Influence of type‐I Interferon receptor expression level on the response to type‐I Interferons in human pancreatic cancer cells

Pancreatic cancer is a highly aggressive malignancy with limited treatment options. Type‐I interferons (e.g. IFN‐α/‐β) have several anti‐tumour activities. Over the past few years, clinical studies evaluating the effect of adjuvant IFN‐α therapy in pancreatic cancer yielded equivocal results. Although IFN‐α and ‐β act via the type‐I IFN receptor, the role of the number of receptors present on tumour cells is still unknown. Therefore, this study associated, for the first time, in a large panel of pancreatic cancer cell lines the effects of IFN‐α/‐β with the expression of type‐I IFN receptors. The anti‐tumour effects of IFN‐α or IFN‐β on cell proliferation and apoptosis were evaluated in 11 human pancreatic cell lines. Type‐I IFN receptor expression was determined on both the mRNA and protein level. After 7 days of incubation, IFN‐α significantly reduced cell growth in eight cell lines by 5–67%. IFN‐β inhibited cell growth statistically significant in all cell lines by 43–100%. After 3 days of treatment, IFN‐β induced significantly more apoptosis than IFN‐α. The cell lines variably expressed the type‐I IFN receptor. The maximal inhibitory effect of IFN‐α was positively correlated with the IFNAR‐1 mRNA (P < 0.05, r = 0.63), IFNAR‐2c mRNA (P < 0.05, r = 0.69) and protein expression (P < 0.05, r = 0.65). Human pancreatic cancer cell lines variably respond to IFN‐α and ‐β. The expression level of the type‐I IFN receptor is of predictive value for the direct anti‐tumour effects of IFN‐α treatment. More importantly, IFN‐β induces anti‐tumour effects already at much lower concentrations, is less dependent on interferon receptor expression and seems, therefore, more promising than IFN‐α.     

Tumor Necrosis Factor‐α Stimulates Cell Proliferation in Adipose Tissue‐Derived Stromal‐Vascular Cell Culture: Promotion of Adipose Tissue Expansion by Paracrine Growth Factors

Objective: Elevated levels of tumor necrosis factor‐α (TNF‐α) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF‐α has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF‐α, alone or together with insulin‐like growth factor‐I (IGF‐I), support preadipocyte development. Research Methods and Procedures: Cultured stromal‐vascular cells from rat inguinal fat depots were exposed to serum‐free media containing insulin and 0.2 nM TNF‐α, 2.0 nM TNF‐α, or 0.2 nM TNF‐α + 1.0 nM IGF‐I at different times during 7 days of culture. Results: TNF‐α inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte‐specific antigen (AD3; early differentiation marker) and glycerol‐3‐phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF‐α did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF‐α from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF‐α + 1 nM IGF‐I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. Discussion: These data suggest that paracrine levels (0.2 nM) of TNF‐α alone or in combination with IGF‐I may support adipose tissue development by increasing the total number of stromal‐vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.     

TNF‐α‐induced cardiomyocyte apoptosis contributes to cardiac dysfunction after coronary microembolization in mini‐pigs

This experimental study was designed to clarify the relationship between cardiomyocyte apoptosis and tumour necrosis factor‐alpha (TNF‐α) expression, and confirm the effect of TNF‐α on cardiac dysfunction after coronary microembolization (CME) in mini‐pigs. Nineteen mini‐pigs were divided into three groups: sham‐operation group (n = 5), CME group (n = 7) and adalimumab pre‐treatment group (n = 7; TNF‐α antibody, 2 mg/kg intracoronary injection before CME). Magnetic resonance imaging (3.0‐T) was performed at baseline, 6th hour and 1 week after procedure. Cardiomyocyte apoptosis was detected by cardiac‐TUNEL staining, and caspase‐3 and caspase‐8 were detected by RT‐PCR and immunohistochemistry. Furthermore, serum TNF‐α, IL‐6 and troponin T were analysed, while myocardial expressions of TNF‐α and IL‐6 were detected. Both TNF‐α expression (serum level and myocardial expression) and average number of apoptotic cardiomyocyte nuclei were significantly increased in CME group compared with the sham‐operation group. Six hours after CME, left ventricular end‐systolic volume (LVESV) was increased and the left ventricular ejection fraction (LVEF) was decreased in CME group. Pre‐treatment with adalimumab not only significantly improved LVEF after CME (6th hour: 54.9 ± 2.3% versus 50.4 ± 3.9%, P = 0.036; 1 week: 56.7 ± 4.2% versus 52.7 ± 2.9%, P = 0.041), but also suppressed cardiomyocyte apoptosis and the expression of caspase‐3 and caspase‐8. Meanwhile, the average number of apoptotic cardiomyocytes nuclei was inversely correlated with LVEF (r = ?0.535, P = 0.022). TNF‐α‐induced cardiomyocyte apoptosis is likely involved in cardiac dysfunction after CME. TNF‐α antibody therapy suppresses cardiomyocyte apoptosis and improves early cardiac function after CME.     

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet‐induced obesity

Mice with diet‐induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN‐γ; IFN‐α receptor 1; CD247 antigen; killer cell lectin‐like receptor subfamily K, member 1; TNF‐α; IL‐12 receptor β1 and IL‐2 receptor β, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.     

Endothelial Progenitor Cell Function,Apoptosis, and Telomere Length in Overweight/Obese Humans

Excess adiposity is associated with increased cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) play an important role in vascular repair. We tested the hypothesis that increased adiposity is associated with EPC dysfunction, characterized by diminished capacity to release angiogenic cytokines, increased apoptotic susceptibility, reduced cell migration, and shorter telomere length. A total of 67 middle‐aged and older adults (42–67 years) were studied: 25 normal weight (normal weight; BMI: 18.5–24.9 kg/m²) and 42 overweight/obese (overweight/obese; BMI: 25.0–34.9 kg/m²). Cells with phenotypic EPC characteristics were isolated from peripheral blood. EPC release of vascular endothelial growth factor (VEGF) and granulocyte colony–stimulating factor (G‐CSF) was determined in the absence and presence of phytohemagglutinin (10 µg/ml). Intracellular active caspase‐3 and cytochrome c concentrations were determined by immunoassay. Migratory activity of EPCs in response to VEGF (2 ng/ml) and stromal cell–derived factor‐1α (SDF‐1α; 10 ng/ml) was determined by Boyden chamber. Telomere length was assessed by Southern hybridization. Phytohemagglutinin‐stimulated release of VEGF (90.6 ± 7.6 vs. 127.2 ± 11.6 pg/ml) and G‐CSF (896.1 ± 77.4 vs. 1,176.3 ± 126.3 pg/ml) was ～25% lower (P < 0.05) in EPCs from overweight/obese vs. normal weight subjects. Staurosporine induced a ～30% greater (P < 0.05) increase in active caspase‐3 in EPCs from overweight/obese (2.8 ± 0.2 ng/ml) compared with normal weight (2.2 ± 0.2) subjects. There were no significant differences in EPC migration to either VEGF or SDF‐1α. Telomere length did not differ between groups. These results indicate that increased adiposity adversely affects the ability of EPCs to release proangiogenic cytokines and resist apoptosis, potentially compromising their reparative potential.     

Activation and promotion of adipose stem cells by tumour necrosis factor‐alpha preconditioning for bone regeneration

There is a major medical need for developing novel and effective approaches for repairing non‐union and critical‐sized bone defects. Although the mechanisms remain to be determined, it is known that inflammation plays a crucial role in initiating bone repair and regeneration. This study investigated the effect of short‐term (3 days) preconditioning with tumor necrosis factor‐alpha (TNF‐α) on proliferation, mobilization, and differentiation of adipose tissue‐derived mesenchymal stem cells (ASCs). We demonstrated that TNF‐α pre‐conditioning increased proliferation, mobilization, and osteogenic differentiation of ASCs and up‐regulated bone morphogenetic protein‐2 (BMP‐2) protein level. BMP‐2 silencing by siRNA partially inhibited osteogenic differentiation of ASCs induced by TNF‐α; BMP‐2 pre‐conditioning also significantly increased osteogenic differentiation of ASCs but the effects were significantly smaller than those observed for TNF‐α preconditioning. Furthermore, TNF‐α treatment promoted extracellular‐signal‐regulated kinases(Erk)1/2 and p38 mitogen‐activated protein kinase (MAPK) signaling pathways, but only Erk1/2 inhibition reduced the BMP‐2 levels and osteogenic differentiation induced by TNF‐α preconditioning. Together, these results support the hypothesis that inflammation contributes to bone regeneration by promoting proliferation, mobilization, and osteogenic differentiation of ASCs; 3 days of TNF‐α preconditioning, mimicking the short boost of inflammation normally occurring after bone injury, might serve as a feasible approach for directing stem cells into osteogenic differentiation. J. Cell. Physiol. 9999: XX–XX, 2013. © 2013 Wiley Periodicals, Inc. J. Cell. Physiol. 228: 1737–1744, 2013. © 2013 Wiley Periodicals, Inc.     

C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: Relevance to disease and ageing?

Reduced muscle mass and increased susceptibility to TNF‐induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C₂ myoblasts display diminished differentiation and increased susceptibility to TNF‐α‐induced cell death versus subcloned C₂C₁₂ cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan‐blue) and anabolic/catabolic parameters (RT‐PCR) over 72 h ± TNF‐α (20 ng ml^?1). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 ± 6.82 vs. 30.71 ± 2.5, P < 0.05; 544.94 ± 27.7 vs. 39.4 ± 3.37 mU mg ml^?1, P < 0.05), fold increases in myoD (21.45 ± 3.12 vs. 3.97 ± 1.76, P < 0.05; 31.07 ± 3.1 vs. 6.82 ± 1.93, P < 0.05) and myogenin mRNA (241.8 ± 40 vs. 36.80 ± 19.3, P < 0.05; 440 ± 100.5 vs. 201.1 ± 86, P < 0.05) were detected in C₂C₁₂ versus C₂. C₂C₁₂ showed significant increases in IGF‐I mRNA (243.05 ± 3.87 vs. 105.75 ± 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 ± 0.28 vs. 1.79 ± 0.29 mg ml^?1, P < 0.05) at 72 h versus C₂ cells. Significant temporal reductions in C₂C₁₂ IGFBP2 mRNA (28.02 ± 15.44, 13.82 ± 8.07, 6.92 ± 4.37, P < 0.05) contrasted increases in C₂s (4.31 ± 3.31, 13.02 ± 9.92, 82.9 ± 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF‐α increased cell death in C₂s (2.67 ± 1.54%, 34.42 ± 5.39%, 29.71 ± 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C₂C₁₂s at 48 h but caused a small significant increase at 72 h (9.88 ± 4.02% (TNF‐α) vs. 6.17 ± 0.749% (DM), 72 h). TNF‐α and TNFRI mRNA were unchanged; however, larger reductions in IGF‐I (8.2‐ and 7.5‐fold vs. 4.5‐ and 4.1‐fold (48, 72 h)), IGF‐IR (2‐fold vs. no‐significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C₂ versus C₂C₁₂ with TNF‐α. This investigation provides insight into regulators of altered basal hypertrophy and TNF‐induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders. J. Cell. Physiol. 225: 240–250, 2010. © 2010 Wiley‐Liss, Inc.     

Determine exogenous human DDAH2 gene function in rabbit bone marrow–derived endothelial progenitor cells in vitro

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow–derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2‐EPCs). Three days after gene transfer, positive transfected‐EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium‐like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk‐1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.     

Short‐term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress

To determine if short‐term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n = 30) and young (Y, n = 10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, approximately 30%) for 8 weeks. Ex vivo carotid artery endothelium‐dependent dilation (EDD) was impaired in old ad libitum (OAL) vs. young ad libitum (YAL) (74 ± 5 vs. 95 ± 2% of maximum dilation, P < 0.05), whereas old calorie‐restricted (OCR) and YCR did not differ (96 ± 1 vs. 94 ± 3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P < 0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P < 0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P < 0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97 ± 2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P < 0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P < 0.05), but unaffected by CR. Carotid artery endothelium‐independent dilation did not differ among groups. Short‐term CR initiated in old age reverses age‐associated vascular endothelial dysfunction by restoring NO bioavailability, reducing oxidative stress (via reduced NADPH oxidase–mediated superoxide production and stimulation of anti‐oxidant enzyme activity), and upregulation of sirtuin‐1.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Molecular mechanism of tumour necrosis factor alpha regulates hypocretin (orexin) expression,sleep and behaviour

Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.     

TNF‐α Promoter Methylation as a Predictive Biomarker for Weight‐loss Response

Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine which is commonly elevated in obese subjects and whose promoter is susceptible to be regulated by cytosine methylation. The aim of this research was to analyze whether epigenetic regulation of human TNF‐α promoter by cytosine methylation could be involved in the predisposition to lose body weight after following a balanced hypocaloric diet. Twenty‐four patients (12 women/12 men) with excessive body weight‐for‐height (BMI: 30.5 ± 0.32 kg/m²; age: 34 ± 4 years old) followed an 8‐week energy‐restricted diet. Blood mononuclear cell DNA, isolated before the nutritional intervention, was treated with bisulfite and a region of TNF‐α gene promoter (from ?360 to +50 bp) was sequenced. Obese men with successful weight loss (≥5% of initial body weight) showed lower levels of total TNF‐α promoter methylation (r = 0.74; P = 0.021), especially in the positions ?170 bp (r = 0.75, P = 0.005) and ?120 bp (r = 0.70, P = 0.011). Baseline TNF‐α circulating levels were positively associated with total promoter methylation (r = 0.84, P = 0.005) and methylation at position ?245 bp (r = 0.75, P = 0.020). TNF‐α promoter methylation could be a good inflammation marker predicting the hypocaloric diet‐induced weight‐loss, and constitutes a first step toward personalized nutrition based on epigenetic criteria.     

Effect of Lifestyle Modification on Adipokine Levels in Obese Subjects with Insulin Resistance

Objective: To study the effect of weight loss in response to a lifestyle modification program on the circulating levels of adipose tissue derived cytokines (adipokines) in obese individuals with insulin resistance. Research Methods and Procedures: Twenty‐four insulin‐resistant obese subjects with varying degrees of glucose tolerance completed a 6‐month program consisting of combined hypocaloric diet and moderate physical activity. Adipokines [leptin, adiponectin, resistin, tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6)] and highly sensitive C‐reactive protein were measured before and after the intervention. Insulin sensitivity index was evaluated by the frequently sampled intravenous glucose tolerance test. Results: Participants had a 6.9 ± 0.1 kg average weight loss, with a significant improvement in sensitivity index and reduction in plasma leptin (27.8 ± 3 vs. 23.6 ± 3 ng/mL, p = 0.01) and IL‐6 (2.75 ± 1.51 vs. 2.3 ± 0.91 pg/mL, p = 0.012). TNF‐α levels tended to decrease (2.3 ± 0.2 vs. 1.9 ± 0.1 pg/mL, p = 0.059). Adiponectin increased significantly only among diabetic subjects. The reductions in leptin were correlated with the decreases in BMI (r = 0.464, p < 0.05) and with changes in highly sensitive C‐reactive protein (r = 0.466, p < 0.05). Discussion: Weight reduction in obese individuals with insulin resistance was associated with a significant decrease in leptin and IL‐6 and a tendency toward a decrease in circulating TNF‐α, whereas adiponectin was increased only in diabetic subjects. Further studies are needed to elucidate the relationship between changes of adipokines and the health benefits of weight loss.     

Roles of cytokines and progesterone in the regulation of the nitric oxide generating system in bovine luteal endothelial cells

Nitric oxide (NO) produced by luteal endothelial cells (LECs) plays important roles in regulating corpus luteum (CL) function, yet the local mechanism regulating NO generation in bovine CL remains unclear. The purpose of the present study was to elucidate if tumor necrosis factor‐α (TNF), interferon γ (IFNG), and/or progesterone (P4) play roles in regulating NO generating system in LECs. Cultured bovine LECs obtained from the CL at the mid‐luteal stage (Days 8–12 of the cycle) were treated for 24 hr with TNF (2.9 nM), IFNG (2.5 nM), or P4 (0.032–32 µM). NO production was increased by TNF and IFNG, but decreased by P4 (P < 0.05). TNF and IFNG stimulated the relative steady‐state amounts of inducible nitric oxide synthase (iNOS) mRNA and iNOS protein expression (P < 0.05), whereas P4 inhibited relative steady‐state amounts of iNOS mRNA and iNOS protein expression (P < 0.05). In contrast, endothelial nitric oxide synthase (eNOS) expression was not affected by any treatment. TNF and IFNG stimulated NOS activity (P < 0.05) and 1400W, a specific inhibitor of iNOS, reduced NO production stimulated by TNF and IFNG in LECs (P < 0.05). Onapristone, a specific P4 receptor antagonist, blocked the inhibitory effect of P4 on NO production in LECs (P < 0.05). The overall findings suggest that TNF and IFNG accelerate luteolysis by increasing NO production via stimulation of iNOS expression and NOS activity in bovine LECs. P4, on the other hand, may act in maintaining CL function by suppressing iNOS expression in bovine LECs. Mol. Reprod. Dev. 79: 689–696, 2012. © 2012 Wiley Periodicals, Inc.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Severely Obese Have Greater LPS‐stimulated TNF‐α Production Than Normal Weight African‐American Women

Obesity is associated with an increase in chronic, low‐grade inflammation which has been implicated in the development of type 2 diabetes mellitus and cardiovascular disease. The purpose of this study was to determine whether obesity was associated with an elevation of whole blood lipopolysaccharide (LPS)‐stimulated tumor necrosis factor‐α (TNF‐α) production. African‐American women were recruited from a larger study and assigned to one of five groups based on BMI: normal weight (NORM; BMI 20–25, n = 7), overweight (OVER; BMI 25–30, n = 12), class 1 obese (OB1; BMI 30–35, n = 19), class 2 obese (OB2; BMI 35–40, n = 10), or class 3 obese (OB3; BMI >40, n = 17). Body composition was determined via a whole body dual‐energy X‐ray absorptiometry (DXA) scan. Venous blood samples were collected following an overnight fast (>8 h), and stimulated with five doses of LPS (Salmonella enteriditis): 80, 40, 20, 10, and 5 µg/ml for 24 h in a 37 °C, 5% CO₂ incubator. Following stimulation, TNF‐α was measured using enzyme‐linked immunosorbent assay. OB3 produced 365% more TNF‐α than NORM at an LPS dose of 20 µg/ml (P < 0.05). When maximal TNF‐α production was assessed regardless of LPS dose, OB3 produced 230% more than NORM and OVER produced 190% more than NW (P = 0.001). Total and trunk fat mass and BMI were significantly correlated with maximal TNF‐α production and LPS = 20 µg/ml. Our findings are consistent with previous reports suggesting a relationship between increased adiposity and inflammatory marker production. This is one of the first studies to focus on African‐American women, who have higher rates of obesity.     

The flow-mediated dilation response to acute exercise in overweight active and inactive men

Objective: Inflammation has been found to play a role in the etiology of cardiovascular disease as well as provoke endothelial dysfunction. Inflammatory cytokines associated with endothelial function are interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α). IL‐6 is exercise intensity dependent and has been shown to inhibit TNF‐α expression directly. The aim of this study was to investigate the interaction of IL‐6 and TNF‐α on endothelial function in response to acute exercise in overweight men exhibiting different physical activity profiles. Methods and Procedures: Using a randomized mixed factorial design, 16 overweight men (8 active, maximal exercise capacity (VO₂peak) = 34.2 ± 1.7, BMI = 27.4 ± 0.7 and 8 inactive, VO₂peak = 30.9 ± 1.2, BMI = 29.3 ± 1.0) performed three different intensity acute exercise treatments. Brachial artery flow‐mediated dilation (FMD) and subsequent blood samples were taken pre‐exercise and 1 h following the cessation of exercise. Results: Independent of exercise intensity, the active group displayed a 24% increase (P = 0.034) in FMD following acute exercise compared to a 32% decrease (P = 0.010) in the inactive group. Elevated (P < 0.001) concentrations of IL‐6 following moderate (50% VO₂) and high (75% VO₂) intensity acute exercise were observed in both groups; however, concentrations of TNF‐ α were unchanged in response to acute exercise (P = 0.584). Discussion: The FMD response to acute exercise is enhanced in active men who are overweight, whereas inactive men who are overweight exhibit an attenuated response. The interaction of IL‐6 and TNF‐ α did not provide insight into the physiological mechanisms associated with the disparity of FMD observed between groups.