Abstract 635: CD40-Filamin A Interactions Are Required for Translocation of CD40 to Lipid Rafts In Endothelial Cells and for Endothelial Cell Activation

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Claudia M van Tiel ◽  
Patrick Burger ◽  
Noam Zelcer ◽  
Peter L Hordijk ◽  
Jaap D van Buul ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lipid Rafts ◽  
Tumor Necrosis ◽  
Cell Activation ◽  
Immune Cell ◽  
Transmembrane Domain ◽  
Tumor Necrosis Factor Receptor ◽  
Site Directed Mutagenesis ◽  
Filamin A ◽  
Endothelial Cell Activation

Background and Aim: CD40 is a member of the co-stimulatory tumor necrosis factor receptor superfamily that is not only constitutively expressed on professional antigen presenting cells like macrophages, dendritic cells and B-cells, but also on endothelial cells. Interactions between CD40 and its ligand, CD40 ligand (CD40L) are crucial for proper immune cell activation. Activation of CD40 signaling plays a role in chronic diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. However, since CD40-CD40L interactions modulate immune reactions as well as thrombosis, blocking CD40(L) can have adverse side-effects. Therefore, in search for new therapeutic targets, we aimed to identify new CD40-binding partners. Methods and Results: We created a cDNA library of murine aortas containing atherosclerotic plaques at various stages and performed a yeast-two-hybrid with the C-terminal domain of CD40 as bait. We identified filamin A as a novel CD40 binding partner in atherosclerosis, which was confirmed by co-immunoprecipitation. Accordingly, using confocal microscopy we showed that, upon activation of CD40, filamin A was recruited to CD40 in endothelial cells. Site directed mutagenesis revealed that Filamin A binds to the intracellular domain of CD40, near the transmembrane domain, at a site distinct from the tumor necrosis receptor associated factor (TRAF) binding sites. In endothelial cells, CD40-signaling is dependent on intact lipid rafts. Interestingly, when we knocked-down filamin in endothelial cells using siRNAs, the translocation of CD40 to lipid rafts upon activation of CD40 signaling was inhibited, resulting in repression of CD40-mediated Akt signaling and subsequent inhibition of VCAM-1 and CCL-2, but not of CD40-mediated JNK signaling. Conclusions: We show that CD40 interacts with filamin A in endothelial cells. This interaction is crucial for translocation of CD40 to the lipid rafts and CD40-mediated activation of the Akt pathway. The reduced upregulation of VCAM-1 and CCL-2 makes CD40-filamin interactions an interesting target for treatment of atherosclerosis.

scholarly journals Epigenetic Dynamics of the Infant Immune System Reveals a Tumor Necrosis Factor Superfamily Signature in Early Human Life

Epigenomes ◽  
2020 ◽  
Vol 4 (3) ◽  
pp. 12
Author(s):  
Maria J. Gutierrez ◽  
Gustavo Nino ◽  
Xiumei Hong ◽  
Xiaobin Wang
Keyword(s):  
Dna Methylation ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Cell Activation ◽  
Immune Cell ◽  
Human Life ◽  
Tumor Necrosis Factor Receptor ◽  
Immune Genes ◽  
Cpg Sites ◽  
Necrosis Factor

DNA methylation (DNAm) is an essential mechanism governing normal development in humans. Although most DNAm patterns in blood cells are established in utero, the genes associated with immune function undergo postnatal DNAm modifications, and the characterization of this subset of genes is incomplete. Accordingly, we used available longitudinal DNAm datasets from a large birth cohort in the U.S. to further identify postnatal DNAm variation in peripheral leukocytes from 105 children (n = 105) between birth and the first two years of life, as determined by postnatal changes in β values (with the percentage of methylation ranging from 0 to 1.0 at individual CpG sites). Our study is an extension of a previous analysis performed by our group and identified that: (1) as previously described, DNAm patterns at most CpG sites were established before birth and only a small group of genes underwent DNAm modifications postnatally, (2) this subset includes multiple immune genes critical for lymphocyte development, and (3) several members of the tumor necrosis factor receptor and cytokine superfamilies with essential roles in immune cell activation, survival, and lymphoid tissue development were among those with a larger postnatal variation. This study describes the precise epigenetic DNA methylation marks in important immune genes that change postnatally and raises relevant questions about the role of DNAm during postnatal immune development in early childhood.

Abstract 495: MicroRNA-155 Modulates Endothelial Intracellular Adhesion Molecule-1 Expression

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Anthony Virtue ◽  
Hong Wang ◽  
Xiao-Feng Yang
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Cell Activation ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Endothelial Activation ◽  
Intercellular Adhesion Molecule ◽  
Phenotypic Change ◽  
Endothelial Cell Activation ◽  
Western Blots

The endothelium is essential in maintaining vascular tone, thrombosis, vascular remodeling, signal transduction, and inflammation by directly producing cytokines and chemokines which act in cell signaling and the recruitment of lymphocytes. In addition, the up-regulation of adhesion molecules such as vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin aid in leukocyte adherence and diapedesis. This collective alteration in the physiological characteristics of endothelial cells is commonly referred to as endothelial activation. Currently, microRNA regulation of endothelial cell activation remains poorly characterized. Specifically, microRNA-155 (miR-155) has been found to play an important role in the development and function of several immune cell types such as dendritic cells, macrophages, B-cells, and T-cells. However, the capacity of miR-155 to participate in a phenotypic change in non-traditional immune cells to a pro-inflammatory state has yet to be examined. In order to address this short-coming, our research focuses on whether the expression miR-155, affects endothelial activation. Thus far, we have determined that the expression of miR-155 can be modulated by pro-inflammatory mediators traditionally associated with endothelial activation such as disturbed flow, TNF-α, IL-1β and lipopolysaccharide. In addition to its inducible expression within endothelial cells, we have found that miR-155 can specifically alter ICAM-1 expression. Western blots with protein from excised aortas of miR-155 -/- mice reveal significantly reduced ICAM-1 expression when compared to WT mice. This data was further confirmed in vitro with protein gathered from cultured mouse aortic endothelial cells. Additionally, in accordance with the modulated ICAM-1 expression, we found monocyte adhesion to endothelial cells over-expressing miR-155 to be increased. In conclusion, these preliminary findings suggest that miR-155 plays a major role in endothelial activation, and therefore regulation of miR-155 expression holds significant therapeutic potential in the treatment of cardiovascular diseases.

scholarly journals Structures of mouse and human GITR–GITRL complexes reveal unique TNF superfamily interactions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Feng Wang ◽  
Bryant Chau ◽  
Sean M. West ◽  
Christopher R. Kimberlin ◽  
Fei Cao ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Signaling ◽  
Tumor Necrosis ◽  
Immune Cell ◽  
Linear Chain ◽  
Tumor Necrosis Factor Receptor ◽  
Higher Order ◽  
Membrane Structures ◽  
Receptor Interface

AbstractGlucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor–ligand interface and receptor–receptor interface; the unique C-terminal receptor–receptor enables higher order structures on the membrane. Human GITR–GITRL has potential to form a hexameric network of membrane complexes, while murine GITR–GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor–receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

Abstract 48: Deletion of the Neuronal Guidance Molecule Epha2 Reduces Inflammation in Experimental Atherosclerosis

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Steven D Funk ◽  
Arif Yurdagul ◽  
Jonette Green ◽  
Patrick Albert ◽  
Marshall McInnis ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Experimental Atherosclerosis ◽  
Marker Genes ◽  
Endothelial Cell Activation ◽  
Enhanced Expression ◽  
Guidance Molecule ◽  
Neuronal Guidance ◽  
Deficient Mice

Neuronal guidance molecules are increasingly implicated in inflammatory responses. Recently, our group demonstrated enhanced expression of the neuronal guidance molecule EphA2 and its ephrinA1 ligand in mouse and human atherosclerotic plaques, and elucidated a novel proinflammatory function for EphA2 perpetuating proinflammatory gene expression during endothelial cell activation. However, a direct role for Eph/ephrins in atherosclerosis has never been demonstrated. We now show that knocking out the EphA2 gene in Western diet-fed ApoE mice blunts atherosclerotic plaque location at multiple sites. This reduction in atherosclerosis is associated with decreased monocyte infiltration and diminished expression of proinflammatory genes. EphA2 reduction may affect monocyte homing through multiple mechanisms, since reducing EphA2 expression in cytokine-activated endothelial cells does not affect endothelial adhesion molecule expression or monocyte rolling but significantly decreases firm adhesion in primary human monocytes. Like endothelial cells, plaque macrophages also express EphA2, and macrophages derived from EphA2 deficient mice show diminished expression of M1 marker genes and enhanced expression of M2 marker genes compared to their ApoE counterparts. Surprisingly, EphA2 deficient mice show significantly elevated plasma cholesterol. However, this elevation does not involve increased LDL levels but instead occurs due to elevations in plasma HDL levels. Taken together, the current data suggest EphA2 inhibition results in a multifaceted protective effect on experimental atherosclerosis characterized by reduced endothelial cell activation, monocyte recruitment, and M1/M2 polarization and enhanced circulating HDL levels.

Inhibition of Tumor-Necrosis-Factor-αDELETEInduced Endothelial Cell Activation by a New Class of PPAR-γ Agonists

2005 ◽  
Vol 42 (6) ◽  
pp. 509-516 ◽  
Author(s):  
Paolo Calabrò ◽  
Ismael Samudio ◽  
Stephen H. Safe ◽  
James T. Willerson ◽  
Edward T.H. Yeh
Keyword(s):  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Cell Activation ◽  
Endothelial Cell Activation ◽  
New Class ◽  
Ppar Γ ◽  
Necrosis Factor

Abstract 327: Mitochondrial Functional Variation Contributes to Endothelial Cell Activation

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
David M Krzywanski ◽  
Bing Cheng ◽  
Xinggui Shen ◽  
Christopher Kevil
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Ethnic Groups ◽  
Mitochondrial Function ◽  
Cell Activation ◽  
Oxidant Stress ◽  
Oxygen Utilization ◽  
Endothelial Cell Activation ◽  
Inflammatory Conditions ◽  
Oxidant Production

Vascular oxidant stress contributes to endothelial dysfunction and plays a critical role in early stage cardiovascular disease (CVD) development. Changes in endothelial function due to oxidant stress may contribute to CVD initiation and progression through the development of a pro-inflammatory environment. Differences in mitochondrial function may contribute to this process and provide insight into why age of onset and clinical outcomes differ amongst individuals form distinct ethnic groups; but no reports demonstrate distinct mitochondrial functional parameters between normal cells. Consequently, we hypothesized that significant variations in normal mitochondrial function and oxidant production exist between endothelial cells from donors representing different ethnic groups. Aspects of mitochondrial oxygen utilization and oxidant production were assessed under basal and inflammatory conditions in human aortic endothelial cells (HAECs) isolated from African Americans (AA) and Caucasians (CA). Bioenergetic analysis indicates that compared to CA, AA HAEC utilized significantly less oxygen for ATP production, possess a lower maximal respiratory capacity, and have reduced electron leak. Significant differences in mitochondrial membrane potential, decreased expression of endothelial nitric oxide synthase, and increased levels of superoxide were also observed and AA HAEC supporting a pro-inflammatory phenotype. As a marker of endothelial cell activation, AA HAEC expressed increased levels of intercellular cell adhesion molecule-1 under both basal and inflammatory conditions that could be partially mitigated but treatment with the mitochondrially targeted antioxidant MitoTEMPO. These data demonstrate that fundamental differences exist in mitochondrial oxygen utilization and oxidant production between CA and AA HAEC and that these changes may affect endothelial cell activation. These findings are consistent with the hypothesis that differences in “normal” mitochondrial function amongst ethnic groups could influence individual susceptibility by contributing to vascular inflammation, providing important insights into the mechanisms that contribute human CVD development.

Abstract 66: The Role of the Viral Nef Protein as a Mediator of HIV-1--Induced Endothelial Dysfunction

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Ting Wang
Keyword(s):  
Cardiovascular Disease ◽  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Binding Site ◽  
Cell Activation ◽  
Endothelial Activation ◽  
Endothelial Cell Activation ◽  
Hiv 1

With the prevalence of antiviral therapy in the developed world, many HIV-1-infected people die of diseases other than AIDS. One of the emerging major causes is cardiovascular disease, leading to the prediction that the majority of HIV-1 patients are expected to develop cardiovascular complications. Endothelial dysfunction is thought to be a key event in the development of cardiovascular diseases, particularly atherosclerosis. Assays testing the effect of HIV-1 on endothelial activation shows that direct contact with HIV-1 infected T cells enhance endothelial cell activation to a greater extent than HIV-1 alone, suggesting an intracellular HIV-1 protein is responsible for endothelial activation. The HIV-1 viral protein Nef, which is responsible for T cell activation and maintenance of high viral loads in vivo , has been shown to mediate its own transfer to bystander cells. We demonstrate here for the first time that Nef induces nanotube-like conduits connecting T cells and endothelial cells. We also show that Nef is transferred from T cells to endothelial cells via these nanotubes, and is necessary and sufficient for endothelial cell activation. Moreover, we show that SIV-infected macaques exhibit endothelial Nef expression in coronary arteries. Nef expression in endothelial cells causes endothelial apoptosis, ROS and MCP-1 production. Interestingly, a Nef SH3 binding site mutant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through an NADPH oxidase- and ROS-dependent mechanism, while Nef-induced MCP-1 production is NF-kB dependent. Taken together, these data suggest that Nef can mediate its transfer from T cells to endothelial cells through nanotubes to enhance endothelial dysfunction.Thus, Nef is a promising new therapeutic target for reducing the risk for cardiovascular disease in the HIV-1 positive population.

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