Kazuhito Morioka

Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma

Osteosarcoma is the most prevalent bone malignant tumor in children and adolescents, and displays heterogeneous histology and high propensity for distant metastasis. Although adjuvant chemotherapy remarkably improved treatment outcome over the past few decades, prognosis for osteosarcoma patients with pulmonary metastasis is still unsatisfactory. To identify novel therapeutic targets for osteosarcoma, we investigated the gene expression profile of osteosarcomas by cDNA microarray analysis and found transactivation of receptor tyrosine kinase‐like orphan receptor 2 (ROR2) expression in the majority of osteosarcoma samples. Treatment of osteosarcoma cell lines with siRNA against ROR2 significantly inhibited cell proliferation and migration. We also identified wingless‐type MMTV integration site family, member 5B (WNT5B) as a putative ROR2 ligand and that the physiological interaction of WNT5B and ROR2 could enhance cell migration, indicating the possible roles of ROR2 and WNT5B in the metastatic property of osteosarcoma cells. Taken together, our findings revealed that the WNT5B/ROR2 signaling pathway is a promising therapeutic target for osteosarcoma. (Cancer Sci 2009; 100: 1227–1233)

Phosphorylated neurofilament subunit NF-H as a biomarker for evaluating the severity of spinal cord injury patients, a pilot study

Study design:A pilot cross-sectional study of patients with acute cervical spinal cord injury (SCI).Objectives:The precise evaluation of the severity of SCI is important for developing novel therapies. Although several biomarkers in cerebrospinal fluid have been tested, few analyses of blood samples have been reported. A novel biomarker for axonal injury, phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), has been reported to be elevated in blood from rodent SCI model. The aim of this study is to investigate whether pNF-H values in blood can serve as a biomarker to evaluate the severity of patients with SCI.Setting:Tokyo Metropolitan Bokutoh Hospital and National Rehabilitation Center, Japan.Methods:This study enrolled 14 patients with acute cervical SCI. Sequential plasma samples were obtained from 6 h to 21 days after injury. Patients were classified according to American Spinal Injury Association impairment scale (AIS) at the end of the follow-up (average, 229.1 days). Plasma pNF-H values were compared between different AIS grades.Results:In patients with complete SCI, pNF-H became detectable at 12 h after injury and remained elevated at 21 days after injury. There was a statistically significant difference between AIS A (complete paralysis) patients and AIS C (incomplete paralysis) patients.Conclusions:Plasma pNF-H was elevated in accordance with the severity of SCI and reflected a greater magnitude of axonal damage. Therefore, pNF-H is a potential biomarker to independently distinguish AIS A patients (complete SCI) from AIS C–E patients (incomplete SCI). However, further studies are required to evaluate its utility in predicting prognosis of patients in the incomplete category.

Lipopolysaccharide preconditioning facilitates M2 activation of resident microglia after spinal cord injury

The inflammatory response following spinal cord injury (SCI) has both harmful and beneficial effects; however, it can be modulated for therapeutic benefit. Endotoxin/lipopolysaccharide (LPS) preconditioning, a well‐established method for modifying the immune reaction, has been shown to attenuate damage induced by stroke and brain trauma in rodent models. Although such effects likely are conveyed by tissue‐repairing functions of the inflammatory response, the mechanisms that control the effects have not yet been elucidated. The present study preconditioned C57BL6/J mice with 0.05 mg/kg of LPS 48 hr before inducing contusion SCI to investigate the effect of LPS preconditioning on the activation of macrophages/microglia. We found that LPS preconditioning promotes the polarization of M1/M2 macrophages/microglia toward an M2 phenotype in the injured spinal cord on quantitative real‐time polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemical analyses. Flow cytometric analyses reveal that LPS preconditioning facilitates M2 activation in resident microglia but not in infiltrating macrophages. Augmented M2 activation was accompanied by vascularization around the injured lesion, resulting in improvement in both tissue reorganization and functional recovery. Furthermore, we found that M2 activation induced by LPS preconditioning is regulated by interleukin‐10 gene expression, which was preceded by the transcriptional activation of interferon regulatory factor (IRF)−3, as demonstrated by Western blotting and an IRF‐3 binding assay. Altogether, our findings demonstrate that LPS preconditioning has a therapeutic effect on SCI through the modulation of M1/M2 polarization of resident microglia. The present study suggests that controlling M1/M2 polarization through endotoxin signal transduction could become a promising therapeutic strategy for various central nervous system diseases.

Hes1 functions downstream of growth factors to maintain oligodendrocyte lineage cells in the early progenitor stage

Expansion of the progenitor pool of oligodendrocytes (OLs) is a critical process for obtaining appropriate amounts of mature myelin-forming OLs in the developing and regenerating central nervous system. In vitro, fibroblast growth factor-2 (FGF2), together with platelet-derived growth factor (PDGF), is required to expand oligodendrocyte progenitor cells (OLPs) in an unlimited manner, maintaining them in the early progenitor stage. However, the intracellular mechanisms that prevent OLP maturation remain elusive. In order to investigate these mechanisms, we established a mouse OLP primary culture, which enabled us to undertake biochemical analyses. We found that the suppressive effects on maturation of early OLP to the late O4(+) progenitor by PDGF+FGF2 treatment was abrogated by Mek inhibitor, while transfecting cells with a constitutively active Mek1 construct prevented OLP maturation, suggesting that the Mek-Erk pathway is implicated in the effects of the growth factor treatment. The activation of Mek-Erk pathway promoted proliferation of OLP suggesting that cell cycle progression has suppressive effects to the maturation of OLP. Furthermore, molecular screening using DNA microarrays revealed that Hes1, a negative regulator of bHLH transcription factors, is one of the downstream molecules induced by PDGF+FGF2 treatment. We confirmed that forced activation of Mek-Erk pathway is sufficient to induce Hes1 expression and that Hes1, in turn, exerts suppressive effects on the maturation of OL lineage by itself. Our observations thus indicate that Mek-Erk pathway plays pivotal role in preventing early OLP maturation to late OLPs and the effect is mediated by cell cycle progression as well as Hes1 induction.

The swimming test is effective for evaluating spasticity after contusive spinal cord injury

Spasticity is a frequent chronic complication in individuals with spinal cord injury (SCI). However, the severity of spasticity varies in patients with SCI. Therefore, an evaluation method is needed to determine the severity of spasticity. We used a contusive SCI model that is suitable for clinical translation. In this study, we examined the feasibility of the swimming test and an EMG for evaluating spasticity in a contusive SCI rat model. Sprague-Dawley rats received an injury at the 8th thoracic vertebra. Swimming tests were performed 3 to 6 weeks after SCI induction. We placed the SCI rats into spasticity-strong or spasticity-weak groups based on the frequency of spastic behavior during the swimming test. Subsequently, we recorded the Hoffman reflex (H-reflex) and examined the immunoreactivity of serotonin (5-HT) and its receptor (5-HT2A) in the spinal tissues of the SCI rats. The spasticity-strong group had significantly decreased rate-dependent depression of the H-reflex compared to the spasticity-weak group. The area of 5-HT2A receptor immunoreactivity was significantly increased in the spasticity-strong group. Thus, both electrophysiological and histological evaluations indicate that the spasticity-strong group presented with a more severe upper motor neuron syndrome. We also observed the groups in their cages for 20 hours. Our results suggest that the swimming test provides an accurate evaluation of spasticity in this contusive SCI model. We believe that the swimming test is an effective method for evaluating spastic behaviors and developing treatments targeting spasticity after SCI.

Assessments of sensory plasticity after spinal cord injury across species

Spinal cord injury (SCI) is a multifaceted phenomenon associated with alterations in both motor function and sensory function. A majority of patients with SCI report sensory disturbances, including not only loss of sensation, but in many cases enhanced abnormal sensation, dysesthesia and pain. Development of therapeutics to treat these abnormal sensory changes require common measurement tools that can enable cross-species translation from animal models to human patients. We review the current literature on translational nociception/pain measurement in SCI and discuss areas for further development. Although a number of tools exist for measuring both segmental and affective sensory changes, we conclude that there is a pressing need for better, integrative measurement of nociception/pain outcomes across species to enhance precise therapeutic innovation for sensory dysfunction in SCI.

MRI and biomechanics multidimensional data analysis reveals R2‐R1ρ as an early predictor of cartilage lesion progression in knee osteoarthritis

To couple quantitative compositional MRI, gait analysis, and machine learning multidimensional data analysis to study osteoarthritis (OA). OA is a multifactorial disorder accompanied by biochemical and morphological changes in the articular cartilage, modulated by skeletal biomechanics and gait. While we can now acquire detailed information about the knee joint structure and function, we are not yet able to leverage the multifactorial factors for diagnosis and disease management of knee OA.

Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells

Endogenous neural stem/progenitor cells (NPCs) can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase (p38) is expressed in doublecortin-positive adult NPCs. Experiments using the p38 inhibitor SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we generated a cell-permeable wild-type p38 protein (PTD-p38WT) in which the HIV protein transduction domain (PTD) was fused to the N-terminus of p38. Treatment with PTD-p38WT significantly promoted the random migration of adult NPCs without affecting cell survival or differentiation; this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-p38WT is a novel and useful tool for unraveling the roles of p38, and that this protein provides a reasonable approach for regenerating the injured brain by enhancing NPC migration.