scholarly journals Oxidized LDL Mediated Upregulation of ADAMTS-4 in Monocytes/Macrophages Involves The ROS-NFκB Pathway

2021 ◽  
Author(s):  
Aswani S. S. ◽  
Mithra. S. Mohan ◽  
Aparna. N. S. ◽  
P. T. Boban ◽  
Saja Kamalamma
Keyword(s):  
Protein Expression ◽  
Morphological Changes ◽  
Oxidized Ldl ◽  
Glucuronidase Activity ◽  
Atherosclerotic Lesions ◽  
Protease Enzyme ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Ros Scavenger ◽  
Nfκb Pathway

Abstract ADAMTS-4 is a protease enzyme which involves in vascular remodeling and atherosclerosis. It was found to be upregulated in macrophages seen in atherosclerotic lesions. The aim of this study was to investigate the expression and regulation of ADAMTS-4 in oxLDL induced human monocytes/macrophages system. PBMCs isolated from human blood(hPBMCs), treated with oxLDL (50μg/ml) were used as the model system for the study. mRNA and protein expressions were studied by qRT-PCR, ELISA, and western blot analysis. ROS production and cell viability were determined by fluorescence imaging and MTT assay respectively. In the presence of oxLDL, monocytes get differentiated into macrophages, which were confirmed by the increased expression of CD-36, b- D glucuronidase activity and by the morphological changes. OxLDL increased the mRNA and protein expression of ADAMTS-4 and TIMP-3 in monocytes/ macrophages. A significant increase in the mRNA and protein expression of TNF-α was also observed in oxLDL treated cells compared to untreated control. In the presence of NAC, the ROS scavenger, the production of NFκB and ADAMTS-4 was decreased significantly. Our study suggests that oxLDL significantly upregulated the expression of ADAMTS-4 in the monocyte/macrophage system. OxLDL mediated upregulation of ADAMTS-4 in hPBMCs involves TNF-α and ROS- NFκB pathway.

scholarly journals Resveratrol improves left ventricular diastolic relaxation in type 2 diabetes by inhibiting oxidative/nitrative stress: in vivo demonstration with magnetic resonance imaging

2010 ◽  
Vol 299 (4) ◽  
pp. H985-H994 ◽  
Author(s):  
Hanrui Zhang ◽  
Brandon Morgan ◽  
Barry J. Potter ◽  
Lixin Ma ◽  
Kevin C. Dellsperger ◽  
...  
Keyword(s):  
Protein Expression ◽  
Left Ventricular ◽  
No Synthase ◽  
No Production ◽  
Enos Expression ◽  
Nitrative Stress ◽  
Tnf Α ◽  
Mrna And Protein Expression

Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control ( m-Lepr db) mice and type 2 diabetic ( Lepr db) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Lepr db mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O2·− production by inhibiting NAD(P)H oxidase activity and gp91phox mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Lepr db mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Lepr db mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Lepr db mice. Both Lepr db mice null for TNF-α ( dbTNF−/ dbTNF− mice) and Lepr db mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Lepr db mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes.

Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-γ and TNF-α and regulation by TGF-β and corticosteroids

2004 ◽  
Vol 287 (6) ◽  
pp. L1230-L1240 ◽  
Author(s):  
Maria B. Sukkar ◽  
Razao Issa ◽  
Shaoping Xie ◽  
Ute Oltmanns ◽  
Robert Newton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Expression ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Ifn Γ ◽  
Sb 203580

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX3C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, CX3CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ, the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-β. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-γ and TNF-α induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-β had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone (10−8–10−6 M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun NH2-terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 μM) and SB-203580 (20 μM), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-γ- and TNF-α-induced JNK phosphorylation remained unaltered in the presence of TGF-β but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-β- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors.

Abstract 584: Allograft Inflammatory Factor-1 is Required for NfκB Pathway Activity in Macrophages and Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Lander Egaña-Gorroño ◽  
Prameladevi Chinnasamy ◽  
Nicholas Sibinga
Keyword(s):  
Oxidized Ldl ◽  
Critical Role ◽  
Immunohistochemical Analysis ◽  
Nuclear Factor Κb ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Atherosclerotic Lesions ◽  
Nfκb Pathway

Introduction: In preliminary studies, we found that Allograft inflammatory factor-1 (AIF1) supports MΦ migration, phagocytosis, survival and pro-inflammatory cytokine secretion. Moreover, AIF1 limits necrotic core formation in atherosclerotic lesions in vivo . Nuclear Factor-κB (NFκB)-mediated signal transduction has been established at different stages of atherosclerosis. We hypothesize that AIF1-regulated processes in atherosclerosis may be mediated through effects on NFκB signaling. Methods: Bone marrow (BM) derived MΦs were isolated and immortalized from wt and Aif1 -/- mice and stimulated with oxidized-LDL (50 ug/ul; oxLDL). Lysates were immunoblotted for total and phosphorylated (active) p65 NFκB, and for total and phosphorylated forms of the IκBα repressor. Aif1 expression in mouse Raw 264.7 cells was knocked down (KD) using siRNA, and NFκB reporter activity, measured by a luciferase reporter, was assessed after adding LPS+IFN-γ. Immunohistochemical analysis for phospho-p65 NFκB was performed in atherosclerotic lesions (aortic roots) from Apoe -/- (SKO) and Apoe -/- ;Aif1 -/- (DKO) mice maintained on high fat diet for 16 weeks. Results: In AIF1-deficient BMMΦs stimulated with oxLDL, we found no differences in the levels of total p65 NFκB and IκBα, but interestingly, phospho-p65 NFκB levels were significantly reduced and phospho-IκBα levels increased compared to wt cells (P<0.05). AIF1 KD using siRNA significantly reduced NFκB activity compared to scrambled control (scrambled control vs. AIF1 KD; 40% vs. 22% luciferase activity, P<0.05), and this impairment was rescued with the addition of AIF1 cDNA. In vivo , NFκB phospho-p65 staining showed that in comparison to SKO samples, DKO aortic roots had decreased phospho-p65 NFκB (SKO vs. DKO; 7.0 vs. 4.0 +nuclei/aortic root, P<0.05). Conclusions: AIF1 is required for NFκB activation in MΦs and moreover, AIF1 enhances NFκB activity in atherosclerotic lesions in vivo . Because NFkB has been closely linked to both cytokine expression and cell survival signaling, these results point to a critical role for AIF1 in pro-inflammatory MΦ functions. Future studies involve identifying the precise steps of the pathway controlled by AIF1, and the mechanisms by which AIF1 affects NFκB signaling.

scholarly journals Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen

2009 ◽  
Vol 296 (5) ◽  
pp. R1376-R1384 ◽  
Author(s):  
Enrique Sánchez-Lemus ◽  
Julius Benicky ◽  
Jaroslav Pavel ◽  
Ignacio M. Larrayoz ◽  
Jin Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Inflammatory Response ◽  
Protein Expression ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Bacterial Endotoxin ◽  
Ang Ii ◽  
Tnf Α ◽  
Mrna And Protein Expression

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

scholarly journals Supervillin contributes to LPS-induced inflammatory response in THP-1 cell-derived macrophages

2021 ◽  
Author(s):  
Jun Zhou ◽  
Yuhui Que ◽  
Lihua Pan ◽  
Xu Li ◽  
Chao Zhu ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Protein Expression ◽  
Mrna Expression ◽  
Inflammatory Cytokines ◽  
Molecular Mechanisms ◽  
Actin Binding ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Underlying Mechanisms

Abstract Supervillin (SVIL), the largest member of villin/gelsolin family, is an actin-binding and membrane-associated protein, that can also be localized to the nucleus. It has been reported that the mRNA expression of SVIL in neutrophils could be increased by lipopolysaccharide (LPS), but the underlying mechanisms remain unknown. Moreover, SVIL was also observed to be involved in the regulation of macrophages’ movement. However, it is not clear whether SVIL is involved in the LPS-induced inflammatory response in macrophages. This work was to investigate the underlying molecular mechanisms of LPS regulating SVIL expression in macrophages and hence the possible role of SVIL in LPS-induced inflammation. Our data showed that in THP-1-derived macrophages, LPS stimulation significantly increased SVIL mRNA and protein expression. Inhibition of TLR4 by Resatorvid (Res) completely reversed the expression of SVIL and inflammatory cytokines (IL-6, IL-1β and TNF-α) induced by LPS. Additionally, ERK1/2 and NF-κB inhibitors (U0126 and BAY) significantly reduced SVIL and IL-6, IL-1β & TNF-α expression. Furthermore, down-regulation of SVIL by SVIL-specific shRNA significantly attenuated the expression of IL-6, IL-1β & TNF-α induced by LPS. Taken together, as a downstream molecule of TLR4/NF-κB and ERK1/2, SVIL was involved in the inflammatory response of LPS-induced elevated IL-6, IL-1β and TNF-α in macrophages.

Protective effects of baicalin on LPS-induced injury in intestinal epithelial cells and intercellular tight junctions

2015 ◽  
Vol 93 (4) ◽  
pp. 233-237 ◽  
Author(s):  
Jian Chen ◽  
Ren Zhang ◽  
Jian Wang ◽  
Peng Yu ◽  
Quan Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Tight Junctions ◽  
Inflammatory Cytokines ◽  
Intestinal Epithelial Cells ◽  
Expression Level ◽  
Protective Effects ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Mrna And Protein Expression

Aims: To investigate the protective effects and mechanisms of baicalin on lipopolysaccharide (LPS)-induced injury in intestinal epithelial cells and intercellular tight junctions. Methods: IEC-6 cells were stimulated with LPS (1.0 μg/mL), with or without baicalin, for 24 h. The levels of the inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were determined using ELISA. Quantitative real-time PCR was used for determining the mRNA expression level of claudin-3, occludin, and ZO-1; Western blot and immunofluorescence analysis were used for analyzing the expression level and the distribution patterns of ZO-1 protein. Results: Pretreatment with baicalin (10.0 μg/mL) improved LPS-stimulated cell viability and repressed IL-6 and TNF-α levels. In addition, pretreatment with baicalin up-regulated mRNA and protein expression levels of ZO-1 and kept the protein intact in IEC-6 cells injured with LPS. Conclusion: Baicalin has the capacity to protect IEC-6 cells and the intercellular tight junctions from LPS-induced injury. The mechanisms may be associated with inhibiting the production of inflammatory cytokines, and up-regulating the mRNA and protein expression of ZO-1.

scholarly journals HIV-1 promoter is gradually silenced when integrated into BACH2

2020 ◽  
Author(s):  
Anne Inderbitzin ◽  
Yik Lim Kok ◽  
Lisa Jörimann ◽  
Audrey Kelley ◽  
Kathrin Neumann ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Expression ◽  
Btb Domain ◽  
Transcriptional Orientation ◽  
Cell Clones ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

AbstractThe persistence of the latent HIV-1 reservoir is a major obstacle to cure HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites in BACH2. The HIV-1-based vector LTatCL[M] contains two fluorophores: 1.) Cerulean, which reports the activity of the HIV-1 promoter, and 2.) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean−/mCherry+) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Our results show that the HIV-1 promoter is silenced when integrated into BACH2 without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.

scholarly journals Molecular Regulation of Ghrelin Expression by Pro-Inflammatory Cytokines TNF-α and IL-6 In Rat Pancreatic AR42J Cell Line

2012 ◽  
Vol 4 (1) ◽  
Author(s):  
Kah-Meng Lao ◽  
Wyi-Sian Lim ◽  
Di-Lin Ng ◽  
Tengku-Sifzizul Tengku-Muhammad ◽  
Quok-Cheong Choo ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Protein Expression ◽  
Cell Line ◽  
Inflammatory Cytokines ◽  
Pancreatic Cell ◽  
Amino Acid Peptide ◽  
Tnf Α ◽  
Densitometry Analysis ◽  
Mrna And Protein Expression ◽  
Pro Inflammatory Cytokines

Ghrelin is a 28 amino acid peptide endogenous ligand for growth hormone secretatgogue receptor (GHS-R) that functions to stimulate growth hormone, regulate inflammation, appetite and energy balance. However, the regulation of ghrelin expression by pro-inflammatory cytokines during inflammation process has never been investigated systematically. This study was carried out to investigate the effects of major pro-inflammatory cytokines such as TNF-α and IL-6 on ghrelin expression using AR42J rat pancreatic cell line as model system. The cells were treated with different concentrations of the cytokines for 24 hours. Real-Time RT-PCR, Western blot and densitometry analysis were carried out to quantify ghrelin mRNA and protein expression, respectively. Although TNF-α and IL-6 stimulation resulted in a general down-regulatory pattern in both mRNA and protein expression of ghrelin, stimulation with 5 ng/ml of TNF-α and IL-6 slightly induced the expression of ghrelin expression. However, higher doses of the cytokines ranging from 10-50 ng/ml suppressed the ghrelin expression in a dose-dependant manner. These results indicate that ghrelin in AR42J pancreatic cell line is regulated by the pro-inflammatory cytokines and that the dosages of the cytokines play an important factor in the regulation of the expression.

scholarly journals Rapid and transient induction of cyclo-oxygenase 2 by epidermal growth factor in human amnion-derived WISH cells

1997 ◽  
Vol 321 (3) ◽  
pp. 677-681 ◽  
Author(s):  
Douglas J. PERKINS ◽  
Douglas A. KNISS
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Protein Expression ◽  
De Novo ◽  
Tnf Α ◽  
Mrna And Protein Expression ◽  
Epidermal Growth ◽  
Cox 2 ◽  
Dose Dependent ◽  
Cox 1

The central enzyme in the prostaglandin (PG) biosynthetic cascade is PGH2 synthase or cyclo-oxygenase (COX). At present, two distinct isoforms of PGH2 synthase/COX have been identified: COX-1 and COX-2. In many systems, COX-1 is a constitutively expressed isoform that is responsible for normal physiological production of PGs, whereas COX-2 is an inducible isoform that responds to cytokines, endotoxin and growth factors by producing high levels of PGs. The regulation of COX-2 mRNA and protein, and the subsequent production of PGE2, were therefore examined in amnion-derived WISH cells stimulated with epidermal growth factor (EGF). Treatment of WISH cells with EGF (0.01Ő100 ng/ml) elicited dose-dependent synthesis of COX-2 mRNA and protein de novo. In addition, stimulation of WISH cells with EGF (10 ng/ml) induced steady-state levels of COX-2 mRNA and protein that appeared within 30 min and then declined rapidly to near baseline levels within 2Ő4 h. In contrast, COX-1 protein was unchanged in response to treatment with EGF. PGE2 production was also rapid and transient. Preincubation of cells with the novel COX-2 enzymic inhibitor NS-398 (10-5Ő10-10 M) completely prevented PGE2 formation in a dose-dependent manner. Preincubation of cells in dexamethasone (Dex; 0.1 ƁM), however, resulted in only a 31% decrease in PGE2 formation in response to EGF (10 ng/ml) while completely attenuating PGE2 biosynthesis in tumour necrosis factor α (TNF-α)-stimulated cells. In addition, Dex (0.1 ƁM) was only partly effective at preventing EGF-induced COX-2 mRNA and protein expression de novo, whereas Dex completely inhibited TNF-α-promoted COX-2 mRNA and protein expression. Thus the results presented here demonstrate that EGF induces the rapid but transient expression of COX-2 mRNA and protein and the subsequent production of PGE2 in WISH cells.

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