Yutaka Oiso

Self-formation of functional adenohypophysis in three-dimensional culture

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke’s pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.

Transplantation of Bone Marrow-derived Mesenchymal Stem Cells Improves Diabetic Polyneuropathy in Rats.

OBJECTIVE—Mesenchymal stem cells (MSCs) have been reported to secrete various cytokines that exhibit angiogenic and neurosupportive effects. This study was conducted to investigate the effects of MSC transplantation on diabetic polyneuropathy (DPN) in rats. RESEARCH DESIGN AND METHODS—MSCs were isolated from bone marrow of adult rats and transplanted into hind limb skeletal muscles of rats with an 8-week duration of streptozotocin (STZ)-induced diabetes or age-matched normal rats by unilateral intramuscular injection. Four weeks after transplantation, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) productions in transplanted sites, current perception threshold, nerve conduction velocity (NCV), sciatic nerve blood flow (SNBF), capillary number–to–muscle fiber ratio in soleus muscles, and sural nerve morphometry were evaluated. RESULTS—VEGF and bFGF mRNA expression were significantly increased in MSC-injected thigh muscles of STZ-induced diabetic rats. Furthermore, colocalization of MSCs with VEGF and bFGF in the transplanted sites was confirmed. STZ-induced diabetic rats showed hypoalgesia, delayed NCV, decreased SNBF, and decreased capillary number–to–muscle fiber ratio in soleus muscles, which were all ameliorated by MSC transplantation. Sural nerve morphometry showed decreased axonal circularity in STZ-induced diabetic rats, which was normalized by MSC transplantation. CONCLUSIONS—These results suggest that MSC transplantation could have therapeutic effects on DPN through paracrine actions of growth factors secreted by MSCs.

Protective effect of dexamethasone on osmotic-induced demyelination in rats

Central pontine myelinolysis (CPM) is a serious demyelination disease commonly associated with the rapid correction of hyponatremia. Although its pathogenesis remains unclear, the disruption of the blood-brain barrier (BBB) as a consequence of a rapid increase in serum sodium concentration is considered to play a critical role. Since glucocorticoids are known to influence BBB permeability and prevent its disruption as a result of hypertension or hyperosmolarity, we investigated whether dexamethasone (DEX) could protect against osmotic demyelination in an animal model of CPM. Hyponatremia was induced in rats by liquid diet feeding and dDAVP infusion. Seven days later, the animals hyponatremia was rapidly corrected by injecting a bolus of hypertonic saline intraperitoneally. Rats subjected to this treatment displayed serious neurological impairment and 77% died within 5 days of rapid correction of their hyponatremia; demyelinative lesions were observed in various brain regions in these animals. On the other hand, rats that were treated with DEX (2 mg/kg, 0 and 6 h after hypertonic saline injection) exhibited minimal neurological impairment and all were alive after 5 days. Demyelinative lesions were rarely seen in the brains of DEX-treated rats. A marked extravasation of endogenous IgG was observed in the demyelinative lesions in the brains of rats that did not receive DEX, indicating disruption of the BBB, but was not observed in DEX-treated rats. Furthermore, Evans blue injection revealed a significant reduction in staining in the brains of DEX-treated rats (P < 0.05). These results indicate that early DEX treatment can prevent the BBB disruption that is caused by the rapid correction of hyponatremia and its associative demyelinative changes, and suggest that DEX might be effective in preventing CPM.

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium‐chain fatty acid receptor

In this study we aimed to identify the physiological roles of G protein‐coupled receptor 84 (GPR84) in adipose tissue, together with medium‐chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high‐fat diet up‐regulated GPR84 expression in fat pads. In 3T3‐L1 adipocytes, co‐culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down‐regulated adiponectin mRNA expression in 3T3‐L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity.

Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons

BACKGROUNDnSchwann cells (SCs) have been supposed to play prominent roles in axonal regeneration under various diseases. Here, to evaluate the direct interaction between SCs and dorsal root ganglion (DRG) neurons under a diabetic condition, the effects of Schwann cell-conditioned media on neurite outgrowth of DRG neurons were investigated.nnnMETHODSnImmortalized mouse Schwann cells (IMS) were cultured under 5.5 mM glucose (NG) or 30 mM glucose (HG) conditions for 4 days. IMS-conditioned media (IMS-media) were added to the culture media of neurons isolated from 8-week-old DDY mice. Neurons were cultured for 48 h with or without mouse recombinant NGF (mrNGF) or nerve growth factor (NGF) neutralizing antibody. The concentrations of NGF in IMS-media by ELISA and neurite outgrowth by a computed image analysis system were evaluated.nnnRESULTSnNeurite outgrowth was significantly enhanced by IMS-media (IMS-media (-): 177+/-177 microm, IMS-media (+): 1648+/-726). The neurite outgrowth cultured with IMS-media obtained under the HG condition was significantly reduced compared with that under the NG condition (NG: 1474+/-652, HG: 734+/-331). The NGF concentrations were significantly lower in IMS-media under the HG condition than in those under the NG condition. The accelerated neurite outgrowth by IMS-media was inhibited by NGF neutralizing antibody.nnnCONCLUSIONSnThese results suggest that SCs play important roles in neurite outgrowth of DRG neurons, and that the decreased NGF secretion by SCs under the diabetic condition would cause a defect of axonal regeneration, resulting in the development of diabetic neuropathy.

Adiponectin promotes migration activities of endothelial progenitor cells via Cdc42/Rac1

MINT‐7217644: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with Rac1 (uniprotkb:P63000) by pull down (MI:0096)

Polymorphism in resistin promoter region at ―420 determines the serum resistin levels and may be a risk marker of stroke in Japanese type 2 diabetic patients

Resistin, which appears to be related to insulin resistance, is secreted mainly from macrophages in human and some of its polymorphisms have been reported. Based on recent in vitro studies, resistin may be associated with atherosclerosis by mediating endothelial hyperactivity. We investigated whether resistin polymorphism at -420C>G is associated with serum resistin levels and diabetic macroangiopathy (coronary heart disease, arteriosclerosis obliterans, and stroke) in 349 Japanese type 2 diabetic patients (DM) and 286 non-diabetic controls (non-DM). Serum resistin levels in DM with a history of stroke were significantly higher than those without, 19.6+/-2.1 and 12.4+/-0.5 ng/ml (P<0.001), respectively. Furthermore, the levels were significantly increased in a genotype-dependent manner (CC, CG, GG) based on the polymorphism at -420C>G (P<0.001) in both DM and non-DM. The prevalence of stroke in DM significantly increased according to the presence of mutations (P<0.035). In multivariate logistic-regression analysis, individuals with the CG or GG genotypes were significantly more likely to have had a stroke than individuals with the CC genotype (vs. CG; OR 2.99, P=0.024, vs. GG; OR 4.49, P=0.010). These data suggested that the genotyping of resistin polymorphism at -420(C>G) can be a risk marker for stroke susceptibility in Japanese type 2 diabetic patients.

Time-dependent changes in proinflammatory and neurotrophic responses of microglia and astrocytes in a rat model of osmotic demyelination syndrome.

Osmotic demyelination syndrome (ODS) is a serious demyelinating disease in the central nervous system usually caused by rapid correction of hyponatremia. In an animal model of ODS, we previously reported microglial accumulation expressing proinflammatory cytokines. Microglia and astrocytes secreting proinflammatory cytokines and neurotrophic factors are reported to be involved in the pathogenesis of demyelinative diseases. Therefore, to clarify the role of microglial and astrocytic function in ODS, we examined the time‐dependent changes in distribution, morphology, proliferation, and mRNA/protein expression of proinflammatory cytokines, neurotrophic factors, and matrix metalloproteinase (MMP) in microglia and astrocytes 2 days (early phase) and 5 days (late phase) after the rapid correction of hyponatremia in ODS rats. The number of microglia time dependently increased at demyelinative lesion sites, proliferated, and expressed tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, inducible nitric oxide synthase, and MMP2, 9, and 12 at the early phase. Microglia also expressed leukemia inhibitory factor (a neurotrophic factor) and phagocytosed myelin debris at the late phase. The number of astrocytes time dependently increased around demyelinative lesions, extended processes to lesions, proliferated, and expressed nerve growth factor and glial cell line‐derived neurotrophic factor at the late phase. Moreover, treatment with infliximab, a monoclonal antibody against TNF‐α, significantly attenuated neurological impairments. Our results suggest that the role of microglia in ODS is time dependently shifted from detrimental to protective and that astrocytes play a protective role at the late phase. Modulation of excessive proinflammatory responses in microglia during the early phase after rapid correction may represent a therapeutic target for ODS.

Minocycline Prevents Osmotic Demyelination Syndrome by Inhibiting the Activation of Microglia

Rapid correction of chronic hyponatremia can lead to osmotic demyelination syndrome (ODS), a severe demyelination disease. The microglia that accumulate in the demyelinative lesions may play a detrimental role in the pathogenesis of ODS by producing proinflammatory cytokines, suggesting that they may be a target for therapeutic intervention. Here, we investigated whether minocycline, a selective and potent inhibitor of microglial activation, could protect against ODS in rats. We induced hyponatremia by liquid diet feeding and dDAVP infusion. Rapid correction of the hyponatremia 7 days later resulted in neurologic impairment with severe demyelinative lesions. Activated microglia accumulated at the site of demyelination. Treatment with minocycline within 24 hours of rapid correction, however, was protective: rats exhibited minimal neurologic impairment, and survival improved. Histologic analysis showed that minocycline inhibited demyelination and suppressed the accumulation of microglia at the site of demyelination. Real-time RT-PCR and immunohistochemical analyses showed that minocycline inhibited the activity of microglia and the expression of inflammatory cytokines (e.g. IL-1β, inducible nitric-oxide synthase, and TNF-α), monocyte chemoattractant protein-1, and matrix metalloproteinase-12 in microglia. These results demonstrate that minocycline can protect against ODS by inhibiting the activation and accumulation of microglia at the site of demyelinative lesions, suggesting its possible use in clinical practice.

Diurnal changes in arginine vasopressin gene transcription in the rat suprachiasmatic nucleus

The diurnal changes in arginine vasopressin (AVP) mRNA and heteronuclear (hn) RNA, an indicator for gene transcription, were examined in the hypothalamus of Sprague-Dawley rats using in situ hybridization. AVP hnRNA levels in the suprachiasmatic nucleus (SCN) varied during a 24-h cycle and showed a peak at day-time [Zeitgeber time (ZT) 5], which preceded the peak in AVP mRNA levels by 4 h. AVP hnRNA was undetectable at ZT 13 and 17, indicating that the gene transcription was almost shut down at these time points. AVP mRNA levels in the SCN continued to decrease at night (ZT 13, 17 and 21) when there were minimal changes in transcription, suggesting rapid turnover of mRNA. Similar diurnal changes in AVP hnRNA levels were observed without photic cues. On the other hand, AVP hnRNA or mRNA levels in the supraoptic nucleus, where AVP is synthesized in response to plasma osmolarity and/or volume, did not show any circadian rhythm. These data suggest that both dynamic changes in AVP gene transcription and rapid turnover of mRNA contribute to the diurnal variation in AVP mRNA levels in the SCN.