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2.  HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression 
Cell Discovery  2016;2:16045-.
Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.
doi:10.1038/celldisc.2016.45
PMCID: PMC5148442  PMID: 27990297
acetyl-CoA binding domain; histone acetylation; hnRNPA2; Mitochondrial retrograde signaling (MtRS); mtDNA depletion; telomerase activation; transcriptional coactivator
3.  Correlation of Scar in Cardiac MRI and High‐Resolution Contact Mapping of Left Ventricle in a Chronic Infarct Model 
Background
Endocardial mapping for scars and abnormal electrograms forms the most essential component of ventricular tachycardia ablation. The utility of ultra‐high resolution mapping of ventricular scar was assessed using a multielectrode contact mapping system in a chronic canine infarct model.
Methods
Chronic infarcts were created in five anesthetized dogs by ligating the left anterior descending coronary artery. Late gadolinium‐enhanced magnetic resonance imaging (LGE MRI) was obtained 4.9 ± 0.9 months after infarction, with three‐dimensional (3D) gadolinium enhancement signal intensity maps at 1‐mm and 5‐mm depths from the endocardium. Ultra‐high resolution electroanatomical maps were created using a novel mapping system (Rhythmia Mapping System, Rhythmia Medical/Boston Scientific, Marlborough, MA, USA) Rhythmia Medical, Boston Scientific, Marlborough, MA, USA with an 8.5F catheter with mini‐basket electrode array (64 tiny electrodes, 2.5‐mm spacing, center‐to‐center).
Results
The maps contained 7,754 ± 1,960 electrograms per animal with a mean resolution of 2.8 ± 0.6 mm. Low bipolar voltage (<2 mV) correlated closely with scar on the LGE MRI and the 3D signal intensity map (1‐mm depth). The scar areas between the MRI signal intensity map and electroanatomic map matched at 87.7% of sites. Bipolar and unipolar voltages, compared in 592 electrograms from four MRI‐defined scar types (endocardial scar, epicardial scar, mottled transmural scar, and dense transmural scar) as well as normal tissue, were significantly different. A unipolar voltage of <13 mV correlated with transmural extension of scar in MRI. Electrograms exhibiting isolated late potentials (ILPs) were manually annotated and ILP maps were created showing ILP location and timing. ILPs were identified in 203 ± 159 electrograms per dog (within low‐voltage areas) and ILP maps showed gradation in timing of ILPs at different locations in the scar.
Conclusions
Ultra‐high resolution contact electroanatomical mapping accurately localizes ventricular scar and abnormal myocardial tissue in this chronic canine infarct model. The high fidelity electrograms provided clear identification of the very low amplitude ILPs within the scar tissue and has the potential to quickly identify targets for ablation.
doi:10.1111/pace.12581
PMCID: PMC5006837  PMID: 25656924
ventricular tachycardia; electroanatomical mapping; magnetic resonance imaging; late potentials; ventricular scar
4.  Nitensidine A, a guanidine alkaloid from Pterogyne nitens, induces osteoclastic cell death 
Cytotechnology  2013;67(4):585-592.
Nitensidine A is a guanidine alkaloid isolated from Pterogyne nitens, a common plant in South America. To gain insight into the biological activity of P. nitens-produced compounds, we examined herein their biological effects on osteoclasts, multinucleated giant cells that regulate bone metabolism by resorbing bone. Among four guanidine alkaloids (i.e., galegine, nitensidine A, pterogynidine, and pterogynine), nitensidine A and pterogynine exhibited anti-osteoclastic effects at 10 μM by reducing the number of osteoclasts on the culture plate whereas galegine and pterogynidine did not. The anti-osteoclastic activities of nitensidine A and pterogynine were exerted in a concentration-dependent manner, whereas nitensidine A exhibited an approximate threefold stronger effect than pterogynine (IC50 values: nitensidine A, 0.93 ± 0.024 μM; pterogynine, 2.7 ± 0.40 μM). In the present study, the anti-osteoclastic effects of two synthetic nitensidine A derivatives (nitensidine AT and AU) were also examined to gain insight into the structural features of nitensidine A that exert an anti-osteoclastic effect. The anti-osteoclastic effect of nitensidine A was greatly reduced by substituting the imino nitrogen atom in nitensidine A with sulfur or oxygen. According to the differences in chemical structures and anti-osteoclastic effects of the four guanidine alkaloids and the two synthetic nitensidine A derivatives, it is suggested that the number, binding site, and polymerization degree of isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their anti-osteoclastic effects.
doi:10.1007/s10616-013-9590-2
PMCID: PMC4474991  PMID: 23892478
Pterogyne nitens; Guanidine alkaloids; Osteoclast; Cytotoxicity; Structure and activity relationship
5.  A pivotal role of Krüppel-like factor 5 in regulation of cancer stem-like cells in hepatocellular carcinoma 
Cancer Biology & Therapy  2015;16(10):1453-1461.
In hepatocellular carcinoma (HCC), there exists a highly tumorigenic subset of cells defined by high expression of CD44 and CD133 that has been reported to contain cancer stem-like cells (CSCs). Krüppel-like factor 5 (KLF5) regulates many factors involved in cell cycle, migration, inflammation, angiogenesis and stemness and has cancer-promoting effects in some cancers. While some reports have indicated that KLF5 may have important roles in regulation of CSCs, what role, if any, KLF5 plays in regulation of CSCs in HCC remains to be elucidated. Flow cytometric analysis of CD44 and CD133 in HCC cell lines revealed subpopulations of CD44High/CD133High and CD44Low/CD133Low cells. We subsequently sorted these subpopulations and identified KLF5 as a gene that is significantly upregulated in CD44High/CD44High cells via RNA sequencing using next generation sequencing technology. Moreover, KLF5 overexpression enriched the CD44High/CD133High subpopulation and, consistent with the up-regulation of CD44High/CD133High cells, KLF5 overexpressing cells were more resistant to anti-cancer drugs and displayed enhanced colony-formation capacity. By contrast, knock-down of KLF5 by siRNA diminished the CD44High/CD133High subpopulation. When KLF5 was acetylated by TGF-β1, the KLF5-mediated CD44High/CD133High subpopulation enrichment was abrogated. Oppositely, ectopic expression of an acetylation-deficient KLF5 mutant further increased CD44High/CD133High subpopulations as compared to cell expressing wild-type KLF5. These findings provide novel mechanistic insight into a pivotal role for KLF5 in the regulation of CSCs in HCC.
doi:10.1080/15384047.2015.1070992
PMCID: PMC4846134  PMID: 26176896
acetylation; cancer stem cell; CD44; CD133; hepatocellular carcinoma; KLF5
6.  ABCB1 polymorphism is associated with atorvastatin-induced liver injury in Japanese population 
BMC Genetics  2016;17:79.
Background
To investigate the associations between atorvastatin-induced liver injury (AILI) and polymorphisms in eight genes possibly involved in the hepatic metabolism (CYP2C9, CYP2C19, CYP3A4, CYP3A5 and UGT1A1) and membrane transport (ABCB1, ABCG2 and SLCO1B1) of atorvastatin, we genotyped 30 AILI and 414 non-AILI patients recruited at BioBank Japan for 15 single nucleotide polymorphisms (SNPs).
Results
An SNP in ABCB1 (rs2032582: 2677G > T/A) was significantly associated with AILI (P = 0.00068, odds ratio (OR) = 2.59 with 95 % confidence interval (CI) of 1.49-4.50, G allele versus T and A alleles), indicating that the G allele might be a risk factor for AILI. The cytotoxicity test demonstrated that IC50 value of atorvastatin to inhibit the growth and/or viability of Flp-In-293/ABCB1 (2677G) cells was 5.44 ± 0.10 mM, which was significantly lower than those in Flp-In-293/ABCB1 (2677 T) (6.02 ± 0.07 mM) and Flp-In-293/ABCB1 (2677A) cells (5.95 ± 0.08 mM).
Conclusions
These results indicate that ABCB1 rs2032582 may predict the risk of AILI in Japanese population.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-016-0390-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-016-0390-5
PMCID: PMC4906899  PMID: 27296832
Atorvastatin-induced adverse reaction; Genetic association; Hepatotoxicity; MDR1 Ala893Ser/Thr/
7.  Disruption of Cytochrome c Oxidase Function Induces Warburg Effect and Metabolic Reprogramming 
Oncogene  2015;35(12):1585-1595.
Defects in mitochondrial oxidative phosphorylation complexes, altered bioenergetics and metabolic shift are often seen in cancers. Here we show a role for the dysfunction of electron transport chain component, cytochrome c oxidase (CcO) in cancer progression. We show that genetic silencing of the CcO complex by shRNA expression and loss of CcO activity in multiple cell types from the mouse and human sources resulted in metabolic shift to glycolysis, loss of anchorage dependent growth and acquired invasive phenotypes. Disruption of CcO complex caused loss of transmembrane potential and induction of Ca2+/Calcineurin-mediated retrograde signaling. Propagation of this signaling, includes activation of PI3-kinase, IGF1R and Akt, Ca2+ sensitive transcription factors and also, TGFβ1, MMP16, periostin that are involved in oncogenic progression. Whole genome expression analysis showed up regulation of genes involved in cell signaling, extracellular matrix interactions, cell morphogenesis, cell motility and migration. The transcription profiles reveal extensive similarity to retrograde signaling initiated by partial mtDNA depletion, though distinct differences are observed in signaling induced by CcO dysfunction. The possible CcO dysfunction as a biomarker for cancer progression was supported by data showing that esophageal tumors from human patients show reduced CcO subunits IVi1 and Vb in regions that were previously shown to be hypoxic core of the tumors. Our results show that mitochondrial electron transport chain defect initiates a retrograde signaling. These results suggest that a defect in CcO complex can potentially induce tumor progression.
doi:10.1038/onc.2015.227
PMCID: PMC4703574  PMID: 26148236
8.  WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma 
Carcinogenesis  2015;36(5):598-606.
Summary
WNT10A is upregulated in invasive esophageal tumor cells versus non-invasive cells in 3D-organotypic cultures. Functionally, WNT10A promotes proliferation, migration, invasion and self-renewal. WNT10A is expressed during development, downregulated postnatally and re-expressed during carcinogenesis, where it correlates with poor prognosis.
Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53R175H-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44High/CD24Low population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.
doi:10.1093/carcin/bgv025
PMCID: PMC4498148  PMID: 25795715
9.  EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition 
Cancer Biology & Therapy  2015;16(6):933-940.
There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.
doi:10.1080/15384047.2015.1040959
PMCID: PMC4623069  PMID: 25897987
cancer stem cell; esophageal squamous cell carcinoma; epithelial-mesenchymal transition; EGFR inhibitor; organotypic 3D culture; ZEB1
10.  The role of the autonomic ganglia in atrial fibrillation 
Recent experimental and clinical studies have shown that the epicardial autonomic ganglia play an important role in the initiation and maintenance of atrial fibrillation (AF). In this review, we present the current data on the role of the autonomic ganglia in the pathogenesis of AF and discuss potential therapeutic implications. Experimental studies have demonstrated that acute autonomic remodeling may play a crucial role in AF maintenance in the very early stages. The benefit of adding ablation of the autonomic ganglia to the standard pulmonary vein (PV) isolation procedure for patients with paroxysmal AF is supported by both experimental and clinical data. The interruption of axons from these hyperactive autonomic ganglia to the PV myocardial sleeves may be an important factor in the success of PV isolation procedures. The vagus nerve exerts an inhibitory control over the autonomic ganglia and attenuation or loss of this control may allow these ganglia to become hyperactive. Autonomic neuromodulation using low-level vagus nerve stimulation inhibits the activity of the autonomic ganglia and reverses acute electrical atrial remodeling during rapid atrial pacing and may provide an alternative non-ablative approach for the treatment of AF, especially in the early stages. This notion is supported by a preliminary human study. Further studies are warranted to confirm these findings.
doi:10.1016/j.jacep.2015.01.005
PMCID: PMC4540352  PMID: 26301262
atrial fibrillation; autonomic nervous system; neuromodulation
11.  Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression 
Oncogene  2015;34(41):5229-5239.
Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44Low-CD24High (CD44L) epithelial phenotype to a CD44High-CD24-/Low (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT while excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference (RNAi) and flow cytometric approaches, we find that transforming growth factor (TGF)-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. This data provides novel mechanistic insights into the dynamic expression of SOD2 during EMT. Additionally, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.
doi:10.1038/onc.2014.449
PMCID: PMC4530096  PMID: 25659582
SOD2; MnSOD; epithelial-mesenchymal transition; esophageal squamous cell carcinoma; reactive oxygen species; CD44
12.  Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE) 
PLoS ONE  2016;11(3):e0150968.
Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl2 differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.
doi:10.1371/journal.pone.0150968
PMCID: PMC4798725  PMID: 26992000
13.  ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds 
A polymorphic mutation in the acetaldehyde dehydrogenase 2 (ALDH2) gene has been epidemiologically linked to the high susceptibility to esophageal carcinogenesis for individuals with alcohol use disorders. Mice subjected to alcohol drinking show increased oxidative stress and DNA adduct formation in esophageal epithelia where Aldh2 loss augments alcohol-induced genotoxic effects; however, it remains elusive as to how esophageal epithelial cells with dysfunctional Aldh2 cope with oxidative stress related to alcohol metabolism. Here, we investigated the role of autophagy in murine esophageal epithelial cells (keratinocytes) exposed to ethanol and acetaldehyde. We find that ethanol and acetaldehyde trigger oxidative stress via mitochondrial superoxide in esophageal keratinocytes. Aldh2-deficient cells appeared to be highly susceptible to ethanol- or acetaldehyde-mediated toxicity. Alcohol dehydrogenase-mediated acetaldehyde production was implicated in ethanol-induced cell injury in Aldh2 deficient cells as ethanol-induced oxidative stress and cell death was partially inhibited by 4-methylpyrazole. Acetaldehyde activated autophagy flux in esophageal keratinocytes where Aldh2 deficiency increased dependence on autophagy to cope with ethanol-induced acetaldehyde-mediated oxidative stress. Pharmacological inhibition of autophagy flux by chloroquine stabilized p62/SQSTM1, and increased basal and acetaldehyde-mediate oxidative stress in Aldh2 deficient cells as documented in monolayer culture as well as single-cell derived three-dimensional esophageal organoids, recapitulating a physiological esophageal epithelial proliferation-differentiation gradient. Our innovative approach indicates, for the first time, that autophagy may provide cytoprotection to esophageal epithelial cells responding to oxidative stress that is induced by ethanol and its major metabolite acetaldehyde. Defining autophagymediated cytoprotection against alcohol-induced genotoxicity in the context of Aldh2 deficiency, our study provides mechanistic insights into the tumor suppressor functions of ALDH2 and autophagy in alcohol-related esophageal carcinogenesis.
PMCID: PMC4859883  PMID: 27186430
Autophagy; ALDH2; acetaldehyde; alcohol; tobacco; esophageal squamous cell carcinoma; reactive oxygen species
14.  Low-level transcutaneous electrical vagus nerve stimulation suppresses atrial fibrillation 
BACKGROUND
Transcutaneous low-level tragus electrical stimulation (LLTS) suppresses atrial fibrillation (AF) in canines.
OBJECTIVES
We examined the antiarrhythmic and anti-inflammatory effects of LLTS in humans.
METHODS
Patients with paroxysmal AF who presented for AF ablation, were randomized to either 1 hour of LLTS (n = 20) or sham control (n = 20). Attaching a flat metal clip onto the tragus produced LLTS (20 Hz) in the right ear (50% lower than the voltage slowing the sinus rate). Under general anesthesia, AF was induced by burst atrial pacing at baseline and after 1 hour of LLTS or sham. Blood samples from the coronary sinus and the femoral vein were collected at those time points and then analyzed for inflammatory cytokines, including tumor necrosis factor (TNF)-α and C-reactive protein (CRP), using a multiplex immunoassay.
RESULTS
There were no differences in baseline characteristics between the 2 groups. Pacing-induced AF duration decreased significantly by 6.3 ± 1.9 min compared to baseline in the LLTS group, but not in the controls (p = 0.002 for comparison between groups). AF cycle length increased significantly from baseline by 28.8 ± 6.5 ms in the LLTS group, but not in controls (p = 0.0002 for comparison between groups). Systemic (femoral vein) but not coronary sinus TNF-α and CRP levels decreased significantly only in the LLTS group.
CONCLUSIONS
LLTS suppresses AF and decreases inflammatory cytokines in patients with paroxysmal AF. Our results support the emerging paradigm of neuromodulation to treat AF.
doi:10.1016/j.jacc.2014.12.026
PMCID: PMC4352201  PMID: 25744003
autonomic nervous system; inflammation; neuromodulation
15.  Diagnosis of Human Axillary Osmidrosis by Genotyping of the Human ABCC11 Gene: Clinical Practice and Basic Scientific Evidence 
BioMed Research International  2016;2016:7670483.
The importance of personalized medicine and healthcare is becoming increasingly recognized. Genetic polymorphisms associated with potential risks of various human genetic diseases as well as drug-induced adverse reactions have recently been well studied, and their underlying molecular mechanisms are being uncovered by functional genomics as well as genome-wide association studies. Knowledge of certain genetic polymorphisms is clinically important for our understanding of interindividual differences in drug response and/or disease risk. As such evidence accumulates, new clinical applications and practices are needed. In this context, the development of new technologies for simple, fast, accurate, and cost-effective genotyping is imperative. Here, we describe a simple isothermal genotyping method capable of detecting single nucleotide polymorphisms (SNPs) in the human ATP-binding cassette (ABC) transporter ABCC11 gene and its application to the clinical diagnosis of axillary osmidrosis. We have recently reported that axillary osmidrosis is linked with one SNP 538G>A in the ABCC11 gene. Our molecular biological and biochemical studies have revealed that this SNP greatly affects the protein expression level and the function of ABCC11. In this review, we highlight the clinical relevance and importance of this diagnostic strategy in axillary osmidrosis therapy.
doi:10.1155/2016/7670483
PMCID: PMC4781944  PMID: 27057547
16.  Mechanisms of Barrett’s esophagus: intestinal differentiation, stem cells, and tissue models 
Barrett’s esophagus (BE) is defined as any metaplastic columnar epithelium in the distal esophagus which replaces normal squamous epithelium and which predisposes to cancer development. It is this second requirement, the predisposition to cancer, which makes this condition both clinically highly relevant and an important area for ongoing research. While BE has been defined pathologically since the 1950’s (Allison and Johnstone, Thorax 1955), and identified as a risk factor for esophageal adenocarcinoma since the 1970’s (Naef A.P., et.al. J Thorac Cardiovasc Surg. 1975), our understanding of the molecular events giving rise to this condition remains limited. Herein we will examine what is known about the intestinal features of BE and how well it recapitulates the intestinal epithelium, including stem identity and function. Finally, we will explore laboratory models of this condition presently in use and under development, to identify new insights they may provide into this important clinical condition.
doi:10.1016/j.bpg.2014.11.001
PMCID: PMC4352719  PMID: 25743452
Gastroesophageal reflux disease (GERD); Barrett’s esophagus (BE); esophageal adenocarcinoma (EAC); tissue engineering; human 3D organotypic model systems (OTC); Multi-layered epithelium (MLE); BMP4; Hedgehog; Cdx2; Sox2
17.  Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4 
Cell Cycle  2015;13(24):3857-3866.
Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.
doi:10.4161/15384101.2014.972875
PMCID: PMC4614677  PMID: 25558829
Barrett's esophagus; KLF4; metaplasia; Notch signaling; transdifferentiation
18.  Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities 
Oncogene  2014;34(18):2347-2359.
Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.
doi:10.1038/onc.2014.169
PMCID: PMC4268095  PMID: 24931169
Notch; Rb; p16; HPV; E7; senescence; squamous cell carcinoma
19.  Immature myeloid progenitors promote disease progression in a mouse model of Barrett's-like metaplasia 
Oncotarget  2015;6(32):32980-33005.
Cdx2, an intestine specific transcription factor, is expressed in Barrett's esophagus (BE). We sought to determine if esophageal Cdx2 expression would accelerate the onset of metaplasia in the L2-IL-1β transgenic mouse model for Barrett's-like metaplasia. The K14-Cdx2::L2-IL-1β double transgenic mice had half as many metaplastic nodules as control L2-IL-1β mice. This effect was not due to a reduction in esophageal IL-1β mRNA levels nor diminished systemic inflammation. The diminished metaplasia was due to an increase in apoptosis in the K14-Cdx2::L2-IL-1β mice. Fluorescence activated cell sorting of immune cells infiltrating the metaplasia identified a population of CD11b+Gr-1+ cells that are significantly reduced in K14-Cdx2::L2-IL-1β mice. These cells have features of immature granulocytes and have immune-suppressing capacity. We demonstrate that the apoptosis in K14-Cdx2::L2-IL-1β mice is CD8+ T cell dependent, which CD11b+Gr-1+ cells are known to inhibit. Lastly, we show that key regulators of CD11b+Gr-1+ cell development, IL-17 and S100A9, are significantly diminished in the esophagus of K14-Cdx2::L2-IL-1β double transgenic mice. We conclude that metaplasia development in this mouse model for Barrett's-like metaplasia requires suppression of CD8+ cell dependent apoptosis, likely mediated by immune-suppressing CD11b+Gr-1+ immature myeloid cells.
PMCID: PMC4741744  PMID: 26460825
Barrett's esophagus; myeloid-derived suppressor cells (MDSC); IL-17; S100A9; IL-1β
20.  Protective role of ALDH2 against acetaldehyde-derived DNA damage in oesophageal squamous epithelium 
Scientific Reports  2015;5:14142.
Acetaldehyde is an ethanol-derived definite carcinogen that causes oesophageal squamous cell carcinoma (ESCC). Aldehyde dehydrogenase 2 (ALDH2) is a key enzyme that eliminates acetaldehyde, and impairment of ALDH2 increases the risk of ESCC. ALDH2 is produced in various tissues including the liver, heart, and kidney, but the generation and functional roles of ALDH2 in the oesophagus remain elusive. Here, we report that ethanol drinking increased ALDH2 production in the oesophagus of wild-type mice. Notably, levels of acetaldehyde-derived DNA damage represented by N2-ethylidene-2′-deoxyguanosine were higher in the oesophagus of Aldh2-knockout mice than in wild-type mice upon ethanol consumption. In vitro experiments revealed that acetaldehyde induced ALDH2 production in both mouse and human oesophageal keratinocytes. Furthermore, the N2-ethylidene-2′-deoxyguanosine levels increased in both Aldh2-knockout mouse keratinocytes and ALDH2-knockdown human keratinocytes treated with acetaldehyde. Conversely, forced production of ALDH2 sharply diminished the N2-ethylidene-2′-deoxyguanosine levels. Our findings provide new insight into the preventive role of oesophageal ALDH2 against acetaldehyde-derived DNA damage.
doi:10.1038/srep14142
PMCID: PMC4570974  PMID: 26374466
21.  Impact of Contact Force Technology on Atrial Fibrillation Ablation: A Meta-Analysis 
Background
Catheter–tissue contact is essential for effective lesion formation, thus there is growing usage of contact force (CF) technology in atrial fibrillation ablation. We conducted a meta-analysis to assess the impact of CF on clinical outcomes and procedural parameters in comparison to conventional catheter for atrial fibrillation ablation.
Methods and Results
An electronic search was performed using major databases. Outcomes of interest were recurrence rate, major complications, total procedure, and fluoroscopic times. Continuous variables were reported as standardized mean difference; odds ratios were reported for dichotomous variables. Eleven studies (2 randomized controlled studies and 9 cohorts) involving 1428 adult patients were identified. CF was deployed in 552 patients. The range of CF used was between 2 to 60 gram-force. The follow-up period ranged between 10 and 53 weeks. In comparing CF and conventional catheter groups, the recurrence rate was lower with CF (35.1% versus 45.5%, odds ratio 0.62 [95% CI 0.45–0.86], P=0.004). Shorter procedure and fluoroscopic times were achieved with CF (procedure time: 156 versus 173 minutes, standardized mean difference −0.85 [95% CI −1.48 to −0.21], P=0.009; fluoroscopic time: 28 versus 36 minutes, standardized mean difference −0.94 [95% CI −1.66; −0.21], P=0.01). Major complication rate was lower numerically in the CF group but not statistically significant (1.3% versus 1.9%, odds ratio 0.71 [95% CI 0.29–1.73], P=0.45).
Conclusions
The use of CF technology results in significant reduction of the atrial fibrillation recurrence rate after atrial fibrillation ablation in comparison to the conventional catheter group. CF technology is able to significantly reduce procedure and fluoroscopic times without compromising complication rate.
doi:10.1161/JAHA.115.002476
PMCID: PMC4599513  PMID: 26391136
ablation; atrial fibrillation; contact force; meta-analysis
22.  PRMT5 is required for lymphomagenesis triggered by multiple oncogenic drivers 
Cancer discovery  2015;5(3):288-303.
Protein arginine transferase 5(PRMT5) has been implicated as a key modulator of lymphomagenesis. Whether PRMT5 has overt oncogenic function in the context of leukemia/lymphoma and whether it represents a therapeutic target remains to be established. We demonstrate that inactivation of PRMT5 inhibits colony-forming activity by multiple oncogenic-drivers including cyclin D1, c-MYC, NOTCH1 and MLL-AF9. Furthermore, we demonstrate that PRMT5 overexpression specifically cooperates with cyclin D1 to drive lymphomagenesis in a mouse model revealing inherent neoplastic activity. Molecular analysis of lymphomas, revealed that arginine methylation of p53 selectively suppresses expression of crucial proapoptotic and anti-proliferative target genes thereby sustaining tumor cell self-renewal and proliferation and bypassing the need for the acquisition of inactivating p53 mutations. Critically, analysis of human tumor specimen reveal a strong correlation between cyclin D1 overexpression and p53 methylation supporting the biomedical relevance of this pathway.
doi:10.1158/2159-8290.CD-14-0625
PMCID: PMC4355177  PMID: 25582697
Cyclin D1; CDK4; PRMT5; MEP50; Arginine Methylation
23.  Novel 5-fluorouracil-resistant human esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase overexpression 
American Journal of Cancer Research  2015;5(8):2431-2440.
5-Fluorouracil (5-FU) is a key drug for the treatment of esophageal squamous cell carcinoma (ESCC); however, resistance to it remains a critical limitation to its clinical use. To clarify the mechanisms of 5-FU resistance of ESCC, we originally established 5-FU-resistant ESCC cells, TE-5R, by step-wise treatment with continuously increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold more resistant to 5-FU in comparison with parental TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation. 5-FU treatment resulted in a significant decrease of the intracellular 5-FU concentration and increase of the concentration of α-fluoro-ureidopropionic acid (FUPA), a metabolite of 5-FU, in TE-5R compared with TE-5 cells in vitro. Conversely, gimeracil, a DPD inhibitor, markedly increased the intracellular 5-FU concentration, decreased the intracellular FUPA concentration, and attenuated 5-FU resistance of TE-5R cells. These results indicate that 5-FU resistance of TE-5R cells is due to the rapid degradation of 5-FU by DPD overexpression. The investigation of 5-FU-resistant ESCC with DPYD gene copy number amplification and consequent DPD overexpression may generate novel biological evidence to explore strategies against ESCC with 5-FU resistance.
PMCID: PMC4568778  PMID: 26396918
Esophageal squamous cell carcinoma; chemotherapy; 5-fluorouracil; drug resistance; dihydropyrimidine dehydrogenase
24.  Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition 
Experimental cell research  2014;330(1):102-110.
Background and Aims
Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro.
Methods and Results
Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production.
Conclusions
Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis.
doi:10.1016/j.yexcr.2014.08.026
PMCID: PMC4489692  PMID: 25183431
25.  BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis 
The Journal of Clinical Investigation  2015;125(4):1557-1568.
Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett’s intestinal differentiation; however, in mice, basal progenitor cell–specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.
doi:10.1172/JCI78850
PMCID: PMC4396468  PMID: 25774506
Gastroenterology; Stem cells

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