Coronary Dilatation and Endothelial Inflammation in Neonates Born to Mothers with Preeclampsia.

OBJECTIVES: To investigate the cardiovascular features and endothelium in neonates born to mothers with preeclampsia. STUDY DESIGN: In this combined observational cohort and case-control study, neonates born to mothers with normotension and mothers with preeclampsia were recruited at a neonatal intensive care unit of a tertiary medical center. Cardiovascular measurements by echocardiography and the clinical measures upon admission were analyzed. Vascular cell adhesion molecule-1 expression in umbilical arteries and in in vitro endothelial cell stimulation with plasma were examined. Continuous data were compared using nonparametric analysis, and their relationships were analyzed using linear regression. Binary logistic regression was performed in the model of adjustment of birth body weight and for multivariate analysis. RESULTS: In the cohort, almost all cardiovascular segments positively correlated to birth weight. Notably, neonates (n = 65) of mothers with preeclampsia had significantly larger coronary arteries at birth than neonates of mothers with normotension (n = 404) (median size of left main coronary artery 1.36 mm versus 1.08 mm, p <0.001; median size of right coronary artery, RCA 1.25 mm versus 1.0 mm, p <0.001). The size of the right coronary artery positively correlated to the maternal antepartum diastolic blood pressure (r = 0.298, P = .018) and was associated with in-hospital death (P < .001). Meanwhile, endothelial vascular cell adhesion molecule-1 expression was significantly increased in the umbilical arteries of the preeclamptic group and following preeclamptic cord-plasma stimulation. The latter also correlated with their relative coronary sizes. CONCLUSIONS: Neonates of mothers with preeclampsia had distinctive coronary dilatation at birth. Coronary size might be useful as a severity index of neonatal endothelial inflammation as a result of maternal preeclampsia.

Targeting mTOR-CCL20 Signaling May Improve Response to Docetaxel in Head and Neck Squamous Cell Carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) usually show a dismal prognosis. It is this worthwhile to develop new, effective therapeutic regimens for these patients, such as molecular targeted therapy, which is promising as an alternative or combination treatment for HNSCC. The mammalian target of rapamycin (mTOR) pathway, which plays an important role in the carcinogenesis of HNSCC, is the most frequently activated, and is thus worthy of further investigation. In this study, two human HNSCC cell lines, FaDu and SAS, were evaluated for cell growth with trypan blue staining and tumor growth using an orthotopic xenograft model. The immunohistochemical expression of mTOR in the subcutaneous xenograft model and the inhibitory effects of docetaxel on the growth and state of activation of the PI3K/mTOR pathway were also evaluated and examined by colony formation and Western blot, respectively. Cell proliferation and migration were measured by water-soluble tetrazolium salt (WST-1) and OrisTM cell migration assay, respectively. Furthermore, the effects of rapamycin and BEZ235, a phosphatidylinositol 3-kinases (PI3K) and mTOR inhibitor in combination with docetaxel or CCL20 were evaluated in the FaDu and SAS cells. The results showed that the expression of mTOR was significantly higher in the SAS and FaDu xenograft models than in the control. Docetaxel treatment significantly suppressed HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. Additionally, when administered in a dose-dependent fashion, mTOR inhibitors inhibited the growth and migration of the HNSCC cells. This combination was synergistic with docetaxel, resulting in almost complete cell growth and migration arrest. In conclusion, docetaxel significantly inhibited HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. The synergistic and additive activity of mTOR inhibitors combined with docetaxel shows potential as a new treatment strategy for HNSCC.

Free Fatty Acids Induce Autophagy and LOX-1 Upregulation in Cultured Aortic Vascular Smooth Muscle Cells.

Elevation of free fatty acids (FFAs) is known to affect microvascular function and contribute to obesity-associated insulin resistance, hypertension, and microangiopathy. Proliferative and synthetic vascular smooth muscle cells (VSMCs) increase intimal thickness and destabilize atheromatous plaques. This study aimed to investigate whether saturated palmitic acid (PA) and monounsaturated oleic acid (OA) modulate autophagy activity, cell proliferation, and vascular tissue remodeling in an aortic VSMC cell line. Exposure to PA and OA suppressed growth of VSMCs without apoptotic induction, but enhanced autophagy flux with elevation of Beclin-1, Atg5, and LC3I/II. Cotreatment with autophagy inhibitors potentiated the FFA-suppressed VSMC growth and showed differential actions of PA and OA in autophagy flux retardation. Both FFAs upregulated lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) but only OA increased LDL uptake by VSMCs. Mechanistically, FFAs induced hyperphosphorylation of Akt, ERK1/2, JNK1/2, and p38 MAPK. All pathways, except OA-activated PI3K/Akt cascade, were involved in the LOX-1 upregulation, whereas blockade of PI3K/Akt and MEK/ERK cascades ameliorated the FFA-induced growth suppression on VSMCs. Moreover, both FFAs exhibited tissue remodeling effect through increasing MMP-2 and MMP-9 expression and their gelatinolytic activities, whereas high-dose OA significantly suppressed collagen type I expression. Conversely, siRNA-mediated LOX-1 knockdown significantly attenuated the OA-induced tissue remodeling effects in VSMCs. In conclusion, OA and PA enhance autophagy flux, suppress aortic VSMC proliferation, and exhibit vascular remodeling effect, thereby leading to the loss of VSMCs and interstitial ECM in vascular walls and eventually the instability of atheromatous plaques. J. Cell. Biochem. 118: 1249-1261, 2017. © 2016 Wiley Periodicals, Inc.

Vascular endothelial growth factor-a in lactobacillus casei cell wall extract-induced coronary arteritis of a murine model.

BACKGROUND: Vascular endothelial growth factor (VEGF) is associated with Kawasaki disease (KD), the most commonly acquired heart disease in developed countries. This study investigated the involvement of VEGF-A expression and its related signaling pathway in Lactobacillus casei cell wall extract (LCWE)-induced murine coronary artery lesions (CALs), and analyzed this in regard to the inhibition of CALs by spleen tyrosine kinase (Syk). METHODS AND RESULTS: Wild-type BALB/C mice were intraperitoneally injected with LCWE (1mg/ml) to induce CALs. The aortic roots, ventricular myocardium, peripheral blood leukocytes (PBLs), spleen, liver, kidneys, and lungs were analyzed for VEGF-A expression. Phosphate buffered saline (PBS)-, lipopolysaccharide (LPS)-, and zymosan-treated mice served as controls, and an oral Syk inhibitor served as an arteritis-ameliorated reagent. In aortic roots and PBLs, LCWE induced an early upregulation and a late downregulation of VEGF-A expression. No differential VEGF-A expression was observed in the other organs. Most importantly, Syk inhibition significantly attenuated the LCWE-induced expression of VEGF-A, dimethylarginine dimethylaminohydrolase (DDAH)-1, and endothelial nitric oxide synthase in aortic roots. However, LCWE-induced aortic DDAH-2 expression remained higher, despite Syk inhibition. CONCLUSIONS: Local VEGF-A and its signaling pathway are associated with the development of LCWE-induced CALs. Therefore, the clinical correlation between VEGF and human KD and the role of the VEGF-A regulation and signaling pathway in murine CALs warrant further investigation.

Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype.

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7 in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-ß1 (transforming growth factor-ß1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-ß1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.

High-mobility group box 1 protein is implicated in advanced glycation end products-induced vascular endothelial growth factor A production in the rat retinal ganglion cell line RGC-5.

PURPOSE: High-mobility group box 1 protein (HMGB1) has been reported to be a potent proangiogenic factor induced by inflammatory stress. In this study, we explore the role of HMGB1 in advanced glycation end products (AGEs)-induced vascular endothelial growth factor A (VEGF-A) production in rat retinal ganglion cell line 5 (RGC-5) cells. METHODS: The VEGF-A protein and mRNA levels in conditioned medium of RGC-5 cells incubated with AGE-modified BSA (AGE-BSA) were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA), and BSA-treated cells were used as controls. The expression of HMGB1, c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) was assessed with immunofluorescence and western blot analysis. Reactive oxidative species (ROS) were detected with flow cytometry measurements of peroxide-dependent oxidation of 2'-7'-dichlorofluorescein-diacetate (DCFH-DA). N-Acetyl-L-cysteine (NAC), glycyrrhizin (GZ), and SP600125 were used to block ROS, HMGB1, and JNK, respectively. RESULTS: Compared with the BSA controls, the RGC-5 cells incubated with AGE-BSA showed a dose- and time-dependent increase in VEGF-A mRNA and VEGF-A protein secretion in the supernatant, with the highest levels achieved at 24 h. AGE-BSA stimulated a significant release of HMGB1 in the supernatant and a significant increase of intracellular ROS production at 3 h. NAC blocked HMGB1 production in a dose-dependent manner. Blocking with GZ, NAC, and JNK significantly suppressed AGE-induced VEGF-A production. CONCLUSIONS: HMGB1 is implicated in the production of VEGF-A in retinal ganglion cell line-5 (RGC-5). Blocking HMGB1, ROS, or the JNK pathway may attenuate VEGF-A production, suggesting HMGB1 and related signaling molecules play a role in diabetic retinopathy.

Systemic and Local Effects Among Patients With Betel Quid-Related Oral Cancer.

The major predisposing factors of developing oral cancer include smoking, alcohol drinking, and betel quid chewing. Betel quid chewing could cause the abrasion and damage of oral mucosa by crude fibers, chemical insults by additive slaked lime, and arecoline from areca nut. These would lead to the local consequence of oral submucosal fibrosis, which is regarded clinically as a precancer lesion and a major cause of trismus. In addition, the components and additives in betel quid contain chemical toxins and carcinogens, which would further affect the oral mucosa and gradually develop a malignancy. Following literature review, aside from having a greater total tumor burden and more local diseases in the oral cavity and digestive tract, patients with betel quid-related oral cancer also have more systemic diseases from metabolic syndrome, hypertension, cardiovascular disease, type II diabetes mellitus, and obesity than those without this habit. In conclusion, those patients who have the history of smoking, alcohol drinking, and betel quid chewing would present much more unique clinical characteristics than those who only have a history of smoking and alcohol drinking. More attention should therefore be paid to pretreatment evaluation, treatment strategy, and posttreatment follow-up among betel quid chewers.

Glucocorticoid modulates high-mobility group box 1 expression and Toll-like receptor activation in obstructive jaundice.

BACKGROUND: Obstructive jaundice is associated with bacterial translocation and inflammatory cytokine induction. It is unknown if toll-like receptors (TLRs) and their upstream molecule high mobility group box-1 (HMGB1) are involved in the pathogenetic mechanism and if glucocorticoid is effective in modulating the process. MATERIALS AND METHODS: A rat model of cholestasis by ligation of the extrahepatic bile duct (BDL) for 2 wk was created. TLRs, interferon regulatory factors (IRFs), IL-6, IL-8, antimicrobial peptide ß-defensin, and cathelicidin, as well as HMGB1 expressions were studied by using real-time quantitative reverse transcription-polymerase chain reaction, immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Glucocorticoid treatment was applied to a group of BDL rats. RESULTS: Obstructive jaundice for 2 wk was associated with significant up-regulation of TLR1, 2, 4, 6, 7, and 9 mRNA expressions. There were significant increases of liver IRF5, IL-6, and ß-defensin 1 mRNA levels in the BDL rats than in the sham and nonoperative control rats, which were associated with significant increase of immunoreactive IRF5 protein staining in the nucleus of Kupffer cells and neutrophils. Hepatic HMGB1 expression and release into serum were significantly elevated in the cholestatic rats than in the sham and control rats. Glucocorticoid treatment significantly decreased hepatic HMGB1 expression and release into serum, which was associated with significantly decreased hepatic TLR4 mRNA expression in the cholestatic rats. CONCLUSIONS: The results indicate that obstructive jaundice may induce hepatic HMGB1 expression with activation of TLR4 and a number of downstream signaling molecules, which can be reversed by glucocorticoid administration.

Oxidized low-density lipoprotein induced hepatoma-derived growth factor upregulation mediates foam cell formation of cultured rat aortic vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) are important vascular components that are essential for the regulation of vascular functions during vascular atherosclerogenesis and vascular injury. Oxidized low-density lipoprotein (oxLDL) is known to induce SMC activation and foam cell transformation. This study characterized the role of hepatoma-derived growth factor (HDGF) in oxLDL-induced foam cell formation in cultured primary rat aortic SMCs. OxLDL exposure significantly increased HDGF expression and extracellular release. It also upregulated atherogenic regulators in SMCs, including TLR4, MyD88, LOX-1, and CD36. Exogenous HDGF stimulation not only increased the expression of cognate receptor nucleolin, but also the innate immunity regulators TLR4/MyD88 and lipid metabolism regulators, including LOX-1 and CD36. Oil red O staining showed that HDGF did not initiate, but enhanced oxLDL-driven foam cell formation in SMCs. Further signaling characterization demonstrated that oxLDL evoked activation of PI3K/Akt and p38 MAPK signaling pathways, both of which were involved in the upregulation of HDGF, LOX-1, and CD36 induced by oxLDL. Gene knockdown experiments using LOX-1 targeted siRNA demonstrated that LOX-1 expression was critical for oxLDL-induced HDGF upregulation, while HDGF gene depletion completely abolished oxLDL-triggered TLR4, LOX-1, and CD36 overexpression and foam cell formation in SMCs. These findings strongly suggest that oxLDL-induced HDGF upregulation participates in subsequent LOX-1 and CD36 expression in aortic SMCs and mechanistically contributes to the formation of SMC-derived foam cells. The oxLDL/LOX-1/HDGF axis may serve as a target for anti-atherogenesis therapy.

Endotoxin and CD14 in the progression of biliary atresia.

BACKGROUND: Biliary atresia (BA) is a typical cholestatic neonatal disease, characterized by obliteration of intra- and/or extra-hepatic bile ducts. However, the mechanisms contributing to the pathogenesis of BA remain uncertain. Because of decreased bile flow, infectious complications and damaging endotoxemia occur frequently in patients with BA. The aim of this study was to investigate endotoxin levels in patients with BA and the relation of these levels with the expression of the endotoxin receptor, CD14. METHODS: The plasma levels of endotoxin and soluble CD14 were measured with a pyrochrome Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay in patients with early-stage BA when they received the Kasai procedure (KP), in patients who were jaundice-free post-KP and followed-up at the outpatient department, in patients with late-stage BA when they received liver transplantation, and in patients with choledochal cysts. The correlation of CD14 expression with endotoxin levels in rats following common bile duct ligation was investigated. RESULTS: The results demonstrated a significantly higher hepatic CD14 mRNA and soluble CD14 plasma levels in patients with early-stage BA relative to those with late-stage BA. However, plasma endotoxin levels were significantly higher in both the early and late stages of BA relative to controls. In rat model, the results demonstrated that both endotoxin and CD14 levels were significantly increased in liver tissues of rats following bile duct ligation. CONCLUSIONS: The significant increase in plasma endotoxin and soluble CD14 levels during BA implies a possible involvement of endotoxin stimulated CD14 production by hepatocytes in the early stage of BA for removal of endotoxin; whereas, endotoxin signaling likely induced liver injury and impaired soluble CD14 synthesis in the late stages of BA.

Hepatoma-derived growth factor enhances osteoblastic transformation of rat aortic vascular smooth muscle cells in vitro.

AIMS: Vascular smooth muscle cells (VSMCs) are important regulators of vascular functions and their conversion to osteoblasts is a key to development of vascular calcification. This study aimed to characterize in vitro effect of hepatoma-derived growth factor (HDGF) on phenotypic conversion of cultured aortic VSMCs into osteoblast-like cells. MATERIALS AND METHODS: Cell proliferation and migration assays were used to examine cell behaviors. Western blotting, alkaline phosphatase activity and calcium staining were used to evaluate osteoblastic marker expression and function, respectively. KEY FINDINGS: Recombinant HDGF treatment enhanced VSMC growth and motility. Treatment of osteogenic medium (OM) increased expression of not only HDGF but also osteoblastic markers, including Runx2 and osteopontin (OPN), while VSMC marker α-smooth muscle actin (α-SMA) declined. Coincidentally, HDGF and OM treatment alone stimulated signaling activities in both PI3K/Akt and MAPK pathways. Conversely, inhibition of Akt and p38 significantly blocked the OM-upregulated HDGF, Runx2, and OPN expression and NF-κB phosphorylation, but did not reversed the α-SMA downregulation, implicating the involvement of Akt and p38 activities in the osteoblastic transformation of VSMCs. Small interfering RNA-mediated HDGF gene silencing effectively prevented the Runx2 and OPN upregulation, alkaline phosphatase activation, and calcium deposition, but did not affect the α-SMA levels in the transformed cells, supporting the involvement of HDGF in regulation of Runx2 and OPN expression. SIGNIFICANCE: In conclusion, in synergism with other osteogenic factor, HDGF may promote the progression of osteobastic transformation of VSMCs via Akt and p38 signaling pathways and contribute to vascular calcification in arteriosclerosis. CHEMICAL COMPOUNDS STUDIED IN THIS STUDY: HDGF (PubChem CID:); LY294002 (PubChem CID: 3973); PD98059 (PubChem CID: 4713); SB203580 (PubChem CID: 176155); SB431542 (PubChem CID: 4521392); SP600125 (PubChem CID: 8515); Wortmannin (PubChem CID: 312145).

The Clinical Impacts of Pretreatment Peripheral Blood Ratio on Lymphocytes, Monocytes, and Neutrophils Among Patients with Laryngeal/Hypopharyngeal Cancer Treated by Chemoradiation/Radiation.

PURPOSE: This study aimed to investigate the clinical impacts of the pretreatment peripheral blood ratios of lymphocytes, monocytes and neutrophils among patients with hypopharyngeal cancer/laryngeal cancer. PATIENTS AND METHODS: A total of 141 people with cases of hypopharyngeal cancer/laryngeal cancer were enrolled to evaluate the clinical impacts of the systemic inflammation response index (SIRI), neutrophil-lymphocyte ratio (NLR) and lymphocyte-monocyte ratio (LMR) in pretreatment blood among patients with laryngeal/hypopharyngeal cancer between January 2012 and December 2014. RESULTS: Those patients with higher pretreatment LMR (>2.99) showed a significantly higher 5-year complete response rate (CR) (69% vs 31%) than those with lower LMR (≤2.99, p = 0.006). Additionally, those patients with lower pretreatment SIRI (<3.26) showed a significantly higher 5-year CR (90% vs 10%) than those with higher SIRI (≥3.26, p < 0.001). Patients with higher LMR had better 5-year overall survival (OS) (p = 0.01) and 5-year progression-free (PFS) (p = 0.005) rates than those with lower LMR in univariate analysis. Patients with lower SIRI had better 5-year OS (p < 0.001) and 5-year PFS (p < 0.001) than those with higher SIRI in univariate analysis. In the Cox regression analysis, SIRI (HR = 1.941, [95% CI: 1.223-3.081], p = 0.005) and N classification (HR = 2.203, [95% CI: 1.327-3.657], p = 0.002) were independent variables of 5-year OS. In addition, SIRI (HR= 2.127, [95% CI: 1.214-3.725], p = 0.008), T classification (HR = 2.18, [95% CI: 1.072-4.433], p = 0.031), and N classification (HR = 2.329, [95% CI: 1.395-3.889], p = 0.001) were independent variables of 5-year PFS. CONCLUSION: Pretreatment SIRI is superior to LMR in predicting treatment response and clinical outcomes among patients with laryngeal/hypopharyngeal cancer treated by CRT/RTO. SIRI may be adopted in the treatment of laryngeal/hypopharyngeal cancer by CRT/RTO.

Infusion of Human Mesenchymal Stem Cells Improves Regenerative Niche in Thioacetamide-Injured Mouse Liver.

BACKGROUND: This study investigated whether xenotransplantation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. METHODS: Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6 weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. RESULTS: WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-ß1-stimulation in Smad2 phosphorylation and RhoA upregulation. CONCLUSION: These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

Dexamethasone restores transforming growth factor-ß activated kinase 1 expression and phagocytosis activity of Kupffer cells in cholestatic liver injury.

The role of transforming growth factor-ß activated kinase 1 (TAK1) in modulating the function of Kupffer cells (KCs) within cholestatic livers remains unclear. This study examined the immunopharmacological action of dexamethasone (DEX) in modulating hepatic TAK1 expression and related signaling activity in a rat model of bile duct ligation-mimicked obstructive jaundice. The in vitro effects of DEX on porcine biliary extract (PBE)-modulated gene expression and phagocytosis of KCs were examined using a rat alveolar macrophage cell line (NR8383 cells). Although DEX therapy did not restore the downregulated TAK1 expression and phosphorylation, it significantly attenuated the upregulation of high-mobility group box 1 expression and caspase-3 activation in whole liver extracts of cholestatic rats, possibly via enhancing extracellular signal-regulated kinase-mediated signaling. Dual immunofluorescence staining of cholestatic livers and western detection on primary KCs isolated from cholestatic livers identified that DEX treatment indeed increased both the expression and phosphorylation levels of TAK1 in the KCs of cholestatic livers. In vitro studies using alveolar NR8383 macrophages with KC-characteristic gene expression further demonstrated that DEX not only repressed the pro-inflammatory cytokine production including with respect to interleukin (IL)-1ß and IL-6, but also enhanced gene expression of TAK1 and a phagocytic marker, natural-resistance-associated macrophage protein 1, under PBE-mimicked cholestatic conditions. However, WST-1 assay showed that DEX did not protect NR8383 macrophages against the PBE-induced cytotoxicity. Immunofluorescence visualization of cellular F-actin by phalloidin suggested that DEX sustained the PBE-induced phagocytosis morphology of NR8383 macrophages. In conclusion, DEX treatment may pharmacologically restore the expression and activity of TAK1 in KCs, and sustain the phagocytic phenotype of KCs in cholestatic livers.

Triggering TLR3 pathway promotes tumor growth and cisplatin resistance in head and neck cancer cells.

Over the last decades, significant advances in targeted therapies have helped provide more effective treatment for head and neck cancer patients. However, chemo-resistance to cisplatin significantly contributes to treatment failure in the clinical management of patients. In response to chemotherapeutic agents, certain molecules inside the cell are released or secreted from damaged or dead/dying cells, named damage-associated molecular patterns (DAMPs), thereby initiating an immune response through interaction with pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs). In present study, we investigated the link between cisplatin-induced DAMPs and TLR3 signaling. We found that cisplatin could be a potential activator of TLR3 and cisplatin treatment results in activation of PRRs' signaling and down-stream associated cytokine/chemokine, IFNß, and CCL5 in TLR3High OC2 cells, but not in TLR3Low FaDu cells. Furthermore, knockdown of the TLR3 gene attenuates the expression of IFNß and CCL5 mRNA and enhances the cytotoxicity of cisplatin in TLR3High OC2 cells. To determine whether TLR3 status affects the stress response of OC2 cells to cisplatin, we generated TLR3 knockdown OC2 cells (psi-TLR3 cells) with a psiRNA-hTLR3 plasmid containing shRNA to TLR3 and control OC2 cells (psi-NT cells) expressing non-silencing shRNA. OC2 cells were more sensitive to cisplatin treatment after TLR3 knockdown. In our animal model, OC2 psi-NT cells were more tumorigenic than were OC2 psi-TLR3 cells. Together, our in vitro and in vivo data imply TLR3 may contribute to tumor development and protect cisplatin-induced DNA damage response leading to cisplatin resistance in head and neck cancer cells.

Characterization of plasma proteome in biliary atresia.

BACKGROUND: Biliary atresia (BA) is a disorder during infancy with unknown etiology in which progression frequently leads to liver cirrhosis. Plasma proteome is characterized in this study. METHODS: Twelve paired plasma samples from 6 children with BA who received surgical correction at early stage and then liver transplantation at late stage of liver cirrhosis were studied. Plasma samples from 2 subjects without liver disorder were used as normal reference for 2-dimensional gel electrophoresis and for identification of protein spots by mass spectrometric analysis. Plasma samples from another 3 normal subjects (with a total of 5) were used for nephelometric quantification of immunoglobulin kappa light chain in comparison with patients' samples. RESULTS: Among the protein spots detected, ranging from 6 to 200 kDa mass with pIs of 3-10, significant up-regulation of immunoglobulin kappa light chain was found at the late stage of BA, which was subsequently confirmed by nephelometric analysis. Conversely, significant decrease of apolipoprotein (Apo) A-I and C-II, haptoglobin alpha2 and beta chain, and transthyretin were detected during the progression of BA. CONCLUSIONS: Increased immunoglobulin kappa light chain detected in late-stage BA characterizes adverse immune modulation in this disorder. Decreased apolipoproteins, haptoglobin and transthyretin levels might be potential markers of progressive liver injury, fibrosis and defective lipid metabolism in BA.

Cobalt chloride induces RhoA/ROCK activation and remodeling effect in H9c2 cardiomyoblasts: Involvement of PI3K/Akt and MAPK pathways.

Chronic heart failure is a serious complication of myocardial infarction, one of the major causes of death worldwide that often leads to adverse cardiac hypertrophy and poor prognosis. Hypoxia-induced cardiac tissue remodeling is considered an important underlying etiology. This study aimed to delineate the signaling profiles of RhoA/ROCK, PI3K/Akt, and MAPK and their involvement in regulation of remodeling events in cultured H9c2 cardiomyoblast cells. In addition to its growth-suppressive effect, the hypoxia-mimetic chemical, cobalt chloride (CoCl2) significantly induced RhoA kinase activation as revealed by increased MBS phosphorylation and ROCK1/2 expression in H9c2 cells. CoCl2 treatment up-regulated type I collagen and MMP-9, but did not affect MMP-2, implicating its role in tissue remodeling. Kinetic signal profiling study showed that CoCl2 also elicited Smad2 hyperphosphorylation and its nuclear translocation in the absence of TGF-ß1. In addition, CoCl2 activated Akt-, ERK1/2-, JNK-, and p38 MAPK-mediated signaling pathways. Kinase inhibition experiments demonstrated that hydroxyfasudil, a RhoA kinase inhibitor, significantly blocked the CoCl2- and lysophosphatidic acid-evoked Smad2 phosphorylation and overexpression of type I collagen and MMP-9, and that PI3K and ERK interplayed with RhoA and its downstream Smad2 signaling cascade. In conclusion, this study demonstrated that RhoA/ROCK, PI3K/Akt, and MAPK pathways are mechanistically involved in the CoCl2-stimulated tissue remodeling in H9c2 cardiomyoblast cells. Targeting signaling mediators might be used to mitigate hypoxia-related Smad2 phosphorylation and cardiac remodeling events in ischemic cardiomyopathy.

2-Deoxyglucose treatment complements the cisplatin- or BH3-only mimetic-induced suppression of neuroblastoma cell growth.

Neuroblastoma (NB) is characterized by pleomorphic molecular characteristics, which may influence cellular metabolism as well as the efficacy of glycolytic inhibitors in suppressing NB cell growth. We studied the metabolic profile of four NB cell lines without or with MYCN amplification and found no unanimous metabolic characteristics. The two NB cell lines with MYCN amplification exhibited a significantly higher HIF-1α expression level and ATP content compared to the two cell lines without MYCN amplification. MYCN amplification was associated with significantly greater inhibition of cellular proliferation and more apoptosis after treatment with the glycolytic inhibitor 2-deoxyglucose (2DG). Further analysis showed that 2DG decreased both PDK1 and the ATP content. [corrected]. In addition, 2DG decreased hexokinase II expression in SK-N-DZ cells and increased HIF-1α, Noxa, and PUMA expression in SK-N-AS cells. Pretreating SK-N-DZ cells with 2DG or cisplatin for 24 h, followed by cisplatin or 2DG for another 24 h, resulted in significantly greater suppression of cellular proliferation compared to treatment with 2DG or cisplatin for 48 h alone. Effective suppression of SK-N-AS proliferation occurred only when the cells were pretreated with cisplatin. Pretreatment of SK-N-DZ, but not SK-N-AS, with 2DG followed by the BH3-only mimetic ABT737 also resulted in significantly greater suppression of cellular proliferation compared to treatment with ABT737 or 2DG alone. A low dose of 2DG (2mM) was as effective as a high dose (20mM) in SK-N-DZ cells. In conclusion, the glycolytic inhibitor 2DG complemented the cisplatin- or ABT737-induced suppression of growth in NB cells, which are sensitive to glycolytic inhibition.

Effects of hepatocyte CD14 upregulation during cholestasis on endotoxin sensitivity.

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats.

Academic achievements of doctors who studied at a domestic institute for clinical medicine.

BACKGROUND: The purpose of this study was to investigate the outcomes of doctors who studied at a graduate degree program at a domestic institute for clinical medicine. METHODS: The academic results of 45 doctors who studied at the Graduate Institute of Clinical Medical Sciences-Kaohsiung Branch (GICMS-KB) of Chang Gung University (CGU) were analyzed and compared with those of 83 doctors who had studied abroad or at domestic institutions other than CGU (non-CGU), as well as with 263 who did not pursue further graduate studies (no GS) during the study period from 2003 to 2007. The 128 doctors who had pursued graduate study were sent a survey consisting of 6 questions about factors which hamper research activity. RESULTS: The average number of Science Citation Index (SCI) articles published by the doctors of GICMS-KB as the first author over five years was 3.16 ± 0.52, which was significantly higher than 1.51 ± 0.22 in the no GS group (p = 0.005) and 2.31 ± 0.39 in the non-CGU group. The average number of research grants was 3.62 ± 0.64 in GICMS-KB group, which was higher than 1.57 ± 0.28 in the non-CGU and 1.56 ± 0.20 in the no GS groups (p < 0.001 in both). The percentage of the doctors with faculty positions was 58% in GICMS-KB group, higher than 43% in the non-CGU and 38% in the no GS groups, with a p value of 0.055 comparing GICMS-KB with the no GS group. The survey indicated that all doctors who pursued post-graduate training had similar problems, such as lack of time and lack of research manpower with no difference between the GICMS-KB and non-CGU groups. CONCLUSION: The academic performance of doctors who did postgraduate study at our graduate institute for clinical medicine was generally better than those who did not pursue further graduate studies or those with postgraduate studies outside CGU. Factors such as adaptation of the GICMS-KB doctors to our system and policies which include enforced grant writing and publication for graduation during the training course may account for the difference.