scholarly journals Dephosphorylation Targets Bcl-2 for Ubiquitin-dependent Degradation: A Link between the Apoptosome and the Proteasome Pathway

1999 ◽  
Vol 189 (11) ◽  
pp. 1815-1822 ◽  
Author(s):  
Stefanie Dimmeler ◽  
Kristin Breitschopf ◽  
Judith Haendeler ◽  
Andreas M. Zeiher
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Inflammatory Diseases ◽  
Apoptotic Cell ◽  
Proteasome Inhibitors ◽  
Inhibitory Function ◽  
Tnf Α ◽  
Proteasome Pathway

Injury of the endothelial cells by the induction of apoptotic cell death may play an important role in the pathophysiology of atherosclerosis and the progression of inflammatory diseases. Here, we demonstrate an essential role for the ubiquitin-dependent proteasome complex in stimulus-induced degradation of the antiapoptotic protein Bcl-2. Bcl-2 is specifically degraded after stimulation of human endothelial cells with tumor necrosis factor (TNF)-α in a process that is inhibited by specific proteasome inhibitors. In addition, the mutation of the potential ubiquitin-acceptor amino acids of Bcl-2 provides protection against TNF-α– and staurosporine-induced degradation in vitro and in vivo. Moreover, mimicking phosphorylation of the putative mitogen-activated protein (MAP) kinase sites of the Bcl-2 protein (Thr 56, Thr 74, and Ser 87) abolishes its degradation, suggesting a link between the MAP kinase pathway to the proteasome pathway. Finally, inhibition of Bcl-2 degradation either by suppressing ubiquitin-dependent proteasomal degradation or by mimicking continuous phosphorylation of the putative MAP kinase sites in the Bcl-2 protein confers resistance against induction of apoptosis. Thus, the degradation of Bcl-2 may unleash the inhibitory function of Bcl-2 over the apoptosome and may thereby amplify the activation of the caspase cascade.

scholarly journals L-Selectin–Dependent Leukocyte Adhesion to Microvascular But Not to Macrovascular Endothelial Cells of the Human Coronary System

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3228-3235 ◽  
Author(s):  
A. Zakrzewicz ◽  
M. Gräfe ◽  
D. Terbeek ◽  
M. Bongrazio ◽  
W. Auch-Schwelk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Leukocyte Adhesion ◽  
Flow Chamber ◽  
Shear Stresses ◽  
Selectin Ligands ◽  
Tnf Α ◽  
Factor Α

Abstract To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

scholarly journals Screening of an anti-inflammatory peptide from Hydrophis cyanocinctus and analysis of its activities and mechanism in DSS-induced acute colitis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zengjie Zheng ◽  
Hailong Jiang ◽  
Yan Huang ◽  
Jie Wang ◽  
Lei Qiu ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Activity Index ◽  
Tumor Necrosis Factor Receptor ◽  
Disease Activity Index ◽  
Acute Colitis ◽  
Surface Plasmon Resonance Analysis ◽  
Tnf Α ◽  
Anti Inflammatory

Abstract Snake has been used for centuries as a traditional Chinese medicine, especially for therapeutic treatment for inflammatory diseases; however, its mechanisms of action and active constituents remain controversial. In our study, a tumor necrosis factor receptor 1 (TNFR1) selective binding peptide, Hydrostatin-SN1 (H-SN1), which was screened from a Hydrophis cyanocinctus venom gland T7 phage display library, was shown to exhibit significant anti-inflammatory activity in vitro and in vivo. As a TNFR1 antagonist, it reduced cytotoxicity mediated by TNF-α in L929 fibroblasts and effectively inhibited the combination between TNF-α with TNFR1 in surface plasmon resonance analysis. H-SN1 was also shown to suppress TNFR1–associated signaling pathways as it minimized TNF-α-induced NF-кB and MAPK activation in HEK293 embryonic kidney and HT29 adenocarcinoma cell lines. We next determined the effect of H-SN1 in vivo using a murine model of acute colitis induced by dextran sodium sulfate, demonstrating that H-SN1 lowered the clinical parameters of acute colitis including the disease activity index and histologic scores. H-SN1 also inhibited TNF/TNFR1 downstream targets at both mRNA and protein levels. These results indicate that H-SN1 might represent a suitable candidate for use in the treatment of TNF-α-associated inflammatory diseases such as inflammatory bowel diseases.

Bidirectional Oscillatory Shear Stress Increases Pro-Atherogenic Gene Expressions (I-CAM1, E-Selectin and IL-6) in Endothelial Cells

2011 ◽  
Author(s):  
Amlan Chakraborty ◽  
Venkatakrishna R. Jala ◽  
Sutirtha Chakraborty ◽  
R. Eric Berson ◽  
M. Keith Sharp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Atherosclerotic Plaque ◽  
Inflammatory Diseases ◽  
Leukotriene B4 ◽  
Oscillatory Shear ◽  
Gene Expressions

Wall shear stress (WSS) plays a key role in altering intracellular pathways and gene expression of endothelial cells, and has significant impacts on atherosclerotic plaque development (1–3). Further, the atherogenic regulators Leukotriene B4 (LTB4) and Lipopolysaccharide (LPS) have significant impacts on the pathophysiology of many inflammatory diseases. This study investigates the effects of oscillatory shear directionality on pro-atherogenic gene expression (I-CAM, E-Selectin, and IL-6) in the presence of LTB4 and LPS. An orbital shaker was used to expose the endothelial cells to oscillatory shear in culture dishes, and Computational fluid dynamics (CFD) was applied to quantify the shear stress on the bottom of the orbiting dish. Directionality of oscillatory shear was characterized by a newly developed hemodynamic parameter — Directional oscillatory shear index (DOSI), which was demonstrated in a previous study to significantly impact cell morphology (4). Results showed that DOSI significantly altered gene expression. Therefore, directionality of shear modulates atherosclerotic gene expression in vitro and thus, may influence the formation of atherosclerotic plaque in vivo.

scholarly journals A Disintegrin and Metalloprotease 15 is Expressed on Rheumatoid Arthritis Synovial Tissue Endothelial Cells and may Mediate Angiogenesis

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 32 ◽  
Author(s):  
Shinichiro Nishimi ◽  
Takeo Isozaki ◽  
Kuninobu Wakabayashi ◽  
Hiroko Takeuchi ◽  
Tsuyoshi Kasama
Keyword(s):  
Rheumatoid Arthritis ◽  
Endothelial Cells ◽  
Conditioned Medium ◽  
Inflammatory Diseases ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Tnf Α ◽  
Adhesion Index

A disintegrin and metalloprotease 15 (ADAM15) is involved in several malignancies. In this study, we investigated the role of ADAM15 in rheumatoid arthritis (RA) angiogenesis. Soluble ADAM15 (s-ADAM15) in serum from RA and normal (NL) subjects was measured using ELISA. To determine membrane-anchored ADAM15 (ADAM15) expression in RA synovial tissues, immunohistochemistry was performed. To examine the role of ADAM15 in angiogenesis, we performed in vitro Matrigel assays and monocyte adhesion assays using human umbilical vein endothelial cells (HUVECs) transfected with ADAM15 siRNA. Finally, to investigate whether angiogenic mediators were affected by ADAM15, cytokines in ADAM15 siRNA-transfected HUVEC-conditioned medium were measured. ADAM15 was significantly higher in RA serum than in NL serum. ADAM15 was also expressed on RAST endothelial cells. ADAM15 siRNA-treated HUVECs had decreased EC tube formation in response to RA synovial fluids compared with non-treated HUVECs. The adhesion index of ADAM15 siRNA-transfected HUVECs was significantly lower than the adhesion index of control siRNA-transfected HUVECs. ENA-78/CXCL5 and ICAM-1 were decreased in tumor necrosis factor (TNF)-α-stimulated ADAM15 siRNA-transfected HUVEC-conditioned medium compared with TNF-α-stimulated control siRNA-transfected HUVEC-conditioned medium. These data show that ADAM15 plays a role in RA angiogenesis, suggesting that ADAM15 might be a potential target in inflammatory diseases such as RA.

TRAF-1 Deficient Mice Show Impaired Monocyte Recruitment and Decreased Atherogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 696-696
Author(s):  
Anna Missiou ◽  
Natascha Köstlin ◽  
Christian Münkel ◽  
Dietmar Pfeifer ◽  
Katja Zirlik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chemokine Receptors ◽  
Inflammatory Diseases ◽  
Inflammatory Cells ◽  
Adaptor Proteins ◽  
High Cholesterol Diet ◽  
Cell Content ◽  
Monocyte Recruitment

Abstract Members of the tumor necrosis factor (TNF) interleukin/toll-like receptor superfamily such as CD40L, TNFa, and IL-1b potently promote atherogenesis in mice and likely also in humans. TNF receptor associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines. We recently reported over-expression of TRAFs in murine and human atheromata and demonstrated dependency of classic inflammatory functions on TRAFs in endothelial cells and macrophages. Here we test the hypothesis that TRAF-1 modulates atherogenesis in vivo. TRAF-1--/LDLR--mice fed a high cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions compared with LDLR--controls. Intimal lesion size decreased by up to 56±6% and 33±5% in sections of the aortic arch and aortic root, respectively (n>10 per group, P<0.01 each). Plaques of TRAF-1-deficient animals contained up to 46±9% and 55±4% fewer macrophages while smooth muscle cell content increased by up to 32±6 and 36±7%, characteristics associated with non disrupted plaques in humans. Lipid content, collagen content, and lymphocyte content remained unchanged. In vitro, gene expression profiling revealed reduced expression of adhesion molecules (VCAM-1, ICAM-1), chemokines (CCL2, CXCL2), and growth factors (M-CSF) in TRAF-1-deficient endothelial cells as well as of integrins (CD29, CD11b) and chemokines/chemokine receptors (CXCL2, CCR1) in TRAF-1-deficient macrophages, verified by siRNA studies in human cells. Finally, both deficiency of TRAF-1 in endothelial cells and neutrophils/monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic adhesion assays. We present the novel finding that TRAF 1 deficiency attenuates atherogenesis in mice, an effect most likely mediated by impaired monocyte recruitment to the vessel wall. These data identify TRAF-1 as potential treatment target for chronic inflammatory diseases such as atherosclerosis.

Membrane-type matrix metalloproteinase-mediated angiogenesis in a fibrin-collagen matrix

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1810-1817 ◽  
Author(s):  
Annemie Collen ◽  
Roeland Hanemaaijer ◽  
Florea Lupu ◽  
Paul H. A. Quax ◽  
Natascha van Lent ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Matrix Metalloproteinase ◽  
Collagen Matrix ◽  
Tubular Structures ◽  
Tubule Formation ◽  
Tnf Α ◽  
Membrane Type

Adult angiogenesis, associated with pathologic conditions, is often accompanied by the formation of a fibrinous exudate. This temporary matrix consists mainly of fibrin but is intermingled with plasma proteins and collagen fibers. The formation of capillary structures in a fibrinous matrix in vivo was mimicked by an in vitro model, in which human microvascular endothelial cells (hMVECs) seeded on top of a fibrin-10% collagen matrix form capillarylike tubular structures after stimulation with basic fibroblast growth factor/tumor necrosis factor α (bFGF/TNF-α) or vascular endothelial growth factor (VEGF)/TNF-α. In the fibrin-collagen matrix the metalloproteinase inhibitor BB94 inhibited tubule formation by 70% to 80%. Simultaneous inhibition of plasmin and metalloproteinases by aprotinin and BB94 caused a nearly complete inhibition of tubule formation. Adenoviral transduction of tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-3 into endothelial cells revealed that TIMP-3 markedly inhibited angiogenesis, whereas TIMP-1 had only a minor effect. Immunohistochemical analysis showed the presence of matrix metalloproteinase 1 (MMP-1), MMP-2, and membrane-type 1 (MT1)–MMP, whereas MMP-9 was absent. The endothelial production of these MMPs was confirmed by antigen assays and real-time polymerase chain reaction (PCR). MT1-MMP mRNA was markedly increased in endothelial cells under conditions that induced tubular structures. The presence of MMP-1, MMP-2, and MT1-MMP was also demonstrated in vivo in the newly formed vessels of a recanalized arterial mural thrombus. These data suggest that MMPs, in particular MT-MMPs, play a pivotal role in the formation of capillarylike tubular structures in a collagen-containing fibrin matrix in vitro and may be involved in angiogenesis in a fibrinous exudate in vivo.

scholarly journals Cimifugin ameliorates imiquimod-induced psoriasis by inhibiting oxidative stress and inflammation via NF-κB/MAPK pathway

2020 ◽  
Vol 40 (6) ◽  
Author(s):  
Aimin Liu ◽  
Wei Zhao ◽  
Buxin Zhang ◽  
Yuanhui Tu ◽  
Qingxing Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Inflammatory Diseases ◽  
Mapk Pathway ◽  
Severity Index ◽  
Mapk Signaling ◽  
Psoriasis Area Severity Index ◽  
Tnf Α ◽  
Necrosis Factor

Abstract Cimifugin is an important component of chromones in the dry roots of Saposhikovia divaricata for treating inflammatory diseases. However, the possible effect of cimifugin in psoriasis needs further investigation. This current work was designed to evaluate the effects of cimifugin in psoriasis in vivo and in vitro, and unravel the underlying molecular mechanism. Here, we used imiquimod (IMQ) or tumor necrosis factor (TNF)-α to induce a psoriasis-like model in mice or keratinocytes. Obviously, the results showed that cimifugin reduced epidermal hyperplasia, psoriasis area severity index (PASI) scores, ear thickness and histological psoriasiform lesions in IMQ-induced mice. The decreased levels of reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), and the accumulation of malondialdehyde (MDA) in skin tissues by IMQ were attenuated by cimifugin. Furthermore, it was observed that cimifugin effectively reversed IMQ-induced up-regulation of proinflammatory cytokines, including TNF-α, IL-6, IL-1β, IL-17A, and IL-22. Mechanically, we noticed that cimifugin inhibited IMQ-activated phosphorylation of NF-κB (IκB and p65) and MAPK (JNK, ERK, and p38) signaling pathways. Similar alterations for oxidative stress and inflammation parameters were also detected in TNF-α-treated HaCaT cells. In addition, cimifugin-induced down-regulation of ICAM-1 were observed in TNF-α-treated cells. Altogether, our findings suggest that cimifugin protects against oxidative stress and inflammation in psoriasis-like pathogenesis by inactivating NF-κB/MAPK signaling pathway, which may develop a novel and effective drug for the therapy of psoriasis.

scholarly journals CB2-receptor stimulation attenuates TNF-α-induced human endothelial cell activation, transendothelial migration of monocytes, and monocyte-endothelial adhesion

2007 ◽  
Vol 293 (4) ◽  
pp. H2210-H2218 ◽  
Author(s):  
Mohanraj Rajesh ◽  
Partha Mukhopadhyay ◽  
Sándor Bátkai ◽  
György Haskó ◽  
Lucas Liaudet ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Cell Activation ◽  
Transendothelial Migration ◽  
Receptor Activation ◽  
Human Coronary Artery ◽  
Endothelial Cell Activation ◽  
Tnf Α

Targeting cannabinoid-2 (CB2) receptors with selective agonists may represent a novel therapeutic avenue in various inflammatory diseases, but the mechanisms by which CB2 activation exerts its anti-inflammatory effects and the cellular targets are elusive. Here, we investigated the effects of CB2-receptor activation on TNF-α-induced signal transduction in human coronary artery endothelial cells in vitro and on endotoxin-induced vascular inflammatory response in vivo. TNF-α induced NF-κB and RhoA activation and upregulation of adhesion molecules ICAM-1 and VCAM-1, increased expression of monocyte chemoattractant protein, enhanced transendothelial migration of monocytes, and augmented monocyte-endothelial adhesion. Remarkably, all of the above-mentioned effects of TNF-α were attenuated by CB2 agonists. CB2 agonists also decreased the TNF-α- and/or endotoxin-induced ICAM-1 and VCAM-1 expression in isolated aortas and the adhesion of monocytes to aortic vascular endothelium. CB1 and CB2 receptors were detectable in human coronary artery endothelial cells by Western blotting, RT-PCR, real-time PCR, and immunofluorescence staining. Because the above-mentioned TNF-α-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that targeting CB2 receptors on endothelial cells may offer a novel approach in the treatment of these pathologies.

scholarly journals Development of a p38 Kinase Binding Assay for High Throughput Screening

1999 ◽  
Vol 4 (3) ◽  
pp. 129-135 ◽  
Author(s):  
Usha Warrior ◽  
X. Grace Chiou ◽  
Michael P. Sheets ◽  
Richard J. Sciotti ◽  
Janet M. Parry ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Binding Assay ◽  
Inflammatory Diseases ◽  
Mitogen Activated Protein Kinase ◽  
P38 Kinase ◽  
Tnf Α ◽  
Pyridinyl Imidazole

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-α and IL-1β secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors.

Two indole-2-carboxamide derivatives attenuate lipopolysaccharide-induced acute lung injury by inhibiting inflammatory response

2018 ◽  
Vol 96 (12) ◽  
pp. 1261-1267
Author(s):  
Wei Dai ◽  
Xiangting Ge ◽  
Tingting Xu ◽  
Chun Lu ◽  
Wangfeng Zhou ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Lung Tissue ◽  
Inflammatory Diseases ◽  
Inflammatory Cells ◽  
Genes Expression ◽  
Tnf Α ◽  
Induced Changes

Acute lung injury (ALI) is the leading cause of mortality in the intensive care unit. Currently, there is no effective pharmacological treatment for ALI. In our previous study, we reported that Lg25 and Lg26, two indole-2-carboxamide derivatives, inhibited the lipopolysaccharide (LPS)-induced inflammatory cytokines in vitro and attenuated LPS-induced sepsis in vivo. In the present study, we confirmed data from previous studies that LPS significantly induced pulmonary edema and pathological changes in lung tissue, increased protein concentration and number of inflammatory cells in bronchoalveolar lavage fluids (BALF), and increased inflammatory cytokine TNF-α expression in serum and BALF, pro-inflammatory genes expression, and macrophages infiltration in lung tissue. However, pretreatment with Lg25 and Lg26 significantly attenuated the LPS-induced changes in mice. Taken together, these data indicate that the newly discovered indole-2-carboxamide derivatives could be particularly useful in the treatment of inflammatory diseases such as ALI.

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