KLF2 and KLF4 Are Essential Mediators of the Anti-Thrombotic Effects of Statins in the Presence of Antiphospholipid/Anti-ß2GPI Antibodies,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3272-3272
Author(s):  
Kristi L Allen ◽  
Mukesh K Jain ◽  
Keith R. McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Statin Treatment ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Endothelial Cell Activation ◽  
Endothelial Cell Surface ◽  
Inflammatory Stimuli

Abstract Abstract 3272 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLA). The majority of APLA are directed against phospholipid binding proteins, particularly β2GPI. Anti-ß2GPI antibodies activate endothelial cells and monocytes in a β2GPI-dependent manner through a pathway that involves NF-κB and leads to increased expression of adhesion molecules, tissue factor and proinflammatory cytokines. Krüppel-like factors (KLFs) regulate endothelial cell and monocyte responses to inflammatory stimuli; increased expression of these transcription factors inhibits proinflammatory and procoagulant gene expression, and maintains vascular homeostasis. We recently reported that anti-ß2GPI antibodies decrease the expression of KLF2 and KLF4 in endothelial cells (Allen et al, Blood 2011), promoting endothelial cell activation. Subsequent studies demonstrate that these antibodies decrease expression of KLF2 in monocytes as well. Statins have been proposed as a potential alternative to anticoagulation for APS patients, and stimulate the expression of KLFs. We hypothesized that the ability of statins to block endothelial cell activation in response to anti-β2GPI antibodies was mediated by KLFs. Treatment of endothelial cells and monocytes with 100 nM fluvastatin, lovastatin, or simvastatin upregulated KLF2 and KLF4 mRNA, even in the presence of anti-ß2GPI antibodies. In parallel, statin treatment inhibited the anti-β2GPI antibody-mediated induction of E-selectin, VCAM-1, and TF mRNA in endothelial cells, and ICAM-1 and TF mRNA in human monocytes. To assess the dependence of these effects on KLF expression, endothelial cells were pretreated with KLF2 or KLF4 siRNA prior to treatment with statins. siRNA-mediated inhibition of KLF expression completely blocked the ability of statins to prevent anti-β2GPI antibody-induced endothelial cell activation, as measured by adhesion molecule and TF mRNA levels and expression of E-selectin on the endothelial cell surface. Taken together, these data demonstrate that KLFs are critical modulators of the effects of statins on endothelial cells, and that increased expression of KLFs may represent a mechanism by which these drugs inhibit the activation of endothelial cells and monocytes by APLA/anti-β2GPI antibodies. Disclosures: No relevant conflicts of interest to declare.

160. THE ROLE OF PHAGOCYTOSIS OF APOPTOTIC SYNCYTIAL KNOTS IN THE PREVENTION OF ENDOTHELIAL CELL ACTIVATION: AN IMPORTANT ADAPTATION FOR NORMAL PREGNANCY

2010 ◽  
Vol 22 (9) ◽  
pp. 78
Author(s):  
Q. Chen ◽  
H. Jin ◽  
P. Stone ◽  
L. Chamley
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Immune Responses ◽  
Cell Activation ◽  
Normal Pregnancy ◽  
Cytochalasin D ◽  
Endothelial Cell Activation ◽  
Endothelial Cell Surface ◽  
Large Numbers

Preeclampsia is characterised by an exaggerated inflammatory response and maternal endothelial cell activation. Syncytial knots, dead multinucleated fetal cells shed from the placenta in large numbers during all pregnancies, may be phagocytosed by maternal endothelial cells. Our previous studies showed that phagocytosis of necrotic but not apoptotic syncytial knots led to endothelial cell activation. It is known that phagocytosis of apoptotic cells leads to active tolerance of immune responses and in this study we questioned whether phagocytosis of apoptotic syncytial knots leads to suppression of the endothelial cells ability to be activated. Syncytial knots were harvested from 1st trimester placental explants. Monolayers of endothelial cells were pre-treated with apoptotic syncytial knots for 24 h. After washing, the endothelial cells were treated with the endothelial cell activators LPS, PMA, IL-6, or necrotic syncytial knots for 24 h. In some experiments the inhibitor of phagocytosis, cytochalasin D, was added into the cultures along with apoptotic syncytial knots. Endothelial cell-surface ICAM-1 was measured using cell based ELISAs. Expression of ICAM-1 by endothelial cells that had phagocytosed apoptotic syncytial knots prior to treatment with LPS, PMA, IL-6, or necrotic syncytial knots was significantly (P =/<0.003) reduced, compared to control endothelial cells that had not phagocytosed apoptotic syncytial knots. Inhibiting phagocytosis of apoptotic syncytial knots with cytochalasin D abolished this protective effect. Our data suggest phagocytosis of apoptotic syncytial knots results in the suppression of the ability of endothelial cells to be activated by a number of potent chemical activators, as well as by the physiologically relevant activator, necrotic syncytial knots. This work suggests that the release of apoptotic syncytial knots from the placenta during normal pregnancy may be a mechanism by which the fetus attempts to protect the maternal vasculature against activation.

Decreased Expression of KLF2 and KLF4 Induced by Antiphospholipid Antibodies Promotes NF-Kb-Mediated Endothelial Cell Activation and Is Modulated by CBP/p300

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4315-4315
Author(s):  
Kristi L Allen ◽  
Mukesh K Jain ◽  
Keith R McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Antiphospholipid Antibodies ◽  
Transcriptional Activity ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Cell Phenotype ◽  
Endothelial Cell Activation

Abstract Abstract 4315 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures: No relevant conflicts of interest to declare.

Gas6 Regulates Thrombin-Induced of VCAM-1 Expression by a FoxO1 Dependent Mechanism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5192-5192
Author(s):  
Richard Robins ◽  
Catherine A. Lemarie ◽  
Mark D. Blostein
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Venous Thrombosis ◽  
Cell Biology ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Vascular Dysfunction ◽  
Soluble Form ◽  
Endothelial Cell Activation

Abstract Abstract 5192 Forkhead proteins play a broad role in endothelial cell biology. These factors mediate cell adhesion to extracellular matrix, regulate the expression of pro-inflammatory and pro-thrombotic genes, and participate in cell repair, proliferation and apoptosis. FoxOs are known downstream targets of the PI3K/Akt signaling pathway. Phosphorylation of FoxO transcription factors results in their translocation from the nucleus to the cytoplasm, thereby inhibiting their transcriptional activity. It has recently been shown that the deletion of the three FoxO isoforms in endothelial cells protects mice from vascular dysfunction. Gas6, a member of the vitamin K-dependent family of proteins, has been shown to protect endothelial cells from apoptosis and promote endothelial cell activation in vivo. It has been shown that the expression of ICAM-1 and VCAM-1 were blunted in the absence of gas6. Interestingly, a role for VCAM-1 in the pathogenesis of venous thrombosis has been proposed. Elevated levels of the soluble form of VCAM-1 have been detected in the serum of patients with venous thrombosis. We previously demonstrated that the anti-apoptotic effect of gas6 was mediated partially through FoxO1, but overall, the signalling mechanisms occurring downstream of gas6 remain largely unknown. We hypothesize that gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO1 in endothelial cells. Western blot analysis demonstrated that thrombin induced time dependent phosphorylation of FoxO1 with a maximum at 30 minutes in WT (p<0. 05) but not in gas6 deficient (−/−) cells. In addition, thrombin reduced the nuclear content of FoxO in WT (p<0. 05) but not in gas6−/− endothelial cells. Using qPCR, we found that mRNA expression of VCAM-1 was increased after 30 minutes of stimulation with thrombin in WT cells (p<0. 05). More importantly, thrombin-mediated induction of VCAM-1 was blunted in gas6−/− endothelial cells. We found that FoxO1 siRNA increased basal VCAM-1 expression in WT endothelial cells. Taken together, our data demonstrate that gas6 is a crucial mediator of FoxO1 that regulates thrombin-induced VCAM-1 expression. This pathway may explain the pro-thrombotic and pro-inflammatory role of gas6. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NADPH Oxidase Mediates Antiphospholipid Antibody-Induced Endothelial Cell Activation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1447-1447
Author(s):  
Meifang Wu ◽  
Keith R. McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Nadph Oxidase ◽  
Conditioned Medium ◽  
Cell Activation ◽  
Fluorescent Dyes ◽  
Ros Generation ◽  
Antiphospholipid Antibody ◽  
Dependent Manner ◽  
Endothelial Cell Activation

Abstract Introduction: Antiphospholipid Syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. APLA/anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner, though the underlying mechanisms are not well defined. Objective: To define the role of NOX1 in the generation of ROS and activation of endothelial cells by anti-β2GPI antibodies. Methods: Endothelial cells were incubated with β2GPI and either control or affinity-purified anti-β2GPI antibodies in the absence or presence of diphenyleneiodonium (DPI), an NADPH oxidase (NOX) inhibitor. Generation of reactive oxygen species (ROS) in treated cells and conditioned medium were measured by using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or luminescent substrate. NOX mRNA and protein expression were assessed using quantitative PCR and immunoblot. Endothelial cell activation was measured by increased expression of E-selectin. Results: Incubation of endothelial cells with β2GPI and anti-β2GPI antibodies stimulated ROS generation in endothelial cells, as well as the release of ROS into conditioned medium. The expression of NOX1 mRNA and protein levels were significantly increased in endothelial cells exposed to anti-β2GPI antibodies, but not control IgG (Figure 1). The ability of β2GPI and anti-β2GPI antibodies to induce endothelial cell E-selectin mRNA expression was blocked by pretreatment of cells with DPI (Figure 2), suggesting that ROS is required for downstream events underlying endothelial cell activation. Conclusions: Endothelial cells exposed to β2GPI and anti-β2GPI antibodies generate ROS, which is subsequently released into the conditioned medium. NOX1 appears to be essential for ROS generation. The impairment of endothelial cell activation by DPI suggests that NOX is also essential for endothelial cell activation by anti-β2GPI antibodies. We hypothesize that production of ROS by NOX plays a central role in APLA-induced endothelial dysfunction. Acknowledgment: This work was supported by an ASH Bridge Grant Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

“Rolling Recruitment” of Endothelial Cell (EC) Activation in the Prothrombotic Nature of Heparin-Induced Thrombocytopenia (HIT)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 536-536
Author(s):  
Vincent M Hayes ◽  
Douglas B. Cines ◽  
Mortimer Poncz ◽  
Lubica Rauova
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Passive Immunization ◽  
Endothelial Cell Activation ◽  
Endothelial Lining ◽  
Platelet Factor ◽  
Laser Injury ◽  
Activated Platelets

Abstract Abstract 536 HIT is an iatrogenic, prothrombotic, immune-mediated disorder associated with antibodies directed against platelet factor 4 (PF4) and heparin complexes. Once these pathogenic anti-PF4/heparin antibodies develop, they recognize and bind to antigenic complexes formed on cell surfaces composed of PF4 released from platelets bound to cell membrane glycosaminoglycans (GAGs). Unlike platelets, which have only chondroitin sulfate on their surface, endothelial cells express mainly heparan sulfate resulting in a very high capacity to form PF4/GAG complexes and become targets for HIT antibodies. In fact, activation of endothelial cells by HIT antibodies was recognized almost 25 years ago (Cines et. al., 1987). We now show direct interaction of the HIT-like monoclonal antibody KKO with perithrombus endothelial cells, which propagates thrombosis following laser induced cremaster vessel injury in a passive immunization murine model of HIT. PF4 released from activated platelets at the site of laser injury binds rapidly and predominantly to the adjacent endothelium and is recognized by KKO in both transgenic mice expressing only human PF4 (hPF4+) and mice expressing hPF4 and human FcgRIIA on platelets (hPF4+/FcgRIIA+). These studies show that HIT-like antigenic complexes are present within thrombi prior to antibody exposure, predominantly localized to endothelial cells, which is consistent with their surface GAGs having higher affinity for PF4 than platelets. These endothelial cell surface PF4/GAG complexes, and with it KKO binding, are dissociated by high concentrations of intravenous heparin. Infusion of KKO in hPF4+/FcgRIIA+ mice but not in control mice, reinitiates growth of previously stable thrombi at sites of laser injury, leading to a significant increase in vascular occlusion (24/33 injuries, 73%, compared to 0/30 in control mice; P<0.0001). Clot extension is followed by dissemination of endothelial activation, demarcated by binding of annexin V and Factor Xa, and extension of the endothelial surface that binds KKO. Based on these studies, we propose a model of feed-forward rolling recruitment to explain how the endothelial lining contributes to the prothrombotic state in HIT: PF4 released from activated platelets at a site of injury or disease binds to the surface of perithrombus endothelial cells, which then binds HIT antibodies followed by endothelial cell activation. These activated endothelial cells then bind and activate additional platelets, leading to the next round of PF4 release, more extensive endothelial cell activation and platelet recruitment into thrombi. Disclosures: No relevant conflicts of interest to declare.

Striatin Is a Novel Regulator Of Aldosterone-Mediated Endothelial Cell Activation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3523-3523 ◽  
Author(s):  
Enrique D. Machado-Fiallo ◽  
Christopher Vega ◽  
Arelys Ramos-Rivera ◽  
Josue A. Benabe-Carlo ◽  
Gregory N. Prado ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Knockout Mice ◽  
Time Course ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Ros Production ◽  
Endothelial Cell Activation ◽  
Caveolin 1

Abstract Activation of the minerolocorticoid receptor (MR) by aldosterone (ALDO) has been shown to play an important role in inflammatory vascular responses in addition to its well described effects on sodium homeostasis. Steroid responses are mediated by well-known genomic and less known rapid/nongenomic responses. However, characterization of the mechanisms underlying ALDO’s rapid/nongenomic actions have been difficult to study and are not clearly understood. We recently reported that in vivo and in vitro activation of MR leads to increases of striatin levels in endothelial cells, aortas and heart tissue (Pojoga, Amer J Hypertens, 2012) and that MR forms a complex with caveolin-1 and striatin within caveolae in endothelial cells. We hypothesized that striatin is a critical intermediary of the rapid effects of ALDO and that striatin serves as a novel link for MR regulation in endothelial cells activation. Endothelial cell activation promotes, among other factors, increased levels of reactive oxygen species (ROS) and protein disulfide isomerase (PDI), a redox modifying enzyme that catalyze disulfide interchange reactions. We studied EA.hy926 cells (EA), a human endothelial cell line that expresses MR, striatin and maintains its caveolae while in culture. We incubated EA cells with ALDO (10–9–10–7M) for 60 min and observed a dose-dependent rise in ROS production (P<0.001, n=4) using the oxidative fluorescent indicator dye 5-6-chloromethyl-2ʼ,7ʼ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) that peaked at around 10-8M ALDO, an event that was blocked by pre-incubation of EA cells for 30 mins with 1μM canrenoic acid (CA), an MR antagonist (P<0.03, n=3). Time course analyses showed ALDO stimulated ROS responses that increased for up to 3 hours following the addition of ALDO. As there are no known inhibitors for striatin we then used siRNA technology to down regulate striatin in these cells. EA cells were transfected with striatin siRNA and subsequently stimulated with ALDO and ROS production measured. The transfection process itself did not modify baseline levels of ROS significantly, as assessed in cells transfected with scrambled siRNA and non-transfected cells, which had nearly identical ROS levels, basally and in response to ALDO. In addition, in the presence of lower levels of striatin protein the ALDO-stimulated ROS response was abrogated, supporting the concept that striatin is necessary for the rapid effects of ALDO. We also measured phosphorylated ERK-1/2 (pERK) levels that peaked within 10 minutes in EA as estimated by western blot analyses. Consistent with these observations pre-incubation of EA cells with 10-6M PD0325901, a selective MEK-1/2 inhibitor was associated with greater than 90% reduction of the ALDO-stimulated ROS responses (7244.3±497 vs 4386.6±586 RFU, P<0.02, n=3). Qualitatively similar responses were observed using another MEK inhibitor, U0126 [10-5M] (P<0.01, n=3). We then tested the effects of ALDO on PDI secretion. Incubation of EA cells with ALDO (10-7M) led to PDI increases when compared to vehicle treated cells (P<0.01, n=3). We also tested the effects of low levels of striatin using siRNA on PDI activity in EA cells. We found that PDI secretion was reduced by 62% in striatin knockdown conditions. We then tested the effects of Methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted 10nM ALDO–stimulated PDI response (530±117 to 215±99 RFU/mg protein, n=3, P<0.01). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from endothelial-specific caveolin-1 knockout mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from caveolin-1 knockout mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). These results suggest that striatin is a novel mediator for ALDO’s rapid effects on PDI and ROS, thereby suggesting a unique level of interaction between the MR and striatin in endothelial cell activation. Supported by NIH R01HL090632 (AR), R01HL104032 (LHP) and R01HL096518 (JRR). Disclosures: No relevant conflicts of interest to declare.

Annexin A2 mediates endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 1964-1969 ◽  
Author(s):  
Jianwei Zhang ◽  
Keith R. McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Endothelial Activation ◽  
Annexin A2 ◽  
Dependent Manner ◽  
Endothelial Cell Activation ◽  
Glycoprotein I ◽  
Endothelial Surface ◽  
Increased Risk

AbstractPatients with antiphospholipid antibodies (APLAs) are at increased risk for arterial and venous thrombosis. Many APLAs associated with these events react with β2 glycoprotein I (β2GPI), and endothelial cell reactive antibodies that activate endothelial cells in a β2GPI-dependent manner occur commonly in these patients. We previously reported that β2GPI binds with high affinity to annexin A2 on the endothelial surface, though the relevance of this interaction to APLA/anti-β2GPI antibody–induced endothelial activation has not been determined. In this report, we confirm that anti-β2GPI antibodies activate endothelial cells in the presence of β2GPI, and demonstrate that anti–annexin A2 antibodies directly cause endothelial cell activation of a similar magnitude and with a similar time course. Moreover, bivalent anti–annexin A2 F(ab′)2 fragments also caused endothelial cell activation, whereas monomeric Fab fragments not only did not cause activation, but blocked activation induced by anti–annexin A2 antibodies and F(ab′)2 fragments, as well as that caused by anti-β2GPI antibodies in the presence of β2GPI. These observations suggest a novel pathway for endothelial activation induced by APLA/anti-β2GPI antibodies that is initiated by cross-linking or clustering of annexin A2 on the endothelial surface.

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.

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.

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