Evaluation of the Effects of Microgravity on Activated Primary Human Hepatic Stellate Cells.

In recent years, research has been conducted to develop new medical treatments by simulating environments existing in space, such as zero-gravity. In this study, we evaluated the cell proliferation and gene expression of activated primary human hepatic stellate cells (HHSteCs) under simulated microgravity (SMG). Under SMG, cell proliferation was slower than in 1 G, and the evaluation of gene expression changes on day 1 of SMG by serial analysis of gene expression revealed the presence of Sirtuin, EIF2 signaling, hippo signaling, and epithelial adherence junction signaling. Moreover, reactive oxygen species were upregulated under SMG, and when N-acetyl-cystein was added, no difference in proliferation between SMG and 1 G was observed, suggesting that the oxidative stress generated by mitochondrial dysfunction caused a decrease in proliferation. Upstream regulators such as smad3, NFkB, and FN were activated, and cell-permeable inhibitors such as Ly294002 and U0126 were inhibited. Immunohistochemistry performed to evaluate cytoskeletal changes showed that more ß-actin was localized in the cortical layer under SMG.

Cryopreserved allogenic fibroblast sheets: development of a promising treatment for refractory skin ulcers.

The purpose of this study was to investigate the therapeutic effect of cryopreserved allogenic fibroblast cell sheets in a mouse model of skin ulcers. It is necessary to reduce the cost of regenerative medicine for it to be widely used. We consider that cell sheets could be applied to various diseases if cryopreservation of allogenic cell sheets was possible. In this study, fibroblasts were frozen using a three-dimensional freezer. Freeze-thawed fibroblasts had ~80% cell viability, secreted ≥ 50% vascular endothelial growth factor, hepatocyte growth factor, and stromal derived factor-1α compared with non-frozen fibroblast sheets, and secreted approximately the same amount of transforming growth factor-ß1. There was no difference in wound-healing rates in the skin ulcer model between non-frozen and freeze-thawed fibroblast sheets regardless of autologous and allogenic cells. The degree of angiogenesis was comparable between autologous and allogenic cells. The number of CD3-positive cells in healed tissues was larger for allogenic fibroblast sheets compared with autologous fibroblast sheets. However, histopathological images showed that the fibrosis, microvascular density, and healing phase of the wound in allogenic freeze-thawed fibroblast sheets were more similar to autologous freeze-thawed fibroblast sheets than to allogenic non-frozen fibroblast sheets. These results suggest that allogenic freeze-thawed fibroblast sheets may be a promising therapeutic option for refractory skin ulcers.

Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces.

BACKGROUND: Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques. RESULTS: Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation. CONCLUSION: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.

Urate transport function of rat sodium-dependent nucleobase transporter 1.

Sodium-dependent nucleobase transporter 1 (SNBT1) is a nucleobase-specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 µmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.

Treatment of Cutaneous Ulcers with Multilayered Mixed Sheets of Autologous Fibroblasts and Peripheral Blood Mononuclear Cells.

BACKGROUND/AIMS: We have developed a mixed-cell sheet consisting of autologous fibroblasts and peripheral blood mononuclear cells with a high potency for angiogenesis and wound healing against refractory cutaneous ulcers in mouse and rabbit models. To increase the effectiveness of the mixed sheet, we developed a multilayered mixed sheet. METHODS: We assessed the therapeutic effects of multilayered sheets on cutaneous ulcers in mice. Growth factors and chemokines were assessed by enzyme-linked immunosorbent assay. Angiogenesis and fibroblast migration were measured by using tube formation and migration assays. Wound healing rate of cutaneous ulcers was evaluated in mice with diabetes mellitus. RESULTS: The concentration of secreted vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor, C-X-C motif chemokine ligand (CXCL)-1, and CXCL-2 in multilayered sheets was much higher than that in single-layered mixed-cell sheets (single-layered sheets) and multilayered sheets of fibroblasts alone (fibroblast sheets). The supernatant in multilayered sheets enhanced angiogenic potency and fibroblast migration compared with single-layered and fibroblast sheets in an in vitro experiment. The wound healing rate in the multilayered sheet-treated group was higher compared with the no-treatment group (control) at the early stage of healing. Moreover, both vessel lumen area and microvessel density in tissues treated with multilayered sheets were significantly increased compared with tissues in the control group. CONCLUSION: Multilayered sheets promoted wound healing and microvascular angiogenesis in the skin by supplying growth factors and cytokines. Accordingly, our data suggest that multilayered sheets may be a promising therapeutic material for refractory cutaneous ulcers.

Development of Novel Mouse Model of Ulcers Induced by Implantation of Magnets.

We developed a novel mouse model of human refractory cutaneous ulcers that more faithfully reflects pathology and evaluated the effects of mixed cell sheets comprising peripheral blood mononuclear cells and fibroblasts, which we previously developed for treating refractory cutaneous ulcers. Model development involved sandwiching the skin between two magnets, one of which was implanted under the skin for 7 consecutive days. This magnet-implanted ulcer model produced persistently large amounts of exudate and induced the infiltration of the ulcer with inflammatory cells. The model mice had a thicker epidermis and impaired transforming growth factor-ß (TGF-ß) signaling followed by SMAD2 down-regulation, which causes epidermal hyperplasia in chronic ulcers. Impaired TGF-ß signaling also occurred in the ulcers of critical limb ischemia patients. Mixed cell implantation in this ulcer model reduced TNF-α and IL-6 levels in the tissues surrounding the mixed cell sheet-treated ulcers compared with controls or mice treated with trafermin (FGF2). Seven days after commencing therapy, the epidermis was thinner in mice treated with the mixed cell sheets than in controls. This model may therefore serve as a clinically relevant model of human ulcers, and our mixed cell sheets may effectively relieve chronic inflammation and inhibit refractoriness mechanisms.

Ulcer healing effect of autologous mixed sheets consisting of fibroblasts and peripheral blood mononuclear cells in rabbit ischemic hind limb.

We developed mixed cell sheets consisting of fibroblasts and peripheral blood mononuclear cells that had high potency for secreting vascular endothelial growth factor. The purpose of this study was to confirm the therapeutic effects of mixed sheets in rabbits suffering from ulcers at the ischemic hind limbs. We used the ulcer model, which was constructed by implantation and sandwiching the skin between two magnets to be a representative of human refractory cutaneous ulcer. The ulcer healing rate of mixed cell sheets was higher than that of the control at an early stage of healing. The calf blood pressure and angiographic score, which were considered to reflect rough collateral blood flow, did not vary amongmixed cell sheets. However, through laser Doppler perfusion image, implantation of mixed cell sheets revealed a significant improvement in microvascular blood flow in the healed skin of the ischemic limb compared to trafermin, a recombinant human basic fibroblast growth factor, and the control. These results suggest that mixed cell may operate predominantly on the surface of the ischemic tissue by their angiogenic potency, thereby promoting healing of the ischemic ulcer. Mixed cell sheets could become a promising therapeutic material for refractory cutaneous ulcers.

Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers.

TLR4 is a critical regulator of angiotensin II-induced vascular remodeling: the roles of extracellular SOD and NADPH oxidase.

Toll-like receptor 4 (TLR4) and angiotensin II (AngII) induce vascular remodeling through the production of reactive oxygen species (ROS). AngII has also been shown to increase antioxidant enzyme extracellular superoxide dismutase (ecSOD). However, the roles of TLR4 in Ang II-induced ROS production, vascular remodeling and hypertension remain unknown. Mice lacking TLR4 function showed significant inhibition of vascular remodeling in response to chronic AngII infusion, with no impact on blood pressure. The increases in ROS level and NADPH oxidase activity in response to AngII infusion were markedly blunted in TLR4-deficient mice. Similar effects were observed in wild-type (WT) mice treated with a sub-depressor dose of the AT1 receptor antagonist irbesartan, which had no effects on TLR4-deficient mice. Intriguingly, the AngII infusion-induced increases in ecSOD activity and expression were rather enhanced in TLR4-deficient mice compared with WT mice, whereas the expression of the proinflammatory chemokine MCP-1 was decreased. Importantly, AngII-induced vascular remodeling was positively correlated with NADPH oxidase activity, ROS levels and MCP-1 expression levels. Notably, chronic norepinephrine infusion, which elevates blood pressure without increasing ROS production, did not induce significant vascular remodeling in WT mice. Taken together, these findings suggest that ROS elevation is required for accelerating vascular remodeling but not for hypertensive effects in this model. We demonstrated that TLR4 plays a pivotal role in regulating AngII-induced vascular ROS levels by inhibiting the expression and activity of the antioxidant enzyme ecSOD, as well as by activating NADPH oxidase, which enhances inflammation to facilitate the progression of vascular remodeling.

Angiotensin â ¡ Activates MCP-1 and Induces Cardiac Hypertrophy and Dysfunction via Toll-like Receptor 4.

AIM: Angiotensin Ⅱ(Ang Ⅱ) produces reactive oxygen species (ROS), thus contributing to the development of cardiac hypertrophy and subsequent heart failure, and stimulates the expression of monocyte chemoattractant protein-1 (MCP-1). In addition, Toll-like receptor 4 (TLR4) is involved in the upregulation of MCP-1. In order to clarify whether TLR4 is involved in the onset of cardiac dysfunction caused by Ang Ⅱ stimulation, we investigated the effects of TLR4 on oxidative stress, the MCP-1 expression and cardiac dysfunction in mice with Ang Ⅱ-induced hypertension. METHODS: TLR4-deficient (Tlr4(lps-d)) and wild-type (WT) mice were randomized into groups treated with Ang Ⅱ, norepinephrine (NE) or a subdepressor dose of the Ang Ⅱreceptor blocker irbesartan (IRB) and Ang Ⅱ for two weeks. RESULTS: Ang Ⅱ and NE similarly increased systolic blood pressure in all drug-treated groups compared to that observed in the control group among both WT and Tlr4(lps-d) mice (p＜0.05). In the WT mice, Ang Ⅱ induced cardiac hypertrophy as well as vascular remodeling and perivascular fibrosis of the intramyocardial arteries and monocyte/macrophage infiltration in the heart (p＜0.05). Furthermore, Ang Ⅱ treatment decreased the left ventricular diastolic function and resulted in a greater left ventricular end-systolic dimension (p＜0.05) in addition to producing a five-fold increase in the NADPH oxidase activity, ROS content and MCP-1 expression (p＜0.05). In contrast, the Tlr4(lps-d) mice showed little effects of Ang Ⅱ on these indices. In the WT mice, IRB treatment reversed these changes compared to that seen in the mice treated with Ang Ⅱ alone. NE produced little effect on any of the indices in either the WT or Tlr4(lps-d) mice. CONCLUSIONS: TLR4 may be involved in the processes underlying the increased oxidative stress, selectively activated MCP-1 expression and cardiac hypertrophy and dysfunction seen in cases of Ang Ⅱ- induced hypertension.

A new Krüppel-like factor 1 mutation (c.947G > A or p.C316Y) in humans causes ß-thalassemia minor.

Here we describe a Japanese patient with mild ß-thalassemia (ß-thal) with an intact ß-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the ß-globin gene. The association of the KLF1 mutation with ß-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the ß-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the ß-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient's ß-thal. Furthermore, the patient's whole-exome analysis using next-generation sequencing revealed that the ß-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

Cardiosphere-derived cell sheet primed with hypoxia improves left ventricular function of chronically infarcted heart.

Cardiosphere-derived cells (CDCs) isolated from postnatal heart tissue are a convenient and efficientresource for the treatment of myocardial infarction. However, poor retention of CDCs in infarcted hearts often causes less than ideal therapeutic outcomes. Cell sheet technology has been developed as a means of permitting longer retention of graft cells, and this therapeutic strategy has opened new avenues of cell-based therapy for severe ischemic diseases. However, there is still scope for improvement before this treatment can be routinely applied in clinical settings. In this study, we investigated whether hypoxic preconditioning enhances the therapeutic efficacy of CDC monolayer sheets. To induce hypoxia priming, CDC monolayer sheets were placed in an incubator adjusted to 2% oxygen for 24 hours, and then preconditioned mouse CDC sheets were implanted into the infarcted heart of old myocardial infarction mouse models. Hypoxic preconditioning of CDC sheets remarkably increased the expression of vascular endothelial growth factor through the PI3-kinase/Akt signaling pathway. Implantation of preconditioned CDC sheets improved left ventricular function inchronically infarcted hearts and reduced fibrosis. The therapeutic efficacy of preconditioned CDC sheets was higher than the CDC sheets that were cultured under normaxia condition. These results suggest that hypoxic preconditioning augments the therapeutic angiogenic and anti-fibrotic activity of CDC sheets. A combination of cell sheets and hypoxic preconditioning offers an attractive therapeutic protocol for CDC transplantation into chronically infarcted hearts.

Haemodynamic unloading increases the survival and affects the differentiation of cardiac stem cells after implantation into an infarcted heart.

OBJECTIVES: It has been anticipated that stem cell therapy is capable of repairing an injured heart but is currently limited by its marginal efficacy. We believe that mechanical stress due to haemodynamic loading may negate the therapeutic potency of stem cells and therefore investigated how haemodynamic unloading affects the survival and differentiation of stem cells after implantation into an infarcted heart. METHODS: A left ventricular (LV) haemodynamic unloading model was implemented by heterotopic transplantation of an infarcted donor heart into another healthy mouse. An in situ infarcted heart with general haemodynamic loading was used as control. A total of 5 million cardiac stem cells expanded from green fluorescence protein (GFP)-transgenic mouse were intramyocardially implanted into the infarcted LVs of haemodynamically unloaded donor heart or general haemodynamic loaded heart. The survival and differentiation of the implanted cardiac stem cells were evaluated by histological analyses at 3 and 21 days after cell implantation (n = 5-6 in each time points per group). RESULTS: Compared with the general haemodynamic loading condition, haemodynamic unloading of the infarcted hearts significantly improved the survival, increased the proliferation and inhibited the apoptosis of cardiac stem cells at 21 days after cell implantation (P < 0.05). In addition, the number of GFP(+)/Sca-1(+) cells was much higher in the unloaded hearts than in the loaded hearts at 21 days after cell implantation, although the difference was not statistically significant (5.67 ± 5.10 vs 0.75 ± 0.50, P = 0.051). Among the surviving GFP(+) donor cells 21 days after implantation, the expressions of platelet endothelial cell adhesion molecule-1, smooth muscle actin and sarcomeric alpha actin were ~7, 38 and 27% in the loaded heart and ~19, 14 and 55% in the unloaded heart, respectively. CONCLUSIONS: Haemodynamic unloading favours the survival/engraftment of donor stem cells and affects their differentiation after implantation into an infarcted heart. Although further studies in a large animal model are required to investigate the functional benefits of haemodynamic unloading on stem cell therapy, we may temporarily unload the damaged heart to enhance cell engraftment and then load the heart again to induce the differentiation of stem cells.

The mobilization and recruitment of c-kit+ cells contribute to wound healing after surgery.

Delayed wound healing is a serious clinical problem in patients after surgery. A recent study has demonstrated that bone marrow-derived c-kit-positive (c-kit(+)) cells play important roles in repairing and regenerating various tissues and organs. To examine the hypothesis that surgical injury induces the mobilization and recruitment of c-kit+ cells to accelerate wound healing. Mice were subjected to a left pneumonectomy. The mobilization of c-kit+ cells was monitored after surgery. Using green fluorescent protein (GFP(+)) bone marrow-transplanted chimera mice, we investigated further whether the mobilized c-kit+ cells were recruited to effect wound healing in a skin puncture model. The group with left pneumonectomies increased the c-kit(+) and CD34(+) stem cells in peripheral blood 24 h after surgery. At 3 days after surgery, the skin wound size was observed to be significantly smaller, and the number of bone marrow-derived GFP(+) cells and GFP(+)/c-kit+ cells in the wound tissue was significantly greater in mice that had received pneumonectomies, as compared with those that had received a sham operation. Furthermore, some of these GFP(+) cells were positively expressed specific markers of macrophages (F4/80), endothelial cells (CD31), and myofibroblasts (αSMA). The administration of AMD3100, an antagonist of a stromal-cell derived factor (SDF)-1/CXCR4 signaling pathway, reduced the number of GFP(+) cells in wound tissue and completely negated the accelerated wound healing. Surgical injury induces the mobilization and recruitment of c-kit+ cells to contribute to wound healing. Regulating c-kit+ cells may provide a new approach that accelerates wound healing after surgery.

Hypoxic preconditioning enhances angiogenic potential of bone marrow cells with aging-related functional impairment.

BACKGROUND: Hypoxic preconditioning of bone marrow cells (BMCs) from young healthy individuals can enhance the cells' therapeutic potential. Considering that the response to hypoxia may differ according to the quality of the cells, we assessed the effect of hypoxic preconditioning on BMCs from aged mice and compared the difference in response between BMCs from aged and young mice. METHODS AND RESULTS: BMCs from young (3 months) and aged (20-22 months) mice were subjected to hypoxic preconditioning by culture for 24 h in 2% O2. Compared with BMCs from young mice, those from aged mice showed significantly fewer CD34- or c-kit-positive stem cells, higher expression of p53, and lower telomerase activity. Adhesion, survival and angiogenic potency were also lower in BMCs from aged mice, indicating an aging-related impairment. Hypoxia-preconditioned BMCs from aged mice showed enhanced adhesion, survival, and angiogenic potency with the in vitro assessments, as well as the in vivo implantation into ischemic hindlimbs. All the enhancements by hypoxic preconditioning were comparable between BMCs from aged and young mice, although the angiogenic potential of BMCs with and without hypoxic preconditioning was lower in old mice compared with young mice. CONCLUSIONS: Similar responses to hypoxia by BMCs from both aged and young mice suggest that hypoxic preconditioning could be a useful method of enhancing the angiogenic potential of BMCs.

TNFR1-mediated signaling is important to induce the improvement of liver fibrosis by bone marrow cell infusion.

The importance of TNF-α signals mediated by tumor necrosis factor receptor type 1 (TNFR1) in inflammation and fibrosis induced by carbon tetrachloride (CCl(4)), and in post-injury liver regeneration including a GFP/CCl(4) model developed as a liver repair model by bone marrow cell (BMC) infusion, was investigated. In mice in which TNFR1 was suppressed by antagonist administration or by knockout, liver fibrosis induced by CCl(4) was significantly decreased. In these mice, intrahepatic macrophage infiltration and TGF-ß1 expression were reduced and stellate cell activity was decreased; however, expression of MMP-9 was also decreased. With GFP-positive BMC (TNFR1 wild-type, WT) infusion in these mice, fibrosis proliferation, including host endogenous intrahepatic macrophage infiltration, TGF-ß1 expression and stellate cell activity, increased significantly. There was no significant increase of MMP-9 expression. In this study, TNFR1 in hosts had a promoting effect on CCl(4)-induced hepatotoxicity and fibrosis, whereas BMC infusion in TNFR1 knockout mice enhanced host-derived intrahepatic inflammation and fibrosis proliferation. These findings differed from those in WT recipient mice, in which improvement in inflammation and fibrosis with BMC infusion had previously been reported. TNFR1-mediated signaling might be important to induce the improvement of liver fibrosis by bone marrow cell infusion.

Operative injury accelerates tumor growth by inducing mobilization and recruitment of bone marrow-derived stem cells.

BACKGROUND: Although operative injury is thought generally to worsen the prognosis of cancer patients, the relevant mechanisms are not yet understood fully. We tested the hypothesis that operative injury induces mobilization and recruitment of bone marrow stem cells, thereby enhancing angiogenesis and accelerating tumor growth. METHODS: Mice were subjected to an open gastrotomy, and naïve mice were used as controls. The mobilization of bone marrow stem cells was monitored after operation. Using an established tumor model in green fluorescent protein (GFP)(+) bone marrow-transplanted chimera mice, we investigated further whether the mobilized stem cells affected tumor growth. RESULTS: Compared with the control, gastrotomy increased the populations of CD34(+) cells (6.9 ± 4.5 % vs 3.3 ± 0.4%, P < .05) and CD34(+)/Flk-1(+) cells (0.08 ± 0.02% vs 0.05 ± 0.01%, P < .05) in peripheral blood 12 h after operation. Twelve days after operation, the tumor volume almost doubled in mice after gastrotomy compared with control (580 ± 106 mm(3) vs 299 ± 162 mm(3), P < .05). A histologic analysis of tumor tissue revealed that the microvessel density and number of proliferating cells were significantly greater, but those of apoptotic cells were significantly less, in mice after gastrotomy as compared with control. Furthermore, the number of GFP(+) cells found in tumor tissue was significantly greater in mice that underwent gastrotomy than in controls. Some of the stained GFP(+) cells were positive for CD34 and had been incorporated into microvessels. Administration of AMD3100, which is an antagonist of stromal-cell-derived factor (SDF)-1/CXCR4 signaling pathway, inhibited the recruitment of GFP(+) cells and negated completely the acceleration in tumor growth after operation (345 ± 172 mm(3), P < .05). CONCLUSION: Operative injury may induce the mobilization and recruitment of bone marrow stem cells, thereby enhancing angiogenesis and accelerating tumor growth. Inhibition of the SDF-1/CXCR4 signals may represent a new therapeutic strategy for preventing acceleration of tumor growth after operation.

Maternal risk factors for small-for-gestational age newborns in Japanese dichorionic twins.

AIM: To investigate the maternal risk factors for small-for-gestational age (SGA) newborns in Japanese dichorionic (DC) twins. METHODS: A retrospective study was conducted from 2003 to 2008 on 340 DC twin pregnancies resulting in two live births. Newborns were classified as SGA if their birth weight was below the 10th percentile according to Japanese singleton norms. Statistical differences were evaluated between pregnancies resulting in appropriate-for-gestational age (AGA) pairs and those resulting in at least one SGA neonate. RESULTS: The study population consisted of AGA/AGA (50.8%), AGA/SGA (37.0%) and SGA/SGA pairs (12.0%). Logistic regression analysis identified significant interrelations for SGA with maternal nulliparity (odds ratio [OR] 0.52, 95% confidence interval [CI] 0.30-0.91), smoking (OR 3.25, 95% CI 1.09-9.66), assisted reproductive technology (OR 0.52, 95% CI 0.30-0.89), pregnancy-induced hypertension (OR 2.00, 95% CI 1.01-4.31), pregravid weight (kg) (unitary OR 0.94, 95% CI 0.91-0.97) and monthly weight gain (kg/month) (unitary OR 0.25, 95% CI 0.14-0.44). Bivariable receiver operating characteristic curves were generated for monthly weight gain (area under the curve [AUC] 0.626, cutoff 1.41 kg/month, P<0.001) and total weight gain (AUC 0.615, cutoff 14.0 kg, P<0.001). CONCLUSION: Cigarette smoking and weight gain control are relatively modifiable factors for which interventional management is necessary to avoid perinatal problems arising from SGA pregnancy. Further studies are needed to investigate optimal nutrition, health guidance and subsequent weight gain control that lead to concrete improvement in maternal and infant prognoses.

Functional characteristics of the human ortholog of riboflavin transporter 2 and riboflavin-responsive expression of its rat ortholog in the small intestine indicate its involvement in riboflavin absorption.

Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 µmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.

Identification and functional characterization of the first nucleobase transporter in mammals: implication in the species difference in the intestinal absorption mechanism of nucleobases and their analogs between higher primates and other mammals.

Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 microM. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use.