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Background: The cell adhesion molecule CHL1, which belongs to the immunoglobulin superfamily, functions in a variety of physiological and pathological processes including neural development, tissue injury and repair. Recently, we found CHL1 was co-localized with GFAP positive cells in mouse colon tissue. Methods: Here, Colon tissues were collected from CHL1+/+ , CHL1+/- and CHL1-/- mice after dextran sodium sulfate (DSS) induced to investigate the effects of CHL1 on the development of DSS-induced colitis. Results: The data showed that CHL1 expression was increased in distal colon in a time-dependent manner after DSS-treatment. CHL1 deficiency induced more pronounced colitis features, an exacerbation of inflammation and damage to colonic tissues in DSS-induced mouse than that of wild type mouse. Moreover, CHL1-/- mice showed a remarkable increase of neutrophil and macrophage infiltration into colonic tissues, and then result in more severe damage to the intestinal epithelial cells and FITC leakage in CHL1 deficiency mice than that in WT mouse. Conclusions: Our results revealed a distinct colonic role for CHL1 in regulating DSS-induced colitis, CHL1 deficiency exacerbates DSS-induced colitis in mice, indicating that CHL1 may be an attractive therapeutic target for IBD.

The kidney is vulnerable to hypoxia-induced injury. One of the mechanisms underlying this phenomenon is cell apoptosis triggered by hypoxia-inducible factor-1- α (HIF-1α) activation. MicroRNA-210 (miR-210) is known to be induced by HIF-1α and can regulate various pathological processes, but its role in hypoxic kidney injury remains unclear. Here, in both rat systemic hypoxia and local kidney hypoxia models, we found miR-210 levels were upregulated significantly in injured kidney, especially in renal tubular cells. A similar increase was observed in hypoxia-treated human renal tubular HK-2 cells. We also verified that miR-210 can directly suppress HIF-1α expression by targeting the 3′ untranslated region of HIF-1α mRNA in HK-2 cells in severe hypoxia. Accordingly, miR-210 overexpression caused significant inhibition of the HIF-1α pathway and attenuated apoptosis caused by hypoxia, while miR-210 knockdown exerted the opposite effect. Taken together, our findings verify that miR-210 is involved in the molecular response in hypoxic kidney lesions in vivo and attenuates hypoxia-induced renal tubular cell apoptosis by targeting HIF-1α directly and suppressing HIF-1α pathway activation in vitro.

WIP1, as a critical phosphatase, plays many important roles in various physiological and pathological processes through dephosphorylating different substrate proteins. However, the functions of WIP1 in adipogenesis and fat accumulation are not clear. Here, we report that WIP1-deficient mice show impaired body weight growth, dramatically decreased fat mass, and significantly reduced triglyceride and leptin levels in circulation. This dysregulation of adipose development caused by the deletion of WIP1 occurs as early as adipogenesis. In contrast, lentivirus-mediated WIP1 phosphatase overexpression significantly increases the adipogenesis of pre-adipocytes via an enzymatic activity-dependent mechanism. PPARγ is a master gene of adipogenesis, and the phosphorylation of PPARγ at serine 112 strongly inhibits adipogenesis; however, very little is known about the negative regulation of this phosphorylation. Here, we show that WIP1 phosphatase plays a pro-adipogenic role by interacting directly with PPARγ and dephosphorylating p-PPARγ S112 in vitro and in vivo.

The hypobaric hypoxic environment in high-altitude areas often aggravates the severity of inflammation and induces brain injury as a consequence. However, the critical genes regulating this process remain largely unknown. The phosphatase wild-type p53-induced phosphatase 1 (WIP1) plays important roles in various physiological and pathological processes, including the regulation of inflammation in normoxia, but its functions in hypoxic inflammation-induced brain injury remain unclear. Here, we established a mouse model of this type of injury and found that WIP1 deficiency augmented the release of inflammatory cytokines in the peripheral circulation and brain tissue, increased the numbers of activated microglia/macrophages in the brain, aggravated cerebral histological lesions, and exacerbated the impairment of motor and cognitive abilities. Collectively, these results provide the first in vivo evidence that WIP1 is a critical neuroprotector against hypoxic inflammation-induced brain injury.

In vitro cell culture has provided a useful model to study the effects of oxygen on cellular behavior. However, it remains unknown whether the in vitro operations themselves affect the medium oxygen levels and the living states of cells. In addition, a prevailing controversy is whether reactive oxygen species (ROS) production is induced by continuous hypoxia or reoxygenation. In this study, we have measured the effects of different types of cell culture containers and the oxygen environment where medium replacement takes place on the actual oxygen tension in the medium. We found that the deviations of oxygen concentrations in the medium are much greater in 25-cm(2) flasks than in 24-well plates and 35-mm dishes. The dissolved oxygen concentrations in the medium were increased after medium replacement in normoxia, but remained unchanged in glove boxes in which the oxygen tension remained at a low level (11.4, 5.7, and 0.5% O2 ). We also found that medium replacement in normoxia increased the number of ROS-positive cells and reduced the cell viability; meanwhile, medium replacement in a glove box did not produce the above effects. Therefore, we conclude that the use of 25-cm(2) flasks should be avoided and demonstrate that continuous hypoxia does not produce ROS, whereas the reoxygenation that occurs during the harvesting of cells leads to ROS and induces cell death.

High altitude cerebral edema (HACE) is a life-threatening illness that develops during the rapid ascent to high altitudes, but its underlying mechanisms remain unclear. Growing evidence has implicated inflammation in the susceptibility to and development of brain edema. In the present study, we investigated the inflammatory response and its roles in HACE in mice following high altitude hypoxic injury. We report that acute hypobaric hypoxia induced a slight inflammatory response or brain edema within 24 h in mice. However, the lipopolysaccharide (LPS)-induced systemic inflammatory response rapidly aggravated brain edema upon acute hypobaric hypoxia exposure by disrupting blood-brain barrier integrity and activating microglia, increasing water permeability via the accumulation of aquaporin-4 (AQP4), and eventually leading to impaired cognitive and motor function. These findings demonstrate that hypoxia augments LPS-induced inflammation and induces the occurrence and development of cerebral edema in mice at high altitude. Here, we provide new information on the impact of systemic inflammation on the susceptibility to and outcomes of HACE.

Notch1 is a transcription factor on the membrane and regulates various stages of neurogenesis. Recently, studies have shown that in vitro neurogenesis is enhanced by hypoxia, and there is cross-coupling between Notch and hypoxia signaling pathways in vitro. However, to date, no data have reported whether Notch1 can be regulated by hypoxia in vivo and mediates hypoxia-induced neurogenesis. To determine causative links between Notch1, neurogenesis and hypoxia, we examined multiple steps of hippocampal neurogenesis followed intermittent hypoxia (IH) in wild type (WT) and Notch1 heterozygous deficient (N+/−) mice. We found that IH increased NSC proliferation, newborn neuron survival and migration, and spine morphogenesis in dentate gyrus of hippocampus, as well as neurogenesis in olfactory bulb in WT mice. However, IH-enhanced neurogenesis was inhibited in N+/− mice. It was shown that Notch1 signaling was activated following IH in WT mice, but not in N+/− mice. Our data indicated that IH, as a novel external stimulus, enhances neurogenesis at multiple stages and that Notch1 is activated by hypoxia in vivo and required for hypoxia-induced neurogenesis. These results suggest IH as a novel therapeutic strategy for degenerative neurological disorders and provide evidence for causative links between Notch1, neurogenesis and hypoxia.

We first reported the role of 5-hydroxymethyl-2-furfural (5-HMF) against hypoxia. Here, we studied the mechanism by using oxygen-dependent degradation domain (ODD)-Luc mice, which are a useful model to probe the stabilization of hypoxia-inducible factor 1α (HIF-1α). Compared with three other compounds that have been reported to have a role in stabilizing HIF-1α, 5-HMF caused stronger bioluminescence, which is indicative of HIF-1α stability in the brain and kidney of ODD-Luc mice. We further demonstrated that the HIF-1α protein accumulated in response to 5-HMF in the brains and kidneys of these mice, as well as in PC12 cells. Additionally, 5-HMF promoted the nuclear translocation of HIF-1α and the transcriptional activity of HIF-1, which was evaluated by detecting vascular endothelial growth factor (VEGF ) mRNA expression. These results suggest that 5-HMF stabilized HIF-1α and increased its activity. Considering the role of proline hydroxylases (PHDs) in negatively regulating HIF-1α stability, we explored whether 5-HMF interacts with the substrates and cofactors of PHDs, such as 2-oxoglutarate (2-OG), Fe(2+) and vitamin C (VC), which affects the activity of PHDs. The result revealed that 5-HMF did not interact with Fe(2+) or 2-OG but interacted with VC. This interaction was confirmed by subsequent experiments, in which 5-HMF entered into cells and reduced the VC content. The enhanced stability of HIF-1α by 5-HMF was reversed by VC supplementation, and the improved survival of mice caused by 5-HMF under hypoxia was abrogated by VC supplementation. Thus, we demonstrated for the first time that 5-HMF increases HIF-1α stability by reducing the VC content, which mediates the protection against hypoxia.

Background and Purpose— NB-3 is a member of the F3/contactin family of neural recognition molecules, which are crucial for cell morphogenesis and motility. NB-3 is expressed in neurons and plays an important role in axonal extension and neuronal survival. However, the role of NB-3 in cerebral ischemic injury remains unknown. Methods— Adult male wild-type and NB-3 knockout mice were subjected to ischemic injury by unilateral middle cerebral carotid artery occlusion for 3 hours, 6 hours, and 12 hours. Ischemic infarction volumes were then determined by 2, 3, 5-triphenyltetrazolium chloride staining. Neurological dysfunction analysis was also performed. Primary culture of neuronal cells from wild-type and knockout animals was also used for analysis of neuronal survival and neurite outgrowth. Results— NB-3 expression in the ischemic hemisphere was decreased after transient middle cerebral artery occlusion (MCAO). NB-3-knockout mice developed a 2.6-fold larger infarct volume and exhibited increased neurological deficit scores after transient middle cerebral artery occlusion compared with control mice. Substrate with NB-3 promoted neuronal survival and neurite outgrowth in vitro, whereas neurite outgrowth and neuronal survival were significantly reduced in NB-3-deficient neurons. In addition, NB-3 deficiency renders neurons more susceptible to oxygen–glucose deprivation-induced damage and NB-3 as substrate could partially through homophilic mechanisms. Conclusions— These data demonstrate that NB-3 deficiency may aggravate brain damage after middle cerebral artery occlusion by impairing neuronal survival and neurite growth.

Hypoxia promotes the proliferation of neural progenitor cells (NPCs), and low oxygen is a useful tool for expansion of NPCs in vitro. To further understand the regulation of the mechanisms involved, we first identified the gene expression profile of NPCs and characterized their metabolic changes in vitro under 3% oxygen. NPCs derived from E13.5 rat mesencephalon were cultured under either normoxia or hypoxia for 24 h and 72 h. Total RNA was subjected to cDNA microarray analysis of 5705 genes. The results showed that approximately 1.24% of gene expression changed under low oxygen at the two time points. Among the 142 differentially expressed genes, the greatest number was involved in glycolysis and metabolism. The metabolic changes of NPCs under low oxygen conditions were also assayed. The glucose content in the conditioned medium incubated in low oxygen decreased significantly; however, the levels of pyruvate and lactic acid increased compared to conditioned medium cultured in normoxia. The NPCs under low oxygen consumed more glucose and produced energy by glycolysis. The information gained from gene expression and metabolic analyses of NPCs under low oxygen conditions will provide new approaches for the evaluation of NPCs as potential in vivo cellular therapeutics.

Recently, the presence of telocytes was demonstrated in human and mammalian tissues and organs (digestive and extra-digestive organs, genitourinary organs, heart, placenta, lungs, pleura, striated muscle). Noteworthy, telocytes seem to play a significant role in the normal function and regeneration of myocardium. By cultures of telocytes in two- and three-dimensional environment we aimed to study the typical morphological features as well as functionality of telocytes, which will provide important support to understand their in vivo roles. Neonatal rat cardiomyocytes were isolated and cultured as seeding cells in vitro in two-dimensional environment. Furthermore, engineered myocardium tissue was constructed from isolated cells in three-dimensional collagen/Matrigel scaffolds. The identification of telocytes was performed by using histological and immunohistochemical methods. The results showed that typical telocytes are distributed among cardiomyocytes, connecting them by long telopodes. Telocytes have a typical fusiform cell body with two or three long moniliform telopodes, as main characteristics. The vital methylene blue staining showed the existence of telocytes in primary culture. Immunohistochemistry demonstrated that some c-kit or CD34 immuno-positive cells in engineered heart tissue had the morphology of telocytes, with a typical fusiform cell body and long moniliform telopodes. Also, a significant number of vimentin+ telocytes were present within engineered heart tissue. We suggest that the model of three-dimensional engineered heart tissue could be useful for the ongoing research on the functional relationships of telocytes with cardiomyocytes. Because the heart has the necessary potential of changing the muscle and non-muscle cells during the lifetime, telocytes might play an active role in the heart regeneration process. Moreover, telocytes might be a useful tool for cardiac tissue engineering.

Small non-coding RNA (ncRNA) plays critical roles in a large number of cellular processes, including neural development, cell survival and cell determination. Our previous work showed that low oxygen promoted the survival and proliferation of neural progenitor cells (NPCs) in vitro. In this study, we examine the expression and regulation of small ncRNAs in the hypoxia-driven proliferation of NPCs. The expression profiles of ncRNAs in NPCs under hypoxia were detected using microarray analysis. Results of significance analysis of microarrays (SAM) revealed that 15 small RNAs were up-regulated at least threefold and 11 were down-regulated under hypoxic conditions. The differentially expressed small ncRNAs were confirmed by quantitative RT-PCR, and miR-210 was observed to be highly expressed in NPCs under hypoxic conditions. Further study showed that hypoxia-inducible factor (HIF)-1α had a direct impact on the putative promoter regions of miR-210. From these results, we conclude that some small ncRNAs participate in the regulation of the proliferation of NPCs under hypoxia and that miR-210 is directly regulated by HIF-1α.

Increasing studies have shown protective effects of intermittent hypoxia on brain injury and heart ischemia. However, the effect of intermittent hypoxia on blood glucose metabolism, especially in diabetic conditions, is rarely observed. The aim of this study was to investigate whether intermittent hypoxia influences blood glucose metabolism in type 1 diabetic rats. Streptozotocin-induced diabetic adult rats and age-matched control rats were treated with intermittent hypoxia (at an altitude of 3 km, 4 h per day for 3 weeks) or normoxia as control. Fasting blood glucose, body weight, plasma fructosamine, plasma insulin, homeostasis model assessment of insulin resistance (HOMA-IR), pancreas β-cell mass, and hepatic and soleus glycogen were measured. Compared with diabetic rats before treatment, the level of fasting blood glucose in diabetic rats after normoxic treatment was increased (19.88 ± 5.69 mmol/L vs. 14.79 ± 5.84 mmol/L, p 0.05). Meanwhile, fasting blood glucose in diabetic rats after hypoxic treatment was also lower than that in diabetic rats after normoxic treatment (13.14 ± 5.77 mmol/L vs. 19.88 ± 5.69 mmol/L, p

In vitro cell culture has provided a useful model to study the effects of oxygen on cellular behavior. However, it remains unknown whether the in vitro operations themselves affect the medium oxygen levels and the living states of cells. In addition, a prevailing controversy is whether reactive oxygen species (ROS) production is induced by continuous hypoxia or reoxygenation. In this study, we have measured the effects of different types of cell culture containers and the oxygen environment where medium replacement takes place on the actual oxygen tension in the medium. We found that the deviations of oxygen concentrations in the medium are much greater in 25-cm(2) flasks than in 24-well plates and 35-mm dishes. The dissolved oxygen concentrations in the medium were increased after medium replacement in normoxia, but remained unchanged in glove boxes in which the oxygen tension remained at a low level (11.4, 5.7, and 0.5% O2 ). We also found that medium replacement in normoxia increased the number of ROS-positive cells and reduced the cell viability; meanwhile, medium replacement in a glove box did not produce the above effects. Therefore, we conclude that the use of 25-cm(2) flasks should be avoided and demonstrate that continuous hypoxia does not produce ROS, whereas the reoxygenation that occurs during the harvesting of cells leads to ROS and induces cell death.

The treatment of stroke is limited by a short therapeutic window and a lack of effective clinical drugs. Methylene blue (MB) has been used in laboratories and clinics since the 1890s. Few studies have reported the neuroprotective role of MB in cerebral ischemia-reperfusion injury. However, whether and how MB protects against acute cerebral ischemia (ACI) injury was unclear. In this study, we investigated the effect of MB on this injury and revealed that MB protected against ACI injury by augmenting mitophagy. Using a rat middle cerebral artery occlusion (MCAO) model, we demonstrated that MB improved neurological function and reduced the infarct volume and necrosis after ACI injury. These improvements depended on the effect of MB on mitochondrial structure and function. ACI caused the disorder and disintegration of mitochondrial structure, while MB ameliorated the destruction of mitochondria. In addition, mitophagy was inhibited at 24 h after stroke and MB augmented mitophagy. In an oxygen-glucose deprivation (OGD) model in vitro, we further revealed that the elevation of mitochondrial membrane potential (MMP) by MB under OGD conditions mediated the augmented mitophagy. In contrast, exacerbating the decline of MMP during OGD abolished the MB-induced activation of mitophagy. Taken together, MB promotes mitophagy by maintaining the MMP at a relatively high level, which contributes to a decrease in necrosis and an improvement in neurological function, thereby protecting against ACI injury.

Background Exposure to acute hypobaric hypoxia (AHH) during ascent to high altitudes (>3500 m) is one of the main causes of acute mountain sickness (AMS) and high-altitude cerebral edema (HACE). Therefore, the aim of this study was to develop a model of HACE. New methods We developed a model of HACE in mice using a decompression chamber with rapid ascent speed. Results Healthy male C57BL/6 mice were randomly divided into the control group and the AHH group. The AHH group was housed in a decompression chamber (at a velocity of 50 m/s within 5 min to 6000 m). Compared with the controls, brain water content was increased in the early stage (within 24 h) in the AHH group. After 72 h of exposure to AHH, there was a higher BBB permeability observed. In addition, the brain structure showed significant widening of the pericellular spaces and a dilatation of the cortical blood vessels after exposure to AHH, and some of the neurons appeared shrunken with darkly stained pyknotic nuclei, resulting in neuronal structural damage. Further, exposure to AHH also decreased cognitive function in the mice. Comparison with existing methods At present, there are no simple and rapid mouse models to study this syndrome in terms of its genetic basis, gene polymorphisms and susceptibility. Conclusion Our findings show that AHH can increase BBB permeability and lead to cerebral edema in mice; thus, we provide an effective and stable model of HACE in mice.

Oxidative injury is involved in many diseases, including ischemic and neurodegenerative diseases. Antioxidant drugs can be used to relieve the oxidative injury caused by these diseases; however, there are very few antioxidant drugs available for clinical use. In this study, we found that 5-(hydroxymethyl)-2-furfural (5-HMF) protects against the oxidative damage induced by cerebral ischemia in rats or by hydrogen peroxide (H2O2) in PC12 cells. We demonstrated that 5-HMF performs this function via apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1). APE/Ref-1 is a multifunctional protein involved in oxidative DNA damage repair through the base excision repair (BER) pathway and in the regulation of the DNA-binding activity of several transcription factors. The current study focused on the role of APE/Ref-1 in the antioxidative properties of 5-HMF. The results show that 5-HMF inhibited the reduction of APE/Ref-1 protein level caused by cerebral ischemia-reperfusion injury in rats or H2O2 treatment in PC12 cells. Treatment with an APE/Ref-1 inhibitor blocked 5-HMF-induced protection, suggesting that APE/Ref-1's DNA repair function contributes to antioxidation. In conclusion, this study suggests that APE/Ref-1 may be a potential target for antioxidant drugs.

MiR-210 is a hypoxia-inducible factor (HIF)-1 target gene and is the most consistently and predominantly upregulated miRNA in response to hypoxia in various cancer cell lines. Our recent study shows that hypoxia increased miR-210 expression in neural progenitor cells (NPCs) in a time-dependent manner. However, the role of miR-210 in NPCs remains unknown. Following the identification of the miR-210 putative target genes, we demonstrated that the Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3), which is regulated by HIF-1 and activates cell death, is regulated by miR-210 in NPCs under hypoxia. Moreover, the over-expression of miR-210 decreased apoptosis in NPCs, and the inhibition of miR-210 expression remarkably increased the number of TUNEL-positive NPCs by 30% in response to hypoxia. Importantly, miR-210 mimics reduced both BNIP3 protein expression and the translocation of AIF into the nucleus, which reduced cell death, whereas miR-210 inhibitors reversed this process, leading to cell death during hypoxia. Taken together, we report a novel feedback loop of BNIP3 regulation in NPCs under hypoxia. HIF-1 is activated under hypoxia and then induces the expression of both BNIP3 and miR-210. The upregulation of miR-210 then directly suppresses BNIP3 expression to maintain the survival of NPCs under hypoxia. This negative feedback regulation might partially contribute to protection against hypoxia-induced cell death via the inhibition of AIF nuclear translocation.

Four and a half LIM domain (FHL) proteins belong to a family of LIM-only proteins that have been implicated in the development and progression of various types of cancers. However, the role of FHL proteins in tumor angiogenesis remains to be elucidated. Herein, we demonstrate that FHL1-3 decrease the promoter activity and expression of vascular endothelial growth factor (VEGF), the key regulator of angiogenesis in cancer growth and progression as well as an important target gene of the transcription factor hypoxia-inducible factor 1 (HIF1α/HIF1β). FHL1-3 interacted with HIF1α both in vitro and in vivo. A single LIM domain of FHL1 was sufficient for its interaction with HIF1α. FHL1 interacted with the HIF1α region containing basic helix-loop-helix (bHLH) motif and PER-ARNT-SIM domain, both of which aid in dimerization with HIF1β and DNA binding. FHL1-3 inhibited HIF1 transcriptional activity and HIF1-mediated VEGF expression in a hypoxia-independent manner. Moreover, FHL1 blocked HIF1α-HIF1β heterodimerization and HIF1α recruitment to the VEGF promoter. These data suggest that FHL proteins are involved in negative regulation of VEGF possibly by interfering with the dimerization and DNA binding of HIF1 subunits and may play an important role in tumor angiogenesis.

Several studies have identified a set of hypoxia-regulated microRNAs, among which is miR-210, whose expression is highly induced by hypoxia in various cancer cell lines. Recent studies have highlighted the importance of miR-210 and its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that the expression of miR-210 was highly induced in neural progenitor cells (NPCs) subjected to hypoxia. Specifically, treating hypoxic NPCs with the DNA demethylating agent 5-aza-2'-deoxycytidine significantly increased the expression of miR-210, even under normoxia; however, the activity of hypoxia-inducible factor-1 was unaffected. Further analysis of the miR-210 sequence revealed that it is embedded in a CpG island. Bisulfite sequencing of the miR-210 CpG island from NPCs grown under hypoxic conditions showed 24% CpG methylation in NPCs exposed to 20% O(2) , 18% in NPCs exposed to 3% O(2) , and 12% in NPCs exposed to 0.3% O(2) . In addition, the activity of DNA methyltransferases (DNMTs) in NPCs decreased after exposure to hypoxia. Specifically, the expression of DNMT3b decreased significantly after exposure to 0.3% O(2) . Thus, these results demonstrate that DNA demethylation regulates miR-210 expression in NPCs under both normoxia and hypoxia.

Our previous study showed that pretreatment with 5-hydroxymethyl-2-furfural (5-HMF) led to protection against hypoxic injury via a p-ERK-mediated pathway in vitro. Whether the protection of 5-HMF against hypoxia is effective in vivo is unknown. The present study is aimed to verify the role of 5-HMF in acute hypobaric hypoxia using Kunming mice as an in vivo model and further investigate the underlying mechanisms. Mice pretreated with or without 5-HMF for 1 h were exposed to acute hypobaric hypoxic condition for 6 h and then the survival time, the survival rate, the permeability of blood-brain barrier (BBB), the histological analysis in hippocampus and cortex, and the phosphorylation level of mitogen-activated protein kinases (ERK, JNK, and p38) were investigated. The results showed that 5-HMF significantly increased the survival time and the survival rate of mice. Accordingly, pretreatment with 5-HMF markedly attenuated acute hypobaric hypoxia-induced permeability of BBB (P < 0.01). In addition, the cellular damage extent of the hippocampus and the cortex induced by hypoxia for 6 h was also attenuated by pretreatment with 5-HMF, especially in the hippocampus CA1 region. Furthermore, the activation of ERK rather than JNK and p38 was involved in the protection of 5-HMF against acute hypobaric hypoxia. In summary, 5-HMF enhanced the survival capability of mice and decreased acute hypoxic damage to the brain, which may be associated with the effects on BBB and p-ERK.

Aims The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts. Methods and results Mouse ESCs (mESCs) were cultured using a bioreactor system to develop embryoid bodies, which were then induced with 1% ascorbic acid to differentiate into cardiomyocytes. The mESCs-derived cardiomyocytes (mESCs-CMs) were enriched by Percoll density gradient separation. The specific markers (OCT-4, Sox2, and Nanog) of undifferentiated ESCs were detected by PCR both in mESCs and in mESCs-CMs, but not in the mESC-derived Percoll-enriched cardiomyocytes (mESC-PE-CMs). Immunosuppressed rats with infarcted hearts were randomly injected with the mESCs, mESC-CMs, or mESC-PE-CMs. Eight weeks after cell transplantation, histological and immunohistochemical analysis showed that the transplantation of both mESCs and mESC-CMs caused the formation of teratomas. The incidence of teratoma was markedly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. The average tumour volume was significantly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. Tumour formation was absent in the mESC-PE-CMs group. Conclusion Enrichment of the mESC-differentiated cardiomyocytes inhibited the development of teratoma after cell transplantation in the infarcted rat hearts. These findings offer a new strategy for eliminating teratoma formation in ESCs transplantation and could be a step forward in the development of human ESCs transplantation therapy in ischaemic heart disease.

Neural recognition molecules of the immunoglobulin superfamily play important roles in the development and regeneration of nervous system. Close Homologue of L1 (CHL1) is a member of the L1 family of recognition molecules which are expressed during neuronal development, suggesting a potential role in neural progenitor cells (NPCs). Here, we investigated the role of CHL1 in the proliferation and differentiation of NPCs both in vivo and in vitro, and the possible mechanism involved. The number of BrdU-positive cells in the subventricular zone (SVZ) significantly increased in CHL1-/- mice compared with CHL1+/+ mice. Moreover, there were more Tuj1-positive cells in the cortical plate region in CHL1-/- mice than in CHL1+/+ controls. To further examine the function of CHL1 in the proliferation and differentiation of NPCs, NPCs from CHL1-/- mice versus littermate wild-type mice were isolated and cultured in vitro. NPCs derived from CHL1-/- mice showed increased proliferation and self-renewal ability compared with CHL1+/+ mice. In the course of differentiation, CHL1 deficiency enhanced neuronal differentiation in the absence of growth factors. Furthermore, CHL1 deficiency on the proliferation of NPCs is accompanied by means of enhanced activation of ERK1/2 mitogen-activated protein kinase (MAPK) and the inhibitor of ERK1/2 MAPK eliminates the effect of CHL1 deficiency on the proliferation of NPCs. Our results first describe the negative modulation of the proliferation and neuronal differentiation of NPCs by CHL1/ERK1/2 MAPK signaling.

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

Cardiac stem cells represent a logical cell type to exploit in cardiac regeneration. The efficient harvest of cardiac stem cells from a suitable source would turn promising in cardiac stem cell therapy. Brown adipose was recently found to be a new source of cardiac stem cells, instrumental to myocardial regeneration. Unfortunately, an efficient method for the cell isolation is unavailable so far. In our study we have developed a new method for the efficient isolation of cardiac stem cells from brown adipose by combining different enzymes. Results showed that the total cell yield dramatically increased (more than 10 times,P<.01) compared with that by previous method. The content of CD133-positive cells (reported to differentiate into cardiomyocytes with a high frequency) was much higher than that in the previous report (22.43% versus 3.5%). Moreover, the isolated cells could be the efficiently differentiated into functional cardiomyocytes in optimized conditions. Thus, the new method we established would be of great use in further exploring cardiac stem cell therapy.

Close homologue of L1 (CHL1) is a transmembrane cell adhesion molecule that is critical for brain development and for the maintenance of neural circuits in adults. Recent studies revealed that CHL1 has diverse roles and is involved in the regulation of recovery after spinal cord injury. CHL1 expression was downregulated in the cerebral cortex, hypothalamus, and brain stem after the induction of acute hypoxia (AH). In the current study, we sought to address the role of CHL1 in regulating homeostasis responses to hypoxia using CHL1-knockout (CHL1(-/-)) mice. We found that, compared with wild-type littermates, CHL1(-/-) mice showed a dramatically lower mortality rate and an augmented ventilatory response after they were subjected to AH. Immunofluorescence staining revealed that CHL1 was expressed in the carotid body (CB), the key oxygen sensor in rodents, and CHL1 expression level in the CB as assayed by western blot was decreased after hypoxic exposure. The number of glomus cells and the expression of tyrosine hydroxylase (a marker for glomus cells) in the CB of CHL1(-/-) mice appeared to be increased compared with CHL1(+/+) mice. In addition, in the ex vivo CB preparation, hypoxia induced a significantly greater afferent nerve discharge in CHL1(-/-) mice compared with CHL1(+/+) mice. Furthermore, the arterial blood pressure and plasma catecholamine levels of CHL1(-/-) mice were also significantly higher than those of CHL1(+/+) mice. Our findings first demonstrate that CHL1 is a novel intrinsic factor that is involved in CB function and in the ventilatory response to AH.