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2.  Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma 
PLoS ONE  2016;11(1):e0145807.
Background
Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC).
Methods
In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined.
Results
Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient’s age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRβ, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRβ (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers.
Conclusion
Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.
doi:10.1371/journal.pone.0145807
PMCID: PMC4701415  PMID: 26731558
3.  Inhibition of Oct 3/4 mitigates the cardiac progenitor-derived myocardial repair in infarcted myocardium 
Background
Recent evidence has demonstrated that cardiac progenitor cells play an essential role in the induction of angiomyogenesis in infarcted myocardium. We and others have shown that engraftment of c-kit+ cardiac stem cells (CSCs) into infarcted hearts led to myocardium regeneration and neovascularization, which was associated with an improvement of ventricular function. The purpose of this study is aimed at investigating the functional role of transcription factor (TF) Oct3/4 in facilitating CSCs to promote myocardium regeneration and preserve cardiac performance in the post-MI heart.
Methods
c-kit+ CSCs were isolated from adult hearts and re-introduced into the infarcted myocardium in which the mouse MI model was created by permanent ligation of the left anterior descending artery (LAD). The Oct3/4 of CSCs was inhibited by transfection of Oct3/4 siRNA, and transfection of CSCs with control siRNA serves as control groups. Myocardial functions were evaluated by echocardiographic measurement. Histological analysis was employed to assess newly formed cardiogenesis, neovascularization, and cell proliferations. Terminal deoxynucleotidyltransferase (TdT) nick-end labeling (TUNEL) was carried out to assess apoptotic cardiomyocytes. Real time polymerase chain reaction and Western blot were carried out to evaluate the level of Oct 3/4 in CSCs.
Results
Two weeks after engraftment, CSCs increased ventricular functional recovery as shown by a serial echocardiographic measurement, which is concomitant with the suppression of cardiac hypertrophy and attenuation of myocardial interstitial fibrosis. Suppression of Oct 3/4 of CSCs abrogated functional improvements and mitigated the hypertrophic response and cardiac remodeling. Transplantation of c-kit+ CSCs into MI hearts promoted cardiac regeneration and neovascularization, which were abolished with the knockdown of Oct3/4. Additionally, suppression of Oct3/4 abrogated myocyte proliferation in the CSC-engrafted myocardium.
Conclusion
Our results indicate that CSCs-derived cardiac regeneration improves the restoration of cardiac function and is mediated through Oct 3/4.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-015-0252-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13287-015-0252-5
PMCID: PMC4690244  PMID: 26704423
Oct3/4; Regeneration; Myocardial infarction; Stem cells
4.  Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability 
ChemMedChem  2014;9(12):2653-2660.
We report herein the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective dopamine-3 (D3) receptor agonists. A number of these new compounds bind to the D3 receptor with subnanomolar affinities and show excellent selectivity (>10,000) for the D3 receptor over the D1 and D2 receptors. Compound 23 for example, binds to the D3 receptor with a Ki value of 0.53 nM and shows a selectivity of >20,000 over the D2 receptor and the D1 receptor in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes and in vitro functional assays showed it to be a full agonist for the human D3 receptor.
doi:10.1002/cmdc.201402398
PMCID: PMC4277207  PMID: 25338762
pramipexole derivatives; dopamine 3; receptor; agonists; microsomal stability
5.  E2F1 suppresses cardiac neovascularization by down-regulating VEGF and PlGF expression 
Cardiovascular Research  2014;104(3):412-422.
Aims
The E2F transcription factors are best characterized for their roles in cell-cycle regulation, cell growth, and cell death. Here we investigated the potential role of E2F1 in cardiac neovascularization.
Methods and results
We induced myocardial infarction (MI) by ligating the left anterior descending artery in wild-type (WT) and E2F1−/− mice. E2F1−/− mice demonstrated a significantly better cardiac function and smaller infarct sizes than WT mice. At infarct border zone, capillary density and endothelial cell (EC) proliferation were greater, apoptotic ECs were fewer, levels of VEGF and placental growth factor (PlGF) were higher, and p53 level was lower in E2F1−/− than in WT mice. Blockade of VEGF receptor 2 (VEGFR2) signalling with the selective inhibitor SU5416 or with the VEGFR2-blocking antibody DC101 abolished the differences between E2F1−/− mice and WT mice in cardiac function, infarct size, capillary density, EC proliferation, and EC apoptosis. In vitro, hypoxia-induced VEGF and PlGF up-regulation was significantly greater in E2F1−/− than in WT cardiac fibroblasts, and E2F1 overexpression suppressed PlGF up-regulation in both WT and p53−/− cells; however, VEGF up-regulation was suppressed only in WT cells. E2F1 interacted with and stabilized p53 under hypoxic conditions, and both E2F1 : p53 binding and the E2F1-induced suppression of VEGF promoter activity were absent in cells that expressed an N-terminally truncated E2F1 mutant.
Conclusion
E2F1 limits cardiac neovascularization and functional recovery after MI by suppressing VEGF and PlGF up-regulation through p53-dependent and -independent mechanisms, respectively.
doi:10.1093/cvr/cvu222
PMCID: PMC4296109  PMID: 25341896
E2F1; VEGF; PlGF; p53; Heart
6.  Histone Deacetylase 1/2 Mediates Proliferation of Renal Interstitial Fibroblasts and Expression of Cell Cycle Proteins 
Journal of cellular biochemistry  2011;112(8):2138-2148.
We recently reported that the histone deacetylase (HDAC) activity is required for activation of renal interstitial fibroblasts. In this study, we further examined the role of HDACs, in particular, HDAC1 and HDAC2, in proliferation of cultured rat renal interstitial fibroblasts (NRK-49F) and expression of cell cycle proteins. Inhibition of HDAC activity with trichostatin A (TSA), blocked cell proliferation, decreased expression of cyclin D1, a positive cell cycle regulator, and increased expression of p27 and p57, two negative cell cycle regulators. Silencing either HDAC1 or HDAC2 with siRNA also significantly inhibited cell proliferation, decreased expression of cyclin D1, and increased expression of p57. However, down-regulation of HDAC2, but not HDAC1 resulted in increased expression of p27. Furthermore, HDAC1 and HDAC2 downregulaton was associated with dephosphorylation and hyperacetylation of STAT3 (signal transducer and activator of transcription 3). Blockade of STAT3 with S3I-201 or siRNA decreased renal fibroblast proliferation. Finally, mouse embryonic fibroblasts (MEFs) lacking STAT3 reduced the inhibitory effect of TSA on cell proliferation, add-back of wild type STAT3 to STAT3−/− MEFs restored the effect of TSA. Collectively, our results reveal an important role of HDAC1 and HDAC2 in regulating proliferation of renal interstitial fibroblasts, expression of cell cycle proteins and activation of STAT3. Further, STAT3 mediates the proliferative action of HDACs.
doi:10.1002/jcb.23135
PMCID: PMC4626014  PMID: 21465537
histone deacetylase; renal interstitial fibroblasts; proliferation; cell cycle; trichostatin A; signal transducer and activator of transcription 3
7.  Activation of mTOR Pathway in Myeloid-derived Suppressor Cells Stimulates Cancer Cell Proliferation and Metastasis in lal−/− Mice 
Oncogene  2014;34(15):1938-1948.
Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal−/−) mouse model, melanoma metastasized massively in allogeneic lal−/− mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Here we report for the first time that MDSCs from lal−/− mice directly stimulated B16 melanoma cell in vitro proliferation, and in vivo growth and metastasis. Cytokines i.e., IL-1β and TNFα from MDSCs are required for B16 melanoma cell proliferation in vitro. Myeloid-specific expression of human LAL (hLAL) in lal−/− mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal−/− MDSCs is mediated, at least in part, through over-activation of the mammalian target of rapamycin (mTOR) pathway. Knockdown of mTOR, Raptor or Rictor in lal−/− MDSCs suppressed their stimulation on proliferation of cancer cells, including B16 melanoma, LLC and Tramp-C2 cancer cells. Our results indicate that LAL plays a critical role in regulating MDSCs ability to directly stimulate cancer cell proliferation, and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore, MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance, and stimulate cancer cell proliferation and growth.
doi:10.1038/onc.2014.143
PMCID: PMC4254377  PMID: 24882582
Lysosomal acid lipase; neutral lipid metabolism; myeloid-derived suppressor cells; mTOR; tumor growth and metastasis
8.  Ameliorating effects of gypenosides on chronic stress-induced anxiety disorders in mice 
Background
Ethanol extract from Gynostemma pentaphyllum (GP) shows anti-stress and anxiolytic functions in mice, and also protects dopamine neurons in 6-hydroxydopamine-lesioned rat model of Parkinson’s disease. In addition, gypenosides (the gypenoside-enriched components of GP, GPS) have a protective effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson’s disease. In this study, the ameliorating effects of GPS on chronic stress-induced anxiety disorders in mice were investigated.
Methods
Mice were orally treated with GPS (100 and 200 mg/kg) once a day for 10 days, followed by exposure to electric footshock (EF) stress (0.6 mA, 1 s every 5 s, 3 min). After the final administration of either GPS, water extract of GP (GP-WX) or ethanol extract of GP (GP-EX, positive control), the behavioral tests such as elevated plus-maze, marble burying and locomotor activity tests, and the biochemical parameters including dopamine, serotonin and corticosterone levels, and c-Fos expression were examined.
Results
Treatment with GPS (100 and 200 mg/kg) increased the number of open arm entries and the time spent on open arms in elevated plus-maze which were reduced by chronic EF stress. GPS (100 and 200 mg/kg) reduced the number of marbles buried which increased by chronic EF stress. In these states, the brain levels of dopamine and serotonin decreased by chronic EF stress and they were recovered by GPS. The serum levels of corticosterone increased by chronic EF stress were also reduced by GPS (100 and 200 mg/kg). Finally, chronic EF stress-induced c-Fos expression was markedly reduced by GPS (100 and 200 mg/kg) in the brain. GPS (100 and 200 mg/kg) also showed an equivalent efficacy on anxiolytic functions, as compared with GP-EX (50 mg/kg). However, GP-WX (50 mg/kg) showed a less effect on anxiety disorders than GP-EX (50 mg/kg) and GPS (100 and 200 mg/kg).
Conclusion
These results suggest that GPS (100 and 200 mg/kg) has anxiolytic effects on chronic EF stress-induced anxiety disorders by modulating dopamine and serotonin neuronal activities, c-Fos expression and corticosterone levels. GPS may serve as a phytonutrient in chronic stress-induced anxiety disorders.
doi:10.1186/s12906-015-0856-4
PMCID: PMC4570649  PMID: 26370834
Gypenosides; Chronic stress-induced anxiety disorders; Elevated plus-maze; Dopamine; Serotonin; Corticosterone
9.  Myeloid-Derived Suppressor Cells Are Involved in Lysosomal Acid Lipase Deficiency-Induced Endothelial Cell Dysfunctions 
The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal−/−) mice. We found that Ly6G+ cells transmigrated more efficiently across lal−/− ECs than wild-type (lal+/+) ECs, which was associated with increased level of platelet endothelial cell adhesion molecule-1 (PECAM-1) and monocyte chemoattractant protein-1 (MCP-1) in lal−/− ECs. In addition, lal−/−ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal−/− ECs also suppressed T cell proliferation in vitro. Interestingly, lal−/− Ly6G+ cells promoted in vivo angiogenesis (including a tumor model), EC tube formation and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal−/− ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G+ cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species (ROS). Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL-deficiency related diseases.
doi:10.4049/jimmunol.1301941
PMCID: PMC4119579  PMID: 25000979
10.  Histone deacetylase (HDAC) inhibition improves myocardial function and prevents cardiac remodeling in diabetic mice 
Background
Recent evidence indicates that inhibition of histone deacetylase (HDAC) protects the heart against myocardial injury and stimulates endogenous angiomyogenesis. However, it remains unknown whether HDAC inhibition produces the protective effect in the diabetic heart. We sought to determine whether HDAC inhibition preserves cardiac performance and suppresses cardiac remodeling in diabetic cardiomyopathy.
Methods
Adult ICR mice received an intraperitoneal injection of either streptozotocin (STZ, 200 mg/kg) to establish the diabetic model or vehicle to serve as control. Once hyperglycemia was confirmed, diabetic mice received sodium butyrate (1%), a specific HDAC inhibitor, in drinking water on a daily basis to inhibit HDAC activity. Mice were randomly divided into following groups, which includes Control, Control + Sodium butyrate (NaBu), STZ and STZ + Sodium butyrate (NaBu), respectively. Myocardial function was serially assessed at 7, 14, 21 weeks following treatments.
Results
Echocardiography demonstrated that cardiac function was depressed in diabetic mice, but HDAC inhibition resulted in a significant functional improvement in STZ-injected mice. Likewise, HDAC inhibition attenuates cardiac hypertrophy, as evidenced by a reduced heart/tibia ratio and areas of cardiomyocytes, which is associated with reduced interstitial fibrosis and decreases in active caspase-3 and apoptotic stainings, but also increased angiogenesis in diabetic myocardium. Notably, glucose transporters (GLUT) 1 and 4 were up-regulated following HDAC inhibition, which was accompanied with increases of GLUT1 acetylation and p38 phosphorylation. Furthermore, myocardial superoxide dismutase, an important antioxidant, was elevated following HDAC inhibition in the diabetic mice.
Conclusion
HDAC inhibition plays a critical role in improving cardiac function and suppressing myocardial remodeling in diabetic heart.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-015-0262-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-015-0262-8
PMCID: PMC4527099  PMID: 26245924
HDAC; Diabetes; Myocardium; Heart failure; Apoptosis; GLUT
11.  HDAC4 Degradation Mediates HDAC Inhibition-Induced Protective Effects Against Hypoxia/Reoxygenation Injury 
Journal of cellular physiology  2015;230(6):1321-1331.
Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition-mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 h of hypoxia (H) (1% O2) and 2 h of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R-elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA-induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4.NMVMtransduced with adenoviral HDAC4 led to an exaggeration of H/R-induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4-induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects.
doi:10.1002/jcp.24871
PMCID: PMC4373665  PMID: 25475100
12.  Selective Histone Deacetylase-6 Inhibition Attenuates Stress Responses and Prevents Immune Organ Atrophy in a Lethal Septic Model 
Surgery  2014;156(2):235-242.
Background
An overproduction of corticosterone during severe sepsis results in increased apoptosis of immune cells, which may result in relative immunosuppression and an impaired ability to fight infections. We have previously demonstrated that administration of Tubastatin A, a selective inhibitor of histone deacetylase-6 (HDAC6), improves survival in a lethal model of cecal ligation and puncture (CLP) in mice. The purpose of this study was to characterize the effects of this treatment on sepsis-induced stress responses and immune function.
Methods
C57BL/6J mice were subjected to CLP, and 1 hour later given an intraperitoneal injection of either Tubastatin A dissolved in dimethyl sulfoxide (DMSO), or DMSO only. Blood samples were collected to measure the levels of circulating corticosterone and adrenocorticotropic hormone (ACTH). Thymus, and long bones (femur and tibia) were subjected to H&E staining, and immunohistochemistry was utilized to detect cleaved-caspase 3 in the splenic follicles as a measure of cellular apoptosis.
Results
All vehicle-treated CLP animals died within 3 days, and displayed increased corticosterone and decreased ACTH levels compared to the sham-operated group. These animals also developed atrophy of thymic cortex with a marked depletion of thymocytes. Tubastatin A treatment significantly attenuated the stress hormone abnormalities. Treated animals also had significantly lower percentages of thymic atrophy (95.0±5.0 vs. 42.5±25.3, p=0.0366), bone marrow depletion and atrophy (58.3±6.5 vs. 25.0±14.4%, p=0.0449), and cellular apoptosis in the splenic follicles (41.2±3.7 vs. 28.5±4.3 per 40× field, p=0.0354).
Conclusions
Selective inhibition of HDAC6 in this lethal septic model was associated with a significant blunting of the stress responses, with attenuated thymic and bone marrow atrophy, and decreased splenic apoptosis. Our findings identify a novel mechanism behind the survival advantage seen with Tubastatin A treatment.
doi:10.1016/j.surg.2014.03.033
PMCID: PMC4267495  PMID: 24947640
13.  Histone Deacetylase Inhibitor Treatment Attenuates Coagulation Imbalance in a Lethal Sepsis Model 
Surgery  2014;156(2):214-220.
Background
Sepsis has a profound impact on the inflammatory and hemostatic systems. In addition to systemic inflammation, it can produce disseminated intravascular coagulation, microvascular thrombosis, consumptive coagulopathy, and multiple organ failure. We have shown that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), improves survival in a lethal model of cecal ligation and puncture (CLP) in mice. However, its effect on coagulation remains unknown. The goal of this study was to quantify the impact of SAHA treatment on coagulopathy in sepsis.
Methods
C57BL/6J mice were subjected to CLP, and 1 hour later given intraperitoneally either SAHA dissolved in dimethyl sulfoxide (DMSO) or DMSO only. Sham-operated animals were handled in similar manner without CLP. Blood samples were collected by cardiac puncture and evaluated using the TEG® 5000 Thrombelastograph® Hemostasis Analyzer System.
Results
Compared to the sham group, all animals in DMSO vehicle group died within 72 hrs, and developed coagulopathy that manifested as prolonged initial fibrin formation and fibrin cross-linkage time, and decreased clot formation speed, platelet function and clot rigidity. SAHA treatment significantly improved survival and was also associated with improvement in fibrin cross-linkage, clot formation, as well as platelet function and clot rigidity, without a significant impact on the clot initiation parameters.
Conclusions
SAHA treatment enhances survival and attenuates sepsis-associated coagulopathy by improving fibrin cross-linkage, rate of clot formation, platelet function and clot strength. HDACI may represent a novel therapeutic strategy for correcting sepsis-associated coagulopathy.
doi:10.1016/j.surg.2014.04.022
PMCID: PMC4267526  PMID: 24957668
14.  Citrullinated Histone H3 – A Novel Target for Treatment of Sepsis 
Surgery  2014;156(2):229-234.
INTRODUCTION
We have recently demonstrated that in a rodent model of lipopolysaccharide (LPS)-induced shock, an increase in circulating citrullinated histone H3 (Cit H3) is associated with lethality of sepsis, and treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor (HDACI), significantly improves survival. However, the role of Cit H3 in pathogenesis and therapeutics of sepsis are largely unknown. The present study was designed to test whether treatment with HDACI could inhibit cellular Cit H3 production, and inhibition of peptidyalarginine deiminase (PAD, an enzyme producing Cit H3) with Cl-amidine (PAD inhibitor) or neutralization of blood Cit H3 with anti-Cit H3 antibody could improve survival in a clinically relevant mouse model of cecal ligation and puncture (CLP) induced septic shock.
METHODS
Three experiments were carried out. In experiment I, HL-60 neutrophilic cells grown on a coverslip were treated with LPS (100 ng/ml) in the presence or absence of SAHA (5 µmol) for 3 h, and subjected to immuno-staining with anti-Cit H3 antibody to assess effect of SAHA on Cit H3 production under a fluorescence microscope. The ratio of Cit H3 positive cells was calculated as mean ± SD (n=3). In experiment II, male C57BL/6J mice were subjected to CLP, and 1 hour later randomly divided into three groups for intraperitoneal injection as follows: (1) dimethyl sulfoxide (DMSO), (2) SAHA (50 mg/kg) in DMSO, and (3) Cl-amidine (80 mg/kg) in DMSO (n=10/group). In experiment III, male C57BL/6J mice were divided into control and treatment groups, and subjected to CLP. Two hours later, immunoglobulin (IgG) and Cit H3 antibody (20 mg/kg iv; n=5/group) were injected into the control and treatment groups, respectively. Survival was monitored for up to 10 days.
RESULTS
In experiment I, LPS induced Cit H3 production in the HL-60 cells, while SAHA treatment inhibited H3 citrullination significantly (p<0.05). In experiment II, all vehicle injected mice died within 3 days with increased circulating Cit H3 levels, whereas treatment with HDACI or Cl-amidine notably improved long-term survival (p< 0.01). In experiment III, administration of IgG did not improve survival, but a single treatment with Cit H3 specific antibody significantly improved survival (p<0.014).
CONCLUSIONS
Inhibition of HDAC or PAD significantly suppresses Cit H3 production in vitro, and improves survival in vivo. Neutralization of Cit H3 significantly improves survival in septic mice. Collectively, our findings indicate for the first time that Cit H3 could not only serve as a potential biomarker but also a novel therapeutic target in sepsis.
doi:10.1016/j.surg.2014.04.009
PMCID: PMC4267527  PMID: 24957671
sepsis; citrullinated histone H3; suberoylanilide hydroxamic acid; peptidyl arginine deiminase inhibitor; Cl-amidine; anti-Cit H3 antibody
15.  Selective Inhibition of Histone Deacetylase-6 Alters the Composition of Circulating Blood Cells in a Lethal Septic Model 
The Journal of surgical research  2014;190(2):647-654.
Background
Phagocytes, especially monocytes, macrophages and dendritic cells, play a pivotal role in the innate as well as adaptive immune responses during sepsis. We have shown that inhibition of histone deacetylase (HDAC)-6 improves survival and increases bacterial clearance in a mouse model of cecal ligation and puncture (CLP). The aim of this study was to determine whether this effect was associated with changes in the number and composition of different blood cell types in the circulation.
Methods
C57BL/6J mice were subjected to CLP, and 1 hour later given an intraperitoneal injecton of either Tubastatin A dissolved in dimethyl sulfoxide (DMSO), or DMSO only. Sham-operated animals were treated in an identical fashion but not subjected to CLP. Forty-eight hours later peripheral blood was obtained via cardiac puncture and analyzed using a veterinary Hematrue hematology analyzer.
Results
Tubastatin A administration increased the number of circulating monocytes in the sham-operated as well as the CLP animals. In comparison to the sham, CLP animals displayed an increase in the granulocyte percentage in white blood cells, decrease in the lymphocyte number and percentage, with a resultant increase in the granulocyte to lymphocyte ratio. Treatment of CLP animals with Tubastatin A decreased the granulocyte percentage, and restored the lymphocyte number and percentage, which decreased the granulocyte to lymphocyte ratio. In the sham animals, Tubastatin A increased red blood cell (RBC) number, hematocrit and hemoglobin. This effect was not seen in CLP animals.
Conclusions
Tubastatin A treatment has significant impact on the composition of circulating blood cells. It increases the number of circulating monocytes and the RBC cell mass in sham-operated animals. In the CLP animals, it increases the monocyte count, decreases the percentage of granulocytes, restores the lymphocyte population, and decreases the granulocyte to lymphocyte ratio. These results may explain why Tubastatin A treatment improves survival in the septic models.
doi:10.1016/j.jss.2014.01.056
PMCID: PMC4267528  PMID: 24613069
16.  NEXN inhibits GATA4 and leads to atrial septal defects in mice and humans 
Cardiovascular Research  2014;103(2):228-237.
Aims
Cardiac structural genes have been implicated as causative factors for congenital heart diseases (CHDs). NEXN is an F-actin binding protein and previously identified as a disease gene causing cardiomyopathies. Whether NEXN contributes to CHDs aetiologically remains unknown. Here, we explored the function of NEXN in cardiac development.
Methods and results
First, we determine the role of NEXN in cardiac differentiation using mouse P19cl6 in vitro model; we demonstrated that NEXN inhibited cardiac contractile markers, serving as a negative regulator. Interestingly, we found this effect was mediated by GATA4, a crucial transcription factor that controls cardiac development by knockdown, overexpression, and rescue experiment, respectively. We then generated transgenic mouse models and surprisingly, we discovered cardiac-selective expression of the NEXN gene caused atrial septal defects (ASDs). Next, to search for the mutations in NEXN gene in patients suffering from ASDs, we sequenced the exon and exon–intron joint regions of the NEXN gene in 150 probands with isolated ASDs and identified three mutations in the conserved region of NEXN (c.-52-78C>A, K199E, and L227S), which were not found in 500 healthy controls. Finally, we characterize the related mechanisms and found all mutations inhibited GATA4 expression.
Conclusion
We identify NEXN as a novel gene for ASD and its function to inhibit GATA4 established a critical regulation of an F-actin binding protein on a transcription factor in cardiac development.
doi:10.1093/cvr/cvu134
PMCID: PMC4498134  PMID: 24866383
Atrial septal defect; NEXN; GATA4; Actin; Mutation
17.  Astragalus polysaccharide promotes the release of mature granulocytes through the L-selectin signaling pathway 
Chinese Medicine  2015;10:17.
Background
This study aims to investigate the leukogenic effect of astragalus polysaccharide (APS), to compare its effect of increasing the numbers of mature granulocytes with that of granulocyte colony-stimulating factor (G-CSF), and to investigate the mechanism.
Methods
Rats were arbitrarily grouped into four groups (control, cyclophosphamide (CTX), CTX + APS, and CTX + G-CSF groups), and each group was then arbitrarily divided into five subgroups according to the time period since CTX infusion (0, 4, 7, 10, and 14 days). The expression of leukocyte selectin (L-selectin), its ligand, and shedding-related protease on granulocytes was analyzed. Leukocyte counts were obtained. Chemotactic capacity of polymorphonuclear leukocytes (PMNLs) was assessed.
Results
Both APS and G-CSF restored the expression of L-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), CD11b/CD18, and ADAM17 to normal levels (P > 0.05 vs. control group on each time point), with APS eliciting a greater effect than G-CSF (P = 0.005 on day 7, P < 0.001 on day 10 and 14 for L-selectin; P = 0.038 on day 7, P = 0.001 on day 10, P < 0.001 on day 14 for PSGL-1; P < 0.001 on day 7, 10 and 14 for ADAM17; P < 0.001 on day 7, 10, and 14 for CD11b/CD18). The percentages of the bands and segmented bone marrow (BM) cells in myeloid neutrophils were higher in the CTX + APS group than in the CTX group on day 7 (P = 0.030) and reached normal levels on day 10 (P = 0.547) and 14 (P = 0.431) vs. control group. The ability of APS to increase numbers of PMNLs in peripheral blood after chemotherapy was significantly superior to that of G-CSF 7 days after chemotherapy (P = 0.029 on day 10, P = 0.006 on day 14). Moreover, APS more significantly improved the chemotactic ability of PMNLs among mature BM granulocytes and peripheral blood neutrophils after chemotherapy than did G-CSF (P < 0.001 on day 7, P = 0.001 on day 10 and P = 0.005 on day 14).
Conclusions
APS promoted the differentiation and chemotactic ability of BM granulocytes via the L-selectin signaling pathway.
Electronic supplementary material
The online version of this article (doi:10.1186/s13020-015-0043-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13020-015-0043-z
PMCID: PMC4497426  PMID: 26161135
18.  Ugi 4–CR Synthesis of γ– and δ–Lactams providing new access to diverse enzyme interactions, a PDB analysis† 
MedChemComm  2014;5(7):949-952.
A three step synthesis of N-unsubstituted tetrazolo γ- and δ-lactams involving a key Ugi-4CR is presented. The compounds, otherwise difficult to access, are conveniently synthesized in overall good yields by our route. PDB analysis of the N-unsubstituted γ- and δ-lactam fragment reveals a strongly tri-directional hydrogen bond donor acceptor interaction with the amino acids of the binding sites.
doi:10.1039/C4MD00162A
PMCID: PMC4084911  PMID: 25013719
19.  Effects of chromium malate on glycometabolism, glycometabolism-related enzyme levels and lipid metabolism in type 2 diabetic rats: A dose–response and curative effects study 
Aims/Introduction
The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzyme levels and lipid metabolism in type 2 diabetic rats, and dose–response and curative effects.
Materials and Methods
The model of type 2 diabetes rats was developed, and daily treatment with chromium malate was given for 4 weeks. A rat enzyme-linked immunosorbent assay kit was used to assay glycometabolism, glycometabolism-related enzyme levels and lipid metabolism changes.
Results
The results showed that the antihyperglycemic activity increased with administration of chromium malate in a dose–dependent manner. The serum insulin level, insulin resistance index and C-peptide level of the chromium malate groups at a dose of 17.5, 20.0 and 20.8 μg chromium/kg bodyweight were significantly lower than that of the model, chromium trichloride and chromium picolinate groups. The hepatic glycogen, glucose-6-phosphate dehydrogenase and glucokinase levels of the chromium malate groups at a dose of 17.5, 20.0 and 20.8 μg chromium/kg bodyweight were significantly higher than that of the model, chromium trichloride and chromium picolinate groups. Chromium malate at a dose of 20.0 and 20.8 μg chromium/kg bodyweight significantly changed the total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides levels compared with the chromium trichloride and chromium picolinate groups.
Conclusions
The results showed that chromium malate exhibits greater benefits in treating type 2 diabetes, and the curative effect of chromium malate is superior to chromium trichloride and chromium picolinate.
doi:10.1111/jdi.12350
PMCID: PMC4511299  PMID: 26221518
Chromium malate; Glycometabolism; Lipid metabolism
20.  Myxoid Epithelial Leiomyoma of the Vulva: A Case Report and Literature Review 
Leiomyoma of vulva is rare, and usually misdiagnosed clinically as Bartholin cyst. It usually presents spindle-shaped tumor cells, but some rare cases consisted mainly of atypical epithelioid tumor cells. We report here a case of 30-year-old woman consulting with a vulvar mass of 7 cm in the Bartholin glands area. The lesion was surgically excised with its capsule completely. Pathological examination and immunochemistry showed characteristic of epithelioid leiomyoma with myxoid stroma with both estrogen receptor (ER) and progesterone receptor (PR) staining negative, which was really rare as only 2 cases of vulvar leiomyoma with both ER and PR were reported before.
doi:10.1155/2015/894830
PMCID: PMC4491551  PMID: 26185695
21.  A New Synthetic Amphiploid (AADDAA) between Gossypium hirsutum and G. arboreum Lays the Foundation for Transferring Resistances to Verticillium and Drought 
PLoS ONE  2015;10(6):e0128981.
Gossypium arboreum, a cultivated cotton species (2n = 26, AA) native to Asia, possesses invaluable characteristics unavailable in the tetraploid cultivated cotton gene pool, such as resistance to pests and diseases and tolerance to abiotic stresses. However, it is quite difficult to transfer favorable traits into Upland cotton through conventional methods due to the cross-incompatibility of G. hirsutum (2n = 52, AADD) and G. arboreum. Here, we improved an embryo rescue technique to overcome the cross-incompatibility between these two parents for transferring favorable genes from G. arboreum into G. hirsutum. Our results indicate that MSB2K supplemented with 0.5 mgl-1 kinetin and 250 mg-1 casein hydrolysate is an efficient initial medium for rescuing early (3 d after pollination) hybrid embryos. Eight putative hybrids were successfully obtained, which were further verified and characterized by cytology, molecular markers and morphological analysis. The putative hybrids were subsequently treated with different concentrations of colchicine solution to double their chromosomes. The results demonstrate that four putative hybrid plants were successfully chromosome-doubled by treatment with 0.1% colchicine for 24 h and become amphiploid, which were confirmed by cytological observation, self-fertilization and backcrossing. Preliminary assessments of resistance at seedling stage indicate that the synthetic amphiploid showed highly resistant to Verticillium and drought. The synthetic amphiploid between G. hirsutum × G. arboreum would lay the foundation for developing G. arboreum-introgressed lines with the uniform genetic background of G. hirsutum acc TM-1, which would greatly enhance and simplify the mining, isolation, characterization, cloning and use of G. arboreum-specific desirable genes in future cotton breeding programs.
doi:10.1371/journal.pone.0128981
PMCID: PMC4465178  PMID: 26061996
22.  Type 2 Diabetic Rats on Diet Supplemented With Chromium Malate Show Improved Glycometabolism, Glycometabolism-Related Enzyme Levels and Lipid Metabolism 
PLoS ONE  2015;10(5):e0125952.
Our previous study showed that chromium malate improved the regulation of blood glucose in mice with alloxan-induced diabetes. The present study was designed to evaluate the effect of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism in type 2 diabetic rats. Our results showed that fasting blood glucose, serum insulin level, insulin resistance index and C-peptide level in the high dose group had a significant downward trend when compared with the model group, chromium picolinate group and chromium trichloride group. The hepatic glycogen, glucose-6-phosphate dehydrogenase, glucokinase, Glut4, phosphor-AMPKβ1 and Akt levels in the high dose group were significantly higher than those of the model, chromium picolinate and chromium trichloride groups. Chromium malate in a high dose group can significantly increase high density lipoprotein cholesterol level while decreasing the total cholesterol, low density lipoprotein cholesterol and triglyceride levels when compared with chromium picolinate and chromium trichloride. The serum chromium content in chromium malate and chromium picolinate group is significantly higher than that of the chromium trichloride group. The results indicated that the curative effects of chromium malate on glycometabolism, glycometabolism-related enzymes and lipid metabolism changes are better than those of chromium picolinate and chromium trichloride. Chromium malate contributes to glucose uptake and transport in order to improved glycometabolism and glycometabolism-related enzymes.
doi:10.1371/journal.pone.0125952
PMCID: PMC4420285  PMID: 25942313
23.  Gypenosides attenuate the development of L-DOPA-induced dyskinesia in 6-hydroxydopamine-lesioned rat model of Parkinson’s disease 
BMC Neuroscience  2015;16:23.
Background
Gypenosides (GPS) and ethanol extract of Gynostemma pentaphyllum (GP-EX) show anxiolytic effects on affective disorders in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse model of Parkinson’s disease (PD). Long-term administration of L-3,4-dihydroxyphenylalanine (L-DOPA) leads to the development of severe motor side effects such as L-DOPA-induced-dyskinesia (LID) in PD. The present study investigated the effects of GPS and GP-EX on LID in a 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD.
Results
Daily administration of L-DOPA (25 mg/kg) in the 6-OHDA-lesioned rat model of PD for 22 days induced expression of LID, which was determined by the body and locomotive AIMs scores and contralateral rotational behaviors. However, co-treatments of GPS (25 and 50 mg/kg) or GP-EX (50 mg/kg) with L-DOPA significantly attenuated the development of LID without compromising the anti-parkinsonian effects of L-DOPA. In addition, the increases in ∆FosB expression and ERK1/2 phosphorylation in 6-OHDA-lesioned rats induced by L-DOPA administration were significantly reduced by co-treatment with GPS (25 and 50 mg/kg) or GP-EX (50 mg/kg).
Conclusion
These results suggest that GPS (25 and 50 mg/kg) and GP-EX (50 mg/kg) effectively attenuate the development of LID by modulating the biomarker activities of ∆FosB expression and ERK1/2 phosphorylation in the 6-OHDA-lesioned rat model of PD. GPS and GP-EX will be useful adjuvant therapeutics for LID in PD.
doi:10.1186/s12868-015-0163-5
PMCID: PMC4431176  PMID: 25896846
Gynostemma pentaphyllum; 6-Hydroxydopamine-lesioned rats; Dyskinesia; Body and locomotive AIMs scores; ∆FosB; ERK1/2; Adjuvant therapeutics
24.  eCROPS-CA: a systematic approach toward effective and sustainable cancer prevention in rural China 
BMC Cancer  2015;15:233.
Background
Effective prevention against cancers depends heavily on sustained individual efforts practicing protective behaviors and avoiding risk factors in a complex sociocultural context, which requires continuous and personalized supports. Contemporary prevention relies primarily on strategies targeting general population with limited attention being paid to individualized approaches. This study tests a novel package called, in acronym of core intervention components, eCROPS-CA that leverages protective behaviors against over 80% leading cancers among high risk individuals via continuous and tailored counseling by village doctors.
Methods/Design
The study utilizes a quesi-RCT design involving 4320 high risk individuals selected, via rapid and detailed risk assessments, from about 72,000 farmers aged 35+ in 36 administrative villages randomized into equal intervention and delayed intervention arms. The intervention arm receives baseline and semiannual follow up evaluations plus eCROPS-CA for 5 years; while the control arm, only the baseline and follow-up evaluations for the first 5 years and eCROPS-CA starting from the 6th year if the intervention is proved effective. eCROPS-CA comprises electronic supports and supervision (e), counseling cancer prevention (C), recipe for objective behaviors (R), operational toolkit (O), performance-based incentives (P), and screening and assessment (S). Evaluation measures include: incidence and stage of the leading cancers, cancer-related knowledge, attitudes and practices; easy biophysical indicators (e.g., body mass index, blood pressure); intervention compliance, acceptance of the package.
Discussion
The prevention package incorporates key success factors in a synergetic way toward cost-effectiveness and long-term sustainability. It targets a set rather than any single cancer; choses village doctors as key solution to the widespread lack of professional manpower in implementing personalized and thus relatively sophisticated prevention; adopts real-time monitoring in reaching continuous improvement; utilizes smart web aids to enable prioritizing complex determinants of objective behaviors, linking counseling sessions happened at different time points and hence delivering highly coordinated prevention; uses 2-stage risk assessment models in identifying high risk individuals so as to focus on the most needed; applies standardized operation procedures in simplifying and smoothing behavior intervention yet ensuring delivery of essential steps and key elements.
Trials registry
ISRCTN33269053
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1253-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1253-6
PMCID: PMC4416351  PMID: 25886568
Cancer; Behavior intervention; Rural communities; Randomized controlled trail; Service integration
25.  Intestinal mucosal barrier dysfunction participates in the progress of nonalcoholic fatty liver disease 
Intestinal mucosal barrier dysfunction is closely related to liver diseases, which implies impaired gut-liver axis may play a role in the pathogenesis of NAFLD. In our study, rats were divided into three groups: normal chow diet (NCD) group, high-fat diet (HFD) group and TNBS-induced colitis with high-fat diet (C-HFD) group. Liver tissues were obtained for histological observation and TNF-α, IL-6 mRNA determination and blood samples were collected for liver enzymes and LPS analysis. Ultrastructural changes of jejuna epithelium, SIBO and amounts of CD103+MHCII+DCs and CD4+CD25+FoxP3+T-regs in terms of percentage in mesenteric lymph nodes (MLN) were observed by electron microscope, bacterial cultivation and flow cytometry, respectively. The results demonstrated the pathological characteristics accorded with nonalcoholic simple fatty liver (NAFL) and NASH in HFD group by week 8 and 12, respectively. Besides, the degree of hepatic steatosis and steatohepatitis was more severe in C-HFD group compared with HFD-group at the same time point. NAFLD activity score (NAS), liver enzymes, concentration of LPS and mRNA expressions of TNF-α, IL-6 were higher significantly in C-HFD group compared with HFD and NCD group at week 4, 8 and 12, respectively. In HFD group, epithelium microvilli atrophy, disruptive tight junctions and SIBO were present, and these changes were more severe in NASH compared with NAFL. The percentage of CD103+MHCII+DCs and CD4+CD25+FoxP3+T-regs decreased significantly in NAFL and NASH compared with NCD group. Our conclusion was that gut-liver axis was impaired in NAFLD, which played crucial role in the pathogenesis of NAFLD.
PMCID: PMC4466933  PMID: 26097546
Nonalcoholic fatty liver disease; intestinal mucosal barrier; TNF-α; IL-6; dendritic cell; regulatory T cell

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