Shiho Kaneko

INVOLVEMENT OF STEM CELL FACTOR AND ITS RECEPTOR TYROSINE KINASE c-kit IN PAIN REGULATION

The c-kit receptor tyrosine kinase is expressed in a subpopulation of small- and medium-sized neurons of the dorsal root ganglia (DRG) and in the superficial layer of the spinal cord. Stem cell factor (SCF), a ligand of the c-kit receptor, induces neurite outgrowth from DRG and supports the survival of c-kit-expressing neurons. To clarify the possible function of the SCF/c-kit receptor system in the adult animal, we investigated the expression of c-kit receptor in the spinal cord and DRG in relation to pain by using H2C7, a newly developed anti-c-kit monoclonal antibody. S.c. and intrathecal injection of SCF markedly reduced the paw withdrawal threshold to mechanical stimuli and intrathecal SCF at 10 pg maximally induced mechanical allodynia in conscious mice. Intrathecal SCF also reduced the paw withdrawal latency to heat stimuli significantly but transiently. The c-kit receptor was co-expressed in 58.4% of calcitonin gene-related peptide (CGRP) -positive, but only 5.1% of isolectin B4-positive, DRG neurons. In the spinal cord, the c-kit receptor was detected in the superficial layer of the dorsal horn and co-localized there with CGRP in central terminals of DRG neurons. Selective elimination of unmyelinated C-fibers by neonatal capsaicin treatment resulted in marked reduction of the c-kit receptor and CGRP expression in the superficial layer of the spinal cord. Cell-size profiles showed that c-kit receptor expression was significantly up-regulated and down-regulated in medium-sized DRG neurons after neonatal capsaicin treatment and nerve injury, respectively. These results suggest that the c-kit receptor is mainly expressed in peptidergic small-sized DRG neurons and may be involved in pain regulation both peripherally and centrally.

Senescence in cultured trabecular meshwork cells

Background: It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases. Aim: To study the process of senescence in trabecular meshwork (TM) cells. Methods: Porcine TM tissues were obtained and placed in primary cultures with Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium. After 2–3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (DiI-Ac-LDL), a marker of TM cells. Staining for senescence-related β-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a 32P-labelled telomere-specific sequence (TTAGGG)3 at each PDL. Results: DiI-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related β-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown. Conclusions: TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.

Induction of arginase II mRNA by nitric oxide using an in vitro model of gyrate atrophy of choroid and retina.

PURPOSE The authors previously reported ornithine cytotoxicity in ornithine-δ-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy of the choroid and retina (GA). Given that RPE cells are severely damaged by arginine combined with ornithine, they investigated the role of arginine metabolism using that in vitro model. METHODS Human telomerase reverse transcriptase (hTERT)-RPE cells were incubated with ornithine or other agents in the presence of 5-fluoromethylornithine (5-FMO), an OAT-specific inhibitor. mRNA expression was determined by quantitative real-time polymerase chain reaction, and the concentration of nitric oxide (NO) was quantified using a Griess assay. Furthermore, cytotoxicity was examined by morphologic observations and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays, with the effect of arginase II examined using short interfering (si) RNA for arginase II and S-(2-boronoethyl)-L-cysteine (BEC), an arginase inhibitor. RESULTS NO production in 5-FMO-treated hTERT-RPE cells was increased by ornithine, and the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione induced cytotoxicity. Ornithine increased the expression of arginase II mRNA in 5-FMO-treated cells. Arginase II upregulation was partially inhibited by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, which was mimicked by SNAP. Arginase II siRNA and BEC enhanced ornithine cytotoxicity, and arginase II silencing resulted in a further increase in NO production. CONCLUSIONS These results demonstrate that NO is produced in our in vitro GA model, which induced cytotoxicity of RPE cells and upregulation of arginase II. NO may be involved in RPE degeneration in GA through the regulation of arginase II mRNA expression.

Controllable Urokinase Gene Expression in Trabecular Meshwork Cells

Purpose: Glaucoma is a type of progressive optic neuropathy that finally leads to blindness related to elevated intraocular pressure (IOP). Accumulation of extracellular matrix in trabecular meshwork (TM) and juxta-canalicular connective tissues, which form an aqueous outflow pathway, may be a major cause of increased IOP, thus the fibrinolytic system may be associated with regulation of IOP. We examined the possibility of controllable urokinase plasminogen activator (uPA) gene transfer into TM cells.Methods: TM cells were freshly isolated from porcine eyes and human TM cells obtained during trabeculectomy procedures and cultured. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse-transcribed polymerase chain reaction (RT-PCR) method was then performed to detect the gene expression of uPA. The cDNA of human uPA was sub-cloned into an expression vector (pEYFP-N1 vector) and controllable expression vector (TRE-Tight vector), then each vector was independently transfected into cultured porcine TM cells. Doxycycline was added to the culture medium to activate the pTet-ON and TRE-Tight vectors. Finally, the expression of uPA was examined using an enzyme linked immunosorbent assay (ELISA).Results: ELISA findings revealed the expression of uPA (3.5 ng/ml) in medium from cultured porcine TM cells that had been transfected with the human uPA gene using the pEYFP-N1 vector. Doxycycline induced human uPA in the pTet-On and TRE-Tight vectors with human uPA gene co-transfected TM cells in a dose-dependent manner.Conclusions: Controllable gene transfer of uPA, which may degrade the extracellular matrix in juxta-canalicular connective tissue, into TM cells was achieved using a Tet-On system. The present method may be useful as a novel therapy for glaucoma.