Hiroshi Iwao

Osaka City University, Ōsaka, Ōsaka, Japan

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Publications (268)933.87 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC–MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers.
    No preview · Article · Jan 2016 · Journal of proteomics
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    ABSTRACT: Background: Highly concentrated carbon dioxide (GO2) is useful for treating ischemic diseases. Therefore, we investigated whether treatment with a few micrometers of CO2 molecules, atomized by two fluid nozzles (CO2 mist), could attenuate the development of right ventricular (RV) dysfunction in pulmonary hypertensive rats. Methods: Six-week-old male Wistar rats were divided into three groups: one that received injected saline; a second that received subcutaneous monocrotaline (MCT; 60 mg/kg) without treatment (PH-UT) group; and a third that received MCT with CO2 mist treatment (PH-CM) after MCT administration. The lower body of each rat was encased in a polyethylene bag, filled with the designated gaseous agent via a gas mist generator, for 30 minutes daily. Hemodynamics and cardiac function were measured at 28 days after beginning MCT administration. Protein levels were measured by western blotting. Results: Rats that received MCT without treatment began to die within 3-4 weeks of the initial administration. However, treatment with CO2 mist extended the survival period of rats in that group. At 28 days after MCT administration, the hemodynamic status, such as the blood pressure and heart rate, involved with left ventricular function, of rats in the PH-UT group were similar to those of rats in the PH-CM group. However, MCT-induced RV weight and RV dysfunction were significantly attenuated by treatment with CO2 mist. Both RV phosphorylated endothelial nitric oxide synthase and heat shock protein 72 levels increased significantly in the PH-CM group, compared to the PH-UT group. Conclusions: Percutaneous CO2 mist therapy may alleviate RV dysfunction in patients with pulmonary hypertension.
    No preview · Article · Oct 2015 · Osaka city medical journal
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    ABSTRACT: Vasodilators, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF), released from the vascular endothelium are important in the maintenance of systemic blood pressure. Some studies have shown that NO affects EDHF-induced vasodilator responses in isolated perfused blood vessel segments. However, the effects of NO on EDHF-mediated dilation, and their contribution to systemic blood pressure, have not been clarified. Therefore, in the present study we investigated the mechanisms underlying acetylcholine- and bradykinin-induced depressor responses, as well as the interaction between NO and EDHF, by measuring systemic blood pressure in anesthetized rats. In the presence of indomethacin and N(G)-nitro-l-arginine (l-NA; an NO synthase inhibitor), apamin plus charybdotoxin significantly inhibited depressor responses to acetylcholine and bradykinin, whereas glibenclamide, iberiotoxin, quinacrine, catalase, and combination of ouabain plus BaCl2 failed to inhibit EDHF-induced depressor responses. 4-Aminopyridine significantly inhibited depressor responses to acetylcholine, but not to bradykinin. In the presence of indomethacin and l-NA, carbenoxolone, a gap junction inhibitor, significantly inhibited depressor responses to agonists. l-NA alone significantly potentiated agonist-induced depressor responses. In contrast, infusion of sodium nitroprusside, an NO donor, or 8-br-cGMP significantly inhibited depressor responses to agonist. The findings of the present study raise the possibility that agonist-induced depressor responses are elicited by propagation of endothelial hyperpolarization via apamin- plus charybdotoxin-sensitive K(+) channels to smooth muscle cells through gap junctions, but not by diffusible substance(s). It is suggested that, in anesthetized rats, the EDHF-induced depressor response is attenuated in the presence of endogenous and exogenous NO via an increment in cGMP. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · May 2015 · European journal of pharmacology
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    ABSTRACT: Percutaneous treatment with carbon dioxide (CO2) mist, CO2 gas dissolved in water, contributes to improved cardiac function after myocardial infarction (MI). In this study, we investigated the effects of repeated pretreatment with CO2 mist on cardiac dysfunction after MI. The CO2 mist was generated by a dry mist production unit. The whole body of rats below the axilla was wrapped in a polyethylene bag, which was sealed and filled with the CO2 mist in the draft cabinet for 30 min daily for 7 days. MI was induced by ligation of the coronary artery in untreated (UT), CO2 gas-pretreated (CG), and CO2 mist-pretreated (CM) rats. The infarct size and the increase in oxidative stress due to MI were significantly smaller in the CM rats than in the UT rats. Furthermore, the expression of inflammation-related genes, such as monocyte chemoattractant protein-1, and fibrosis-related genes, such as transforming growth factor-β1, was significantly suppressed in the CM rats. The CM rats had a better left ventricular ejection fraction than the UT rats 7 days after MI. These parameters in the CG rats were the same as in the UT group. Thus, CO2 mist preparative treatment may be potentially useful for the reduction of MI. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.
    Full-text · Article · Apr 2015 · Journal of Pharmacological Sciences
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    ABSTRACT: Remote ischemic conditioning (RIC) by repeated treatment of transient limb ischemia is a clinically applicable method for protecting the heart against injury at the time of reperfusion. In this study, we investigated the effects of repeated RIC on cardiac dysfunction after myocardial infarction (MI). At 4weeks after MI, rats were separated into the untreated (UT) group or the RIC-treated group. RIC treatment was performed by 5cycles of 5min of bilateral hindlimb ischemia and 5min of reperfusion once a day for 4weeks. Despite comparable MI size, left ventricular (LV) ejection fraction (LVEF) was significantly improved in the RIC group compared with the UT group. Furthermore, the LVEF in the RIC group was improved, although not significantly, after treatment. RIC treatment also prevented the deterioration of LV diastolic function. MI-induced LV interstitial fibrosis in the boundary region and oxidant stress were significantly attenuated by RIC treatment. MicroRNA-29a (miR-29a), a key regulator of tissue fibrosis, was highly expressed in the exosomes and the marginal area of the RIC group. Even in the differentiated C2C12-derived exosomes, miR-29a expression was significantly increased under hypoxic condition. As well as miR-29a, insulin-like growth factor 1 receptor (IGF-1R) was highly expressed both in the exosomes and remote non-infarcted myocardium of the RIC group. IGF-1R expression was also increased in the C2C12-derived exosomes under hypoxic conditions. Repeated RIC reduces adverse LV remodeling and oxidative stress by MI. Exosome-mediated intercellular communication may contribute to the beneficial effect of RIC treatment. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
    Full-text · Article · Oct 2014 · International Journal of Cardiology
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    ABSTRACT: Metabolic syndrome (MetS) induces serious complications; therefore, we developed a noninvasive MetS model using an extremely small minipig, the Microminipig. For 8 weeks, Microminipigs were administrated a high-fat and high-cholesterol diet (HFCD) for atherosclerosis and N(G)-nitro-l-arginine methyl ester (l-NAME) for inhibiting nitric oxide synthase. HFCD significantly increased serum low-density lipoprotein levels, l-NAME increased blood pressure and cardiac hypertrophy, and HFCD-induced aortal arteriosclerosis was accelerated by l-NAME administration. Endothelium-dependent relaxation of the coronary artery was remarkably decreased by l-NAME administration. This model may be useful for elucidating the mechanisms of MetS and developing new therapeutic medicines for its treatment.
    No preview · Article · Sep 2014 · Journal of Pharmacological Sciences
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    ABSTRACT: Aim: Highly concentrated carbon dioxide (CO2) is thought to be useful for ischemic diseases. We investigated whether treatment with a few micrometers of CO2 molecules atomized via two fluidnozzles (CO2 mist) exerts an angiogenic effect in a mouse ischemic hindlimb model. Methods: Mice with unilateral hindlimb ischemia were divided into untreated (UT), 100% CO2 gas alone-treated (CG), mixed air (O2; 20%, N2; 80%) mist-treated (AM) and 100% CO2 mist-treated (CM) groups. The lower body of the mice was encased in a polyethylene bag filled with each gaseous agent using a gas mist generator for 10 minutes daily. Results: According to a laser Doppler analysis, the ischemic hindlimb blood flow was persistently higher after the seventh day of induction of ischemia in the CM group than in the UT group. The capillary density was also greater in the CM group on day 28 compared with that observed in the UT group. In addition, the parameters in the AM and CG groups were similar to those obtained in the UT group. The observed effects were abolished by the administration of an inhibitor of nitric oxide synthase (NOS). The vascular endothelial growth factor mRNA expression and protein levels and the phosphorylated endothelial NOS level were increased in the CM group compared with that observed in the UT group. A proteomic analysis using liquid chromatography-tandem mass spectrometry identified novel protein candidates regulated by CO2 mist. Conclusion: Percutaneous CO2 mist therapy may be useful for treating ischemia-induced angiogenesis.
    Full-text · Article · Aug 2014 · Journal of atherosclerosis and thrombosis
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    ABSTRACT: Fibroblast growth factor-2 (FGF-2) plays a critical role in endothelial survival, proliferation, and angiogenesis and is localized on the cell membrane by binding to heparan sulfate proteoglycans. Here we established a neutralizing monoclonal antibody, 1B9B9, against FGF-2 using the rat medial iliac lymph node method. 1B9B9 blocked the binding of FGF-2 to its receptor, inhibiting FGF-2-induced proliferation and corresponding downstream signaling in endothelial cells. Treatment of human umbilical vein endothelial cells with 1B9B9 reduced the basal phosphorylation levels of Akt and MAPK. Furthermore, continued treatment with 1B9B9 induced cell death by apoptosis. Compared with FGF-2 knockdown, 1B9B9 significantly reduced cell survival. In addition, the combination of FGF-2 siRNA and 1B9B9 showed a synergistic effect. The data indicate that 1B9B9 established by the rat iliac lymph node method is a fully compatible neutralizing antibody.
    Preview · Article · Aug 2014 · Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
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    ABSTRACT: Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.
    Full-text · Article · May 2014 · PLoS ONE

  • No preview · Article · Apr 2014 · Hypertension Research

  • No preview · Article · Apr 2014 · Hypertension Research
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    ABSTRACT: Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3-4 fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.
    No preview · Article · Mar 2014 · Biochemical and Biophysical Research Communications
  • Yasukatsu Izumi · Katsuyuki Miura · Hiroshi Iwao
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    ABSTRACT: Arginine vasopressin (AVP) is a 9-amino acid peptide that is secreted from the posterior pituitary in response to high plasma osmolality and hypotension. AVP has important roles in circulatory and water homoeostasis, which are mediated by oxytocin receptors and by AVP receptor subtypes: V1a (mainly vascular), V1b (pituitary), and V2 (renal). Vaptans are orally and intravenously active nonpeptide vasopressin-receptor antagonists. Recently, subtype-selective nonpeptide vasopressin-receptor agonists have been developed. A selective V1a-receptor antagonist, relcovaptan, has shown initial positive results in the treatment of Raynaud's disease, dysmenorrhea, and tocolysis. A selective V1b-receptor antagonist, nelivaptan, has beneficial effects in the treatment of psychiatric disorders. Selective V2-receptor antagonists including mozavaptan, lixivaptan, satavaptan, and tolvaptan induce highly hypotonic diuresis without substantially affecting the excretion of electrolytes. A nonselective V1a/V2-receptor antagonist, conivaptan, is used in the treatment for euvolaemic or hypervolemic hyponatremia. Recent basic and clinical studies have shown that AVP-receptor antagonists, especially V2-receptor antagonists, may have therapeutic potential for heart failure. This review presents current information about AVP and its antagonists.
    No preview · Article · Jan 2014 · Journal of Pharmacological Sciences
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    ABSTRACT: Triple-negative breast cancers (TNBCs) are defined as tumors that lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Clinically, TNBC patients are treated with cytotoxic drugs including 5-fluorouracil (5-FU). However, TNBCs develop resistance to such drugs after a series of treatments. To elucidate the mechanisms of drug resistance, establishment of drug-resistant cancer cell lines should be one of the most useful model systems. However, 5-FU-resistant TNBC cell lines have not been previously reported. In this study, we established a 5-FU-resistant cell line, MDA-MB-231/5-FU, from the human TNBC cell line MDA-MB-231, by repeated exposure to stepwise increases in the concentration of 5-FU. The IC50 value of 5-FU for MDA-MB-231/5-FU was 5.5-fold that for the parental cells. The MDA-MB-231/5-FU cell line acquired resistance to not only 5-FU, but also vinorelbine, paclitaxel and gemcitabine. Additionally, we performed iTRAQ-based quantitative proteomics in MDA-MB-231/5-FU cells and the parental cells in order to characterize MDA-MB-231/5-FU. The proteins upregulated in the newly established cells were mainly classified into the categories of 'DNA recombination', 'cell cycle', 'complex assembly', 'cytoskeleton organization', 'transport' and 'negative regulation of cell death'. These proteins may be related to mechanisms of drug resistance in TNBCs. Our established MDA-MB-231/5-FU cell line should be a useful tool for identifying new mechanisms of drug resistance and new drug targets in TNBCs.
    Preview · Article · Oct 2013 · International Journal of Oncology
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    ABSTRACT: Tolvaptan, a non-peptide V2-receptor antagonist, is a newly developed diuretic agent. Recently, we reported that tolvaptan has diuretic as well as anti-inflammatory and anti-fibrotic actions in chronic heart failure. In this study, we investigated whether tolvaptan has a cardioprotective effect in acute heart failure after myocardial infarction (MI). After MI induction, rats were randomized into 6 groups as follows: vehicle group, group treated with 15 mg∙kg(-1)∙day(-1) furosemide, 2 groups treated with 3 or 10 mg∙kg(-1)∙day(-1) tolvaptan, and 2 groups treated with 15 mg∙kg(-1)∙day(-1) furosemide combined with 3 or 10 mg∙kg(-1)∙day(-1) tolvaptan. Each agent was administered for 2 weeks, and blood pressure levels and infarct sizes were similar in all MI groups. Lower left ventricular end-systolic volumes and greater improvement of left ventricular ejection fraction were observed in the tolvaptan-treated groups compared with the vehicle group. In contrast, furosemide alone did not improve them. Sirius red staining revealed that tolvaptan significantly repressed MI-induced interstitial fibrosis in the left ventricle. MI-induced mRNA expressions related to cardiac load, inflammation, and fibrosis were significantly attenuated in the combination group. The combination treatment also repressed MI-induced mineralocorticoid receptor expression. Tolvaptan, or combination of furosemide and tolvaptan, may improve cardiac function in acute MI.
    No preview · Article · Sep 2013 · Journal of Pharmacological Sciences

  • No preview · Conference Paper · Jan 2013

  • No preview · Article · Jan 2013
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    ABSTRACT: Background: Arginine vasopressin, which promotes the reabsorption of renal water is increased in chronic heart failure. Here, we compared the effects of tolvaptan, a newly developed nonpeptide V(2) receptor antagonist, with those of furosemide, a loop diuretic, and a combination of these 2 agents in rats with left ventricular dysfunction after myocardial infarction (MI). Methods and results: After 10 weeks of MI induction, the rats were separated them into the following 6 groups adjusted to the infarct size: a vehicle group, a group treated with 15 mg·kg(-1)·day(-1) of furosemide, 2 groups treated with 3 or 10 mg·kg(-1)·day(-1) of tolvaptan; and 2 groups treated with 15 mg·kg(-1)·day(-1) of furosemide plus 3 or 10 mg·kg(-1)·day(-1) tolvaptan. Each treatment agent was administered for 4 weeks, and all groups had similar blood pressure levels and infarct size. The tolvaptan-treated groups were found to have lower levels of left ventricular end-diastolic and systolic cardiac volumes than the vehicle group did. Furthermore, the improvement in the ejection fraction in the tolvaptan-treated groups was significantly greater than those in the vehicle group. ED-1 immunostaining and Sirius red staining revealed that tolvaptan significantly repressed MI-induced macrophage infiltration and interstitial fibrosis in the left ventricle, respectively. Tolvaptan attenuated the MI-induced mRNA expressions of atrial and brain natriuretic peptides, monocyte chemotactic protein-1, transforming growth factor-β1, arginine vasopressin V(1a) receptor, and endothelin-1 in the marginal infarct region. Conclusions: Tolvaptan may improve cardiac dysfunction after MI, which is partially mediated by the suppression of V(1a) receptor, neurohumoral activation and inflammation.
    No preview · Article · Sep 2012 · Circulation Heart Failure
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    ABSTRACT: Although angiotensin II potently affects blood pressure and fluid balance, it is also involved in deterioration in atherosclerotic cardiovascular disease. Recently, angiotensin AT(1) receptor blockers have been demonstrated to be effective in patients with atherosclerotic disease, but the exact mechanisms of these blockers are still controversial. Atherosclerotic plaques are characterized by cholesterol ester accumulation and acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) expression, which are both parameters of degeneration of macrophage-derived foam cells. We examined the effects of angiotensin AT(1) receptor blockers on the formation of foam cells from macrophages. When macrophages from a human cell line were stimulated with oxidized low-density lipoprotein (oxLDL), the angiotensin AT(1) receptor blockers candesartan and losartan attenuated the intracellular accumulation of cholesterol ester and the increases in mRNA and protein levels of ACAT-1. Moreover, the increase in oxLDL-induced ACAT-1 was reduced by AG1478, an inhibitor of the epidermal growth factor (EGF) receptor. Additionally, oxLDL up-regulated the protein level of heparin-binding EGF-like growth factor (HB-EGF), a ligand of the EGF receptor. Inhibitors of angiotensin-converting enzyme affected neither cholesterol ester accumulation nor the expression of ACAT-1. Although oxLDL itself increased the secretion of angiotensin II, the amount of secreted angiotensin II was insufficient to induce expression of ACAT-1 protein. Thus, we first demonstrated that angiotensin AT(1) receptor blockers suppress ACAT-1 expression and cholesterol ester accumulation through an oxLDL-activated EGF receptor, but it is unclear how oxLDL activates angiotensin AT1 receptor in an angiotensin II-independent manner. The therapeutic mechanism of angiotensin AT(1) receptor blockers for atherosclerosis may be at least partially explained by our present results.
    No preview · Article · Mar 2012 · European journal of pharmacology
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    ABSTRACT: Pruritus is a severe symptom that is difficult to treat in atopic dermatitis patients. Red ginseng (RG), a natural medicine, has various biological activities such as anti-inflammatory effects. In this study, we examined the efficacy of RG extract (RGE) and its mechanism on experimental atopic dermatitis in mice. The effects of RGE on vascular permeability and itching were first evaluated. Histamine-induced permeability and itching were significantly inhibited by embrocation with RGE as well as diphenhydramine, an antihistamine drug. Next, we assessed the therapeutic effect of topical RGE in a mouse model of atopic dermatitis. Dermatitis was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB) acetone solution to the mouse ear. The effects of tacrolimus (a calcineurin blocker), dexamethasone (a corticosteroid), and RGE on dermatitis and associated scratching behavior were compared. Repeated DNFB application caused frequent scratching behaviors and ear swelling. Topical treatment with tacrolimus, dexamethasone, and RGE for 8 days before the final challenge with DNFB significantly inhibited ear swelling. Tacrolimus and RGE significantly inhibited scratching behavior, whereas dexamethasone failed to do so. DNFB-induced nerve growth factor expression and nerve fiber extension were significantly attenuated by tacrolimus and RGE, but not by dexamethasone. RGE may have the potential for treatment of atopic dermatitis.
    No preview · Article · Mar 2012 · Journal of Pharmacological Sciences

Publication Stats

7k Citations
933.87 Total Impact Points


  • 1982-2015
    • Osaka City University
      • • Department of Pharmacology
      • • Graduate School of Medicine
      • • Department of Gastroenterology
      • • Third Department of Internal Medicine
      Ōsaka, Ōsaka, Japan
  • 2004
    • The University of Tokyo
      • Department of Pharmaceutical Sciences
      白山, Tōkyō, Japan
  • 2003
    • University of Tsukuba
      Tsukuba, Ibaraki, Japan