the molecular mechanism underlying the anti‐inflammatory effect of AF, we studied the effects of AF on cellular responses to interleukin‐6 (IL‐6). In HepG2 human hepatoma cells, AF markedly inhibited IL‐6‐induced phosphorylation of janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) and STAT3 translocation into the nucleus. Consistent with this, AF diminished IL‐6‐induced production of the acute‐phase proteins, haptoglobin, fibrinogen, C3 complement and α1‐acid glycoprotein, and gene expression of vascular endothelial growth factor, all of whose transcriptional activities are regulated by STAT3. The inhibitory activity of AF on STAT3 phosphorylation was also demonstrated in primary cells, i.e. fibroblast‐like synoviocytes from rheumatoid arthritis patients, human umbilical vein endothelial cells and rat astrocytes. Auranofin‐mediated inhibition of STAT3 phosphorylation was recovered by pretreatment with antioxidants containing thiol groups. These findings suggest that the anti‐inflammatory action of AF is associated with a blockade of JAK1/STAT3 signalling. Thiol‐group‐reactive proteins may be involved in AF‐induced suppression of JAK1/STAT3 phosphorylation.

Ischemic preconditioning-induced activation of ERK1/2 in the rat hippocampus

We investigated the activation and cellular localization of the extracellular signal-regulated kinases ERK1/2 in a rat model of ischemic tolerance induction. Adult male Sprague-Dawley rats were subjected to 3 min of sublethal ischemic preconditioning. Activation of ERK1/2 showed the characteristic time- and cell-dependent patterns. Rapid and short-lasting activation of ERK after 3 min of cerebral ischemia was noted immediately in the dentate granule cells and mossy fibers of the hippocampus, and then occurred sequentially in CA3 and CA1 neurons and dentate hilar neurons at 10 min. Phosphorylation of ERK1/2 in hippocampal neurons returned to the basal level in an ordered manner. Basal level phosphorylation was attained first, at 30 min, by the CA1 neurons, and was then observed in CA3 and granule cells by 1 h and noted in some dentate hilar neurons at 12 h. By contrast, phosphorylation of ERK1/2 in mossy fibers and the CA1 dendritic field was sustained for at least 3 d. Transient activation of ERK1/2 was induced also in astrocytes of the dentate hilar region at 1 d post-stimulation. These data demonstrate that the short cerebral-ischemic preconditioning induced rapid and transient activation of ERK1/2 in tolerance-acquired CA1 neurons as well as in ischemia-resistant CA3 and dentate granule cells, and that the short preconditioning sustained activation in mossy fibers and neuropil areas, suggesting that ERK1/2 activation may be involved in the mechanism of ischemic tolerance in the rat hippocampus.

Induction of Bis, a Bcl-2-binding protein, in reactive astrocytes of the rat hippocampus following kainic acid-induced seizure

The expression of Bis (also called Bag-3), a Bcl-2-binding protein, was investigated in the rat kainic acid (KA) model of temporal lobe epilepsy. Western blot analysis showed a significant increase in the expression levels of Bis protein in the hippocampus following the systemic administration of KA. Bis immunoreactivity increased preferentially in the CA1 and CA3 regions, as well as in the hilar region of the dentate gyrus. Experiments with double immunofluorescence revealed that, in KA-administered rats, the cells expressing Bis were GFAP-expressing reactive astrocytes. The increase in Bis immunoreactivity was accompanied by increased Bcl-2 in reactive astrocytes in the striatum radiatum, whereas Bcl-2 immunoreactivity in pyramidal neurons was not affected. These results of the co-expression of Bis and Bcl-2 in reactive astrocytes in this seizure model suggest that Bis might modulate the glial reaction under excitotoxic brain injury, probably by interacting with Bcl-2.

Upregulation of haptoglobin in reactive astrocytes after transient forebrain ischemia in rats.

Immunohistochemistry for haptoglobin (Hp) in the postischemic hippocampus demonstrated an immunoreactivity visible one day after reperfusion and continuing to increase until 14 days after ischemia. The immunoreactivity was most prominent in CA1 and the dentate hilar region, especially in cells with astroglial morphology. Double immunofluorescence histochemistry confirmed colocalization of the Hp and glial fibrillary acidic protein. Furthermore, a reverse transcription-polymerase chain reaction study confirmed an elevated Hp mRNA level in the postischemic hippocampus. The Hp gene expression was also upregulated in C6 and A-172 glioblastoma cell lines after H2O2 treatment. These findings suggest that Hp is synthesized in reactive astrocytes in response to ischemia-reperfusion injury.

Transient microglial and prolonged astroglial upregulation of osteopontin following transient forebrain ischemia in rats

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes, with neuroprotective properties in ischemic injury. We examined the postischemic expression and localization of OPN in the rat brain after transient forebrain ischemia. The semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that OPN expression in the hippocampal CA1 region was biphasic, peaking at day 3 after reperfusion and again between days 14 and 28. The two phases of OPN induction occurred in a time- and cell-dependent manner in the ischemic hippocampus. OPN mRNA expression in activated microglia was first induced 1 day after reperfusion, reached a peak at 3 days, and returned to basal levels by 7 days. In contrast, OPN expression in reactive astrocytes was first induced by 10 days after reperfusion in the hippocampal CA1. Astroglial OPN expression further increased, reaching a peak at day 14 and was maintained up to day 28, the latest time point we examined. OPN immunoreactivity in the ischemic hippocampus matched the mRNA induction patterns. OPN protein was first localized in the astroglial cytoplasm and later in the extracellular matrix of the hippocampal CA1. The temporal and cellular patterns of OPN induction in the ischemic hippocampus suggest a multifunctional capacity in the pathogenesis of ischemic injury, with the increased OPN production and secretion by reactive astrocytes being involved in subsequent tissue repair and reorganization.

Upregulation of Vascular Endothelial Growth Factor Receptors Flt-1 and Flk-1 Following Acute Spinal Cord Contusion in Rats:

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors. (J Histochem Cytochem 55: 821–830, 2007)

Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats

To identify whether vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, is involved in pathophysiology of stroke, we investigated the spatiotemporal regulation of VEGFR-3 mRNA after transient focal cerebral ischemia. Most of the increase in VEGFR-3 expression in the ischemic core could be attributed to brain macrophages, whereas VEGFR-3 in the peri-infarct penumbra region was predominantly expressed in reactive astrocytes. A subpopulation of VEGFR-3-expressing brain macrophages was positive for NG2 proteoglycan and showed proliferative activity. In addition, in vitro model of stroke revealed no significant induction of VEGFR-3 in activated microglial cells, indicating that infiltrating exogenous macrophages expressed VEGFR-3 after focal ischemia. These data suggest that VEGFR-3 may be involved in the glial reaction and possibly in the recruitment of monocytic macrophages during ischemic insults.

Differential regulation of osteopontin receptors, CD44 and the αv and β3 integrin subunits, in the rat hippocampus following transient forebrain ischemia

We have examined the spatiotemporal regulation of CD44 and the alpha(v)beta(3) integrin subunits, which have been identified as receptors for osteopontin (OPN), in the rat hippocampus following transient forebrain ischemia. Immunoreactivity for CD44 and the integrin subunits, alpha(v) and beta(3), showed characteristic time- and cell-dependent patterns in the ischemic hippocampus. CD44 immunoreactivity was induced at day 1 after reperfusion, reached a peak at day 3, and returned to basal levels by day 7. CD44 was induced in a subset of activated microglial cells within sites of intense neural damage, and the concomitant induction of OPN and CD44 was observed in the same cells in the ischemic hippocampus. In contrast, increased immunoreactivity for alpha(v) and beta(3), which shared overlapping expression patterns in the ischemic hippocampus, occurred in the majority of reactive astrocytes and only a few microglia at day 3 after reperfusion, and was sustained for more than 2 weeks. Immunoreactivity for both integrin subunits colocalized with OPN immunoreactivity in reactive astrocytes, and OPN immunoreactivity was also diffusely localized over the extracellular matrix around the reactive astrocytes. These data indicated that the rapid and transient induction of CD44 and OPN occurred in activated microglia/macrophages, whereas the long-lasting induction of alpha(v) and beta(3) integrin subunits and OPN occurred in reactive astrocytes, suggesting that the multifunctional role of OPN in the ischemic brain may be attributed, in part, to a time- and cell-dependent interaction with CD44 or integrin alpha(v)beta(3).

Enhanced expression of vascular endothelial growth factor receptor-3 in the subventricular zone of stroke-lesioned rats ☆

Vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D, has recently been proposed to be involved in adult hippocampal neurogenesis in response to cerebral ischemia. To identify whether VEGFR-3 is involved in poststroke neurogenesis, we investigated the temporal regulation of VEGFR-3 mRNA expression in the subventricular zone (SVZ) of rats with transient focal cerebral ischemia by in situ hybridization analysis, and identified the phenotypes of cells expressing VEGFR-3 by double- and triple-labeling techniques. In sham-operated rats, hybridization signals for VEGFR-3 mRNA were evident at a weaker intensity in the SVZ of the lateral ventricle. VEGFR-3 was transiently increased in the dorsolateral SVZ of the infarcted hemisphere on days 3-7 after reperfusion. Almost all VEGFR-3-expressing cells in the ipsilateral SVZ were colabeled with glial fibrillary acidic protein and the neural progenitor marker nestin, and were highly proliferative. In addition, a subset of VEGFR-3-labeled cells in the ipsilateral SVZ expressed the immature neuronal marker, polysialic acid-neural cell adhesion molecule. These data indicate that VEGFR-3 is upregulated in SVZ astrocytes and immature neurons after focal ischemia, suggesting that VEGFR-3 might mediate the adult neurogenesis after ischemic stroke.