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1.  Molecular Mechanism Underlying Lymphatic Metastasis in Pancreatic Cancer 
BioMed Research International  2014;2014:925845.
As the most challenging human malignancies, pancreatic cancer is characterized by its insidious symptoms, low rate of surgical resection, high risk of local invasion, metastasis and recurrence, and overall dismal prognosis. Lymphatic metastasis, above all, is recognized as an early adverse event in progression of pancreatic cancer and has been described to be an independent poor prognostic factor. It should be noted that the occurrence of lymphatic metastasis is not a casual or stochastic but an ineluctable and designed event. Increasing evidences suggest that metastasis-initiating cells (MICs) and the microenvironments may act as a double-reed style in this crime. However, the exact mechanisms on how they function synergistically for this dismal clinical course remain largely elusive. Therefore, a better understanding of its molecular and cellular mechanisms involved in pancreatic lymphatic metastasis is urgently required. In this review, we will summarize the latest advances on lymphatic metastasis in pancreatic cancer.
doi:10.1155/2014/925845
PMCID: PMC3919106  PMID: 24587996
2.  Aggressive vertebral hemangioma of the thoracic spine without typical radiological appearance 
European Spine Journal  2012;21(10):1994-1999.
Purpose
Vertebral hemangioma (VH) is virtually vascular malformation, which is usually asymptomatic. Only 3.7 % of VH may become active and symptomatic, and 1 % may invade the spinal canal and/or paravertebral space. Treatment protocols for active or aggressive VHs are still in controversy. Reported treatments include radiotherapy, vertebroplasty, direct alcohol injection, embolization, surgery and a combination of these modalities.
Methods
A 41-year-old lady was presented with 18 month history of intermittent back pain. CT revealed T5 osteolytic lesion with epidural and paravertebral extension. The first CT guided biopsy yielded little information.
Results
Histopathological diagnosis of the second biopsy was VH. Vertebroplasty, posterior decompression and fixation were performed followed by postoperative radiotherapy. Her symptoms were resolved immediately after the operation. At 12 months follow-up, no recurrence was detected by CT with contrast enhancement.
Conclusion
Surgical decompression, vertebroplasty and fixation are safe and effective for aggressive VH. More attention is needed in determining the algorithm for the diagnosis and treatment of aggressive VH.
doi:10.1007/s00586-012-2349-1
PMCID: PMC3463694  PMID: 22732826
Vertebral hemangioma; Diagnosis; Radiotherapy; Vertebroplasty; Surgery
4.  Curcumin prevents and reverses murine cardiac hypertrophy 
Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase–dependent (p300-HAT–dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT–dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-κB, and TGF-β–Smad signaling pathways.
doi:10.1172/JCI32865
PMCID: PMC2248327  PMID: 18292803
5.  Id1 expression promotes peripheral CD4+ T cell proliferation and survival upon TCR activation without co-stimulation 
Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4+ cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.
doi:10.1016/j.bbrc.2013.05.054
PMCID: PMC3955101  PMID: 23707719
Inhibitor of differentiation; E protein; CD4+ T cell; co-stimulation; T cell activation
7.  DNA Aptamer-mediated Cell Targeting 
One important issue using cells as therapeutics is targeted delivery. Engineering cell surfaces to improve delivery efficiency is thus of great interest. Here we report a simple, efficient and effective way to modify the cell surface with target-specific ligands, i.e., DNA aptamers, while minimizing the effects on the modified cells. We demonstrated that after incubating with lipo-aptamer probes (shown in expansion), immune cells (red) recognize cancer cells (blue) in the cell mixture, and kill cancer cells.
doi:10.1002/anie.201207063
PMCID: PMC3793636  PMID: 23233389
DNA aptamer; cell modification; cell targeting; cell-based immunotherapy
8.  Mechanism of faster NO scavenging by older stored red blood cells☆ 
Redox Biology  2014;2:211-219.
The blood storage lesion involves morphological and biochemical changes of red blood cells (RBCs) that occur during storage. These include conversion of the biconcave disc morphology to a spherical one, decreased mean corpuscular hemoglobin concentration, varied mean corpuscular volume, reduced integrity of the erythrocyte membrane with formation of microparticles, and increased cell-free hemoglobin. We studied the extent that older stored red blood cells scavenge nitric oxide (NO) faster than fresher stored red blood cells. Using electron paramagnetic resonance spectroscopy and stopped-flow absorption spectroscopy to measure the rate of NO uptake and reaction with hemoglobin in red cells, we found that older stored red blood cells scavenge NO about 1.8 times faster than fresher ones. Based on these experimental data, we simulated NO scavenging by fresher or older stored red blood cells with a biconcave or spherical geometry, respectively, in order to explore the mechanism of NO scavenging related to changes that occur during blood storage. We found that red blood cells with a spherical geometry scavenges NO about 2 times slower than ones with a biconcave geometry, and a smaller RBC hemoglobin concentration or volume increases NO scavenging by red blood cells. Our simulations demonstrate that even the most extreme possible changes in mean corpuscular hemoglobin concentration and mean corpuscular volume that favor increased NO scavenging are insufficient to account for what is observed experimentally. Therefore, RBC membrane permeability must increase during storage and we find that the permeability is likely to increase between 5 and 70 fold. Simulations using a two-dimensional blood vessel show that even a 5-fold increase in membrane permeability to NO can reduce NO bioavailability at the smooth muscle.
Background
Transfusion of older stored blood may be harmful.
Results
Older stored red blood cells scavenge nitric oxide more than fresher cells.
Conclusion
As stored red blood cells age, structural and biochemical changes occur that lead to faster scavenging.
Significance
Increased nitric oxide scavenging by red blood cells as a function of storage age contributes to deleterious effects upon transfusion.
Graphical abstract
Highlights
•Older stored red blood cells scavenge NO faster.•The biconcave geometry of fresh red cells favors faster NO scavenging.•A smaller RBC MCHC or MCV leads to increased NO scavenging.•RBC membrane permeability to NO needs to increase in order to explain our experimental results.•Even a 5-fold increase of RBC membrane permeability to NO can reduce NO bioavailability.
doi:10.1016/j.redox.2013.12.014
PMCID: PMC3909782  PMID: 24494195
NO, nitric oxide; RBC, red blood cell; Hb, hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular hemoglobin volume; PBS, phosphate buffered saline; MetHb, methemoglobin; Nitric oxide; Erythrocyte; Blood; Kinetics; Electron paramagnetic resonance (EPR); Blood storage lesion
9.  Serotonin 2C receptors in pro-opiomelanocortin neurons regulate energy and glucose homeostasis 
The Journal of Clinical Investigation  2013;123(12):5061-5070.
Energy and glucose homeostasis are regulated by central serotonin 2C receptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2C receptor–expressing neurons that mediate the effects of serotonin and serotonin 2C receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking serotonin 2C receptors (Htr2c) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of serotonin 2C receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2c loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that serotonin 2C receptor–expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of serotonin 2C receptor agonists on weight-loss induction and improved glycemic control.
doi:10.1172/JCI70338
PMCID: PMC3859401  PMID: 24177424
10.  Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin★ 
Molecular Metabolism  2013;3(1):64-72.
The hormone ghrelin stimulates eating and helps maintain blood glucose upon caloric restriction. While previous studies have demonstrated that hypothalamic arcuate AgRP neurons are targets of ghrelin, the overall relevance of ghrelin signaling within intact AgRP neurons is unclear. Here, we tested the functional significance of ghrelin action on AgRP neurons using a new, tamoxifen-inducible AgRP-CreERT2 transgenic mouse model that allows spatiotemporally-controlled re-expression of physiological levels of ghrelin receptors (GHSRs) specifically in AgRP neurons of adult GHSR-null mice that otherwise lack GHSR expression. AgRP neuron-selective GHSR re-expression partially restored the orexigenic response to administered ghrelin and fully restored the lowered blood glucose levels observed upon caloric restriction. The normalizing glucoregulatory effect of AgRP neuron-selective GHSR expression was linked to glucagon rises and hepatic gluconeogenesis induction. Thus, our data indicate that GHSR-containing AgRP neurons are not solely responsible for ghrelin's orexigenic effects but are sufficient to mediate ghrelin's effects on glycemia.
doi:10.1016/j.molmet.2013.10.001
PMCID: PMC3929914  PMID: 24567905
GHSR, growth hormone secretagogue receptor, ghrelin receptor; GOAT, ghrelin O-acyltransferase; CNS, central nervous system; Phox2b, paired-like homeobox 2b; AgRP, Agouti-related peptide; NPY, neuropeptide Y; GABA, gamma-aminobutyric acid; VGAT, vesicular GABA transporter; POMC, pro-opiomelanocortin; ARC, arcuate nucleus; BAC, bacterial artificial chromosome; VTA, ventral tegmental area; DG, dentate gyrus; NAc, nucleus accumbens; DVC, dorsal vagal complex; G6p, glucose-6 phosphatase; Pepck, phosphoenolpyruvate carboxykinase; Hnf4α, hepatocyte nuclear factor 4α; Foxo1, Forkhead box protein O1; Pcx, pyruvate carboxylase; GHRH, Growth-hormone-releasing hormone; AgRP; Ghrelin; Ghrelin receptor; Food intake; Blood glucose homeostasis
11.  Identification of c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland 
The CD117 (KIT) protein is overexpressed in many human neoplasms including adenoid cystic carcinoma of salivary glands. To evaluate the function of c-kit-activating mutations in adenoid cystic carcinoma of the salivary gland, we studied 14 cases (13 primary, 1 cervical lymph node metastasis) from our institution. KIT protein expression was evaluated by immunohistochemistry using formalin-fixed paraffin-embedded tissue. Mutational analyses of c-kit extracellular (exon 9), juxtamembrane (exon 11) and tyrosine kinase domains (exons 13 and 17) were performed by polymerase chain reaction, clonal selection and DNA sequencing. All 14 cases demonstrated strong KIT expression by immunohistochemistry. Molecular analysis was successful in 8 of 14 cases, and c-kit missense point mutations were detected in seven of eight cases (88%) including seven in exon 11, two in exon 9, two in exon 13 and two in exon 17. Eight silent point mutations were detected in five cases. Two cases contained missense mutations in more than one exon. Different mutations were found in the primary tumor and the cervical lymph node metastasis of one patient. Point mutations in domains similar to those described in gastrointestinal stromal tumors were detected, including Pro551Leu and Lys558Glu (5′ end of exon 11), Leu576Phe (3′ end of exon 11), Val643Ala (exon 13) and Asn822Ser (exon 17). Additional novel point mutations in exons 9, 11, 13 and 17 were also identified. This study is the first to report c-kit gene mutations in primary adenoid cystic carcinoma of the salivary gland. Identification of such potential gain-of-function mutations in exon 11, and less frequently in exons 9, 13 and 17, suggests that KIT may be involved in the pathogenesis of adenoid cystic carcinoma of salivary glands. Our study raises a prospect of correlation of c-kit mutation and a potential treatment of adenoid cystic carcinoma with tyrosine kinase inhibitor (imatinib).
doi:10.1038/modpathol.2009.95
PMCID: PMC3746033  PMID: 19617878
adenoid cystic carcinoma; c-kit gene mutations; KIT protein; salivary gland
12.  Disruption of mindin exacerbates cardiac hypertrophy and fibrosis 
Cardiac hypertrophy is a response of the myocardium to increased workload and is characterised by an increase of myocardial mass and an accumulation of extracellular matrix (ECM). As an ECM protein, an integrin ligand, and an angiogenesis inhibitor, all of which are key players in cardiac hypertrophy, mindin is an attractive target for therapeutic intervention to treat or prevent cardiac hypertrophy and heart failure. In this study, we investigated the role of mindin in cardiac hypertrophy using littermate Mindin knockout (Mindin−/−) and wild-type (WT) mice. Cardiac hypertrophy was induced by aortic banding (AB) or angiotensin II (Ang II) infusion in Mindin−/− and WT mice. The extent of cardiac hypertrophy was quantitated by echocardiography and by pathological and molecular analyses of heart samples. Mindin−/− mice were more susceptible to cardiac hypertrophy and fibrosis in response to AB or Ang II stimulation than wild type. Cardiac function was also markedly exacerbated during both systole and diastole in Mindin−/− mice in response to hypertrophic stimuli. Western blot assays further showed that the activation of AKT/glycogen synthase kinase 3β (GSK3β) signalling in response to hypertrophic stimuli was significantly increased in Mindin−/− mice. Moreover, blocking AKT/GSK3β signalling with a pharmacological AKT inhibitor reversed cardiac abnormalities in Mindin−/− mice. Our data show that mindin, as an intrinsic cardioprotective factor, prevents maladaptive remodelling and the transition to heart failure by blocking AKT/GSK3β signalling.
doi:10.1007/s00109-012-0883-2
PMCID: PMC3734934  PMID: 22367478
Mindin; Hypertrophy; Remodelling; Signal transduction; AKT
13.  Intratumoral α-SMA Enhances the Prognostic Potency of CD34 Associated with Maintenance of Microvessel Integrity in Hepatocellular Carcinoma and Pancreatic Cancer 
PLoS ONE  2013;8(8):e71189.
Microvessel density (MVD) as an angiogenesis predictor is inefficient per se in cancer prognosis. We evaluated prognostic values of combining intratumoral alpha-smooth muscle actin (α-SMA)-positive stromal cell density and MVD after curative resection in hypervascular hepatocellular carcinoma (HCC) and hypovascular pancreatic cancer (PC). Tissue microarrays were constructed from tumors of 305 HCC and 57 PC patients who underwent curative resection and analyzed for α-SMA and CD34 expression by immunostaining. Prognostic values of these two proteins and other clinicopathological features were examined. Both low α-SMA density and high MVD-CD34 were associated in HCC with the presence of intrahepatic metastasis and microvascular invasion, and they were related to lymph node involvement and microvascular invasion in PC (p<0.05). Although CD34 alone, but not α-SMA, was an independent prognostic factor for overall survival and recurrence-free survival, the combination of low α-SMA and high CD34 was a predictor of worst prognosis for both types of tumors and had a better power to predict patient death and early recurrence (p<0.01). Furthermore, the results show that distribution of most of the α-SMA-positive cells and vascular endothelial cells overlap, showing major colocalization on vascular walls. Poor microvessel integrity, as indicated by high MVD, together with low perivascular α-SMA-positive cell coverage is associated with early recurrence, unfavorable metastasis, and short survival after tumor resection. This finding highlights the significance of vascular quality in tumor progression, which provides an optimized complement to vascular quantity in prognosis of postoperative patients.
doi:10.1371/journal.pone.0071189
PMCID: PMC3734294  PMID: 23940715
14.  Application of “Papillary-Like Main Pancreatic Duct Invaginated” Pancreaticojejunostomy for Normal Soft Pancreas Cases 
Scientific Reports  2013;3:2068.
Pancreaticojejunostomy is the key procedure of pancreaticoduodenectomy. Our study introduced a new pancreaticojejunal (PJ) anastomosis named “papillary-like main pancreatic duct invaginated” pancreaticojejunostomy. Nighty-two patients underwent pancreaticojejunostomy with either conventional duct-to-mucosa pancreaticojejunostomy or the new “papillary-like main pancreatic duct invaginated” techniques were analyzed retrospectively from January 2010 to September 2012. The incidence of pancreatic fistula was 15.7% (8/51) for the “papillary-like main pancreatic duct invaginated” group and 19.5% (8/41) for the duct-to-mucosa fashion respectively. It is noteworthy that the rate of grade B/C postoperative pancreatic fistula (POPF) in the “papillary-like main pancreatic duct invaginated” group was significantly lower than that of the duct-to-mucosa group (P = 0.039). There were no differences in the incidence of postoperative morbidity and mortality such as postoperative hemorrhage, delayed gastric emptying or remnant pancreatitis. The “papillary-like main pancreatic duct invaginated” pancreaticojejunostomy could provide a feasible option to pancreatic surgeons for patients with normal soft pancreas.
doi:10.1038/srep02068
PMCID: PMC3691567  PMID: 23797701
15.  2-Octynoic Acid Inhibits Hepatitis C Virus Infection through Activation of AMP-Activated Protein Kinase 
PLoS ONE  2013;8(5):e64932.
Many chronic hepatitis C virus (HCV)-infected patients with current therapy do not clear the virus. It is necessary to find novel treatments. The effect of 2-octynoic acid (2-OA) on HCV infection in human hepatocytes was examined. The mechanism of 2-OA antiviral activity was explored. Our data showed that 2-OA abrogated lipid accumulation in HCV replicon cells and virus-infected hepatocytes. It suppressed HCV RNA replication and infectious virus production with no cytotoxicity to the host cells. 2-OA did not affect hepatitis B virus replication in HepG2.2.15 cells derived from HepG2 cells transfected with full genome of HBV. Further study demonstrated that 2-OA activated AMP-activated protein kinase (AMPK) and inhibited acetyl-CoA carboxylase in viral-infected cells. Compound C, a specific inhibitor of AMPK, inhibited AMPK activity and reversed the reduction of intracellular lipid accumulation and the antiviral effect of 2-OA. Knockdown of AMPK expression by RNA interference abolished the activation of AMPK by 2-OA and blocked 2-OA antiviral activity. Interestingly, 2-OA induced interferon-stimulated genes (ISGs) and inhibited microRNA-122 (miR-122) expression in virus-infected hepatocytes. MiR-122 overexpression reversed the antiviral effect of 2-OA. Furthermore, knockdown of AMPK expression reversed both the induction of ISGs and suppression of miR-122 by 2-OA, implying that activated AMPK induces the intracellular innate response through the induction of ISGs and inhibiting miR-122 expression. 2-OA inhibits HCV infection through regulation of innate immune response by activated AMPK. These findings reveal a novel mechanism by which active AMPK inhibits HCV infection. 2-OA and its derivatives hold promise for novel drug development for chronic hepatitis C.
doi:10.1371/journal.pone.0064932
PMCID: PMC3669134  PMID: 23741428
16.  Oxidized Low-Density Lipoprotein Cholesterol and the Ratio in the Diagnosis and Evaluation of Therapeutic Effect In Patients with Coronary Artery Disease 
Disease markers  2012;33(6):295-302.
Objective: The purpose of the present study was to investigate the value of ox-LDL and oxidation ratio of LDL (ox-LDL/TC, ox-LDL/HDL-C and ox-LDL/LDL-C) in diagnosis and prognosis evaluation in CAD patients. Also, we aimed to observe the effect of statins on reducing level of ox-LDL and oxidation ratio of LDL, and explore whether statins still have similar effect on ox-LDL in a short period of therapy (within 2 weeks).
Methods: Blood ox-LDL, TC, HDL-C, LDL-C, and TG were measured in cases with acute myocardial infarction (AMI, n = 177), unstable angina pectoris (UAP, n = 195), stable angina pectoris (SAP, n = 228), normal control (n = 120), and high risk control (n = 140).
Results: Mean value of ox-LDL and oxidation ratio of LDL was significantly higher in the CAD group than in the two control groups. The AUC of ROC curve of ox-LDL, ox-LDL/TC, ox-LDL/HDL-C, ox-LDL/LDL-C and apoA1/apoB were more than 0.50 (P < 0.001). Multivariate logistic regression analysis showed that age and ox-LDL/LDL-C related with short-term, while ox-LDL/LDL-C and ox-LDL/TC related with long-term prognosis (P < 0.05). Furthermore, after treatment with statins for 2 weeks, TC, LDL-C, ox-LDL, ox-LDL/TC, ox-LDL/HDL-C and ox-LDL/LDL-C decreased by 22%, 28%, 38%, 29%, 23% and 25% respectively. And the reduction of ox-LDL by statins is independent of lowering of LDL-C and TC.
Conclusions: Ox-LDL and oxidation ratio of LDL are closely related with AS, and they are better biomarkers for discriminating between patients with coronary artery disease and healthy subjects. In addition, statins can decrease level of ox-LDL significantly, which is independent of lowering of LDL-C and TC.
doi:10.3233/DMA-2012-00941
PMCID: PMC3810697  PMID: 23089925
Coronary artery disease; oxidized low density lipoprotein; diagnosis; prognosis
17.  MicroRNA-450a-3p Represses Cell Proliferation and Regulates Embryo Development by Regulating Bub1 Expression in Mouse 
PLoS ONE  2012;7(10):e47914.
Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3′-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.
doi:10.1371/journal.pone.0047914
PMCID: PMC3478270  PMID: 23110129
18.  Iron Regulator Hepcidin Exhibits Antiviral Activity against Hepatitis C Virus 
PLoS ONE  2012;7(10):e46631.
Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.
doi:10.1371/journal.pone.0046631
PMCID: PMC3478283  PMID: 23110054
19.  Cardiac-specific mindin overexpression attenuates cardiac hypertrophy via blocking AKT/GSK3β and TGF-β1–Smad signalling 
Cardiovascular Research  2011;92(1):85-94.
Aims
Mindin is a secreted extracellular matrix protein, an integrin ligand, and an angiogenesis inhibitor, other examples of which are all key players in the progression of cardiac hypertrophy. However, its function during cardiac hypertrophy remains unclear. This study was aimed to identify the effect of mindin on cardiac hypertrophy and the underlying mechanisms.
Methods and results
A significant down-regulation of mindin expression was observed in human failing hearts. To further investigate the role of mindin in cardiac hypertrophy, we used cultured neonatal rat cardiomyocytes with gain and loss of mindin function and cardiac-specific Mindin-overexpressing transgenic (TG) mice. In cultured cardiomyocytes, mindin negatively regulated angiotensin II (Ang II)-mediated hypertrophic growth, as detected by [3H]-Leucine incorporation, cardiac myocyte area, and hypertrophic marker protein levels. Cardiac hypertrophy in vivo was produced by aortic banding (AB) or Ang II infusion in TG mice and their wild-type controls. The extent of cardiac hypertrophy was evaluated by echocardiography as well as by pathological and molecular analyses of heart samples. Mindin overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and left ventricular dysfunction in mice in response to AB or Ang II. Further analysis of the signalling events in vitro and in vivo indicated that these beneficial effects of mindin were associated with the interruption of AKT/glycogen synthase kinase 3β (GSK3β) and transforming growth factor (TGF)-β1–Smad signalling.
Conclusion
The present study demonstrates for the first time that mindin serves as a novel mediator that protects against cardiac hypertrophy and the transition to heart failure by blocking AKT/GSK3β and TGF-β1–Smad signalling.
doi:10.1093/cvr/cvr159
PMCID: PMC3657538  PMID: 21632881
Mindin; Hypertrophy; Remodelling; Signal transduction; AKT
20.  Biodegradation, Biocompatibility, and Drug Delivery in Poly(ω-pentadecalactone-co-p-dioxanone) Copolyesters 
Biomaterials  2011;32(27):6646-6654.
Poly(ω-pentadecalactone-co-p-dioxanone) [poly(PDL-co-DO)] copolyesters are copolymers of an isodimorphic system, which remain semicrystalline over the whole range of compositions. Here, we evaluated enzymatically synthesized poly(PDL-co-DO) copolymers as new materials for biomedical applications. In vivo experiments using mice showed that the copolyesters are well tolerated, with tissue responses that are comparable to poly(p-dioxanone). In addition, the copolymers were found to degrade hydrolytically at controlled rates over a period of several months under physiological conditions. The poly(PDL-co-DO) copolymers with up to 69 mol% DO units were successfully transformed to free-standing nanoparticles that are capable of encapsulating an anticancer drug, doxorubicin, or a polynucleotide, siRNA. Drug- or siRNA-loaded nanoparticles exhibited controlled and continuous release of agent over many weeks. In addition, siLUC-encapsulated poly(PDL-co-DO) nanoparticles were active in inhibiting luciferase gene expression in LUC-RKO cells. Because of substantial differences in structure and hydrophobicity between PDL and DO units, poly(PDL-co-DO) biodegradation rate and physical properties can be tuned over a wide range depending on the copolymer composition. Our results demonstrate that the semicrystalline and biodegradable poly(PDL-co-DO) copolyesters are promising biomaterials to serve as drug carriers, as well as potential raw materials for constructing bioabsorbable sutures and other medical devices.
doi:10.1016/j.biomaterials.2011.05.046
PMCID: PMC3133836  PMID: 21641030
Poly(ω-pentadecalactone-co-p-dioxanone); doxorubicin; siRNA; biodegradable nanoparticles; drug delivery
21.  Targeted Delivery of Chemotherapy Agents Using a Liver Cancer-Specific Aptamer 
PLoS ONE  2012;7(4):e33434.
Background
Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.
Methodology/Principal Findings
In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.
Conclusions/Significance
Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.
doi:10.1371/journal.pone.0033434
PMCID: PMC3338807  PMID: 22558072
22.  Innate Host Response in Primary Human Hepatocytes with Hepatitis C Virus Infection 
PLoS ONE  2011;6(11):e27552.
Background and Aim
The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.
Methods
An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.
Results
Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.
Conclusion
Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection.
doi:10.1371/journal.pone.0027552
PMCID: PMC3210809  PMID: 22087337
23.  Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells 
PLoS ONE  2011;6(6):e21186.
Background and Aim
The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.
Methods
The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.
Results
HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.
Conclusion
These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection.
doi:10.1371/journal.pone.0021186
PMCID: PMC3117876  PMID: 21695051
24.  Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells 
PLoS ONE  2011;6(2):e16966.
This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.
doi:10.1371/journal.pone.0016966
PMCID: PMC3037406  PMID: 21347335
25.  NOD2 regulates hematopoietic cell function during graft-versus-host disease 
The Journal of Experimental Medicine  2009;206(10):2101-2110.
Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2−/− hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function.
doi:10.1084/jem.20090623
PMCID: PMC2757869  PMID: 19737867

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