Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.     

Multiple Doses of Erythropoietin Impair Liver Regeneration by Increasing TNF-α, the Bax to Bcl-xL Ratio and Apoptotic Cell Death

Background

Liver resection and the use of small-for-size grafts are restricted by the necessity to provide a sufficient amount of functional liver mass. Only few promising strategies to maximize liver regeneration are available. Apart from its erythropoiesis-stimulating effect, erythropoietin (EPO) has meanwhile been recognized as mitogenic, tissue-protective, and anti-apoptotic pleiotropic cytokine. Thus, EPO may support regeneration of hepatic tissue.

Methodology

Rats undergoing 68% hepatectomy received daily either high dose (5000 IU/kg bw iv) or low dose (500 IU/kg bw iv) recombinant human EPO or equal amounts of physiologic saline. Parameters of liver regeneration and hepatocellular apoptosis were assessed at 24 h, 48 h and 5 d after resection. In addition, red blood cell count, hematocrit and serum EPO levels as well as plasma concentrations of TNF-α and IL-6 were evaluated. Further, hepatic Bcl-x_L and Bax protein expression were analyzed by Western blot.

Principal Findings

Administration of EPO significantly reduced the expression of PCNA at 24 h followed by a significant decrease in restitution of liver mass at day 5 after partial hepatectomy. EPO increased TNF-α levels and shifted the Bcl-x_L to Bax ratio towards the pro-apoptotic Bax resulting in significantly increased hepatocellular apoptosis.

Conclusions

Multiple doses of EPO after partial hepatectomy increase hepatocellular apoptosis and impair liver regeneration in rats. Thus, careful consideration should be made in pre- and post-operative recombinant human EPO administration in the setting of liver resection and transplantation.     

Gremlin-1 Is an Inhibitor of Macrophage Migration Inhibitory Factor and Attenuates Atherosclerotic Plaque Growth in ApoE?/? Mice

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE^−/− mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (K_D = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE^−/− mice with a dimeric recombinant fusion protein, _mGremlin1-Fc, but not with equimolar control Fc or inactivated _mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.     

Progression of coronary atherosclerosis relates to the onset of myocardial infarction in an animal model of spontaneous myocardial infarction (WHHLMI rabbits).

Recently, we developed myocardial infarction-prone WHHLMI rabbits from coronary atherosclerosis-prone WHHL rabbits (WHHLCA rabbits) by selective breeding. In this study, we examined the relation of atherosclerotic plaques to the onset of myocardial infarction. We examined myocardial lesions of 378 WHHL rabbits born between 1992 and 2000, and atherosclerosis lesions of 93 WHHLCA and 82 WHHLMI rabbits. The aortic lesions were evaluated as percent surface lesion area. The coronary lesions were evaluated as cross sectional narrowing using sections prepared at 500 or 1,000 microm intervals. Serum lipid levels were assayed with enzymatic methods. The cumulative incidence of fatal myocardial infarction between 11 and 35 months old was 90% in WHHLMI rabbits and 21% in WHHLCA rabbits, respectively. Selective breeding increased the serum cholesterol levels by about 200 mg/dl despite there being no changes in triglyceride levels. Aortic and coronary atherosclerosis progressed markedly in WHHLMI rabbits compared to WHHLCA rabbits. Especially, WHHLMI rabbits over 15 months old showed more than 90% cross sectional narrowing of the left circumflex arteries, main stem of the left coronary artery, and the origin portion of the right coronary artery. In addition, there were no gender differences in atherosclerotic lesions of both aortas and coronary arteries. In conclusion, the present study showed that marked progression of coronary atherosclerosis was probably associated with spontaneous development of myocardial infarction in WHHLMI rabbits.     

Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions

Repressor activator protein 1 (Rap1) is essential for maintaining telomere length and structural integrity, but it also exerts other non-telomeric functions. The present study tested the hypothesis that Rap1 is released into the cytoplasm and induces production of pro-inflammatory cytokines via nuclear factor kappa B (NFκB) signaling in macrophages, a cell type involved in the development and progression of atherosclerotic lesions. Western blotting analysis confirmed that Rap1 was present in the cytoplasm of differentiated human monocytic leukemia cells (THP-1, a macrophage-like cell line). Co-immunoprecipitation assay revealed a direct interaction between Rap1 and I kappa B kinase (IKK). Knockdown of Rap1 suppressed lipopolysaccharide-mediated activation of NFκB, and phosphorylation of inhibitor of kappa B α (IκBα) and p65 in THP-1 macrophages. The reduction of NFκB activity was paralleled by a decreased production of NFκB-dependent pro-inflammatory cytokines and an increased expression of IκBα (native NFκB inhibitor) in various macrophage models with pro-inflammatory phenotype, including THP-1, mouse peritoneal macrophages and bone marrow-derived M1 macrophages. These changes were observed selectively in pro-inflammatory macrophages but not in bone marrow-derived M2 macrophages (with an anti-inflammatory phenotype), mouse lung endothelial cells, human umbilical vein endothelial cells or human aortic smooth muscle cells. Immunostaining revealed that Rap1 was localized mainly in macrophage-rich areas in human atherosclerotic plaques and that the presence of Rap1 was positively correlated with the advancement of the disease process. In pro-inflammatory macrophages, Rap1 promotes cytokine production via NFκB activation favoring a pro-inflammatory environment which may contribute to the development and progression of atherosclerosis.     

Insulin regulates monocyte trans-endothelial migration through surface expression of macrophage-1 antigen

During the pathogenesis of atherosclerosis, adhesion of monocytes to vascular endothelium and subsequent migration across the endothelium has been recognized as a key process in the chronic inflammatory response in atherosclerosis. As type 2 diabetes is closely associated with the pathogenesis of atherosclerosis, we investigated whether monocyte adhesion and migration were affected by insulin. We found that insulin activated Akt and induced subsequent migration in THP-1. However, glucose and insulin-like growth factor-1, which is a growth factor that is structurally similar to insulin, were not effective. Insulin-dependent migration of THP-1 was blocked by inhibition of PI3K or Akt and by silencing of Akt1. Insulin-dependent migration of bone marrow-derived monocytic cells (BDMCs) was attenuated by inhibition of PI3K and Akt. In addition, BDMCs from Akt1^−/− mice showed defects in insulin-dependent migration. Stimulation of THP-1 with insulin caused adhesion with human vein endothelial cells (HUVECs) that was blocked by silencing of Akt1. However, stimulation of HUVECs did not cause adhesion with THP-1. Moreover, BDMCs from Akt1^−/− mice showed defects in insulin-dependent adhesion with HUVECs. Insulin induced surface expression of Mac-1, and neutralization of Mac-1 blocked insulin-induced adhesion of THP-1 as well as BDMCs. Surface expression of Mac-1 was blocked in THP-1 with silenced Akt1, and in BDMCs isolated from mice lacking Akt1. Finally, trans-endothelial migration of THP-1 and BDMCs was blocked by Mac-1-neutralizing antibody, in THP-1 with silenced Akt1 and in BDMCs from Akt1^−/− mice. These results suggest that insulin stimulates monocyte trans-endothelial migration through Akt-dependent surface expression of Mac-1, which may be part of the atherogenesis in type 2 diabetes.     

Reduced Size and Macrophage Content of Advanced Atherosclerotic Lesions in Mice with Bone Marrow Specific Deficiency of Alpha 7 Nicotinic Acetylcholine Receptor

In macrophages the α7 nicotinic acetylcholine receptor (α7nAChR) modulates production of inflammatory cytokines, cholesterol accumulation and lipoprotein uptake. Recently, our laboratory showed that selective stimulation of the α7nAChR protects macrophages from apoptosis, an effect that is absent in α7nAChR-deficient macrophages. All these observations are suggestive of a potential role of macrophage α7nAChR in atherosclerosis. Mouse models of the disease with bone marrow deletion of α7nAChR represent an attractive approach to address the in vivo relevance of these in vitro findings. However, recent studies that focused on the impact of hematopoietic deficiency of α7nAChR on early atherosclerotic lesions of low density lipoprotein receptor knockout (LDLRKO) mice, yielded controversial results. The question also remained whether macrophage α7nAChR modulates the characteristics of advanced lesions. Here we used LDLR knockout mice transplanted with bone marrow from wild-type or α7nAChR knockout animals to revisit the effect of hematopoietic deficiency of α7nAChR on early lesions and to examine, for the first time, its impact on advanced plaques. Aortic sinus atherosclerotic lesions were analyzed following 8 and 14 weeks on a high fat diet. Early lesions in mice with α7nAChR deficient bone marrow were not different from those in control animals. However, advanced lesions of mice with bone marrow deletion of α7nAChR exhibited reduction in size, macrophage content and cell proliferation. These studies are the first in examining the impact of hematopoietic deficiency of α7nAChR on the characteristics of advanced atherosclerotic lesions in a mouse model of the disease and provide novel evidence underscoring a potential pro-atherogenic role of macrophage α7nAChR.     

Ubiquitination-Deubiquitination by the TRIM27-USP7 Complex Regulates Tumor Necrosis Factor Alpha-Induced Apoptosis

Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α–d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α–cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.     

The protective effect of Prunella vulgaris ethanol extract against vascular inflammation in TNF-α-stimulated human aortic smooth muscle cells

Atherosclerosis, which manifests as acute coronary syndrome, stroke, and peripheral arterial diseases, is a chronic inflammatory disease of the arterial wall. Prunella vulgaris, a perennial herb with a worldwide distribution, has been used as a traditional medicine in inflammatory disease. Here, we investigated the effects of P. vulgaris ethanol extract on TNF-α-induced inflammatory responses in human aortic smooth muscle cells (HASMCs). We found that P. vulgaris ethanol extract inhibited adhesion of monocyte/macrophage-like THP-1 cells to activated HASMCs. It also decreased expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin and ROS, No production in TNF-α-induced HASMCs and reduced NF-kB activation. Furthermore, P. vulgaris extract suppressed TNF-α-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK). These results demonstrate that P. vulgaris possesses antiinflammatory properties and can regulate TNF-α-induced expression of adhesion molecules by inhibiting the p38 MAPK/ ERK signaling pathway. [BMB Reports 2013; 46(7): 352-357]     

N-Acetylcysteine Attenuates Hexavalent Chromium-Induced Hypersensitivity through Inhibition of Cell Death,ROS-Related Signaling and Cytokine Expression

Chromium hypersensitivity (chromium-induced allergic contact dermatitis) is an important issue in occupational skin disease. Hexavalent chromium (Cr (VI)) can activate the Akt, Nuclear factor κB (NF-κB), and Mitogen-activated protein kinase (MAPK) pathways and induce cell death, via the effects of reactive oxygen species (ROS). Recently, cell death stimuli have been proposed to regulate the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). However, the exact effects of ROS on the signaling molecules and cytotoxicity involved in Cr(VI)-induced hypersensitivity have not yet been fully demonstrated. N-acetylcysteine (NAC) could increase glutathione levels in the skin and act as an antioxidant. In this study, we investigated the effects of NAC on attenuating the Cr(VI)-triggered ROS signaling in both normal keratinocyte cells (HaCaT cells) and a guinea pig (GP) model. The results showed the induction of apoptosis, autophagy and ROS were observed after different concentrations of Cr(VI) treatment. HaCaT cells pretreated with NAC exhibited a decrease in apoptosis and autophagy, which could affect cell viability. In addition, Cr (VI) activated the Akt, NF-κB and MAPK pathways thereby increasing IL-1α and TNF-α production. However, all of these stimulation phenomena could be inhibited by NAC in both of in vitro and in vivo studies. These novel findings indicate that NAC may prevent the development of chromium hypersensitivity by inhibiting of ROS-induced cell death and cytokine expression.     

Role of protein kinase C δ in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells

During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.     

PI3K p110γ Deletion Attenuates Murine Atherosclerosis by Reducing Macrophage Proliferation but Not Polarization or Apoptosis in Lesions

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR^−/−p110γ^+/− and LDLR^−/−p110γ^−/− mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ^+/− and p110γ^−/− mice. Lack of p110γ in LDLR^−/− mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR^−/−p110γ^−/− mice were smaller than in LDLR^−/−p110γ^+/− controls, which coincided with decreased macrophage proliferation in LDLR^−/−p110γ^−/− mouse lesions. This proliferation defect was also observed in p110γ^−/− bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR^−/−p110γ^−/− mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR^−/− mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.     

TLR4/NF-κB Signaling Contributes to Chronic Unpredictable Mild Stress-Induced Atherosclerosis in ApoE-/- Mice

Objective

Chronic stress is an important risk factor for atherosclerotic diseases. Our previous studies have shown that chronic unpredictable mild stress (CUMS) accelerates atherosclerosis and up-regulates TLR4/NF-κB expression in apoE^-/- mice. However, TLR4/NF-κB signaling whether directly contributes to the development of atherosclerosis in CUMS mice is unclear. We hypothesized that the interference of TLR4/NF-κB can ameliorate CUMS-induced inflammation and atherosclerosis in apoE^-/- mice.

Methods

ApoE^-/- mice were exposed to 12 weeks CUMS. Ad-siRNA TLR4 was given by tail vein injection (10 μl/mouse, every 5 days), and PDTC (an inhibitor of NF-κB) was given by intraperitoneal injection (60 mg/kg, once a day). Plasma corticosterone concentrations were determined by solid-phase ¹²⁵I radioimmunoassay. Atherosclerosis lesions in aortic sinuses were evaluated and quantified by IMAGEPRO PLUS. Western blotting was used to detect the expression of TLR4, NF-κB, and IL-1β in aortas of the mice. Plasma lipid profiles, IL-1β, TNF-α, and MCP-1 were measured by ELISA.

Results

Our results indicated that CUMS apoE^-/- mice treatment with siRNA TLR4 significantly decreased atherosclerosis and down-regulated TLR4, NF-κB, and inflammatory cytokines. PDTC also remarkably reduced atherosclerosis and the levels of IL-1β, TNF-α and MCP-1 in plasma. However, Treatment with siRNA TLR4 or PDTC had no effect on plasma corticosterone levels, and lipid profiles.

Conclusions

TLR4/NF-κB pathway may participate in CUMS-induced atherosclerosis through activation of proinflammatory cytokines in apoE^-/- mice. Our data may provide a new potential therapeutic target for prevention of CUMS -induced atherosclerosis.     

Salusin-β, but Not Salusin-α, Promotes Human Umbilical Vein Endothelial Cell Inflammation via the p38 MAPK/JNK-NF-κB Pathway

Objective

Recently, salusin-β has been reported to have pro-atherosclerotic effects, but salusin-α has anti-atherosclerotic effects. Our previous study has shown that salusin-β but not salusin-α promotes vascular inflammation in apoE-deficient mice. However, the underlying mechanism remains unknown. In this study, we observed the effect of salusins on inflammatory responses and the MAPK-NF-κB signaling pathway in human umbilical vein endothelial cells (HUVECs).

Methods and Results

HUVECs were incubated with different concentrations of salusin-α and salusin-β. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were quantified using quantitative real-time polymerase chain reaction (PCR). The protein expressions of VCAM-1, MCP-1, I-κBα, NF-κB, p-JNK and p-p38 MAPK were measured using western blotting analysis. Our results showed that in HUVECs, salusin-β could up-regulate the levels of IL-6, TNF-α, VCAM-1 and MCP-1, promote I-κBα degradation and NF-κB activation, and increase the phosphorylation of JNK and p38 MAPK. These effects could be inhibited by p38 MAPK inhibitor SB203580 and/or JNK inhibitor SP600125. In contrast, salusin-α could selectively decrease VCAM-1 protein, but did not show any effect on the expressions of VCAM-1 mRNA, TNF-α, IL-6, MCP-1, I-κBα, NF-κB, p-JNK or p-p38 MAPK.

Conclusion

Salusin-β was able to promote inflammatory responses in HUVECs via the p38 MAPK-NF-κB and JNK-NF-κB pathways. In contrast, salusin-α failed to show any significant effects on the inflammatory responses in HUVECs. These results provide further insight into the mechanisms behind salusins in vascular inflammation and offer a potential target for the prevention and treatment of atherosclerosis.     

Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.     

Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells

Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1 and IL-8) production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin (THA, an ERS inducer) attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS.     

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.     

Folic Acid Represses Hypoxia-Induced Inflammation in THP-1 Cells through Inhibition of the PI3K/Akt/HIF-1α Pathway

Though hypoxia has been implicated as a cause of inflammation, the underlying mechanism is not well understood. Folic acid has been shown to provide protection against oxidative stress and inflammation in patients with cardiovascular disease and various models approximating insult to tissue via inflammation. It has been reported that hypoxia-induced inflammation is associated with oxidative stress, upregulation of hypoxia-inducible factor 1-alpha (HIF-1α), and production of pro-inflammatory molecules. Whether folic acid protects human monocytic cells (THP-1 cells) against hypoxia-induced damage, however, remains unknown. We used THP-1 cells to establish a hypoxia-induced cellular injury model. Pretreating THP-1 cells with folic acid attenuated hypoxia-induced inflammatory responses, including a decrease in protein and mRNA levels of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), coupled with increased levels of IL-10. Folic acid also reduced hypoxia-induced Akt phosphorylation and decreased nuclear accumulation of HIF-1α protein. Both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a selective inhibitor of phosphatidyl inositol-3 kinase, PI3K) and KC7F2 (a HIF-1α inhibitor) reduced levels of hypoxia-induced inflammatory cytokines. We also found that insulin (an Akt activator) and dimethyloxallyl glycine (DMOG, a HIF-1α activator) induced over-expression of inflammatory cytokines, which could be blocked by folic acid. Taken together, these findings demonstrate how folic acid attenuates the hypoxia-induced inflammatory responses of THP-1 cells through inhibition of the PI3K/Akt/HIF-1α pathway.     

A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis

Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE^−/− mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE^−/− mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1β, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE^−/− mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE^−/− mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.