Hyperbaric oxygen treatment increases intestinal stem cell proliferation through the mTORC1/S6K1 signaling pathway in Mus musculus.

BACKGROUND: Hyperbaric oxygen treatment (HBOT) has been reported to modulate the proliferation of neural and mesenchymal stem cell populations, but the molecular mechanisms underlying these effects are not completely understood. In this study, we aimed to assess HBOT somatic stem cell modulation by evaluating the role of the mTOR complex 1 (mTORC1), a key regulator of cell metabolism whose activity is modified depending on oxygen levels, as a potential mediator of HBOT in murine intestinal stem cells (ISCs). RESULTS: We discovered that acute HBOT synchronously increases the proliferation of ISCs without affecting the animal's oxidative metabolism through activation of the mTORC1/S6K1 axis. mTORC1 inhibition by rapamycin administration for 20 days also increases ISCs proliferation, generating a paradoxical response in mice intestines, and has been proposed to mimic a partial starvation state. Interestingly, the combination of HBOT and rapamycin does not have a synergic effect, possibly due to their differential impact on the mTORC1/S6K1 axis. CONCLUSIONS: HBOT can induce an increase in ISCs proliferation along with other cell populations within the crypt through mTORC1/S6K1 modulation without altering the oxidative metabolism of the animal's small intestine. These results shed light on the molecular mechanisms underlying HBOT therapeutic action, laying the groundwork for future studies.

Downregulation of the Netrin-1 Receptor UNC5b Underlies Increased Placental Angiogenesis in Human Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a common metabolic disorder, defined by high blood glucose levels during pregnancy, which affects foetal and post-natal development. However, the cellular and molecular mechanisms of this detrimental condition are still poorly understood. A dysregulation in circulating angiogenic trophic factors, due to a dysfunction of the feto-placental unit, has been proposed to underlie GDM. But even the detailed study of canonical pro-angiogenic factors like vascular endothelial growth factor (VEGF) or basic Fibroblast Growth Factor (bFGF) has not been able to fully explain this detrimental condition during pregnancy. Netrins are non-canonical angiogenic ligands produced by the stroma have shown to be important in placental angiogenesis. In order to address the potential role of Netrin signalling in GDM, we tested the effect of Netrin-1, the most investigated member of the family, produced by Wharton's Jelly Mesenchymal Stem Cells (WJ-MSC), on Human Umbilical Vein Endothelial Cells (HUVEC) angiogenesis. WJ-MSC and HUVEC primary cell cultures from either healthy or GDM pregnancies were exposed to physiological (5 mM) or high (25 mM) d-glucose. Our results reveal that Netrin-1 is secreted by WJ-MSC from healthy and GDM and both expression and secretion of the ligand do not change with distinct experimental glucose conditions. Noteworthy, the expression of its anti-angiogenic receptor UNC5b is reduced in GDM HUVEC compared with its expression in healthy HUVEC, accounting for an increased Netrin-1 signalling in these cells. Consistently, in healthy HUVEC, UNC5b overexpression induces cell retraction of the sprouting phenotype.

The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin-1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts in vitro However, the participation of histatin-1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin-1 promotes angiogenesis, as shown in vivo, using the chick chorioallantoic membrane model, and by an in vitro tube formation assay, using both human primary cultured endothelial cells (HUVECs) and the EA.hy926 cell line. Specifically, histatin-1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin-1 increased the recruitment of RIN2, a Rab5-guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin-1-dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin-1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin-1 is a novel proangiogenic factor that may contribute to oral wound healing.-Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina-Berríos, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-ß1 and focal adhesion kinase activation.

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin ß1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin ß1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin ß1 and promotes neuroblastoma cell migration.

Costs of exploratory behavior: the energy trade-off hypothesis and the allocation model tested under caloric restriction.

In order to maintain the energy balance, animals often exhibit several physiological adjustments when subjected to a decrease in resource availability. Specifically, some rodents show increases in behavioral activity in response to food restriction; a response regarded as a paradox because it would imply an investment in locomotor activity, despite the lack of trophic resources. Here, we aim to explore the possible existence of trade-offs between metabolic variables and behavioral responses when rodents are faced to stochastic deprivation of food and caloric restriction. Adult BALB/c mice were acclimatized for four weeks to four food treatments: two caloric regimens (ad libitum and 60% restriction) and two periodicities (continuous and stochastic). In these mice, we analyzed: exploratory behavior and home-cage behavior, basal metabolic rate, citrate synthase and cytochrome oxidase c enzyme activity (in liver and skeletal muscle), body temperature and non-shivering thermogenesis. Our results support the model of allocation, which indicates commitments between metabolic rates and exploratory behavior, in a caloric restricted environment. Specifically, we identify the role of thermogenesis as a pivotal budget item, modulating the reallocation of energy between behavior and basal metabolic rate. We conclude that brown adipose tissue and liver play a key role in the development of paradoxical responses when facing decreased dietary availability.

Correction: Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source.

[This corrects the article DOI: 10.1371/journal.pone.0065818.].

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells.

Adaptive Physiological and Morphological Adjustments Mediated by Intestinal Stem Cells in Response to Food Availability in Mice.

Several studies have evaluated plastic changes in the morphology of the digestive tract in rodents subjected to caloric restriction or restricted availability. Nevertheless, studies that link these morphological responses to physiological consequences are scarce. In order to investigate short-term plastic responses in the intestine, we acclimated adult Mus musculus (BALB/c) males for 20 days to four distinctive treatments: two caloric regimens (ad libitum and 60% of calorie ingestion) and two levels of periodicity of the regimens (continuous and stochastic treatment). At the end of the treatment we analyzed the cell proliferation and cell death dynamics of small intestinal crypts in these animals. In addition, we measured organ masses and lengths, hydrolytic digestive enzyme activities, and energy output from feces. Finally, in order to explore the metabolic changes generated by these dietary conditions we assessed the catabolic activity (i.e., enzymes) of the liver. Our results show that individuals acclimated to a continuous and 60% regimen presented longer intestines in comparison to the other treatments. Indeed, their intestines grew with a rate of 0.22 cm/day, generating a significant caloric reduction in the content of their feces. Besides, both mass and intestinal lengths were predicted strongly by the stabilization coefficient of BrdU+ proliferating cells per crypt, the latter correlating positively with the activity of n-aminopeptidases. Interestingly, by using pharmacological inhibition of the kinase mammalian target of rapamycin complex 1 (mTORC1) by Rapamycin, we were able to recapitulate similar changes in the proliferation dynamics of intestinal stem cells. Based on our results, we propose that the impact of caloric restriction on macroscopic variation in morphology and functional changes in digestive n-aminopeptidases occurs through synchronization in the proliferation rate of stem and/or progenitor cells located in the small intestinal crypts and requires mTORC1 as a key mediator. Hence, we suggest that an excessive stem and progenitor activity could result in increased crypts branching and might therefore underlie the reported intestinal tissue expansion in response to short-term caloric restriction. Summarizing, we demonstrate for the first time that short-term caloric restriction induces changes in the level of cell proliferation dynamics explaining in part digestive tract plasticity in adaptive performance.

Hyperbaric Oxygen Increases Stem Cell Proliferation, Angiogenesis and Wound-Healing Ability of WJ-MSCs in Diabetic Mice.

Hyperbaric oxygen therapy (HBOT) is effective for the medical treatment of diverse diseases, infections, and tissue injury. In fact, in recent years there is growing evidence on the beneficial effect of HBOT on non-healing ischemic wounds. However, there is still yet discussion on how this treatment could benefit from combination with regenerative medicine strategies. Here we analyzed the effects of HBOT on three specific aspects of tissue growth, maintenance, and regeneration: (i) modulation of adult rodent (Mus musculus) intestinal stem cell turnover rates; (ii) angiogenesis dynamics during the development of the chorio-allantoic membrane (CAM) in Gallus gallus embryos; (iii) and wound-healing in a spontaneous type II diabetic mouse model with a low capacity to regenerate skin. To analyze these aspects of tissue growth, maintenance, and regeneration, we used HBOT alone or in combination with cellular therapy. Specifically, Wharton Jelly Mesenchymal Stem cells (WJ-MSC) were embedded in a commercial collagen-scaffold. HBOT did not affect the metabolic rate of adult mice nor of chicken embryos. Notwithstanding, HBOT modified the proliferation rate of stem cells in the mice small intestinal crypts, increased angiogenesis in the CAM, and improved wound-healing and tissue repair in diabetic mice. Moreover, our study demonstrates that combining stem cell therapy and HBOT has a collaborative effect on wound-healing. In summary, our data underscore the importance of oxygen tension as a regulator of stem cell biology and support the potential use of oxygenation in clinical treatments.

Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC).

BACKGROUND: Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. METHODS: Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. RESULTS: The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6ß1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: Thus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.

Shh Signaling through the Primary Cilium Modulates Rat Oligodendrocyte Differentiation.

Primary Cilia (PC) are a very likely place for signal integration where multiple signaling pathways converge. Two major signaling pathways clearly shown to signal through the PC, Sonic Hedgehog (Shh) and PDGF-Rα, are particularly important for the proliferation and differentiation of oligodendrocytes, suggesting that their interaction occurs in or around this organelle. We identified PC in rat oligodendrocyte precursor cells (OPCs) and found that, while easily detectable in early OPCs, PC are lost as these cells progress to terminal differentiation. We confirmed the interaction between these pathways, as cyclopamine inhibition of Hedgehog function impairs both PDGF-mediated OPC proliferation and Shh-dependent cell branching. However, we failed to detect PDGF-Rα localization into the PC. Remarkably, ciliobrevin-mediated disruption of PC and reduction of OPC process extension was counteracted by recombinant Shh treatment, while PDGF had no effect. Therefore, while PDGF-Rα-dependent OPC proliferation and survival most probably does not initiate at the PC, still the integrity of this organelle and cilium-centered pathway is necessary for OPC survival and differentiation.

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation.

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors.

Proliferation of murine midbrain neural stem cells depends upon an endogenous sonic hedgehog (Shh) source.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

Cell membrane regionalization and cytoplasm polarization in the rat early embryo.

In rat pre-implantation embryos, we compared the polarization of cytoplasmic organelles with cell membrane regionalization as revealed by the cytochemical demonstration of 5'-nucleotidase and alkaline phosphatase. The polarization is shown at the eight-cell stage by columns of organelles extending from the nuclei to the embryo periphery. No consistent segregation of cytoplasmic components was recognized prior to the eight-cell stage. As to regionalization, both enzyme activities were demonstrated, from the late four-cell stage onwards on the cell surface between blastomeres of early and late morulae, while the external surface of the embryo lacked these activities. In early blastocysts, these enzyme activities were lost on the inner surface of the blastocyst cavity and in late blastocysts; 5'-nucleotidase activity appeared on the external surface of the embryonal trophoplast and extended to the abembryonal pole. From these observations we conclude that cell membrane regionalization precedes the polarization of the cytoplasm; however, this temporal relationship does not necessarily imply a causal relationship, since both phenomena may be independent expressions of an underlying morphonetic process.