THP-1 Monocyte Adhesion Research Articles

Galectin-3 inhibition ameliorates hypoxia-induced pulmonary artery hypertension.

Galectin-3 (Gal-3) is a β-galactoside-binding lectin, which is important in inflammation, fibrosis and heart failure. The present study aimed to investigate the role and mechanism of Gal-3 in hypoxia-induced pulmonary arterial hypertension (PAH). Male C57BL/6J and Gal-3−/− mice were exposed to hypoxia, then the right ventricular systolic pressure (RVSP) and Fulton's index were measured, and Gal-3 mRNA and protein expression in the pulmonary arteries was analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. Compared with the control, hypoxia increased the mRNA and protein expression levels of Gal-3 in wild type murine pulmonary arteries. Gal-3 deletion reduced the hypoxia-induced upregulation of RVSP and Fulton's index. Furthermore, human pulmonary arterial endothelial cells (HPAECs) and human pulmonary arterial smooth muscle cells (HPASMCs) were stimulated by hypoxia in vitro, and Gal-3 expression was inhibited by small interfering RNA. The inflammatory response of HPAECs, and the proliferation and cell cycle distribution of HPASMCs was also analyzed. Gal-3 inhibition alleviated the hypoxia-induced inflammatory response in HPAECs, including tumor necrosis factor-α and interleukin-1 secretion, expression of intercellular adhesion molecule-1 and adhesion of THP-1 monocytes. Gal-3 inhibition also reduced hypoxia-induced proliferation of HPASMCs, partially by reducing cyclin D1 expression and increasing p27 expression. Furthermore, Gal-3 inhibition suppressed HPASMC switching from a ‘contractile’ to a ‘synthetic’ phenotype. In conclusion, Gal-3 serves a fundamental role in hypoxia-induced PAH, and inhibition of Gal-3 may represent a novel therapeutic target for the treatment of hypoxia-induced PAH.

Cytotoxicity, oxidative stress and inflammation induced by ZnO nanoparticles in endothelial cells: interaction with palmitate or lipopolysaccharide.

Recent studies showed that ZnO nanoparticles (NPs) might induce the toxicity to human endothelial cells. However, little is known about the interaction between ZnO NPs and circulatory components, which is likely to occur when NPs enter the blood. In this study, we evaluated ZnO NP-induced cytotoxicity, oxidative stress and inflammation in human umbilical vein endothelial cells (HUVECs), with the emphasis on the interaction with palmitate (PA) or lipopolysaccharide (LPS), because PA and LPS are normal components in human blood that increase in metabolic diseases. Overall, ZnO NPs induced cytotoxicity and intracellular reactive oxygen species (ROS) at a concentration of 32 μg ml-1 , but did not significantly affect the release of inflammatory cytokines or adhesion of THP-1 monocytes to HUVECs. In addition, exposure to ZnO NPs dose-dependently promoted intracellular Zn ions in HUVECs. PA and LPS have different effects. Two hundred μm PA significantly induced cytotoxicity and THP-1 monocyte adhesion, but did not affect ROS or release of inflammatory cytokines. In contrast, 1 μg ml-1 LPS significantly induced ROS, release of inflammatory cytokines and THP-1 monocyte adhesion, but not cytotoxicity. The presence of ZnO NPs did not significantly affect the toxicity induced by PA or LPS. In addition, the accumulation of Zn ions after ZnO NP exposure was not significantly affected by the presence of PA or LPS. We concluded that there was no interaction between ZnO NPs and PA or LPS on toxicity to HUVECs in vitro. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract 087: Mitochondrial Fission Inducer, Dynamin-Related Protein 1 (DRP1), is Required for Endothelial Inflammatory Responses

Among various cardiovascular diseases, hypertension (HTN) is considered to be a disease plagued by chronic low-grade inflammation associated with endothelial dysfunction. Interestingly, recent studies have identified mitochondrial adaptation and/or dysfunction as components to hypertensive vascular dysfunction. While mitochondria are indispensable to maintain cellular metabolism, they also participate in adaptive and maladaptive cell/tissue responses via several retro grade signaling pathways. DRP1 plays a major role in mitochondrial quality control. However, whether DRP1 is involved in mitochondrial dysfunction and endothelial inflammation during development of HTN remains unknown. In the present study, we tested the hypothesis that inflammatory stimuli, through DRP1-dependent mitochondrial alteration, enhance endothelial inflammation. In cultured rat aortic endothelial cells (RAECs), TNFα (10 μg/mL) transiently induced mitochondrial fission maximally at 3h which was inhibited using a mitochondrial fission inhibitor, Mdivi1 (10 μM) (0.16±0.04 vs 0.10±0.02 mitochondria fragmentation count with MitoTracker, p<.01 ). TNFα and FCCP (a fission agonist, 10 μM) increased THP-1 monocyte adhesion to RAECs, which was also inhibited with Mdivi1 (256±17 vs 139±16 for TNFα, 238±30 vs 156±14 for FCCP, attached cells per field scanned, p<.01 ). Likewise, mdivi1 and adenoviruses encoding siRNA for DRP1 or dominant-negative K38A DRP1 (50 moi) attenuated TNFα-induced VCAM-1 induction in RAECs. TNFα increased aerobic respiration, which was prevented by mdivi1 or ER stress inhibitor PBA (10 mM). Inhibition of ER stress, glycolysis or mitochondrial respiration using PBA, 2-DG (1 mg/mL) or oligomycin (1 μM) prevented VCAM-1 induction. However, suppression of TNFα-induced mitochondrial ROS production by mito-Tempo (25 nM) was unable to prevent VCAM-1 induction. In C57BL6 mice receiving AngII (1000 ng/kg/min, 2 weeks) infusion, treatment with Mdivi-1 (25 mg/kg ip every other day) or PBA (1g/kg/day) prevented vascular VCAM-1 induction. In conclusion, our data suggests a critical role for ER stress and subsequent functional and structural remodeling of mitochondria induced by DRP1 in mediating endothelial inflammatory activation in HTN.

Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion.

ObjectiveIt is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab–which is approved for several inflammatory disorders–on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions.Methods and ResultsPhorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice.ConclusionOur results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.

Antiatherogenic effects of ellagic acid and urolithins in vitro

Atherosclerosis, one of the leading causes of death worldwide, is characterized by impaired endothelial function and lipid metabolism, among other factors. Ellagitannins are a class of phenolic compounds that may play a role in cardiovascular health. This work aimed to study the potential atheroprotective effects of urolithins, ellagitannin-derived gut microbiota metabolites, on different key factors in atherosclerosis development: the ability of monocytes to adhere to endothelial cells and the uptake and efflux of cholesterol by macrophages. The biotransformations urolithins undergo in peripheral cells were also evaluated. Results indicated that some urolithins and ellagic acid were able to reduce the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the secretion of a cellular adhesion molecule (sVCAM-1) and pro-inflammatory cytokine (IL-6). Urolithin C, a combination of urolithins A and B, and ellagic acid also decreased the accumulation of cholesterol in THP-1-derived macrophages, but they were not able to promote cholesterol efflux. The analysis of cell media by UHPLC-ESI-MSn indicated urolithins and ellagic underwent extensive metabolism, with sulfate and methyl conjugation. This evidence indicates that atherosclerotic processes may be attenuated by urolithins, but future human intervention trials are required to establish if is translated in vivo.

The Anti-Atherosclerotic Effect of Naringin Is Associated with Reduced Expressions of Cell Adhesion Molecules and Chemokines through NF-κB Pathway.

Naringin has been reported to have an anti-atherosclerosis effect but the underlying mechanism is not fully understood. The aim of this study is to investigate the impact of naringin on the TNF-α-induced expressions of cell adhesion molecules, chemokines and NF-κB signaling pathway in human umbilical vein endothelial cells (HUVECs). The experiments revealed that naringin, at concentrations without cytotoxicity, dose-dependently inhibited the adhesion of THP-1 monocytes to the TNF-α-stimulated HUVECs. The TNF-α-induced expressions of cell adhesion molecules, including VCAM-1, ICAM-1 and E-selectin, at both the mRNA and protein levels, were significantly suppressed by naringin in a dose dependent manner. In addition, the TNF-α-induced mRNA and protein levels of chemokines, including fractalkine/CX3CL1, MCP-1 and RANTES, were also reduced by naringin. Naringin significantly inhibited TNF-α-induced nuclear translocation of NF-κB, which resulted from the inhibited phosphorylation of IKKα/β, IκB-α and NF-κB. Altogether, we proposed that naringin modulated TNF-α-induced expressions of cell adhesion molecules and chemokines through the inhibition of TNF-α-induced activation of IKK/NF-κB signaling pathway to exert the anti-atherosclerotic effect.

Single- and double-walled carbon nanotubes enhance atherosclerogenesis by promoting monocyte adhesion to endothelial cells and endothelial progenitor cell dysfunction.

BackgroundThe use of carbon nanotubes has increased lately. However, the cardiovascular effect of exposure to carbon nanotubes remains elusive. The present study investigated the effects of pulmonary exposure to single-walled carbon nanotubes (SWCNTs) and double-walled carbon nanotubes (DWCNTs) on atherosclerogenesis using normal human aortic endothelial cells (HAECs) and apolipoprotein E-deficient (ApoE−/−) mice, a model of human atherosclerosis.MethodsHAECs were cultured and exposed to SWCNTs or DWCNTs for 16 h. ApoE−/− mice were exposed to SWCNTs or DWCNTs (10 or 40 μg/mouse) once every other week for 10 weeks by pharyngeal aspiration.ResultsExposure to CNTs increased the expression level of adhesion molecule (ICAM-1) and enhanced THP-1 monocyte adhesion to HAECs. ApoE−/− mice exposed to CNTs showed increased plaque area in the aorta by oil red O staining and up-regulation of ICAM-1 expression in the aorta, compared with vehicle-treated ApoE−/− mice. Endothelial progenitor cells (EPCs) are mobilized from the bone marrow into the circulation and subsequently migrate to the site of endothelial damage and repair. Exposure of ApoE−/− mice to high-dose SWCNTs or DWCNTs reduced the colony-forming units of EPCs in the bone marrow and diminished their migration function.ConclusionThe results suggested that SWCNTs and DWCNTs enhanced atherosclerogenesis by promoting monocyte adhesion to endothelial cells and inducing EPC dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0166-0) contains supplementary material, which is available to authorized users.

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

Abstract 17078: A Potent Vasoconstrictor Kisspeptin-10 Induces Atherosclerotic Plaque Progression and Instability

Background: Recently, there is a great deal of research interest regarding the role and effects of kisspeptin-10 (KP-10), a potent vasoconstrictor and inhibitor of angiogenesis expressed in vascular endothelial cells (ECs), in cardiovascular disease. However, it still remains unknown whether KP-10 could affect atherogenesis. Objective and Methods: We aimed to clarify the effects of KP-10 on atherosclerosis. We evaluated the effects of KP-10 on human umbilical vein endothelial cells (HUVECs), human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro , and aortic lesions in ApoE -/- mice in vivo . Results: GPR54, a KP-10 receptor, was expressed at higher levels in human monocytes, macrophages, HASMCs, and HUVECs. KP-10 significantly suppressed the proliferation of EAhy.926 ECs. KP-10 significantly increased the adhesion of THP1 monocytes to HUVECs associated with increased endothelial expressions of IL-6, MCP-1, TNF-α, ICAM-1, VCAM-1, and E-selectin. KP-10 significantly enhanced oxidized low-density lipoprotein-induced foam cell formation associated with increased expressions of CD36 and acyl-CoA:cholesterol acyltransferase-1 in human monocyte-derived macrophages. KP-10 significantly suppressed the migration and proliferation of HASMCs with inducing apoptosis via AKT, p38, and bax upregulation, but significantly enhanced the activities of MMP-2 and MMP-9 in HASMCs. Four-week-infusion of KP-10 into ApoE -/- mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration. Conclusions: Our results indicate that KP-10 accelerates atherogenesis by enhancing EC inflammatory responses and macrophage foam cell formation, In addition, KP-10 suppresses EC proliferation, VSMC migration and proliferation, but enhances MMP-2 and MMP-9 activities in VSMCs, which may contribute to plaque instability, leading to plaque rupture. Thus, KP-10 may serve as a novel therapeutic target for acute coronary syndrome.

Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar–endothelial co-cultures

Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar–endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect measured in the lower chamber on HUVECs and THP-1 cells. The exposure to MWCNTs, including a short (NM400) and long (NM402) type of entangled fibers, was associated with elevated levels of reactive oxygen species as well as a decrease in the intracellular glutathione concentration in HUVEC and A549 monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did not promote THP-1 adhesion to HUVECs in the lower chamber. When THP-1 macrophages were present on the cell culture inserts, there was a modest increase in the adhesion and an increase in interleukin-6 and interleukin-8 levels in the lower chamber whereas no tumor necrosis factor was detected. Overall, this study showed that direct exposure of HUVECs to MWCNTs was associated with oxidative stress and monocyte adhesion and the presence of PA increased the adhesion when exposed to NM400.

Abstract 223: Inhibition of STAT3 Phosphorylation by Sulforaphane Reduces Adhesion Molecule Expression in Vascular Endothelial Cell

This study explored the mechanism(s) by which sulforaphane inhibits intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressions stimulated with lipopolysaccharide (LPS) in human umbilical vein endothelial cell (HUVEC). The protein levels of ICAM-1 and VCAM-1 were investigated by primary antibodies of anti-human ICAM-1, anti-human VCAM-1. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) were investigated by phospho-STAT3 antibody. The STAT3-specific small interfering RNA (siRNA) was used to knockdown the expression of STAT3. THP-1 monocyte adhesion photograph images were obtained using a fluorescence microscope. Sulforaphane prevented LPS-mediated increase of ICAM-1 and VCAM-1 expression. Sulforaphane prevented LPS-mediated increase of STAT3 phosphorylation. AG490, Janus kinase (JAK)-2 inhibitor and Stattic, STAT3 inhibitor, reduced LPS-induced the expressions of ICAM-1 and VCAM-1, respectively. STAT3 small interfering RNA (siRNA) treatment reduced ICAM-1 and VCAM-1 expression stimulated with LPS. Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC. These data provide a novel mechanism where sulforaphane decreases an expression of ICAM-1 and VCAM-1 onto vascular wall through an inhibition of STAT3 phosphorylation.

An ethanol root extract of Cynanchum wilfordii containing acetophenones suppresses the expression of VCAM-1 and ICAM-1 in TNF-α-stimulated human aortic smooth muscle cells through the NF-κB pathway.

The root of Cynanchum wilfordii (C. wilfordii) contains several biologically active compounds which have been used as traditional medicines in Asia. In the present study, we evaluated the anti-inflammatory effects of an ethanol root extract of C. wilfordii (CWE) on tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs). The inhibitory effects of CWE on vascular cell adhesion molecule (VCAM)-1 expression under an optimum extraction condition were examined. CWE suppressed the expression of VCAM-1 and ICAM-1 and the adhesion of THP-1 monocytes to the TNF-α-stimulated HASMCs. Consistent with the in vitro observations, CWE inhibited the aortic expression of ICAM-1 and VCAM-1 in atherogenic diet-fed mice. CWE also downregulated the expression of nuclear factor-κB (NF-κB p65) and its uclear translocation in the stimulated HASMCs. In order to identify the active components in CWE, we re-extracted CWE using several solvents, and found that the ethyl acetate fraction was the most effective in suppressing the expression of VCAM-1 and ICAM-1. Four major acetophenones were purified from the ethyl acetate fraction, and two components, p-hydroxyacetophenone and cynandione A, potently inhibited the expression of ICAM-1 and VCAM-1 in the stimulated HASMCs. We assessed and determined the amounts of these two active components from CWE, and our results suggested that the root of C. wilfordii and its two bioactive acetophenones may be used for the prevention and treatment of atherosclerosis and vascular inflammatory diseases.

Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion.

Antiplatelet drugs, frequently used for cardiovascular events with thrombotic involvement, are also regarded as possible promising agents for cardiovascular primary prevention. The roles of P2Y12, an ADP receptor and the target of thienopyridine antiplatelet drugs, are not satisfactorily known in the vascular wall. We investigated the hypothesis that vascular smooth muscle cell (VSMC) P2Y12 is involved in vascular wall inflammatory changes by upregulating monocyte chemoattractant protein-1 (MCP-1) and promoting monocyte adhesion. ADP at 10(-5) M induced a 3.6 ± 0.3-fold upregulation of MCP-1 mRNA in cultured rat VSMCs, which was significantly inhibited by R-138727, the active metabolite of P2Y12 inhibitor prasugrel and siRNAs against P2Y12. ADP also induced MCP-1 protein upregulation, which was diminished by R-138727 and P2Y12 siRNAs. JNK (c-Jun NH2-terminal kinase) inhibition attenuated ADP-induced MCP-1 mRNA and protein upregulation. R-138727 and P2Y12 siRNAs inhibited ADP-induced JNK activation. The reactive oxygen species (ROS) inhibitors N-acetylcysteine (NAC), diphenyleneiodonium (DPI), and Tempol also diminished MCP-1 upregulation and JNK activation induced by ADP. ADP induced MCP-1 promoter activation, which was inhibited by R-138727 and P2Y12 siRNAs. Nuclear factor-κB (NF-κB) consensus sites in the MCP-1 promoter region were involved in this activation. ADP-induced NF-κB pathway activation, examined by a plasmid containing multiple NF-κB sites, was diminished by P2Y12 inhibition. For cellular function analysis, stimulation of VSMC with ADP increased subsequent THP-1 monocyte adhesion. P2Y12 siRNAs and CCR2 antagonism diminished this ADP-induced monocyte adhesion. These data suggested that ADP, via the VSMC P2Y12 receptor, induces vascular inflammatory changes by upregulating MCP-1 and promoting monocyte adhesion.

Role of Hic-5 in the formation of microvilli-like structures and the monocyte-endothelial interaction that accelerates atherosclerosis.

The adhesion of circulating monocytes to endothelial cells (ECs) is an early and critical event in the formation of atherosclerotic plaques. Hydrogen peroxide-inducible clone 5 (Hic-5) serves as an adaptor molecule in cell adhesion complexes. However, the role of endothelial Hic-5 in monocyte-EC interaction and atherogenesis remains unclear. We examined the roles of endothelial Hic-5 in monocyte-EC interaction and atherogenesis using mouse models of atherosclerosis and cultured human umbilical vein endothelial cells (HUVECs). Hic-5 was expressed in ECs, but not in monocytes/macrophages. An ex vivo monocyte adhesion assay revealed that adhesion of THP-1 monocytes to aortas isolated from Apoe(-/-) and LDLR(-/-) mice stimulated by TNF-α or oxidized LDL was suppressed by Hic-5 deficiency. Scanning electron microscopic observations of aortas harvested from Apoe(-/-) mice revealed that TNF-α- or oxidized LDL-induced microvilli-like structures were markedly suppressed by Hic-5 deficiency. Relative Hic-5 deficiency suppressed 60% of the atherosclerotic lesions in aortas from Apoe(-/-) and LDLR(-/-) mice. In contrast, overexpression of Hic-5 in HUVECs promoted induction of microvilli-like structures and adherence of THP-1 cells in an adhesion receptor such as intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-dependent manner. Hic-5 in ECs plays an important role in the formation of microvilli-like structures and in the interaction between ECs and monocytes, leading to monocyte recruitment and subsequent development of atherosclerosis.

Placental fractalkine mediates adhesion of THP-1 monocytes to villous trophoblast.

The chemokine fractalkine (CX3CL1) recently attracted increasing attention in the field of placenta research due to its dual nature, acting both as membrane-bound and soluble forms. While the membrane-bound form mediates flow-resistant adhesion of leukocytes to endothelial and epithelial cells via its corresponding receptor CX3CR1, the soluble form arises from metalloprotease-dependent shedding and bears chemoattractive activity for monocytes, natural killer cells and T cells. In human placenta, fractalkine is expressed at the apical microvillous plasma membrane of the syncytiotrophoblast, which may enable close physical contact with circulating maternal leukocytes. Based on these observations, we tested the hypothesis that fractalkine mediates adhesion of monocytes to the villous trophoblast. Forskolin-induced differentiation and syncytialization of the trophoblast cell line BeWo was accompanied with a substantial upregulation in fractalkine expression and led to increased adhesion of the monocyte cell line THP-1, which preferentially bound to syncytia. Blocking as well as silencing of the fractalkine receptor CX3CR1 proved involvement of the fractalkine/CX3CR1 system in adherence of THP-1 monocytes to villous trophoblast. Pre-incubation of THP-1 monocytes with human recombinant fractalkine as well as silencing of CX3CR1 expression in THP-1 monocytes significantly impaired their adherence to BeWo cells and primary term trophoblasts. The present study suggests fractalkine as another candidate among the panel of adhesion molecules enabling stable interaction between leukocytes and the syncytiotrophoblast.

Cycling Hypoxia Induces a Specific Amplified Inflammatory Phenotype in Endothelial Cells and Enhances Tumor-Promoting Inflammation In Vivo

Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, leads to temporal fluctuations in tissue oxygen tension exposing tumor and stromal cells to cycling hypoxia. Inflammation is another feature of tumor microenvironment and is considered as a new enabling characteristic of tumor progression. As cycling hypoxia is known to participate in tumor aggressiveness, the purpose of this study was to evaluate its role in tumor-promoting inflammation. Firstly, we assessed the impact of cycling hypoxia in vitro on endothelial inflammatory response induced by tumor necrosis factor α. Results showed that endothelial cells exposed to cycling hypoxia displayed an amplified proinflammatory phenotype, characterized by an increased expression of inflammatory cytokines, namely, interleukin (IL)-6 and IL-8; by an increased expression of adhesion molecules, in particular intercellular adhesion molecule–1 (ICAM-1); and consequently by an increase in THP-1 monocyte adhesion. This exacerbation of endothelial inflammatory phenotype occurs through nuclear factor–κB overactivation. Secondly, the role of cycling hypoxia was studied on overall tumor inflammation in vivo in tumor-bearing mice. Results showed that cycling hypoxia led to an enhanced inflammation in tumors as prostaglandin-endoperoxide synthase 2 (PTGS2), IL-6, CXCL1 (C-X-C motif ligand 1), and macrophage inflammatory protein 2 (murine IL-8 functional homologs) mRNA expression was increased and as a higher leukocyte infiltration was evidenced. Furthermore, cycling hypoxia–specific inflammatory phenotype, characterized by a simultaneous (baculoviral inhibitor of apoptosis repeat-containing 5)low/PTGS2high/ICAM-1high/IL-6high/IL-8high expression, is associated with a poor prognosis in human colon cancer. This new phenotype could thus be used in clinic to more precisely define prognosis for colon cancer patients. In conclusion, our findings evidenced for the first time the involvement of cycling hypoxia in tumor-promoting inflammation amplification.

Glucagon-like peptide-1 (GLP-1) analog liraglutide inhibits endothelial cell inflammation through a calcium and AMPK dependent mechanism.

Liraglutide is a glucagon-like peptide-1 (GLP-1) mimetic used for the treatment of Type 2 diabetes. Similar to the actions of endogenous GLP-1, liraglutide potentiates the post-prandial release of insulin, inhibits glucagon release and increases satiety. Recent epidemiological studies and clinical trials have suggested that treatment with GLP-1 mimetics may also diminish the risk of cardiovascular disease in diabetic patients. The mechanism responsible for this effect has yet to be determined; however, one possibility is that they might do so by a direct effect on vascular endothelium. Since low grade inflammation of the endothelium is an early event in the pathogenesis of atherosclerotic cardiovascular disease (ASCVD), we determined the effects of liraglutide on inflammation in cultured human aortic endothelial cells (HAECs). Liraglutide reduced the inflammatory responses to TNFα and LPS stimulation, as evidenced by both reduced protein expression of the adhesion molecules VCAM-1 and E-Selectin, and THP-1 monocyte adhesion. This was found to result from increased cell Ca2+ and several molecules sensitive to Ca2+ with known anti inflammatory actions in endothelial cells, including CaMKKβ, CaMKI, AMPK, eNOS and CREB. Treatment of the cells with STO-609, a CaMKK inhibitor, diminished both the activation of AMPK, CaMKI and the inhibition of TNFα and LPS-induced monocyte adhesion by liraglutide. Likewise, expression of an shRNA against AMPK nullified the anti-inflammatory effects of liraglutide. The results indicate that liraglutide exerts a strong anti-inflammatory effect on HAECs. They also demonstrate that this is due to its ability to increase intracellular Ca2+ and activate CAMKKβ, which in turn activates AMPK.

Chinese Yam Extracts Containing β‐Sitosterol and Ethyl Linoleate Protect against Atherosclerosis in Apolipoprotein E‐Deficient Mice and Inhibit Muscular Expression of VCAM‐1 In Vitro

Atherosclerosis is a chronic inflammatory disease, which is associated with increased expression of adhesion molecules and monocyte recruitment into the arterial wall. This study evaluated whether hexane extracts from the edible part (DB-H1) or bark region (DB-H2) of Dioscorea. batatas Decne have anti-atherosclerotic properties in vivo and in vitro experiments. We also identified bioactive components in the hexane extracts. Thirty-six apolipoprotein E (ApoE(-/-) ) mice and 12 control (C57BL/6J) mice were given a Western-type diet for 11 or 21 wk. To examine the effects of yam extracts on lesion development, ApoE(-/-) mice were orally administered DB-H1 or DB-H2 for the duration of the study (200 mg/kg b.w./day, 3 times per wk). Both DB-H1 and DB-H2 significantly reduced the total atherosclerotic lesion area in the aortic root. In addition, plasma concentrations of total cholesterol, oxidized-low-density lipoprotein, and c-reactive protein were decreased by administration of DB-H1 and DB-H2. Consistent with the in vivo observations, DB-H1 and DB-H2 inhibited tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule-1 expression and adhesion of THP-1 monocytes to TNF-α-activated vascular smooth muscle cells. It was also found that treatment with DB-H1 or DB-H2 resulted in the inhibition nitric oxide (NO) and reactive oxygen species production and iNOS expression in macrophages. Thus, DB-H1 and DB-H2 seem to influence atherosclerosis by affecting the production of inflammatory mediators in vivo. Our results suggest that yam extracts have the potential to be used in the prevention of atherosclerosis.

Effect of PIP3 on Adhesion Molecules and Adhesion of THP-1 Monocytes to HUVEC Treated with High Glucose

Background: Phosphatidylinositol-3,4,5-triphosphate (PIP3), a well-known lipid second messenger, plays a key role in insulin signaling and glucose homeostasis. Using human umbilical vein endothelial cells (HUVEC) and THP-1 monocytes, we tested the hypothesis that PIP3 can downregulate adhesion molecules and monocyte adhesion to endothelial cells. Methods: HUVEC and monocytes were exposed to high glucose (HG, 25 mM, 20 h) with or without PIP3 (0-20 nM), or PIT-1 (25 µM), an inhibitor of PIP3. Results: Both HG and PIT-1 caused a decrease in cellular PIP3 in monocytes and HUVEC compared to controls. Treatment with PIT-1 and HG also increased the ICAM-1 (intercellular adhesion molecule 1) total protein expression as well as its surface expression in HUVEC, CD11a (a subunit of lymphocyte function-associated antigen 1, LFA-1) total protein expression as well as its surface expression in monocytes, and adhesion of monocytes to HUVEC. Exogenous PIP3 supplementation restored the intracellular PIP3 concentrations, downregulated the expression of adhesion molecules, and reduced the adhesion of monocytes to HUVEC treated with HG. Conclusion: This study reports that a decrease in cellular PIP3 is associated with increased expression of adhesion molecules and monocyte-endothelial cell adhesion, and may play a role in the endothelial dysfunction associated with diabetes.

Phloretin suppresses thrombin‐mediated leukocyte‐platelet‐endothelial interactions

Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. Human endothelial cells were pre-treated with 1-20 μM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin β3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis.