Far-infrared irradiation increases the uptake of LDL cholesterol by downregulating PCSK9 through the activation of TRPV4 calcium channels in HepG2 cells.

This study investigated the effects of far-infrared (FIR) irradiation on low-density lipoprotein cholesterol (LDL-C) uptake by human hepatocellular carcinoma G2 (HepG2) cells via the regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). FIR irradiation for 30 min significantly decreased PCSK9 expression (p < 0.01) in HepG2 cells. FIR irradiation substantially increased the low-density lipoprotein receptor (p < 0.0001) and LDL-C uptake (p < 0.01). Activation of transient receptor potential vanilloid (TRPV) channels mimicked the effects of FIR irradiation, significantly decreasing the protein expression of PCSK9 (p < 0.05). Conversely, inhibition of TRP channels using ruthenium red reversed the reduction in PCSK9 protein expression following FIR irradiation (p < 0.01). The specific activation of TRPV4 using 4α-PDD mimicked the effect of FIR irradiation (p < 0.01), whereas PCSK9 reduction by FIR irradiation was significantly reversed by the inhibition of TRPV4 using RN1734 (p < 0.05). These findings implied that FIR irradiation emitted from a ceramic lamp specifically increased TRPV4 activity. These findings provide insights into a novel therapeutic approach using FIR irradiation for LDL-C regulation and its implications for cardiovascular health.

Nucleus-targeted delivery of nitric oxide in human mesenchymal stem cells enhances osteogenic differentiation.

Nitric oxide (NO) is an important gaseous signaling molecule in various physiological processes, which functions through interactions with its acceptor molecules located in organelles. NO has an extremely short half-life, making it challenging to experimentally achieve effective NO levels in organelles to study these interactions. Here we developed an organelle-specific, peptide-based NO delivery material that targets the nucleus. NO was attached to the SH group of a cysteine residue inserted into the N-terminus of a cell-penetrating peptide (CPP) conjugated to varying repeats of the nuclear localization signal (NLS), which we denoted NO-CysCPP-NLS, through S-nitrosylation. NO-CysCPP-NLS strongly induced osteogenic differentiation of mesenchymal stem cells. This delivery concept can be extended to cells other than stem cells to elucidate the effects of NO release in the nucleus. Furthermore, conjugation of NO to CysCPP fused to mitochondria- or lysosome-targeting signals can be used to deliver NO to other organelles such as mitochondria and lysosomes, respectively.

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death.

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 µM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Mortality Risk within 14 Days after Coronavirus Disease 2019 Diagnosis in Dementia Patients: A Nationwide Analysis.

INTRODUCTION: The study evaluated the increased mortality risk within 14 days of coronavirus disease 2019 (COVID-19) diagnosis in dementia patients. METHODS: This retrospective study was conducted from February to April 2020 using the COVID-19 patients' database from the Korea Disease Control and Prevention Agency. The risk factors for early death within 14 days were determined using generalized logistic regression performed in a stepwise manner. Dementia patients diagnosed with COVID-19 were used for the study. The propensity score-matched cohort was included as controls. The differences in mortality within 14 days after COVID-19 diagnosis between the dementia patients and controls were evaluated. RESULTS: We enrolled 5,349 COVID-19 patients from the database; 224 had dementia as comorbidity. The mortality rate within 14 days after COVID-19 diagnosis in dementia patients and the controls was 23.7% versus 1.7%, respectively, before propensity score matching (PSM) (p < 0.001), and 23.7% versus 9.2% after PSM (p < 0.001). The hazard ratio (HR) for mortality within 14 days in COVID-19 patients with dementia was significant even after PSM (HR 5.104, 95% confidence interval 2.889-5.673, p < 0.001). The survival curve of dementia patients was steeply inclined within 14 days after COVID-19 diagnosis, resulting in 70.7% of all deaths in dementia patients. CONCLUSIONS: COVID-19 patients with dementia had a higher risk of early death within 14 days. Thus, prompt intervention is necessary for dementia patients after COVID-19 diagnosis.

Application of Tonsil-Derived Mesenchymal Stem Cells in Tissue Regeneration: Concise Review.

Since the discovery of stem cells and multipotency characteristics of mesenchymal stem cells (MSCs), there has been tremendous development in regenerative medicine. MSCs derived from bone marrow have been widely used in various research applications, yet there are limitations such as invasiveness of obtaining samples, low yield and proliferation rate, and questions regarding their practicality in clinical applications. Some have suggested that MSCs from other sources, specifically those derived from palatine tonsil tissues, that is, tonsil-derived MSCs (TMSCs), could be considered as a new potential therapeutic tool in regenerative medicine due to their superior proliferation rate and differentiation capabilities with low immunogenicity and ease of obtaining. Several studies have determined that TMSCs have differentiation potential not only into the mesodermal lineage but also into the endodermal as well as ectodermal lineages, expanding their potential usage and placing them as an appealing option to consider for future studies in regenerative medicine. In this review, the differentiation capacities of TMSCs and their therapeutic competencies from past studies are addressed. Stem Cells 2019;37:1252-1260.

Far-Infrared Irradiation Inhibits Adipogenic Differentiation and Stimulates Osteogenic Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells: Role of Protein Phosphatase 2B.

BACKGROUND/AIMS: Far-infrared (FIR) irradiation has been reported to exhibit various biological effects including improvement of cardiovascular function. However, its effect on the differentiation of stem cells has not been studied. Using tonsil-derived mesenchymal stem cells (TMSC), we examined whether and how FIR irradiation affects adipogenic or osteogenic differentiation. METHODS: TMSC were exposed to FIR irradiation (3-25 µm wavelength) for various times (0, 30, or 60 min), and then adipogenic or osteogenic differentiation was induced for 14 days with its respective commercially available differentiation medium. At the end of the differentiation, the cells were stained using Oil red O or Alizarin red S solution, and the expression of differentiation-specific proteins was analyzed by western blotting. RESULTS: FIR irradiation did not alter cell viability or the expression of MSC-specific surface antigens (CD14, CD34, CD45, CD73, CD90, and CD105) in TMSC. However, FIR irradiation significantly inhibited adipogenic differentiation of TMSC, as evidenced by decreased Oil red O staining as well as protein expression of peroxisome proliferator-activated receptor γ and fatty acid binding protein 4. In contrast, FIR irradiation induced osteogenic differentiation, as evidenced by increased Alizarin red S staining as well as protein expression of osteocalcin and alkaline phosphatase. Treatment with heat alone did not inhibit the adipogenic differentiation of TMSC, suggesting that the inhibitory effect on adipogenic differentiation was not due to heat induced by FIR irradiation. However, heat alone did stimulate osteogenic differentiation, but to a lesser extent than FIR irradiation. Furthermore, FIR irradiation increased intracellular Ca²âº levels and the activity of protein phosphatase 2B (PP2B) in TMSC. Treatment with cyclosporin A, a specific PP2B inhibitor, reversed the inhibitory effect of FIR irradiation on adipogenic differentiation of TMSC, but had no effect on osteogenic differentiation. CONCLUSION: Our data demonstrate that FIR irradiation inhibits adipogenic differentiation but enhances osteogenic differentiation of TMSC; the inhibitory effect on adipogenic differentiation is non-thermal and mediated at least in part by activation of Ca²âº-dependent PP2B.

Double intratibial injection of human tonsil-derived mesenchymal stromal cells recovers postmenopausal osteoporotic bone mass.

BACKGROUND AND AIMS: Osteoporosis, which is a disease characterized by weakening of the bone, affects a large portion of the senior population. The current therapeutic options for osteoporosis have side effects, and there is no effective treatment for severe osteoporosis. Thus, we urgently need new treatment strategies, such as topical therapies and/or safe and effective stem cell therapies. METHODS: We investigated the therapeutic potential of directly injecting human tonsil-derived mesenchymal stem cells (TMSC) into the right proximal tibias of ovariectomized postmenopausal osteoporosis model mice. Injections were given once (1×) or twice (2×) during the 3-month experimental period. At the end of the experiment, micro-computed tomographic images revealed some improvement in the proximal tibias and more significant improvement in the femoral heads of treated mice. RESULTS: Osteogenic effect was qualitatively and quantitatively more pronounced in TMSC/2×-treated mice. Furthermore, TMSC/2× mice exhibited significant recovery of the serum osteocalcin level, which is pathologically elevated in osteoporosis, and increased serum alkaline phosphatase, which indicates bone formation. TMSC therapy was generally well tolerated and caused no apparent toxicity in the experimental mice. Moreover, TMSC therapy reduced visceral fat. CONCLUSION: Our results demonstrate that double injection of TMSC directly into the proximal tibia triggers recovery of osteoporosis, and thus could be a potential therapeutic approach for severe bone loss.

Telmisartan mitigates hyperglycemia-induced vascular inflammation by increasing GSK3ß-Ser9 phosphorylation in endothelial cells and mouse aortas.

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), attenuates hyperglycemia-aggravated vascular inflammation by decreasing IκB kinase ß (IKKß) expression in endothelial cells. Because glycogen synthase 3ß (GSK3ß) is involved in inflammatory process by regulating nuclear factor-κB (NF-κB) activity, we investigated whether GSK3ß mediates telmisartan-ameliorated vascular inflammation in hyperglycemia-treated endothelial cells and high-fat diet (HFD)-fed mice. Telmisartan remarkably induced GSK3ß-Ser9 phosphorylation in hyperglycemia-treated endothelial cells that accompanied a decrease in hyperglycemia-induced NF-κB p65-Ser536 phosphorylation, vascular cell adhesion molecule-1 (VCAM-1) expression, and THP-1 monocyte adhesion. Ectopic expression of GSK3ß-S9A, a constitutively active mutant of GSK3ß, significantly restored complete telmisartan-inhibited NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and THP-1 monocyte adhesion. In addition, it reversed telmisartan-repressed IKKß expression. Among the ARB, including losartan and fimasartan, only telmisartan increased GSK3ß-Ser9 phosphorylation, and telmisartan-induced GSK3ß-Ser9 phosphorylation remained unchanged by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Finally, in the aortas of HFD-fed mice, telmisartan treatment significantly attenuated HFD-induced upregulation of NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and IKKß expression and downregulation of GSK3ß-Ser9 phosphorylation. Taken together, our findings demonstrated that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by inducing GSK3ß-Ser9 phosphorylation, which consequently inhibits IKKß expression, NF-κB p65-Ser536 phosphorylation, and VCAM-1 expression in a PPARγ-independent manner.

Effect of Individual and District-level Socioeconomic Disparities on Cognitive Decline in Community-dwelling Elderly in Seoul.

This study was to investigate the effects of individual and district-level socioeconomic status (SES) on the development of cognitive impairment among the elderly. A 3-year retrospective observational analysis (2010-2013) was conducted which included 136,217 community-dwelling healthy elderly who participated in the Seoul Dementia Management Project. Cognitive impairment was defined as 1.5 standard deviations below the norms on the Mini-mental status examination. In the individual lower SES group, the cumulative incidence rate (CIR) of cognitive impairment was 8.7% (95% confidence interval [CI], 8.64-8.70), whereas the CIR in the individual higher SES group was 4.1% (95% CI, 4.08-4.10). The CIR for lower district-level SES was 4.7% (95% CI, 4.52-4.86), while that in the higher district-level SES was 4.3% (95% CI, 4.06-4.44). There were no additive or synergistic effects between individual and district-level SES. From this study, the individual SES contributed 1.9 times greater to the development of cognitive impairment than the district-level SES, which suggests that individual SES disparities could be considered as one of the important factors in public health related to cognitive impairment in the elderly.

Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation.

We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on ß-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

Maternal Food Restriction during Pregnancy and Lactation Adversely Affect Hepatic Growth and Lipid Metabolism in Three-Week-Old Rat Offspring.

Maternal malnutrition influences the early development of foetal adaptive changes for survival. We explored the effects of maternal undernutrition during gestation and lactation on hepatic growth and function. Sprague-Dawley rats were fed a normal or a food-restricted (FR) diet during gestation and/or lactation. We performed analyses of covariance (adjusting for the liver weight/body weight ratio) to compare hepatic growth and lipid metabolism among the offspring. Maternal FR during gestation triggered the development of wide spaces between hepatic cells and increased the expression of mammalian target of rapamycin (mTOR) in three-week-old male offspring compared with controls (both p < 0.05). Offspring nursed by FR dams exhibited wider spaces between hepatic cells and a lower liver weight/body weight ratio than control offspring, and increased mTOR expression (p < 0.05). Interestingly, the significant decrease in expression of lipogenic-related genes was dependent on carbohydrate-responsive element-binding protein, despite the increased expression of sterol regulatory element-binding protein 1 (SREBP1) (p < 0.05). This study demonstrated increased expression of key metabolic regulators (mTOR and SREBP1), alterations in lipid metabolism, and deficits in hepatic growth in the offspring of FR-treated dams.

Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration.

Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

CCN1 secreted by tonsil-derived mesenchymal stem cells promotes endothelial cell angiogenesis via integrin αv ß3 and AMPK.

CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin α(v) ß(3) with anti-integrin α(v) ß(3) antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin α(v) ß(3) and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration.

B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production.

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179 phosphorylation, aphidicolin decreased eNOS-Ser116 phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116 dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566 phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116 and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury.

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.

Uric acid induces fat accumulation via generation of endoplasmic reticulum stress and SREBP-1c activation in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common types of chronic liver injury. Elevated serum uric acid is a strong predictor of the development of fatty liver as well as metabolic syndrome. Here we demonstrate that uric acid induces triglyceride accumulation by SREBP-1c activation via induction of endoplasmic reticulum (ER) stress in hepatocytes. Uric acid-induced ER stress resulted in an increase of glucose-regulated protein (GRP78/94), splicing of the X-box-binding protein-1 (XBP-1), the phosphorylation of protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor-2α (eIF-2α) in cultured hepatocytes. Uric acid promoted hepatic lipogenesis through overexpression of the lipogenic enzyme, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) via activation of SREBP-1c, which was blocked by probenecid, an organic anion transport blocker in HepG2 cells and primary hepatocytes. A blocker of ER stress, tauroursodeoxycholic acid (TUDCA), and an inhibitor of SREBP-1c, metformin, blocked hepatic fat accumulation, suggesting that uric acid promoted fat synthesis in hepatocytes via ER stress-induced activation of SREBP-1c. Uric acid-induced activation of NADPH oxidase preceded ER stress, which further induced mitochondrial ROS production in hepatocytes. These studies provide new insights into the mechanisms by which uric acid stimulates fat accumulation in the liver.

Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: role for CCN1 in replicative senescence-associated increase in osteogenic differentiation.

Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture-related alterations in their stem cell properties, such data have not been reported in human tonsil-derived MSC (T-MSC). Here, we investigated the culture-related changes of phenotypes, the senescence, and the differentiation potential of T-MSC. T-MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC-specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC-specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4-A and Sox-2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ~ 23% at passage 3 to ~ 1% at passage 15. The average doubling time increased significantly from ~ 38 h at passage 10 to ~ 46 h at passage 15. From passage 10, the cell size increased slightly and SA-ß-gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage-dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage-dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long-term in vitro cultured T-MSC and that CCN1 may be involved in mediating a passage-dependent increase in osteogenic potential of T-MSC.

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease.

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acid-induced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin.