The glucagon-like peptide 1 analog liraglutide reduces TNF-α-induced oxidative stress and inflammation in endothelial cells

ObjectiveGlucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells.Methods and resultsThe effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H₂DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until 30 nM while liraglutide inhibited the induction of gp91^phox and p22^phox, subunit of NADPH oxidase, by TNF-α? In addition, protein levels of SOD-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-3 induced by TNF-α.ConclusionLiraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes.     

Estrogen receptor agonists and estrogen attenuate TNF-α-induced apoptosis in VSC4.1 motoneurons

Tumor necrosis factor-alpha (TNF-α) may cause apoptosis and inflammation in amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI). Recent studies suggest that estrogen (EST) provides neuroprotection against SCI. We tested whether 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (EST receptor alpha (ERα) agonist), 2,3-bis (4-hydroxyphenyl) propionitrile (DPN) (EST receptor beta (ERβ) agonist), or EST itself would prevent apoptosis in VSC4.1 motoneurons following exposure to TNF-α. Cells were exposed to TNF-α and 15?min later treated with PPT, DPN, or EST. Posttreatment with 50?nM PPT, 50?nM DPN, or 150?nM EST prevented cell death in VSC4.1 motoneurons. Treatment of VSC4.1 motoneurons with PPT, DPN, or EST induced overexpression of ERα, ERβ, or both, which contributed to neuroprotection by upregulating expression of anti-apoptotic proteins (p-AKT, p-CREB, Bcl-2, and p-Src). Our analyses also revealed that EST agonists and EST increased phosphorylation of extracellular signal-regulated kinase (ERK). The L-type Ca(2+) channel inhibitor, nifedipine (10?μM), partially inhibited EST agonist and EST-induced increase in phosphorylated ERK expression. The mitogen-activated protein kinase inhibitor, PD98059 (5?μM), partially prevented ER agonists and EST from providing neuroprotection to TNF-α toxicity. Presence of the nuclear ER antagonist, ICI 182?780 (10?μM), blocked the neuroprotection provided by all three ER agonists tested. Taken together, our data indicate that both ERα and ERβ contribute to PPT, DPN, or EST-mediated neuroprotection with similar signaling profiles. Our data strongly imply that PPT, DPN, or EST can be used as effective neuroprotective agents to attenuate motoneuron death in ALS and SCI.     

Alternatively spliced tissue factor and full-length tissue factor protect cardiomyocytes against TNF-α-induced apoptosis

Tissue Factor (TF) is expressed in various cell types of the heart, such as cardiomyocytes. In addition to its role in the initiation of blood coagulation, the TF:FVIIa complex protects cells from apoptosis. There are two isoforms of Tissue Factor (TF): "full length" (fl)TF--an integral membrane protein, and alternatively spliced (as)TF--a protein that lacks a transmembrane domain and can thus be secreted in a soluble form. Whether asTF or flTF affects apoptosis of cardiomyocytes is unknown. In this study, we examined whether asTF or flTF protects murine cardiomyocytes from TNF-α-induced apoptosis. We used murine cardiomyocytic HL-1 cells and primary murine embryonic cardiomyocytes that overexpressed either murine asTF or murine flTF, and stimulated them with TNF-α to initiate cell death. Apoptosis was assessed by annexin-V assay, propidium iodide assay, as well as activation of caspase-3 and -9. In addition, signaling via integrins, Akt, NFκB and Erk1/2, and gene-expression of Bcl-2 family members were analyzed. We here report that overexpression of asTF reduced phosphatidylserine exposure upon TNF-α-stimulation. asTF overexpression led to an increased expression and phosphorylation of Akt, as well as up-regulation of the anti-apoptotic protein Bcl-x(L). The anti-apoptotic effects of asTF overexpression were mediated via α(V)β(3)/Akt/NFκB signaling and were dependent on Bcl-x(L) expression in HL-1 cells. The anti-apoptotic activity of asTF was also observed using primary cardiomyocytes. Analogous yet less pronounced anti-apoptotic sequelae were observed due to overexpression of flTF. Importantly, cardiomyocytes deficient in TF exhibited increased apoptosis compared to wild type cells. We propose that asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis via activation of specific signaling pathways, and up-regulation of anti-apoptotic members of the Bcl-2 protein family.     

The preventive effect of low molecular weight heparin on CCL₄-induced necrosis and apoptosis in rat liver

Introduction. Heparin having anti-inflammatory and anti-fibrotic properties may have therapeutic effect on liver injury. The present study investigated the effect of low molecular weight heparin (Enoxaparin) on carbon tetrachloride (CCl₄) induced hepatic necrosis and apoptosis in rats.Material and methods. Thirty male rats were divided into 5 groups. Group I: Control; Group II: Olive oil dissolved CCl₄ at dose of 1 mL/kg, ip, twice per week; Group III: CCl₄ and Enoxaparin at dose of 180 lU/kg, sc, daily; Group IV: Enoxaparin; Group V: Olive oil at dose of 1 mL, ip, twice per week. The liver histology at the forth week was examined by haematoxylin-eosin, Masson’s trichrome, Toluidine blue and Periodic acid schiff stains. Proliferative and apoptotic activities were assessed semi-quantitatively by proliferating cell nuclear antigen (PCNA) and cas-pase-3 immune staining and TUNEL method. Semi-quantitative values formulated by the equation HSCORE = ΣP₍ including both distribution and intensity of staining. Additionally, nidogen and α-smooth muscle actin were labeled by immunohistochemistry.Results. CCl₄ group had marked hepatocelluar necrosis around the vena centralis and increased inflammatory cells and mast cells. Hepatocytes showed deposition of lipid droplets, decrease in glycogen, apoptosis, and picnotic or enlarged nuclei. Enoxaparin reduced necrosis, apoptosis, and number of mast cells but had no effect on lipid droplets in hepatocytes. HSCORE’s of caspase-3 and PCNA were also significantly decreased by administration.Conclusion. Enoxaparin have beneficial effects against necrosis as well as apoptosis at the early stage of CCL₄ induced liver injury.     

The role of apoptosis in childhood Henoch–Schonlein purpura

The pathogenesis of vasculitis is complex and is yet to be fully elucidated, although it is known that inflammatory cells play a major role. Dysregulation of apoptosis and defective clearance of inflammatory cells could lead to the persistence of inflammation and excessive tissue injury. In this study we aimed to investigate Fas (CD95) and apoptosis on peripheral blood (PB) neutrophil and lymphocytes in Henoch–Schonlein purpura, both in the acute phase and after resolution to determine the role of apoptosis in this self-limited vasculitis. Leukocytoclastic vasculitis presenting with Henoch–Schonlein purpura (HSP) was diagnosed according to ACR 1990 criteria and confirmed by skin biopsy. Thirty-seven patients (22 boys, 15 girls) aged 2.5–17 years (9 ± 3.3) were enrolled in the study. Expression of CD95 and apoptosis were investigated by the annexin/PI method on peripheral blood neutrophils and lymphocytes in both the acute and the resolution phases of the disease. The mean neutrophil and lymphocyte CD95 expression was 65.4 ± 37.6% and 33.3 ± 7.3%, respectively, in the acute stage and 62.8 ± 44.2% and 41 ± 20%, respectively, in the resolution (P > 0.05). The percentage of apoptotic peripheral blood neutrophils and lymphocytes as determined by annexin positivity was 13.3 ± 11.31% and 8.6 ± 9.5%, respectively, during the acute phase and 4.6 ± 3.4% and 3.1 ± 3.1%, respectively, in the resolution (P = 0.002, P = 0.008). These results suggest that increased apoptotic process in the immune effector cells in the acute phase of the disease may play an important role in the early control of inflammatory response and repair in leukocytoclastic vasculitis, thereby contributing to the self-limited nature of the disease.     

Long-term treatment with interleukin-1β induces insulin resistance in murine and human adipocytes

Aims/hypothesis Adipose tissue inflammation has recently been implicated in the pathogenesis of insulin resistance and is probably linked to high local levels of cytokines. IL1B, a proinflammatory cytokine, may participate in this alteration.Materials and methods We evaluated the chronic effect (1–10 days) of IL1B (0.1–20 ng/ml) on insulin signalling in differentiating 3T3-F442A and differentiated 3T3-L1 murine adipocytes and in human adipocytes. We also assessed expression of the gene encoding IL1B in adipose tissue of wild-type and insulin-resistant mice (diet-induced and genetically obese ob/ob mice).Results IL1B inhibited insulin-induced phosphorylation of the insulin receptor β subunit, insulin receptor substrate 1, Akt/protein kinase B and extracellular regulated kinase 1/2 in murine and human adipocytes. Accordingly, IL1B suppressed insulin-induced glucose transport and lipogenesis. Long-term treatment of adipose cells with IL1B decreased cellular lipid content. This could result from enhanced lipolysis and/or decreased expression of genes involved in lipid metabolism (acetyl-CoA carboxylase, fatty acid synthase). Down-regulation of peroxisome proliferating-activated receptor γ and CCAAT/enhancer-binding protein α in response to IL1B may have contributed to the altered phenotype of IL1B-treated adipocytes. Moreover, IL1B altered adipocyte differentiation status in long-term cultures. IL1B also decreased the production of adiponectin, an adipocyte-specific protein that plays a positive role in insulin sensitivity. Expression of the gene encoding IL1B was increased in epididymal adipose tissue of obese insulin-resistant mice.Conclusions/interpretation IL1B is upregulated in adipose tissue of obese and insulin-resistant mouse models and may play an important role in the development of insulin resistance in murine and human adipose cells.Electronic supplementary material Supplementary material is available in the online version for this article at and is accessible for authorized users.     

TNF-α production and apoptosis in hepatocytes after Listeria monocytogenes and Salmonella Typhimurium invasion

Invasion of hepatocytes by Listeria monocytogenes (LM) and Salmonella Typhimurium (ST) can stimulate tumor necrosis factor alpha (TNF-α) release and induce apoptosis. In this study, we compared the behavior of hepatocytes invaded by three L. monocytogenes serotypes (LM-4a, LM-4b and LM-1/2a) and by ST to understand which bacterium is more effective in the infectious process. We quantified TNF-α release by ELISA, apoptosis rates by annexin V (early apoptosis) and TUNEL (late apoptosis) techniques. The cell morphology was studied too. TNF-α release rate was highest in ST-invaded hepatocytes. ST and LM-1/2a induced the highest apoptosis production rates evaluated by TUNEL. LM-4b produced the highest apoptosis rate measured by annexin. Invaded hepatocytes presented various morphological alterations. Overall, LM-4b and LM-1/2a proved to be the most efficient at cell invasion, although ST adapted faster to the environment and induced earlier hepatocyte TNF-α release.     

The role of autophagy in β-cell lipotoxicity and type 2 diabetes

Autophagy, a ubiquitous catabolic pathway involved in both cell survival and cell death, has been implicated in many age-associated diseases. Recent findings have shown autophagy to be crucial for proper insulin secretion and β-cell viability. Transgenic mice lacking autophagy in their β-cells showed decreased β-cell mass and suppressed glucose-stimulated insulin secretion. Several studies showed that stress can stimulate autophagy in β-cells: the number of autophagosomes is increased in different in vivo models for diabetes, such as db/db mice, mice fed high-fat diet, pdx-1 knockout mice, as well as in in vitro models of glucotoxicity and lipotoxicity. Pharmacological and molecular inhibition of autophagy increases the susceptibility to cell stress, suggesting that autophagy protects against diabetes-relevant stresses. Recent findings, however, question these conclusions. Pancreases of diabetics and β-cells exposed to fatty acids show accumulation of abnormal autophagosome morphology and suppression of lysosomal gene expression suggesting impairment in autophagic turnover. In this review we attempt to give an overview of the data generated by others and by us in view of the possible role of autophagy in diabetes, a role which depending on the conditions, could be beneficial or detrimental in coping with stress.     

The renin–angiotensin system in Barrett’s esophagus

Objective: Barrett’s esophagus (BE) is a risk factor for esophageal adenocarcinoma. In addition to its classical endocrine character known for hemodynamic regulation, the renin–angiotensin system (RAS) can be associated with inflammation, wound healing, and cancer. The aim of this study was to explore a potential expression of the RAS in BE, with or without the presence of dysplasia.

Material and methods: Biopsy material was prepared for western blotting and immunohistochemistry. Non-BE patients (controls) were compared with BE patients regarding RAS in the squamous epithelium. In the columnar BE mucosa, RAS expression was studied in patients with and without dysplasia. Key components of the ‘classical’ RAS were assessed: the angiotensin-converting enzyme (ACE) and the angiotensin II subtype 1 and 2 receptors (AT1R and AT2R).

Results: The presence of RAS factors was confirmed in the esophageal mucosa of both control and BE patients. ACE protein expression was 48% lower (p?=?0.001) whereas AT1R was 45% higher (p?=?0.039) in the squamous epithelium of BE patients compared to epithelia from non-BE controls. In the metaplastic intestinal-like epithelium, AT1R expression was 37% higher in BE patients with confirmed dysplasia than in patients without dysplasia (p?=?0.009). Immunohistochemistry showed an altered distribution of RAS proteins in BE patients with dysplasia.

Conclusions: The differential RAS expression observed may prove to be useful as a biomarker or a pharmaceutical target.     

Serum Levels of TNF-α, TNF-αRI,TNF-αRII and IL-12 in Treated Rheumatoid Arthritis Patients

Background: Rheumatoid arthritis (RA) is a chronic multisystem autoimmune disease common in all races and ethnics. Cytokines and cytokines receptors play an important role in RA pathogenesis and clinical presentation. Objective: To investigate the serum levels of TNF-α, TNF-α RI, TNF-α RII and IL-12 in RA patients and healthy control group. Methods: In this study 43 patients fulfilling the revised criteria of American College of Rheumatology (ACR) for RA and 13 healthy cases as a control group were selected for TNF-α, TNF-αRI, TNF-αRII and IL-12 serum level analysis. The patients' age was 42.2 ± 22 and the age of healthy group was 40.1 ± 19.2 years (p=0.1). The pa-tients had an active disease with at least six swollen and ten tender joints. Minimum ESR was 28 mm at first hours of the morning. Early morning stiffness in patients lasted longer than 45 minutes. Results: Our study showed that IL-12 serum level of the pa-tients (91.69 ± 43.07 ρg/ml) and control (61.79 ± 40.08 ρg/ml) group was significantly different (p<0.001). The serum level of TNF-αRI was 2.36 ± 0.77 ng/ml in the patient and 1.73 ± 0.37 ng/ml in the control group (p<0.01). TNF-αRII serum concentration in patients was 8.89 ± 2.3 ng/ml, while that of control group was 7.06±1.30 ng/ml (p=0.03). The serum level of TNF-α in patients was 32.90 ± 19.27 ρg/ml and that of the control group was 24.27± 8.28 ρg/ml (p=0.08) with no significant difference between the two. Conclusions: It is concluded that IL-12, TNF-αRI and TNF- αRII serum con-centrations are more important and better predictive factors than TNF-α in RA course and in the active forms of the disease.     

TNF-α-induced up-regulation of pro-inflammatory cytokines is reduced by phosphatidylcholine in intestinal epithelial cells

Background  

Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties.