Shunya Takizawa

Administration of Hematopoietic Cytokines in the Subacute Phase After Cerebral Infarction Is Effective for Functional Recovery Facilitating Proliferation of Intrinsic Neural Stem/Progenitor Cells and Transition of Bone Marrow-Derived Neuronal Cells

Background— Hematopoietic cytokines, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) were reported to show a neuroprotective effect or to support neurogenesis. These cytokines also mobilize bone marrow (BM) cells into the brain, and the BM-derived cells differentiate into neuronal cells. We administered these hematopoietic cytokines after focal cerebral ischemia and assessed their effects and the therapeutic time window for neuronal regeneration. Methods and Results— We induced permanent middle cerebral artery occlusion in mice whose BM had been replaced with BM cells from green fluorescent protein (GFP)-transgenic mice. The occluded mice were treated with G-CSF and SCF in the acute phase (days 1 to 10) or subacute phase (days 11 to 20), and the brain functions and histological changes were evaluated. Separately, we injected bromodeoxyuridine during cytokine treatment to assess cell kinetics in the brain. Six mice were prepared for each experimental group. Administration of G-CSF and SCF in the subacute phase effectively improved not only motor performance but also higher brain function, compared with acute-phase treatment. Acute-phase and subacute-phase treatments identically reduced the infarct volume relative to vehicle treatment. However, subacute-phase treatment significantly induced transition of BM-derived neuronal cells into the peri-infarct area and stimulated proliferation of intrinsic neural stem/progenitor cells in the neuroproliferative zone. Conclusions— Administration of G-CSF and SCF in the subacute phase after focal cerebral ischemia is effective for functional recovery, enhancing cytokine-induced generation of neuronal cells from both BM-derived cells and intrinsic neural stem/progenitor cells. Because G-CSF and SCF are available for clinical use, these findings suggest a new therapeutic strategy for stroke.

A Novel Class of Prolyl Hydroxylase Inhibitors Induces Angiogenesis and Exerts Organ Protection Against Ischemia

Objective—Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD). Methods and Results—We discovered 2 unique compounds (TM6008 and TM6089), which inhibited PHD and stabilized HIF activity in vitro. Our docking simulation studies based on the 3-dimensional structure of human PHD2 disclosed that they preferentially bind to the active site of PHD. Whereas PHD inhibitors previously reported inhibit PHD activity via iron chelation, TM6089 does not share an iron chelating motif and is devoid of iron chelating activity. In vitro Matrigel assays and in vivo sponge assays demonstrated enhancement of angiogenesis by local administration of TM6008 and TM6089. Their oral administration stimulated HIF activity in various organs of transgenic rats expressing a hypoxia-responsive reporter vector. No acute toxicity was observed up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008. Oral administration of TM6008 protected neurons in a model of cerebrovascular disease. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis. Conclusions—The present study uncovered beneficial effects of novel PHD inhibitors preferentially binding to the active site of PHD.

Peroxynitrite Formation in Focal Cerebral Ischemia—Reperfusion in Rats Occurs Predominantly in the Peri-Infarct Region

Peroxynitrite (ONOO−) exhibits potent neurotoxicity and plays an important role in neu ronal death, but no evidence shows that it is formed in the brain during ischemia or subsequent reperfusion. To detect the formation of ONOO−, we used a hydrolysis/HPLC procedure to measure the formation of 3-nitro-l-tyrosine (NO2-Tyr), which is considered to reflect attack of ONOO− on l-tyrosine residues of cellular components in the brain. Focal ischemia was produced by occluding the right common carotid and right middle cerebral arteries for 2 hours, and the ischemic area was reperfused by reopening the middle cerebral artery. After 2 hours of ischemia, the values of the ratio of NO2-Tyr to l-tyrosine were 0% ± 0%, 0.42% ± 0.13% and 0.29% ± 0.10% in the noninfarct, periinfarct, and core-of-infarct regions, respectively. After 3 hours of reperfusion following 2 hours of ischemia, the ratio in the periinfarct region reached 0.89 ± 0.22%, which was significantly higher than that in the core-of-infarct region (0.35 ± 0.09%). The NO2-Tyr was not detected in 50 mg/kg of N-monomethyl-l-arginine–treated or sham-operated rats. Regional CBF in the periinfarct region decreased to 30.8 ± 15.9 mL/100 g/min during occlusion, but recovered more rapidly than did that in the core-of-infarct region.

Inhibition of Plasminogen Activator Inhibitor-1: Its Mechanism and Effectiveness on Coagulation and Fibrosis

Objective—Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. Methods and Results—We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A β-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride–induced testicular artery thrombosis. It also prevents the fibrotic process initiated by bleomycin in mouse lung. Conclusions—The present study demonstrates beneficial in vitro and in vivo effects of novel PAI-1 inhibitors. Our methodology proves to be a useful tool to obtain effective inhibitors of serpin activity.

Stem cell therapy for cerebral ischemia: from basic science to clinical applications

Recent stem cell technology provides a strong therapeutic potential not only for acute ischemic stroke but also for chronic progressive neurodegenerative diseases such as Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis with neuroregenerative neural cell replenishment and replacement. In addition to resident neural stem cell activation in the brain by neurotrophic factors, bone marrow stem cells (BMSCs) can be mobilized by granulocyte-colony stimulating factor for homing into the brain for both neurorepair and neuroregeneration in acute stroke and neurodegenerative diseases in both basic science and clinical settings. Exogenous stem cell transplantation is also emerging into a clinical scene from bench side experiments. Early clinical trials of intravenous transplantation of autologous BMSCs are showing safe and effective results in stroke patients. Further basic sciences of stem cell therapy on a neurovascular unit and neuroregeneration, and further clinical advancements on scaffold technology for supporting stem cells and stem cell tracking technology such as magnetic resonance imaging, single photon emission tomography or optical imaging with near-infrared could allow stem cell therapy to be applied in daily clinical applications in the near future.

Hypoxia. 1. Intracellular sensors for oxygen and oxidative stress: novel therapeutic targets.

A variety of human disorders, e.g., ischemic heart disease, stroke, kidney disease, eventually share the deleterious consequences of a common, hypoxic and oxidative stress pathway. In this review, we utilize recent information on the cellular defense mechanisms against hypoxia and oxidative stress with the hope to propose new therapeutic tools. The hypoxia-inducible factor (HIF) is a key player as it activates a broad range of genes protecting cells against hypoxia. Its level is determined by its degradation rate by intracellular oxygen sensors prolyl hydroxylases (PHDs). There are three different PHD isoforms (PHD1-3). Small molecule PHD inhibitors improve hypoxic injury in experimental animals but, unfortunately, may induce adverse effects associated with PHD2 inhibition, e.g., angiogenesis. As yet, no inhibitor specific for a distinct PHD isoform is currently available. Still, the specific disruption of the PHD1 gene is known to induce hypoxic tolerance, without angiogenesis and erythrocytosis, by reprogramming basal oxygen metabolism with an attendant decreased oxidative stress in hypoxic mitochondria. A specific PHD1 inhibitor might therefore offer a novel therapy against hypoxia. The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and inducible expression of numerous antioxidant stress genes. Disruption of its gene exacerbates oxidative tissue injury. Nrf2 activity is modulated by Kelch-like ECH-associated protein 1 (Keap1), an intracellular sensor for oxidative stress. Inhibitors of Keap 1 may prove therapeutic against oxidative tissue injury.

Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death

Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.

Induction of Heme Oxygenase Protein Protects Neurons in Cortex and Striatum, But Not in Hippocampus, against Transient Forebrain Ischemia

To clarify whether heme oxygenase-1 (HO-1) protein plays a protective role against cerebral ischemia, we investigated the effects of an HO inhibitor (tin mesoporphyrin IX [SnMP] three doses of 30 μmol/kg, intraperitoneally) and an HO inducer (hemin, three doses of 30 μmol/kg, intraperitoneally) on the pathologic outcome and on the immunohistochemical reaction for HO-1 after 20-minute transient forebrain ischemia followed by 3-day reperfusion in rats. Hemin significantly increased viable neurons in the cortex (compared to the SnMP-treated group, P<.05) and striatum (compared to the saline-treated group at P<.01 and SnMP-treated group at P<.05), and intense HO-1 immunoreactivity was observed in cortex and striatum, whereas the administration of SnMP tended to decrease viable neurons in the parietal cortex. In contrast, neither hemin nor SnMP affected the pathologic outcome in the CA1 and CA3 hippocampi, in which HO-1 immunoreactivity was weak. These results suggest that induction of HO-1 protein may contribute to cellular defense against ischemic damage in brain regions where potential ability to synthesize HO-1 is retained in ischemia.

A Selective N-Type Calcium Channel Antagonist Reduces Extracellular Glutamate Release and Infarct Volume in Focal Cerebral Ischemia

Although a number of studies have demonstrated the neuroprotective effects of antagonists of postsynaptic N-methyl-d-aspartate (NMDA) and non-NMDA receptors in cerebral ischemia, little is known about the treatment of cerebral infarction through presynaptic blocking of extracellular glutamate release. We evaluated the effects of a presynaptic selective N-type calcium channel antagonist (SNX-111, given intravenously by continuous infusion at 5 mg/kg/h from 20 min prior to occlusion until 2 h postocclusion) on blood flow, extracellular glutamate, and infarct volume in rats with permanent occlusions of the right middle cerebral and right common carotid arteries plus 1-h transient occlusion of the left common carotid artery. There was no significant difference in CBF in the occluded cortex during the experiment between the treated and vehicle groups. SNX-111 significantly reduced total amount of extracellular glutamate during the experiment and the peak value of the glutamate after occlusion from 44.2 ± 15.8 μM (mean ± SD) to 21.4 ± 11.4 μM (p < 0.01). Infusion of SNX-111 also significantly reduced the cortical volume of infarction from 47.2 ± 5.8 to 19.9 ± 7.3% (p < 0.0001). These results suggest that SNX-111 has a protective effect against focal ischemia through the inhibition of glutamate release from presynaptic sites, although SNX-111 may also affect the release of other neurotransmitters.

A novel inhibitor of plasminogen activator inhibitor-1 provides antithrombotic benefits devoid of bleeding effect in nonhuman primates.

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterialvenous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latters therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.