Metabotropic glutamate receptor 5 activation enhances tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor and NMDA-induced cell death in hippocampal cultured neurons.

The activation of group I metabotropic glutamate receptors (mGluRs), which are coupled with Gq-protein, initiates a variety physiological responses in different types of cells. While Gq-protein-coupled receptors can upregulate N-methyl-D-aspartate (NMDA) receptor function, group I mGluR-mediated regulations of NMDA receptor function are not fully understood. To determine biochemical roles of group I mGluRs in the regulation of the NMDA receptor, we have investigated changes in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B induced by a selective mGluR5 agonist, (RS)-chloro-5-hydroxyphenylglycine (CHPG) in hippocampal neuronal cultures. Activation of mGluR5 by CHPG increased active-forms of Src. CHPG also enhanced tyrosine phosphorylation of NR2A and NR2B in hippocampal neuronal cultures. In addition, NMDA-induced cell death was enhanced by CHPG-induced mGluR5 stimulation at the concentration, which increased tyrosine phosphorylation of Src and NR2A/2B but did not induce cell death. This effect was inhibited by selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The results suggest that in hippocampal neurons, mGluR5 may regulate NMDA receptor activity, involving tyrosine phosphorylation of NR2A and NR2B and may be involved in NMDA receptor-mediated cell injury.

Possible pathway of Na(+) flux into mitochondria in ischemic heart.

Previous studies showed that myocardial Na(+) overload during ischemia directly induced mitochondrial damage. The pathway for Na(+) flux into mitochondria remains unclear. We examined possible routes for Na(+) flux into mitochondria in the ischemic heart. Isolated perfused rat hearts were subjected to 15- to 35-min ischemia followed by 60-min reperfusion and then Na(+) content and respiratory function in mitochondria of the ischemic heart were determined. The mitochondrial Na(+) content of the ischemic heart was ischemic duration-dependently increased, associated with a reduction in mitochondrial respiratory function. To mimic induction of mitochondrial Na(+) overload in vitro, isolated mitochondria were incubated with 6.25 to 50 mM NaCl or sodium lactate, a metabolite of anaerobic glycolysis, in the presence and absence of a mitochondrial Na(+)/Ca(2+) exchanger inhibitor CGP37157 and a monocarboxylate transporter (MCT) inhibitor α-cyano-4-hydroxy cinnamic acid (CHCA). Incubation of mitochondria with NaCl or sodium lactate increased the mitochondrial Na(+) concentration. This increase in mitochondrial Na(+) was partially attenuated by the presence of either inhibitor. Combined treatment of mitochondria with both inhibitors attenuated sodium lactate-induced increase in Na(+) content to a greater degree than that treated with either agent. These results suggest that mitochondrial Na(+)/Ca(2+) exchanger and MCT inhibitor-sensitive Na(+) transporter are possible pathways for the mitochondrial Na(+) overload in the ischemic myocardium.

NADPH oxidase-mediated oxidative damage to proteins in the postsynaptic density after transient cerebral ischemia and reperfusion.

NADPH oxidase is an important source of superoxide in the central nervous system. Although NADPH oxidase is localized near the postsynaptic site in neurons, little is known about the pathophysiological role of NADPH oxidase in synapses after cerebral ischemia and reperfusion. In the present study, we sought to determine the role of NADPH oxidase in oxidative damage to postsynaptic density (PSD) proteins, which were isolated from rats subjected to transient focal cerebral ischemia and reperfusion. The amounts of carbonylated PSD proteins were increased after transient focal cerebral ischemia and reperfusion. This change was accompanied by an increase in the level of NADPH oxidase subunits in the PSD. The administration of apocynin, an NADPH oxidase inhibitor, attenuated both the protein carbonylation in the PSD and cerebral infarct volume. We further demonstrated that the decreases seen in the amounts of PSD-associated proteins, such as neuroligin, N-cadherin, and SAP102, in the PSD were prevented by treatment with apocynin. These results suggest that pronounced activation of NADPH oxidase in the PSD after cerebral ischemia and reperfusion may be related to the focal oxidative damage to synaptic functions and subsequent development of ischemia and reperfusion-induced cerebral injury.

Stable interaction between the human proliferating cell nuclear antigen loader complex Ctf18-replication factor C (RFC) and DNA polymerase {epsilon} is mediated by the cohesion-specific subunits, Ctf18, Dcc1, and Ctf8.

One of the proliferating cell nuclear antigen loader complexes, Ctf18-replication factor C (RFC), is involved in sister chromatid cohesion. To examine its relationship with factors involved in DNA replication, we performed a proteomics analysis of Ctf18-interacting proteins. We found that Ctf18 interacts with a replicative DNA polymerase, DNA polymerase ε (pol ε). Co-immunoprecipitation with recombinant Ctf18-RFC and pol ε demonstrated that their binding is direct and mediated by two distinct interactions, one weak and one stable. Three subunits that are specifically required for cohesion in yeast, Ctf18, Dcc1, and Ctf8, formed a trimeric complex (18-1-8) and together enabled stable binding with pol ε. The C-terminal 23-amino acid stretch of Ctf18 was necessary for the trimeric association of 18-1-8 and was required for the stable interaction. The weak interaction was observed with alternative loader complexes including Ctf18-RFC(5), which lacks Dcc1 and Ctf8, suggesting that the common loader structures, including the RFC small subunits (RFC2-5), are responsible for the weak interaction. The two interaction modes, mediated through distinguishable structures of Ctf18-RFC, both occurred through the N-terminal half of pol ε, which includes the catalytic domain. The addition of Ctf18-RFC or Ctf18-RFC(5) to the DNA synthesis reaction caused partial inhibition and stimulation, respectively. Thus, Ctf18-RFC has multiple interactions with pol ε that promote polymorphic modulation of DNA synthesis. We propose that their interaction alters the DNA synthesis mode to enable the replication fork to cooperate with the establishment of cohesion.

Cardioprotective effects of 2-octynyladenosine (YT-146) in ischemic/reperfused rat hearts.

The present study was aimed at investigating the cardiac receptor subtypes involved in the cardioprotective effects of 2-octynyladenosine (YT-146), a novel adenosine receptor (AR) agonist. Isolated rat hearts were perfused in the Langendorff manner, and the hearts were exposed to 30 minute of ischemia followed by 60 minutes of reperfusion. YT-146 was infused for 10 minutes just before ischemia, and selective antagonists for AR subtypes were coadministered with YT-146. YT-146 (0.03­0.3 µM) dose dependently improved postischemic recovery of the left ventricular developed pressure (LVDP) of the ischemic/reperfused rat heart (maximum 59.7% ± 2.3% of the preischemic value). Coadministration of 8-(3-chlorostyryl) caffeine (A(2A) AR antagonist), alloxazine (A(2B)AR antagonist), or MRS-1191 (A(3) AR antagonist) with YT-146 failed to alter the cardioprotective effects of YT-146, and their LVDP recoveries were 55.9% ± 5.1%, 52.1% ± 1.9%, and 47.5% ± 1.7%, respectively, at the end of the reperfusion. On the other hand, coadministration of 8-cyclopentyl-1,3-dipropylxanthine (A(1) AR antagonist) abolished the YT-146­induced enhancement of postischemic LVDP recovery (31.7% ± 4.6%). The protein kinase C inhibitor chelerythrine also abolished the YT-146­induced enhancement of postischemic LVDP recovery (22.2% ± 4.5%). YT-146 has been known as an A(2) AR agonist, but our findings suggest that the cardioprotective effects of YT-146 are exerted via cardiac A(1) AR, not A(2) AR, stimulation and the activation of protein kinase C by preischemic treatment in isolated and crystalloid-perfused rat hearts.

Intravenous injection of neural progenitor cells improves cerebral ischemia-induced learning dysfunction.

The ability of stem cells to enhance neurological recovery seen after cerebral ischemia has been reported. However, it remains to be clarified whether neural progenitor cells (NPCs) improve cerebral ischemia-induced learning dysfunction. We found in an earlier study that the direct injection of NPCs into the hippocampus prevents spatial learning dysfunction after cerebral ischemia. As the intravascular injection of cells represents a minimally invasive therapeutic approach, we sought to determine whether the intravenous injection of NPCs also would improve ischemia-induced spatial learning dysfunction. Cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of rats. The injection of NPCs via a femoral vein on day 7 after the induction of ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task on days 12-28 after cerebral ischemia. The intravenous injection of NPCs on day 7 did not affect the viable area of the ipsilateral hemisphere on day 28 compared with that of the non-treated ischemic rats. Furthermore, the NPCs injected via the vein were detected in the ipsilateral hemisphere; and they expressed brain-derived neurotrophic factor (BDNF) on day 28. The decrease in the BDNF level in the ipsilateral hemisphere was also inhibited by the injection of NPCs. These results suggest that the NPCs injected via the vein after cerebral ischemia improved spatial learning dysfunction, but without having any restorative effect on the damaged areas, possibly by acting as a source of neurotrophic factors.

mGluR1 antagonist decreased NADPH oxidase activity and superoxide production after transient focal cerebral ischemia.

NADPH oxidase, which is activated by PKC and signaling via the NMDA receptor, is one of the crucial enzymes for superoxide production in the CNS. We showed earlier that the metabotropic glutamate receptor 1 (mGluR1) plays an important role in the activation of PKC and tyrosine phosphorylation of the NMDA receptor, which has been implicated in enhancement of the channel activity, after cerebral ischemia. In this study, we sought to determine the role of mGluR1 in the activation of NADPH oxidase and subsequent superoxide production after transient focal cerebral ischemia. The amounts of NADPH oxidase subunits in the membrane fraction were increased after the start of reperfusion. These changes were accompanied by increased NADPH oxidase activity followed by superoxide production. The administration of an mGluR1 antagonist attenuated NADPH oxidase activity, which was coincident with inhibition of superoxide production. We further showed that the increase in the amount of PKCδ, but not of PKCζ, as well as the increase in those of NADPH oxidase subunits, was attenuated by the mGluR1 antagonist. These results suggest that mGluR1 may be linked to the increase in NADPH oxidase activity that is mediated by PKCδ and subsequent superoxide production after cerebral ischemia.

Alterations in dystrophin-related glycoproteins in development of right ventricular failure in rats.

Genetic depletion of the dystrophin-related glycoprotein (DRGP) complex causes cardiomyopathy in animals and humans. The present study was undertaken to explore the possible involvement of alterations in DRGP in the development of the right ventricular failure in monocrotaline-administered rats (MCT rats). At the 6th and 8th weeks after subcutaneous administration of 60 mg/kg monocrotaline, echocardiographic examination showed that cardiac output indices were decreased and that the right ventricular Tei indices were increased, suggesting that right ventricular failure occurs, at the latest, by 6 weeks after monocrotaline-administration. The levels of alpha- and beta-sarcoglycan and beta-dystroglycan in the right ventricle of the MCT rats at the 6th and 8th weeks were markedly decreased, and these decreases were inversely related to the increase in the right ventricular Tei index of the MCT-administered animals. The content and activity of the Ca(2+)-activated neutral protease m-calpain in the right ventricle of the MCT rats were increased at the 4th to 8th weeks and those of matrix metalloproteinase-2, at the 6th and 8th weeks. These results suggest that m-calpain- and/or matrix metalloproteinase-2-mediated alterations in the contents of alpha-sarcoglycan, beta-sarcoglycan, and beta-dystroglycan may be involved in the development of right ventricular failure in MCT rats.

Alterations in pharmacological action of the right ventricle of monocrotaline-induced pulmonary hypertensive rats.

The present study was undertaken to elucidate pathophysiological and pharmacological alterations in the right ventricle in monocrotaline-administered (MCT) rats. Examination of tissue weights of the MCT and age-matched control (CON) rats indicated the right ventricular (RV) hypertrophy until 8 weeks after a single subcutaneous administration of 60 mg/kg MCT. Apparent fibrosis in the right ventricle of the MCT rat at the 6th week (6w-MCT) was observed. Echocardiographic measurement of the cardiac and hemodynamic parameters of the MCT rat showed decreases in cardiac output and stroke volume indices at the 6th and 8th weeks. The RV Tei index, which increase represents aggravation of RV function, was augmented at the 4th to 8th week. The results suggest the genesis of cardiac and RV failure until 6 weeks after MCT administration. Injection of dobutamine (300 ng) or colforsin daropate (1 microg) into the perfused right ventricle isolated from CON rat at the 6th week resulted in a marked increase in cardiac double product, whereas injection of either agent into the right ventricle from the 6w-MCT rat elicited a small increase in the double product, followed by a sustained decrease in the developed tension. Infusion of acetylcholine (1 microg) into the RV muscle of the 6w-MCT rat resulted in prolongation of the periods for cardiac arrest and for bradycardia of the right ventricle. The results suggest that MCT administration causes the RV hypertrophy and eventually leads to the RV failure, accompanied by abnormal inotropic and chronotropic actions of the RV muscle.

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia.

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCgamma and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.

Delayed injection of neural progenitor cells improved spatial learning dysfunction after cerebral ischemia.

Although stem cells are likely to improve neurological deficits seen after cerebral ischemia, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning dysfunction remain to be clarified. We tested whether the delayed injection of exogenous NPCs could prevent learning dysfunction after cerebral ischemia. Cerebral ischemia was produced by the injection of microspheres into the right hemisphere of each rat. Injection of NPCs obtained from green fluorescent protein transgenic rats into the hippocampus on Day 7 after the induction of cerebral ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task. A few of the injected NPCs were positive for mature neuronal markers. In addition, the injected NPCs expressed BDNF on Day 28 after cerebral ischemia. Thus, the exogenous NPCs delivered by injection could act as a source of neurotrophic factors and prevent cerebral ischemia-induced learning dysfunction.

Effects of tanshinone VI on phosphorylation of ERK and Akt in isolated cardiomyocytes and cardiac fibroblasts.

Effects of tanshinone VI, a diterpene from Tan-Shen, on humoral factor-induced phosphorylation of ERK and Akt during hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts isolated from neonatal rats were examined. Treatment of cultured cardiomyocytes with 10 nM insulin-like growth factor-1 (IGF-1) or 10 nM endothelin-1 resulted in an increase in leucine incorporation into acid-insoluble fraction. Treatment of cultured cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II increased incorporation of proline. IGF-1 increased phosphorylated extracellular signal-regulated kinase (pERK) and protein kinase B (pAkt) of cardiomyocytes, whereas endothelin-1 increased pERK, but not pAkt. Treatment of cardiac fibroblasts with 10 nM IGF-1 or 10 nM angiotensin II also increased pERK, whereas pAkt was increased by treatment with IGF-1 alone. When the cardiomyocytes were incubated in the presence of 10 microM tanshinone VI, IGF-1- and endothelin-1-induced increases in pERK, but not pAkt, were partially attenuated. Treatment of cardiac fibroblasts with 10 microM tanshinone VI also attenuated IGF-1-induced increases in pERK and pAkt. Tanshinone VI also partially attenuated angiotensin II-induced increase in proline incorporation into cardiac fibroblasts. PD98059, an inhibitor for phosphorylation of extracellular signal-regulated kinase (ERK), but not wortmannin, that for protein kinase B phosphorylation, attenuated an increase in leucine incorporation into cardiomyocytes in the presence of either IGF-1 or endothelin-1. These results suggest that tanshinone VI is a possible agent that can attenuate the humoral factor-induced hypertrophy of cardiomyocytes and fibrosis of cardiac fibroblasts via an attenuation of ERK phosphorylation in these cells.

Hepatocyte growth factor suppresses ischemic cerebral edema in rats with microsphere embolism.

The present study was aimed at determining whether human recombinant hepatocyte growth factor (HGF) ameliorates cerebral edema induced by microsphere embolism (ME). Rats were injected with 700 microspheres (48 microm in diameter). Continuous administration of HGF at 13 microg/3 days/animal into the right ventricle was started from 10 min after embolism to the end of the experiment by using an osmotic pump. On day 3 after the ME, the rats were anesthetized, and their brains were perfused with an isotonic mannitol solution to eliminate constituents in the vascular and extracellular spaces. Thereafter, tissue water and cation contents were determined. A significant increase in tissue water content of the right hemisphere by ME was seen. This ME-induced increase in water content was associated with increases in tissue sodium and calcium ion contents and decreases in tissue potassium and magnesium ion contents of the right hemisphere. The treatment of the animal with HGF suppressed the increases in water and sodium and calcium ion contents, but not the decreases in potassium and magnesium ion contents. These results suggest that HGF suppresses the formation of ischemic cerebral edema provoked intracellularly in rats with ME.

The alpha(1D)-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction.

To investigate the physiological role of the alpha(1D)-adrenergic receptor (alpha(1D)-AR) subtype, we created mice lacking the alpha(1D)-AR (alpha(1D)(-/-)) by gene targeting and characterized their cardiovascular function. In alpha(1D)-/- mice, the RT-PCR did not detect any transcript of the alpha(1D)-AR in any tissue examined, and there was no apparent upregulation of other alpha(1)-AR subtypes. Radioligand binding studies showed that alpha(1)-AR binding capacity in the aorta was lost, while that in the heart was unaltered in alpha(1D)-/- mice. Non-anesthetized alpha(1D)-/- mice maintained significantly lower basal systolic and mean arterial blood pressure conditions, relative to wild-type mice, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. Besides hypotension, the pressor responses to phenylephrine and norepinephrine were decreased by 30-40% in alpha(1D)-/- mice. Furthermore, the contractile response of the aorta and the pressor response of isolated perfused mesenteric arterial beds to alpha(1)-AR stimulation were markedly reduced in alpha(1D)-/- mice. We conclude that the alpha(1D)-AR participates directly in sympathetic regulation of systemic blood pressure by vasoconstriction.

Vasopressin promotes cardiomyocyte hypertrophy via the vasopressin V1A receptor in neonatal mice.

[Arg8]-vasopressin (AVP) is an essential hormone for maintaining osmotic homeostasis and is known to be a potent vasoconstrictor that regulates the cardiovascular system. In the present study, cardiomyocytes were isolated from neonatal mice and used to investigate the effects of AVP on cardiac hypertrophy. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that vasopressin V1A receptor mRNA, but not V1B or V2 receptor mRNA, was expressed in primary cultured neonatal mouse cardiomyocytes. By exposing the cultured neonatal cardiomyocytes to AVP for 24 h, cell surface areas were significantly increased, suggesting that AVP could induce cardiomyocyte growth. We then investigated the expression level of the atrial natriuretic peptide (ANP), which is a marker of cardiac hypertrophy. Stimulation with AVP increased the expression of cardiomyocyte ANP mRNA in a dose- and time-dependent manner. Immunocytochemical studies showed that stimulation with AVP significantly increased the expression of the ANP protein as well. Furthermore, AVP administration activated extracellular signal-regulated kinase (ERK)1/2 in cardiomyocytes. The effects of AVP on these parameters were significantly inhibited by a selective vasopressin V1A receptor antagonist, OPC-21268, and were not observed in cardiomyocytes from mice lacking the vasopressin V1A receptor. In vivo cardiac hypertrophy in response to pressure overload was attenuated in vasopressin V1A receptor-deficient (V1AR-KO) mice. Taken together, our data suggest that AVP promotes cardiomyocyte hypertrophy via the vasopressin V1A receptor, which is in part regulated by the pathway of ERK1/2 signaling.

Vasopressin V1A receptor enhances baroreflex via the central component of the reflex arc.

The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts its physiological actions via 3 distinct receptor isoforms designated V1A, V1B, and V2. We recently showed that V1A receptor was involved in the baroreflex control of heart rate using V1A receptor knockout mice. The present study was undertaken to further clarify this finding. In conscious mice, resting blood pressure of the knockout group was lower than that of the wild-type group (wild-type, 108+/-2.0 mm Hg; knockout, 98+/-3.8 mm Hg; n=6-7) without notable change in heart rate. Although phenylephrine and nitroprusside-induced changes in blood pressure did not differ in these strains, the subsequent bradycardia and tachycardia were markedly blunted in the knockout mice (mean slopes for baroreflex curve after phenylephrine treatment; wild-type, -5.65+/-0.30 bpm/mm Hg; knockout, -3.97+/-0.52 bpm/mm Hg; those after nitroprusside treatment; wild-type, -0.51+/-0.10 bpm/mm Hg; knockout, -0.18+/-0.05 bpm/mm Hg; n=6-7). Under urethane anesthesia (1.0-1.2 g/kg, i.p.), electrical stimulation of the vagal afferent nerve evoked frequency-dependent hypotension and bradycardia in the wild-type mice. In contrast, in the knockout mice such stimulation induced a pressor, not a depressor, response and diminished bradycardia. Moreover, electrical stimulation-induced hemodynamic changes through the vagal afferent nerve in the wild-type mice were significantly attenuated by pretreatment with intravenously administered V1A receptor antagonist d(CH(2))(5)Tyr(Me)AVP. Electrical stimulation of the vagal efferent nerve-induced hemodynamic changes (depressor and bradycardia) and chronotropic responses to adrenergic and cholinergic stimuli were not different between the 2 strains. These results suggest that the V1A receptor in the central nervous system is involved in the regulation of the heart rate via the baroreflex arc.

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain.

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 microM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 microM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.

[Ischemic brain injury and hepatocyte growth factor].

Cerebral ischemia causes an irreversible and neurodegenerative disorder that may lead to progressive dementia and global cognitive deterioration. Since the overall process of ischemic brain injuries is extremely complex, treatment with endogenous multifunctional factors would be better choices for preventing complicated ischemic brain injuries. Hepatocyte growth factor, HGF, is a multifunctional cytokine originally identified and purified as a potent mitogen for hepatocyte. The activation of the c-Met/HGF receptor evokes diverse cellular responses, including mitogenic, morphogenic, angiogenic and anti-apoptotic activities in various types of cell. Previous studies showed that HGF and c-Met were expressed in various brain regions under normal conditions and that HGF enhanced the survival of hippocampal and cortical neurons during the aging of cells in culture. The protective effects of HGF on in vivo ischemic brain injuries and their mechanisms have not fully understood. To elucidate therapeutic potencies of HGF for ischemic brain injuries, we examined effects of HGF on ischemia-induced learning and memory dysfunction, neuronal cell death and endothelial cell damage by using the 4-vessel occlusion model and the microsphere embolism model in rats. Our findings suggested that treatment with HGF was capable of protecting hippocampal neurons against ischemia-induced cell death through the prevention of apoptosis-inducing factor translocation to the nucleus. Furthermore, we demonstrated that HGF had the ability to prevent tissue degeneration and improved learning and memory function after cerebral embolism, possibly through prevention of cerebral vessel injuries. As HGF has a potent cerebroprotective effect, it could be a prospective agent for the therapy against complicated ischemic brain diseases.

Prevention of apoptosis-inducing factor translocation is a possible mechanism for protective effects of hepatocyte growth factor against neuronal cell death in the hippocampus after transient forebrain ischemia.

Hepatocyte growth factor (HGF) is one of the prospective agents for therapy against a variety of neurologic and neurodegenerative disorders, although the precise mechanisms for the effect of HGF remain to be elucidated. We showed that treatment with HGF protected hippocampal cornu ammonis (CA) subregion 1 neurons from apoptotic cell death after transient forebrain ischemia. Accumulating evidence indicates that ischemia-induced neuronal damage occurs via caspase-independent pathways. In the present study, we focused on the localization of apoptosis-inducing factor (AIF), which is an important protein in the signal-transduction system through caspase-independent pathways, to investigate the possible mechanism for the protective effect of HGF after transient forebrain ischemia. Hepatocyte growth factor attenuated the increase in the expression of AIF protein in the nucleus after transient forebrain ischemia. We further explored the upstream components of AIF translocation. Primary DNA damage induced by Ca(2+) influx and subsequent NO formation are thought to be the initial events for AIF translocation, which results in the subsequent DNA damage by AIF. Hepatocyte growth factor prevented the primary oxidative DNA damage, as was estimated by using anti-8-OHdG (8-hydroxy-2'-deoxyguanosine) antibody. Oxidative DNA damage after ischemia is known to lead to the activation of poly(ADP-ribose) polymerase (PARP) and p53, resulting in AIF translocation. Marked increases in the PAR polymer formation and the expression of p53 protein after ischemia were effectively prevented by HGF treatment. In the present study, we first showed that HGF was capable of preventing neuronal cell death by inhibiting the primary oxidative DNA damage and then preventing the activation of the PARP/p53/AIF pathway.

Effects of hepatocyte growth factor on phosphorylation of extracellular signal-regulated kinase and hippocampal cell death in rats with transient forebrain ischemia.

Hepatocyte growth factor (HGF) has been implicated in protection against several types of cell injuries. We investigated the effects of human recombinant HGF (hrHGF) on the selective neuronal cell death in the hippocampal CA1 region after transient forebrain ischemia in rats and explored the nature of the intracellular signaling pathway for the protection against this neuronal injury. hrHGF was injected continuously into the hippocampal CA1 region directly using an osmotic pump from 10 min to 72 h after the start of reperfusion. The marked increase in the number of TUNEL-positive cells found in the CA1 region after ischemia was almost completely abolished by the hrHGF treatment. Akt phosphorylation as well as IkappaB phosphorylation, which has been implicated in events downstream of the Akt, was not affected by hrHGF treatment. Extracellular signal-regulated kinase (ERK) phosphorylation was decreased in the CA1 region with time after ischemia. hrHGF increased or recovered ERK phosphorylation without changing the total amount of ERK protein. Immunohistochemical analysis demonstrated that phosphorylated ERK was colocalized with a neuronal nucleus marker NeuN in the hippocampal CA1 region of ischemic rats with hrHGF treatment at the early period after reperfusion. These results suggest that the protective effects of hrHGF against neuronal death in the hippocampal CA1 after transient forebrain ischemia could be related to an ERK-dependent pathway.