Low level laser irradiation precondition to create friendly milieu of infarcted myocardium and enhance early survival of transplanted bone marrow cells

We suggested that low‐level laser irradiation (LLLI) precondition prior to cell transplantation might remodel the hostile milieu of infarcted myocardium and subsequently enhance early survival and therapeutic potential of implanted bone marrow mesenchymal stem cells (BMSCs). Therefore, in this study we wanted to address: (1) whether LLLI pre‐treatment change the local cardiac micro‐environment after myocardial infarction (MI) and (2) whether the LLLI preconditions enhance early cell survival and thus improve therapeutic angiogenesis and heart function. MI was induced by left anterior descending artery ligation in female rats. A 635 nm, 5 mW diode laser was performed with energy density of 0.96 J/cm² for 150 sec. for the purpose of myocardial precondition. Three weeks later, qualified rats were randomly received with LLLI precondition (n= 26) or without LLLI precondition (n= 27) for LLLI precondition study. Rats that received thoracotomy without coronary ligation were served as sham group (n= 24). In the cell survival study, rats were randomly divided into 4 groups: serum‐free culture media injection (n= 8), LLLI precondition and culture media injection (n= 8), 2 million male BMSCs transplantation without LLLI pre‐treatment (n= 26) and 2 million male BMSCs transplantation with LLLI precondition (n= 25) group, respectively. Vascular endothelial growth factor (VEGF), glucose‐regulated protein 78 (GRP78), superoxide dismutase (SOD) and malondialdehyde (MDA) in the infarcted myocardium were evaluated by Western blotting, real‐time PCR and colorimetry, respectively, at 1 hr, 1 day and 1 week after laser irradiation. Cell survival was assayed with quantitative real‐time PCR to identify Y chromosome gene and apoptosis was assayed with transferase‐mediated dUTP end labelling staining. Capillary density, myogenic differentiation and left ventricular function were tested by immunohistochemistry and echocardiography, respectively, at 1 week. After LLLI precondition, increased VEGF and GRP78 expression, as well as the enhanced SOD activity and inhibited MDA production, was observed. Compared with BMSC transplantation and culture media injection group, although there was no difference in the improved heart function and myogenic differentiation, LLLI precondition significantly enhanced early cell survival rate by 2‐fold, decreased the apoptotic percentage of implanted BMSCs in infarcted myocardium and thus increased the number of newly formed capillaries. Taken together, LLLI precondition could be a novel non‐invasive approach for intraoperative cell transplantation to enhance cell early survival and therapeutic potential.     

Identification of TREK-2 K+ channels in human mesenchymal stromal cells

The maintenance of pluripotency of mesenchymal stromal cells (MSCs), their proliferation and initiation of differentiation may critically depend on functional expression of ion channels. Despite such a possibility, mechanisms of electrogenesis in MSCs remain poorly understood. In particular, little is known about a variety of ion channels active in resting MSCs or activated upon MSC stimulation. Here we aimed at uncovering ion channels operating in MSCs, including those being active at rest, using the patch clamp technique and inhibitory analysis. In trying to evaluate a contribution of anion channels in MSC resting potential, we employed a number of diverse inhibitors of anion channels and transporters, including niflumic acid (NFA). Basically, NFA caused hyperpolarization of MSCs that was accompanied by a marked increase in ion conductance of their plasma membranes. The blockage of Cl^? channels could not underlie such a NFA effect, given that cells dialyzed with a CsCl solution were weakly or negligibly sensitive to this blocker. This and other findings indicated that NFA affected the MSC ion permeability not by targeting Cl^? channels but by stimulating K⁺ channels. NFA-activated K⁺ current was TEA and diltiazem blockable, and K⁺ channels involved were potentiated from outside by solution acidification and Cu²⁺ ions. Taken together, the data obtained implicated two-pore domain K⁺ channels of the TREK-2 subtype in mediating stimulatory effects of NFA on MSCs. The notable inference from our work is that TREK-2 channels should be expressed and functional virtually in every MSC, given that all cells examined by us (n > 100) similarly responded to NFA by increasing their TREK-2-like K⁺ conductance.     

Gamma irradiation preserves immunosuppressive potential and inhibits clonogenic capacity of human bone marrow‐derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are promising candidates for the treatment of graft‐versus‐host and autoimmune diseases. Here, by virtue of their immunosuppressive effects, they are discussed to exhibit inhibitory actions on various immune effector cells, including T lymphocytes that promote the underlying pathology. While it becomes apparent that MSCs exhibit their therapeutic effect in a transient manner, they are usually transplanted from third party donors into heavily immunocompromised patients. However, little is known about potential late complications of persisting third party MSCs in these patients. We therefore analysed the effect of gamma irradiation on the potency and proliferation of MSCs to elucidate an irradiation dose, which would allow inhibition of MSC proliferation while at the same time preserving their immunosuppressive function. Bone marrow‐derived MSCs (BM‐MSCs) were gamma‐irradiated at increasing doses of 5, 10 and 30 Gy and subsequently assessed by colony formation unit (CFU)‐assay, Annexin V‐staining and in a mixed lymphocyte reaction, to assess colony growth, apoptosis and the immunosuppressive capacity, respectively. Complete loss of proliferative capacity measured by colony formation was observed after irradiation with a dose equal to or greater than 10 Gy. No significant decrease of viable cells was detected, as compared to non‐irradiated BM‐MSCs. Notably, irradiated BM‐MSCs remained highly immunosuppressive in vitro for at least 5 days after irradiation. Gamma irradiation does not impair the immunosuppressive capacity of BM‐MSCs in vitro and thus might increase the safety of MSC‐based cell products in clinical applications.     

Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE^?/?) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE^?/? mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6c^high monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.     

Kir1.1 expression in embryonic kidney epithelia

K(+) channels may regulate cell cycling, cell volume, and cell proliferation. We have recently shown a role for an inwardly rectifying K(+) channel, Kir6.1/SUR2(B), in the regulation of cell proliferation during early kidney development. Here, we show that the protein of a further K(+) channel, Kir1.1 (ROMK), is also developmentally expressed in prenatal rat kidney epithelia. In the embryonic stage, Kir1.1 protein was localized to the plasma membrane of ureteric buds and collecting ducts, and of nephron stages up to the comma-shaped body. Experimental increase in cAMP upregulated Kir1.1b (ROMK2) mRNA abundance in ureteric buds. Kir1.1 protein was restricted to the distal nephron during later postnatal development and adulthood, as has been reported. In conclusion, we demonstrate redundancy of Kir channel expression in early embryonic kidney which could suggest that Kir1.1 acts in a similar way as Kir6.1/SUR2(B) to promote cell proliferation or other developmental functions.     

A critical role of the KCa3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca²⁺-activated K⁺ channel, K_Ca3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the K_Ca3.1 channel expression and its role in rat bone marrow-derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up-regulated the K_Ca3.1 channel expression and pharmacological or genetic inhibition of the K_Ca3.1 channel strongly suppressed stretch-induced increase in cell proliferation and cell cycle progression. These results support that the K_Ca3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.     

Biological effects of Er:YAG laser irradiation on the proliferation of primary human gingival fibroblasts

We investigated the biological effects of Er:YAG laser (2940‐nm; DELight, HOYA ConBio, Fremont, California) irradiation at fluences of 3.6, 4.2, 4.9, 6.3, 8.1 or 9.7 J cm^?2 at 20 or 30 Hz for 20 or 30 seconds on primary human gingival fibroblasts (HGFs). Irradiation at 6.3 J cm^?2 promoted maximal cell proliferation, determined by WST‐8 assay and crystal violet staining, but was accompanied by lactate dehydrogenase release, on day 3 post‐irradiation. Elevation of ATP level, Ki67 staining, and cyclin‐A2 mRNA expression confirmed that Er:YAG affected the cell cycle and increased the number of proliferating cells. Transmission electron microscopy showed alterations of mitochondria and ribosomal endoplasmic reticulum (ER) at 3 hours post‐irradiation at 6.3 J cm^?2, and the changes subsided after 24 hours, suggesting transient cellular injury. Microarray analysis revealed up‐regulation of 21 genes involved in heat‐related biological responses and ER‐associated degradation. The mRNA expression of heat shock protein 70 family was increased, as validated by Real‐time PCR. Surface temperature measurement confirmed that 6.3 J cm^?2 generated heat (40.9°C post‐irradiation). Treatment with 40°C‐warmed medium increased proliferation. Laser‐induced proliferation was suppressed by inhibition of thermosensory transient receptor potential channels. Thus, despite causing transient cellular damage, Er:YAG laser irradiation at 6.3 J cm^?2 strongly potentiated HGF proliferation via photo‐thermal stress, suggesting potential wound‐healing benefit.      

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self‐renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real‐time RT‐PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16^INK4A and p18^INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16^INK4A, p18^INK4C, and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental‐tissue‐derived MSCs.     

Dual regulation of renal Kir7.1 potassium channels by protein Kinase A and protein Kinase C

The renal inward rectifier potassium channel Kir7.1 has been proposed to be functionally important for tubular K⁺ recycling and secretion. This study investigated the regulation of Kir7.1 by PKA and PKC.Cloned human Kir7.1 channels were expressed heterologously in Xenopus oocytes. After pharmacological PKC activation, Kir7.1 currents were strongly inhibited. Co-application of PKC inhibitors attenuated this effect. Inactivation of PKC consensus sites also strongly attenuated the effect with a single site (²⁰¹S) being essential for almost the total PKC sensitivity. In contrast, PKA activation induced an increase of Kir7.1 currents. This effect was absent in mutant Kir7.1 channels lacking PKA consensus site ²⁸⁷S.In summary, this study demonstrates the dual regulation of Kir7.1 channel function by PKA and PKC. Structurally, these regulations depend on two key residues in the C-terminal channel domain (^Ser201 for PKC and ^Ser287 for PKA).     

iPSC‐derived mesenchymal stem cells exert SCF‐dependent recovery of cigarette smoke‐induced apoptosis/proliferation imbalance in airway cells

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.     

Haemin pre‐treatment augments the cardiac protection of mesenchymal stem cells by inhibiting mitochondrial fission and improving survival

The cardiac protection of mesenchymal stem cell (MSC) transplantation for myocardial infarction (MI) is largely hampered by low cell survival. Haem oxygenase 1 (HO‐1) plays a critical role in regulation of cell survival under many stress conditions. This study aimed to investigate whether pre‐treatment with haemin, a potent HO‐1 inducer, would promote the survival of MSCs under serum deprivation and hypoxia (SD/H) and enhance the cardioprotective effects of MSCs in MI. Bone marrow (BM)‐MSCs were pretreated with or without haemin and then exposed to SD/H. The mitochondrial morphology of MSCs was determined by MitoTracker staining. BM‐MSCs and haemin‐pretreated BM‐MSCs were transplanted into the peri‐infarct region in MI mice. SD/H induced mitochondrial fragmentation, as shown by increased mitochondrial fission and apoptosis of BM‐MSCs. Pre‐treatment with haemin greatly inhibited SD/H‐induced mitochondrial fragmentation and apoptosis of BM‐MSCs. These effects were partially abrogated by knocking down HO‐1. At 4 weeks after transplantation, compared with BM‐MSCs, haemin‐pretreated BM‐MSCs had greatly improved the heart function of mice with MI. These cardioprotective effects were associated with increased cell survival, decreased cardiomyocytes apoptosis and enhanced angiogenesis. Collectively, our study identifies haemin as a regulator of MSC survival and suggests a novel strategy for improving MSC‐based therapy for MI.     

Effect of the He-Ne laser irradiation on resistance of the isolated heart to the ischemic and reperfusion injury

The aim of this work was to investigate the myocardial protection against ischemia/reperfusion using low level laser irradiation (LLLI). It has been shown that pulse pressure was higher in the period of post-ischemic reperfusion as compared with the control group. It provided a better restoration of myocardial contractility as well as increasing of coronary flow in the reperfusion period. The amount of ventricular rhythm disorder episodes decreased. These effects of laser application were registered in conditions of coronary flow reduction less than 50%. One of the suggested mechanisms of laser effect is an ATP-sensitive channel activation.     

Dissecting the mechanisms of Notch induced hyperplasia

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch‐CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non‐autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed‐forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross‐regulatory relationships are important in coordinating the pattern of hyperplasia.     

TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Exosomes from mesenchymal stem cells expressing miR‐125b inhibit neointimal hyperplasia via myosin IE

Intercellular communication between mesenchymal stem cells (MSCs) and their target cells in the perivascular environment is modulated by exosomes derived from MSCs. However, the potential role of exosome‐mediated microRNA transfer in neointimal hyperplasia remains to be investigated. To evaluate the effects of MSC‐derived exosomes (MSC‐Exo) on neointimal hyperplasia, their effects upon vascular smooth muscle cell (VSMC) growth in vitro and neointimal hyperplasia in vivo were assessed in a model of balloon‐induced vascular injury. Our results showed that MSC‐Exo were internalised by VSMCs and inhibited proliferation and migration in vitro. Further analysis revealed that miR‐125b was enriched in MSC‐Exo, and repressed the expression of myosin 1E (Myo1e) by targeting its 3? untranslated region. Additionally, MSC‐Exo and exosomally transferred miR‐125b repressed Myo1e expression and suppressed VSMC proliferation and migration and neointimal hyperplasia in vivo. In summary, our findings revealed that MSC‐Exo can transfer miR‐125b to VSMCs and inhibit VSMC proliferation and migration in vitro and neointimal hyperplasia in vivo by repressing Myo1e, indicating that miR‐125b may be a therapeutic target in the treatment of vascular diseases.     

The Effects of Hedgehog on the RNA-Binding Protein Msi1 in the Proliferation and Apoptosis of Mesenchymal Stem Cells

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are essential tools for regenerative medicine due to their capacity for self-renewal and multi-lineage differentiation. As MSCs are found in very small numbers in various tissues, in vitro cell expansion is an essential step that is needed before these cells can be used in clinical applications. Therefore, it is important to identify and characterize factors that are involved in MSC proliferation and apoptosis. In the present study, we focused on Hedgehog (Hh) signaling because several studies have proposed that Hh signaling plays a critical role in controlling the proliferation of stem and progenitor cells. However, the molecular mechanisms underlying the effects on the proliferation and apoptosis of MSCs remain unclear. In this study, we evaluated the direct effects of Hh signaling on the proliferation and apoptosis of hUCB-MSCs as well as investigated potential downstream regulatory mechanisms that may be responsible for Hh signaling. We observed that the Hedgehog agonist purmorphamine enhanced cell proliferation and suppressed apoptosis through the RNA-binding protein Msi1 by regulating the expression of an oncoprotein (i.e., c-Myc), a cell cycle regulatory molecule (i.e., p21^CIP1,WAF1 ) and two microRNAs (i.e., miRNA-148a and miRNA-148b). This study provides novel insights into the molecular mechanisms regulating the self-renewal capability of MSCs with relevance to clinical applications.     

Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3

Background

Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABA_A) receptors, and σ₁ receptors, and voltage-gated Ca²⁺ and K⁺ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K⁺ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K⁺ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.

Methodology/Principal Findings

The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.

Conclusions/Significance

The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.     

Hypoxia‐induced proliferation in mesenchymal stem cells and angiotensin II‐mediated PI3K/AKT pathway

Hypoxia could stimulate proliferation of mesenchymal stem cells (MSCs) under certain conditions. This study determined angiotensin II mechanisms and PI3K/AKT pathway in hypoxia‐induced proliferation of MSCs. Hypoxia (3% oxygen) induced cellular proliferation in mouse MSCs and upregulated endogenous angiotensin II and angiotensin‐converting enzyme in the cell culture and expression of AT1 receptors. The expressions of Sox2, not Oct4 and Rex1, were significantly increased by the hypoxia. The blockade of AT1 receptors, not AT2 receptors, depressed hypoxia induced the proliferative effects. Both hypoxia and exogenous angiotensin II activated p‐AKT. Moreover, AT1 receptor inhibitor blocked the effects of hypoxia‐mediated p‐AKT upregulation. The data demonstrated that the hypoxia at 3% oxygen level could induce mouse MSC proliferation, probably as a result of the activation of PI3K signalling pathways via AT1 receptors. Copyright © 2015 John Wiley & Sons, Ltd.