Rapamycin and Starvation Mitigate Indomethacin-Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

The cytotoxicity of gefitinib on patient­derived induced pluripotent stem cells reflects gefitinib­induced liver injury in the clinical setting.

Gefitinib is a key drug used in the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations. Gefitinib therapy is superior to conventional chemotherapy for the progression-free survival rate of patients with EGFR mutations. However, 10-26% of patients develop grade 3 or higher hepatotoxicity during gefitinib treatment; therefore, the development of preclinical tests for hepatotoxicity prior to clinical use is desirable. The present study evaluated the use of induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iPSC-heps), as a platform for preclinical test development. Patient-derived iPSCs were generated by reprogramming peripheral blood mononuclear cells obtained from two groups of gefitinib-treated patients with severe hepatotoxicity [toxicity group (T group)] or mild hepatotoxicity [no clinical toxicity group (N group)]. To examine the hepatotoxicity, the iPSCs from both T and N groups were differentiated into hepatocytes to obtain iPSC-heps. Differentiation was confirmed by measuring the expression levels of hepatocyte markers, such as albumin or α-fetoprotein, via western blotting and quantitative PCR analyses. Cytotoxicity in iPSCs and iPSC-heps after gefitinib treatment was evaluated using a lactate dehydrogenase release assay. The gefitinib-induced cytotoxicity in iPSCs from the T group was significantly higher than that from the N group, whereas there were no significant differences between the groups of iPSC-heps. These results suggested that using iPSCs in preclinical assessment may be a good indicator for the prediction of gefitinib-induced cytotoxicity in clinical use.

O-GlcNAcylation-induced GSK-3ß activation deteriorates pressure overload-induced heart failure via lack of compensatory cardiac hypertrophy in mice.

O-GlcNAc transferase (OGT) modulates many functions of proteins via O-GlcNAcylation that adds O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine/threonine residues of proteins. However, the role of O-GlcNAcylation in cardiac remodeling and function is not fully understood. To examine the effect of O-GlcNAcylation on pressure overload-induced cardiac hypertrophy and subsequent heart failure, transverse aortic constriction (TAC) surgery was performed in wild type (WT) and Ogt transgenic (Ogt-Tg) mice. Four weeks after TAC (TAC4W), the heart function of Ogt-Tg mice was significantly lower than that of WT mice (reduced fractional shortening and increased ANP levels). The myocardium of left ventricle (LV) in Ogt-Tg mice became much thinner than that in WT mice. Moreover, compared to the heart tissues of WT mice, O-GlcNAcylation of GSK-3ß at Ser9 was increased and phosphorylation of GSK-3ß at Ser9 was reduced in the heart tissues of Ogt-Tg mice, resulting in its activation and subsequent inactivation of nuclear factor of activated T cell (NFAT) activity. Finally, the thinned LV wall and reduced cardiac function induced by TAC4W in Ogt-Tg mice was reversed by the treatment of a GSK-3ß inhibitor, TDZD-8. These results imply that augmented O-GlcNAcylation exacerbates pressure overload-induced heart failure due to a lack of compensatory cardiac hypertrophy via O-GlcNAcylation of GSK-3ß, which deprives the phosphorylation site of GSK-3ß to constantly inactivate NFAT activity to prevent cardiac hypertrophy. Our findings may provide a new therapeutic strategy for cardiac hypertrophy and subsequent heart failure.

Augmented O-GlcNAcylation exacerbates right ventricular dysfunction and remodeling via enhancement of hypertrophy, mitophagy, and fibrosis in mice exposed to long-term intermittent hypoxia.

Previously, we showed that augmented O-linked N-acetylglucosaminylation (O-GlcNAcylation) mitigates cardiac remodeling in O-GlcNAc transferase-transgenic (Ogt-Tg) mice exposed to acute (2-week) intermittent hypoxia (IH) by suppressing nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF-κB) via the O-GlcNAcylation of glycogen synthase kinase 3 beta (GSK-3ß) and NF-κB p65. Because this effect is time dependent, we exposed Ogt-Tg mice to IH for 4 weeks (IH4W) in the present study. O-GlcNAcylation was significantly enhanced in Ogt-Tg mice vs. wild-type (WT) mice exposed to normoxia and IH4W. Total O-GlcNAcylation levels were significantly increased in WT and Ogt-Tg mice after IH4W vs. normoxia. After IH4W, Ogt-Tg mice displayed significantly exacerbated signs of cardiac hypertrophy and fibrosis in the right ventricles (RVs) but not the left ventricles (LVs). Echocardiography revealed IH4W-induced right ventricular dysfunction. Phosphorylated GSK-3ß levels were increased in Ogt-Tg mice vs. WT mice after IH4W, whereas phosphorylated NF-κB p65 levels were unaffected. Mitophagy, which is associated with cardiac dysfunction, was increased in the RVs of Ogt-Tg mice after IH4W. Furthermore, the levels of phosphorylated dynamin-related protein 1 (p-Drp1) were significantly increased, and the expression of mitofusin-2 (MFN2) was significantly decreased. In human embryonic kidney cells, mitochondrial uncoupler-induced mitochondrial dysfunction was accelerated in Ogt-overexpressing cells. In addition to increasing the levels of phosphorylated Smad2, IH4W promoted cardiac fibrosis in the RVs of Ogt-Tg mice. Thus, augmented O-GlcNAcylation may aggravate IH4W-induced right ventricular dysfunction and remodeling by promoting hypertrophy, mitophagy, and fibrosis via GSK-3ß inactivation, an increased p-Drp-1/MFN2 ratio, and Smad2 activation, respectively.

Non-thermal atmospheric-pressure plasma potentiates mesodermal differentiation of human induced pluripotent stem cells.

Non-thermal atmospheric-pressure plasma has been used for biological applications, including sterilization and stimulation of cell growth and differentiation. Here, we demonstrate that plasma exposure influences the differentiation pattern of human induced pluripotent stem cells (hiPSCs). We treated hiPSCs with dielectric barrier-discharge air plasma and found an exposure dose that does not kill hiPSCs. Immunohistochemical staining for E-CADHERIN showed that the exposure affected cell-cell attachment and doubled the average size of the hiPSCs. Analysis of mRNAs in embryoid bodies (EBs) from plasma-treated hiPSCs revealed repression of ectoderm genes, including WNT1, and increased expression of mesoderm genes. Importantly, hiPSCs deficient in DNA repair only displayed minimal damage after plasma exposure. Collectively, our results suggest that plasma treatment can be another tool for directing the fate of pluripotent stem cells without disrupting their genomic integrity.

BDNF/TRKB axis provokes EMT progression to induce cell aggressiveness via crosstalk with cancer-associated fibroblasts in human parotid gland cancer.

Parotid gland cancer (PGC) is a rare malignancy and its molecular characteristics remain poorly understood, which has precluded the development of effective drug therapies. Given the poor prognosis of many human cancers in which tropomyosin receptor kinase B (TRKB) is highly expressed, we investigated the involvement of brain-derived neurotrophic factor (BDNF)/TRKB pathway in PGC cells using clinical specimens and observed upregulation of TRKB and BDNF. In primary culture systems of patient-derived PGC cells and cancer-associated fibroblasts (CAFs), PGC cells co-cultured with CAFs exhibited significant upregulation of BDNF and epithelial-mesenchymal transition (EMT). Similar results were observed in PGC cells treated with conditioned medium from co-cultures of PGC cells with CAFs. Administration of TRK inhibitors suppressed BDNF-induced cell migration in PGC cells. Immunohistochemical and clinicopathological analyses of tumors from patients with PGC revealed that BDNF and TRKB were highly expressed in both tumor cells and stromal cells such as CAFs, and TRKB expression levels in PGC cells were significantly correlated with aggressive features, including vascular invasion, nodal metastasis, and poor prognosis. Collectively, these data suggest that the BDNF/TRKB pathway regulates PGC cell aggressiveness via crosstalk with CAFs and is a potential therapeutic target for PGC harboring invasive and metastatic features.

Rivaroxaban Attenuates Right Ventricular Remodeling in Rats with Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition that frequently results in right ventricular (RV) remodeling. The objectives of this study are to investigate effects of rivaroxaban on RV remodeling in a rat model of PAH, created with Sugen5416 and chronic hypoxia, and the in vitro effects of rivaroxaban on human cardiac microvascular endothelial cells (HCMECs). To create the PAH model, male Sprague-Dawley rats were subcutaneously injected with Sugen5416 (20 mg/kg) and exposed to 2 weeks of hypoxia (10% O2), followed by 2 weeks of exposure to normoxia. The animals were then divided into 2 groups with or without administration of rivaroxaban (12 mg/kg/d) for a further 4 weeks. HCMECs were cultured under hypoxic conditions (37 °C, 1% O2, 5% CO2) with Sugen5416 and with or without rivaroxaban. In the model rats, RV systolic pressure and Fulton index increased by hypoxia with Sugen5416 were significantly decreased when treated with rivaroxaban. In HCMECs, hypoxia with Sugen5416 increased the expression of protease-activated receptor-2 (PAR-2) and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB), while treatment with rivaroxaban significantly suppressed the expression of these proteins. Rivaroxaban attenuated RV remodeling in a rat model of PAH by reducing ERK, JNK and NF-κB activation. Rivaroxaban has the possibility of providing additive effects on RV remodeling in patients with PAH.

Iron derived from autophagy-mediated ferritin degradation induces cardiomyocyte death and heart failure in mice.

Heart failure is a major public health problem, and abnormal iron metabolism is common in patients with heart failure. Although iron is necessary for metabolic homeostasis, it induces a programmed necrosis. Iron release from ferritin storage is through nuclear receptor coactivator 4 (NCOA4)-mediated autophagic degradation, known as ferritinophagy. However, the role of ferritinophagy in the stressed heart remains unclear. Deletion of Ncoa4 in mouse hearts reduced left ventricular chamber size and improved cardiac function along with the attenuation of the upregulation of ferritinophagy-mediated ferritin degradation 4 weeks after pressure overload. Free ferrous iron overload and increased lipid peroxidation were suppressed in NCOA4-deficient hearts. A potent inhibitor of lipid peroxidation, ferrostatin-1, significantly mitigated the development of pressure overload-induced dilated cardiomyopathy in wild-type mice. Thus, the activation of ferritinophagy results in the development of heart failure, whereas inhibition of this process protects the heart against hemodynamic stress.

Intravenously Injected Pluripotent Stem Cell-derived Cells Form Fetomaternal Vasculature and Prevent Miscarriage in Mouse.

Miscarriage is the most common complication of pregnancy, and about 1% of pregnant women suffer a recurrence. Using a widely used mouse miscarriage model, we previously showed that intravenous injection of bone marrow (BM)-derived endothelial progenitor cells (EPCs) may prevent miscarriage. However, preparing enough BM-derived EPCs to treat a patient might be problematic. Here, we demonstrated the generation of mouse pluripotent stem cells (PSCs), propagation of sufficient PSC-derived cells with endothelial potential (PSC-EPs), and intravenous injection of the PSC-EPs into the mouse miscarriage model. We found that the injection prevented miscarriage. Three-dimensional reconstruction images of the decidua after tissue cleaning revealed robust fetomaternal neovascularization induced by the PSC-EP injection. Additionally, the injected PSC-EPs directly formed spiral arteries. These findings suggest that intravenous injection of PSC-EPs could become a promising remedy for recurrent miscarriage.

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.

Enhancement of O-linked N-acetylglucosamine modification promotes metastasis in patients with colorectal cancer and concurrent type 2 diabetes mellitus.

Reversible post-translational modification of serine and threonine residues by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation has been indicated to regulate the activities of a number of different proteins. Augmented O-GlcNAcylation contributes to the etiologies of type 2 diabetes mellitus (T2DM) and cancer. Moreover, diabetic conditions increase the risk of colorectal cancer. However, the effect of O-GlcNAcylation in patients with colorectal cancer and concurrent T2DM has not been elucidated. The current study evaluated the level of O-GlcNAcylation in patients with colorectal cancer with or without T2DM. Notably, O-GlcNAcylation levels were significantly higher in tissues from patients with T2DM compared with those in patients without T2DM, and higher in cancer tissues compared with corresponding adjacent tissues. O-GlcNAcylation and cancer stage were more strongly correlated in cancer tissues from patients with T2DM compared with those from patients without T2DM. Additionally, distant metastasis was significantly correlated with O-GlcNAcylation in cancer tissues from patients with T2DM. ß-catenin levels in colorectal cancer tissues were the highest in patients with advanced-stage cancer and concurrent T2DM. In SW480 human colon cancer cells, thiamet G (TMG) treatment and OGA silencing, which increased O-GlcNAcylation, significantly increased ß-catenin and SNAIL in high-glucose, but not during normal-glucose conditions. These data suggest that O-GlcNAcylation is closely associated with distant metastasis, most likely through upregulation of the ß-catenin/SNAIL signaling pathway in colorectal cancer patients with T2DM.

Exacerbation of autoimmune myocarditis by an immune checkpoint inhibitor is dependent on its time of administration in mice.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have made an immense breakthrough in cancer therapeutics, they can exert unique, immune-related adverse events. Among them, myocarditis is less frequent, but it is serious and often follows a lethal course. METHODS: To examine the changes in cardiac autoimmunity after ICI administration, we developed a mouse experimental autoimmune myocarditis (EAM) model via intraperitoneal administration of murine α-cardiac myosin heavy chain (MyHC-α) fragment. Thereafter, the mouse anti-PD-1 antibody (mPD1ab) was administered at two time points, subsequent to and concurrent with MyHC-α fragment administration. RESULTS: Severe EAM developed in 3 weeks; wide inflammatory lesions were observed in the cardiac sections. Furthermore, inflammatory/fibrotic genes, such as interleukin 1ß, interleukin 6, and collagen 1, were upregulated, although the cardiac function was not significantly affected. The subsequent administration of mPD1ab at 2 weeks post administration of the first MyHC-α fragment exacerbated EAM, whereas the administration of mPD1ab concurrent with MyHC-α fragment administration did not exacerbate EAM. The subsequent administration of mPD1ab significantly increased the infiltration of cluster of differentiation (CD)4- and F4/80-positive cells, whereas the concurrent administration of mPD1ab significantly decreased the infiltration of CD4-positive cells, indicating that the concurrent and subsequent administration of mPD1ab had opposite effects on immune/inflammatory cell infiltration. CONCLUSIONS: These data suggest that the appearance of ICI-induced autoimmune myocarditis might be related to autoimmune system activity before ICI administration. Although ICIs do not adversely affect patients with normal immune systems, we propose that ICI administration should be avoided in patients with autoimmune disorders.

O-GlcNAcylation-mediated degradation of FBXL2 stabilizes FOXM1 to induce cancer progression.

O-GlcNAcylation is a dynamic and reversible post-translational modification of cytonuclear molecules that regulates cellular signaling. Elevated O-GlcNAcylation is a general property of cancer and plays a critical role in cancer progression. We previously showed that the expression of FOXM1, a critical oncogenic transcription factor widely overexpressed in solid tumors, was elevated in MKN45 cells, a human gastric cancer cell line, by the O-GlcNAcase inhibitor Thiamet G (TMG), which induces augmented O-GlcNAcylation. Here, we identified FBXL2 E3 ubiquitin ligase as a new target of O-GlcNAcylation. Consistent with the results in MKN45 cells, FOXM1 expression was increased, accompanied by its decreased ubiquitination and degradation by TMG in the other gastric cancer cell lines, including NUGC-3 cells. We found that FBXL2 ubiquitinated FOXM1, and the interaction with FBXL2 and ubiquitination of FOXM1 were reduced by TMG in NUGC-3 cells. Interestingly, FBXL2 was also ubiquitinated, which was promoted by TMG in the cells. Moreover, FOXM1 expression and cell proliferation were reduced in FBXL2-induced NUGC-3 cells, and the reductions were attenuated by TMG, indicating that FOXM1 was stabilized by O-GlcNAcylation-mediated degradation of FBXL2 to induce cancer progression. These data suggest that elevated O-GlcNAcylation contributes to cancer progression by suppressing FBXL2-mediated degradation of FOXM1.

Autophagy deficiency exacerbates colitis through excessive oxidative stress and MAPK signaling pathway activation.

BACKGROUND AND AIM: Autophagy is an essential process involved in the pathogenesis of inflammatory bowel disease (IBD). Although there are many data showing the roles of autophagy in intestinal epithelial cells (IECs), the mechanisms involved remain to be fully elucidated. We investigated the influence of autophagy in IECs on gastrointestinal tract inflammation. METHODS: Mice with conditional knockout of Atg5 in IECs (Atg5flox/flox/villin-Cre mice) were subjected to dextran sulfate sodium (DSS)-induced colitis and analyzed for colitis susceptibility. Additionally, we used Atg5-silenced rat IECs (IEC6shAtg5 cells) for in vitro assays. RESULTS: Sensitivity to DSS markedly increased in Atg5flox/flox/villin-Cre mice compared to that in wild-type mice. In IEC6shAtg5 cells, apoptosis was enhanced, and cell viability significantly decreased compared to IEC-6 cells. The expression of proinflammatory cytokines increased upon suppression of autophagy. Furthermore, silencing of Atg5 was associated with inflammation of IECs, activation of the mitogen-activated protein kinase (MAPK) signaling pathway by the intracellular reactive oxygen species accumulation, and NF-κB p65 phosphorylation. CONCLUSIONS: Autophagy in IECs plays an essential role in the maintenance of intestinal homeostasis, and autophagy deficiency triggers inflammation. Development of methods targeting autophagy might be beneficial in the treatment of IBD.

Overexpression of Na+/H+ exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the Rho kinase ROCK.

Understanding the specific properties of human induced pluripotent stem cells (iPSCs) is important for quality control of iPSCs. Having incidentally discovered that overexpression of plasma membrane Na+/H+ exchanger 1 (NHE1) induces cell death in iPSCs, we investigated the mechanism of NHE1-induced cell death. Doxycycline-induced NHE1 overexpression arrested cell growth, and nearly all cells were killed by a necrotic process within 72 h. NHE1 overexpression led to sustained activation of Rho-associated coiled-coil kinase (ROCK), accompanied by dramatic changes in cell shape, cell elongation, and swelling of peripheral cells in iPSC colonies, as well as marked stress fiber formation. The ROCK inhibitor Y27632 reduced NHE1-induced cell death. ROCK-dependent phenotypes were suppressed by a loss-of-function mutation of NHE1 and inhibited by an inhibitor of NHE1 activity, indicating that NHE1-mediated transport activity is required. Moreover, ROCK was activated by trimethylamine treatment-mediated cytosolic alkalinization and accumulated in the plasma membrane near NHE1 in peripheral iPSCs of cell colonies. By contrast, cell death did not occur in mesendoderm-like cells that had differentiated from iPSCs, indicating that the NHE1-mediated effects were specific for iPSCs. These results suggest that NHE1 overexpression specifically induces death of iPSCs via sustained ROCK activation, probably caused by an increase in local pH near NHE1. Finally, monensin, a Na+/H+ exchange ionophore, selectively killed iPSCs, suggesting that monensin could help eliminate iPSCs that remain after differentiation, a strategy that might be useful for improving regenerative medicine.

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells.

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.

Augmented O-GlcNAcylation attenuates intermittent hypoxia-induced cardiac remodeling through the suppression of NFAT and NF-κB activities in mice.

Type 2 diabetes mellitus (T2DM) has been reported to be associated with cardiac remodeling. Although O-GlcNAcylation is known to be elevated in diabetic and ischemic hearts, the effects of O-GlcNAcylation on cardiac remodeling induced by intermittent hypoxia (IH), such as sleep apnea syndrome (SAS), remain unknown. To evaluate the effects, we induced IH in wild-type (WT) and transgenic O-GlcNAc transferase (Ogt-Tg) mice. Two weeks of IH increased O-GlcNAcylation in the heart tissues of both strains of mice, whereas O-GlcNAcylation in Ogt-Tg mice was significantly higher than that in WT mice under both normoxic and IH conditions. WT mice exhibited cardiac remodeling after IH, whereas cardiac remodeling was significantly attenuated in Ogt-Tg mice. Oxidative stress and apoptosis increased after IH in both strains of mice, whereas the rate of increase in these processes in Ogt-Tg mice was significantly lower than that in WT mice. To examine the mechanism of cardiac remodeling attenuation in Ogt-Tg mice after IH, the effects of O-GlcNAcylation on the activities of the master regulators nuclear factor of activated T cells (NFAT) and NF-κB were determined. The O-GlcNAcylation of GSK-3ß, a negative regulator of NFAT, was significantly increased in Ogt-Tg mice, whereas the phosphorylation of GSK-3ß was reciprocally reduced. The same result was observed for NF-κB p65. An in vitro reporter assay showed that the augmentation of O-GlcNAcylation by an O-GlcNAcase inhibitor suppressed NFAT and NF-κB promoter activity. These data suggest that augmented O-GlcNAcylation mitigates IH-induced cardiac remodeling by suppressing NFAT and NF-κB activities through the O-GlcNAcylation of GSK-3ß and NF-κB p65.

Cardiac Regeneration by Statin-Polymer Nanoparticle-Loaded Adipose-Derived Stem Cell Therapy in Myocardial Infarction.

Clinical trials with autologous adipose-derived stem cell (AdSC) therapy for ischemic heart diseases (IHDs) are ongoing. However, little is known about combinational therapeutic effect of AdSCs and statin poly(lactic-co-glycolic) acid (PLGA) nanoparticles on the ischemic myocardium. We investigated the hypothesis that statins, which have pleiotropic effects, augment the therapeutic potential of AdSCs and that AdSCs also act as drug delivery tools. Simvastatin-conjugated nanoparticles (SimNPs) significantly promoted migration activity without changing proliferation activity and upregulated growth factor gene expression in vitro. A small number of intravenously administered SimNP-loaded AdSCs (10,000 cells per mouse) improved cardiac function following myocardial infarction, inducing endogenous cardiac regeneration in the infarcted myocardium. The de novo regenerated myocardium was thought to be derived from epicardial cells, which were positive for Wilms' tumor protein 1 expression. These findings were attributed to the sustained, local simvastatin release from the recruited SimNP-loaded AdSCs in the infarcted myocardium rather than to the direct contribution of recruited AdSCs to tissue regeneration. SimNP-loaded AdSCs may lead to a novel somatic stem cell therapy for IHDs. Stem Cells Translational Medicine 2019;8:1055-1067.

Sufficient therapeutic effect of cryopreserved frozen adipose-derived regenerative cells on burn wounds.

INTRODUCTION: The purpose of this study was to evaluate whether cryopreserved (frozen) adipose-derived regenerative cells (ADRCs) have a therapeutic effect on burn wound healing as well as freshly isolated (fresh) ADRCs. METHODS: Full thickness burns were created on dorsum of nude mice and burn wound was excised. The wound was covered by artificial dermis with; (i) fresh ADRCs, (ii) frozen ADRCs, and (iii) PBS (control). The assessment for wound healing was performed by morphological, histopathological and immunohistochemical analyses. RESULTS: In vivo analyses exhibited the significant therapeutic effect of frozen ADRCs on burn wound healing up to the similar or higher level of fresh ADRCs. There were significant differences of wound closure, epithelized tissue thickness, and neovascularization between the treatment groups and control group. Although there was no significant difference of therapeutic efficacy between fresh ADRC group and frozen ADRC group, frozen ADRCs improved burn wound healing process in dermal regeneration with increased great type I collagen synthesis compared with fresh ADRCs. CONCLUSIONS: These findings indicate that frozen ADRCs allow us to apply not only quickly but also for multiple times, and the cryopreserved ADRCs could therefore be useful for the treatment of burn wounds in clinical settings.

Establishment of a novel mouse xenograft model of human uterine leiomyoma.

Uterine leiomyoma is the most common benign tumour in women, and an appropriate animal model for leiomyoma would be useful for exploring new therapeutic strategies. Therefore, we have been challenged to develop a new simple mouse model for human leiomyoma. Leiomyoma tissues were harvested from myomas resected by different surgical procedures with or without gonadotropin-releasing hormone agonist (GnRHa) treatment and were subcutaneously implanted into BALB/c nude mice with an estradiol/progesterone-releasing pellet. The implanted leiomyoma tissues that were obtained from the marginal site of large myomas resected by abdominal myomectomy with GnRHa treatment exhibited sufficient tumour growth in the transplanted mice. The leiomyomas that were treated with GnRHa highly expressed the estrogen/progesterone receptor genes, insulin-like growth factor 2 (IGF2) and embryonic smooth muscle myosin heavy chain (SMemb), which suggests that these factors are critical in the establishment of a mouse model of growing leiomyoma. As a result, this model will be useful for the development of new therapeutic strategies.