Cytokine-stimulated Endothelial Cells Research Articles

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Aspirin modulates production of pro-inflammatory and pro-resolving mediators in endothelial cells.

Endothelial cells synthesize biochemical signals to coordinate a response to insults, resolve inflammation and restore barrier integrity. Vascular cells release a variety of vasoactive bioactive lipid metabolites during the inflammatory response and produce pro-resolving mediators (e.g., Lipoxin A4, LXA4) in cooperation with leukocytes and platelets to bring a halt to inflammation. Aspirin, used in a variety of cardiovascular and pro-thrombotic disorders (e.g., atherosclerosis, angina, preeclampsia), potently inhibits proinflammatory eicosanoid formation. Moreover, aspirin stimulates the synthesis of pro-resolving lipid mediators (SPM), so-called Aspirin-Triggered Lipoxins (ATL). We demonstrate that cytokines stimulated a time- and dose-dependent increase in PGI2 (6-ketoPGF1α) and PGE2 formation that is blocked by aspirin. Eicosanoid production was caused by cytokine-induced expression of cyclooxygenase-2 (COX-2). We also detected increased production of pro-resolving LXA4 in cytokine-stimulated endothelial cells. The R-enantiomer of LXA4, 15-epi-LXA4, was enhanced by aspirin, but only in the presence of cytokine challenge, indicating dependence on COX-2 expression. In contrast to previous reports, we detected arachidonate 5-lipoxygenase (ALOX5) mRNA expression and its cognate protein (5-lipoxygenase, 5-LOX), suggesting that endothelial cells possess the enzymatic machinery necessary to synthesize both pro-inflammatory and pro-resolving lipid mediators independent of added leukocytes or platelets. Finally, we observed that, endothelial cells produced LTB4 in the absence of leukocytes. These results indicate that endothelial cells produce both pro-inflammatory and pro-resolving lipid mediators in the absence of other cell types and aspirin exerts pleiotropic actions influencing both COX and LOX pathways.

NDRG1 Signaling Is Essential for Endothelial Inflammation and Vascular Remodeling.

NDRG-1 (N-myc downstream-regulated gene 1) is a member of NDRG family that plays essential roles in cell differentiation, proliferation, and stress responses. Although the expression of NDRG1 is regulated by fluid shear stress, its roles in vascular biology remain poorly understood. The purpose of the study is to determine the functional significance of NDRG1 in vascular inflammation and remodeling. By using quantitative polymerase chain reaction, western blot, and immunohistochemistry, we demonstrate that the expression of NDRG1 is markedly increased in cytokine-stimulated endothelial cells and in human and mouse atherosclerotic lesions. To determine the role of NDRG1 in endothelial activation, we performed loss-of-function studies using NDRG1 short hairpin RNA. Our results demonstrate that NDRG1 knockdown by lentivirus bearing NDRG1 short hairpin RNA substantially attenuates both IL-1β (interleukin-1β) and TNF-α (tumor necrosis factor-α)-induced expression of cytokines/chemokines and adhesion molecules. Intriguingly, inhibition of NDRG1 also significantly attenuates the expression of procoagulant molecules, such as PAI-1 (plasminogen activator inhibitor type 1) and TF (tissue factor), and increases the expression of TM (thrombomodulin) and t-PA (tissue-type plasminogen activator), thus exerting potent antithrombotic effects in endothelial cells. Mechanistically, we showed that NDRG1 interacts with orphan Nur77 (nuclear receptor) and functionally inhibits the transcriptional activity of Nur77 and NF-κB (nuclear factor Kappa B) in endothelial cells. Moreover, in NDRG1 knockdown cells, both cytokine-induced mitogen-activated protein kinase activation, c-Jun phosphorylation, and AP-1 (activator protein 1) transcriptional activity are substantially inhibited. Neointima and atherosclerosis formation induced by carotid artery ligation and arterial thrombosis were markedly attenuated in endothelial cell-specific NDRG1 knockout mice compared with their wild-type littermates. Our results for the first time identify NDRG1 as a critical mediator implicated in regulating endothelial inflammation, thrombotic responses, and vascular remodeling, and suggest that inhibition of NDRG1 may represent a novel therapeutic strategy for inflammatory vascular diseases, such as atherothrombosis and restenosis.

17R/S-Benzo-RvD1, a synthetic resolvin D1 analogue, attenuates neointimal hyperplasia in a rat model of acute vascular injury.

Persistent inflammation following vascular injury drives neointimal hyperplasia (NIH). Specialized lipid mediators (SPM) mediate resolution which attenuates inflammation and downstream NIH. We investigated the effects of a synthetic analogue of resolvin D1 (RvD1) on vascular cells and in a model of rat carotid angioplasty. Human venous VSMC and endothelial cells (EC) were employed in migration, cell shape, toxicity, proliferation and p65 nuclear translocation assays. Murine RAW 264.7 cells were utilized to test the effect of pro-resolving compounds on phagocytic activity. A model of rat carotid angioplasty was used to evaluate the effects of 17R/S-benzo-RvD1 (benzo-RvD1) and 17R-RvD1 applied to the adventitia via 25% Pluronic gel. Immunostaining was utilized to examine Ki67 expression and leukocyte recruitment. Morphometric analysis was performed on arteries harvested 14 days after injury. Exposure to benzo-RvD1 attenuated PDGF- stimulated VSMC migration across a range of concentrations (0.1-100 nM), similar to that observed with 17R-RvD1. Pre-treatment with either Benzo-RvD1 or 17R-RvD1 (10, 100nM) attenuated PDGF-BB-induced VSMC cytoskeletal changes to nearly baseline dimensions. Benzo-RvD1 demonstrated modest anti-proliferative activity on VSMC and EC at various concentrations, without significant cytotoxicity. Benzo-RvD1 (10nM) inhibited p65 nuclear translocation in cytokine-stimulated EC by 21% (p<0.05), similar to 17R-RvD1. Consistent with pro-resolving activities of other SPM, both 17R-RvD1 and benzo-RvD1 increased the phagocytic activity of RAW 264.7 cells against S. Aureus and Zymosan particles. There were no significant differences in Ki-67 or CD45 staining observed on day 3 after angioplasty. Periadventitial treatment with benzo-RvD1 reduced carotid neointimal area at 14 days compared to control (0.08 mm2 v. 0.18 mm2; p<0.05), with similar efficacy to 17R-RvD1. 17R/S-benzo-RvD1 and 17R-RvD1 exhibit similar pro-resolving and anti-migratory activity in cell-based assays, and both compounds attenuated NIH following acute arterial injury in rats. Further studies of the mechanisms of resolution following vascular injury, and the translational potential of SPM analogues, are indicated.

Characterization of putative regulatory isoforms of porcine tumor necrosis factor receptor 2 in endothelial cells.

Tumor necrosis factor α (TNFα) and its receptors contribute to rejection of transplanted cells and organs. To elucidate how TNFα affects xenograft rejection, we previously cloned the cDNA of pig TNF-receptor 2 (pTNFR2) and found four isoforms: one comprising the full receptor with four cysteine-rich domains (CRD), a shorter variant (pTNFR2ΔE7-10) encoding for a soluble isoform, another lacking exon 4 (pTNFR2ΔE4) displaying only 3 CRD and poor ligand binding, and the smallest one generated by the two alternative splicings. All isoforms contained the pre-ligand assembly domain (PLAD) responsible for receptor trimerization. We now investigated their roles by structural, expression, and subcellular localization studies. Structural in silico analyses identified four amino acids potentially involved in TNFα binding and lacking in pTNFR2ΔE4. Quantitative RT-PCR determined regulated expression affecting the two pTNFR2 alternative splicings in cytokine-stimulated porcine aortic endothelial cells (PAEC). Particularly, human IL-1α and TNFα produced a strong mRNA upregulation of all isoforms, being the full receptor the predominant one. However, expression of pTNFR2 on PAEC did not correlate with mRNA and decreased after 24-hour exposure to IL-1α or TNFα. Notably, confocal microscopy confirmed the presence of pTNFR2 inside and on the plasma membrane, whereas pTNFR2ΔE4 located only intracellularly. Most interestingly, FRET analyses showed that membrane-bound isoforms pTNFR2 and pTNFR2ΔE4 colocalized intracellularly and associated through the PLAD. Our data show that pTNFR2ΔE4 bind and may retain the full receptor intracellularly. This mechanism has not been described in other species and represents a particularity that may affect the pathophysiology of pig xenografts.

Molecular Imaging of a New Multimodal Microbubble for Adhesion Molecule Targeting

IntroductionInflammation is an important risk-associated component of many diseases and can be diagnosed by molecular imaging of specific molecules. The aim of this study was to evaluate the possibility of targeting adhesion molecules on inflammation-activated endothelial cells and macrophages using an innovative multimodal polyvinyl alcohol-based microbubble (MB) contrast agent developed for diagnostic use in ultrasound, magnetic resonance, and nuclear imaging.MethodsWe assessed the binding efficiency of antibody-conjugated multimodal contrast to inflamed murine or human endothelial cells (ECs), and to peritoneal macrophages isolated from rats with peritonitis, utilizing the fluorescence characteristics of the MBs. Single-photon emission tomography (SPECT) was used to illustrate 99mTc-labeled MB targeting and distribution in an experimental in vivo model of inflammation.ResultsFlow cytometry and confocal microscopy showed that binding of antibody-targeted MBs to the adhesion molecules ICAM-1, VCAM-1, or E-selectin, expressed on cytokine-stimulated ECs, was up to sixfold higher for human and 12-fold higher for mouse ECs, compared with that of non-targeted MBs. Under flow conditions, both VCAM-1- and E-selectin-targeted MBs adhered more firmly to stimulated human ECs than to untreated cells, while VCAM-1-targeted MBs adhered best to stimulated murine ECs. SPECT imaging showed an approximate doubling of signal intensity from the abdomen of rats with peritonitis, compared with healthy controls, after injection of anti-ICAM-1-MBs.ConclusionsThis novel multilayer contrast agent can specifically target adhesion molecules expressed as a result of inflammatory stimuli in vitro, and has potential for use in disease-specific multimodal diagnostics in vivo using antibodies against targets of interest.

Bradykinin B2 receptor and dopamine D2 receptor cooperatively contribute to the regulation of neutrophil adhesion to endothelial cells.

Leukocyte adhesion to the vascular endothelium contributes to many immunological and inflammatory disorders. These processes have been shown to be mediated by bradykinin receptor type 2 (B2R) and dopamine receptor type 2 (D2R). In a previous study, we reported the formation of a B2R-D2R heterodimer, possibly altering cellular functions. Hence, in the present study, we examined the effect of co-activation of endothelial cells with B2R and D2R agonists on the interaction of these cells with neutrophils. Bradykinin, the main B2R agonist, significantly increased cell adhesion, and this effect was reversed when the endothelial cells were additionally co-treated with a selective D2R agonist, sumanirole. These results were dependent on the incubation time, showing an opposite tendency after prolonged stimulation. Significant changes in the expression of adhesion proteins, such as E-selectin and intercellular adhesion molecule 1 in endothelial cells were observed. Additionally, the cells preincubated with tumor necrosis factor-α showed decreased cell adhesion and IL-8 release after long incubation with both agonists. The modulation of cell adhesion by D2R and B2R seem to be mediated via STAT3 phosphorylation. In summary, this study demonstrated a protective role of D2R in neutrophil-endothelial cell adhesion induced by bradykinin, especially in cytokine-stimulated endothelial cells.

3D matrix-embedding inhibits cycloheximide-mediated sensitization to TNF-alpha-induced apoptosis of human endothelial cells.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Others and we previously demonstrated endothelial biology being intertwined with biochemical and structural composition of the subendothelial basement membrane. We now demonstrate that a three-dimensional growing environment significantly shields endothelial cells from cytokine-induced apoptosis. Detailed analysis reveals differences in intracellular signaling pathways in naive endothelial cells and cytokine-stimulated endothelial cells when cells are grown within a three-dimensional collagen-based matrix compared to cells grown on two-dimensional tissue culture plates. Main findings are significantly reduced p53 expression and level of p38-phosphorylation in three-dimensional grown endothelial cells. Despite similar concentrations of focal adhesion kinase, three-dimensional matrix-embedded endothelial cells express significantly less tyrosine-phosphorylated focal adhesion kinase. Pretreatment with antibodies against integrin αv β3 partially reversed the protective effect of three-dimensional matrix-embedding on endothelial apoptosis. Our findings provide detailed insights into the mechanisms of endothelial apoptosis with respect to the spatial matrix environment. These results enhance our understanding of endothelial biology and may otherwise help in the design of tissue-engineered materials. Furthermore, findings on focal adhesion kinase phosphorylation might enhance our understanding of clinical studies with tyrosine kinase inhibitors.

Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells to Assess Lymphocyte-Endothelial Interactions.

A major impediment when studying primary human endothelial cell function is the resistance of these cells to gene transfer. Here we describe methods for transferring genes into primary endothelial cells prior to incorporation into a static adhesion assay to analyze the adhesion and migration of isolated lymphocytes. Human embryonic kidney (HEK)-293T (HEK-293 cells expressing the large T-antigen of simian virus 40) cells are initially transfected with plasmids containing the lentiviral packaging and envelope genes and the target sequence, such as a gene of interest or short hairpin loop RNA (shRNA). These cells produce lentivirus packaged with this target sequence and are used to transduce primary human umbilical vein endothelial cells (HUVECs). Human peripheral blood lymphocytes (PBLs) isolated from venous blood are co-incubated with lentivirally transduced cytokine-stimulated endothelial cells to assess lymphocyte adhesion in a static adhesion assay. Direct observations of lymphocyte adhesion and migration over a time course can also be made. In general, lentiviral transduction of primary endothelial cells provides an invaluable system to manipulate gene expression levels when studying the cellular adhesion dynamics that regulate leukocyte adhesion and extravasation.

Estradiol Inhibits Cytokine-Induced Expression of VCAM-1 and ICAM-1 in Cultured Human Endothelial Cells Via AMPK/PPARα Activation.

Expression of cell adhesion molecules by vascular endothelial cells (ECs) plays an important role in migration and adhesion of leukocytes into the surrounding tissues, which is a vital step in inflammation and atherogenesis. Estradiol (E2), a human sex hormone and steroid, has differential anti-inflammatory properties, increases arterial vasodilation, inhibits the cellular response to vascular injury, and prevents atherosclerosis. However, the related mechanisms are poorly understood. Here, we studied the effect of E2 on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human aortic endothelial cells (HAECs). Pre-treatment of HAEC with E2 inhibited tumor necrosis factor-α (TNFα)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time- and dose-dependent manner. E2 at 10nmol/L inhibited interleukin-1 (IL-1)-stimulated expression of VCAM-1 and ICAM-1. The effect of E2 on cytokine-stimulated VCAM-1 expression was more prominent than that of ICAM-1. E2 reduced TNF-induced mRNA expression of VCAM-1 and ICAM-1. Repressed expression of VCAM-1 and ICAM-1 was associated with decreased adherence of lymphocytes and monocytes to cytokine-stimulated HAEC. E2 suppressed TNF-induced activation of nuclear factor-κB (NF-κB) in HAEC. Importantly, E2 improved AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα) expression in HAEC. Pharmaceutical inhibition of AMPK and PPARα blocks the effect of E2 on NF-κB, VCAM-1, and ICAM-1. These results indicate that E2 may have anti-inflammatory properties in ECs. The anti-inflammatory and anti-atherogenic properties of E2 may partly be ascribed to an effect on activation of AMPK and PPARα and subsequent inhibition of TNF-induced NF-κB activation, leading to the decreased expression of VCAM-1 and ICAM-1. Our findings support further investigations on the therapeutic benefits of E2 in several pathological events involving endothelial dysfunction and atherosclerosis.

Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization.

Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endothelial progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/cytokine gradients. Some growth factors are less stable under acidic conditions of tissue ischemia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke.

A Low-Molecular-Weight Compound K7174 Represses Hepcidin: Possible Therapeutic Strategy Against Anemia Of Chronic Disease

BackgroundHepcidin (HAMP) is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of HAMP increased in patients with anemia of chronic disease. Previously, a synthesized compound K7174 (Kowa Company Ltd., Tokyo, Japan) was identified through chemical screening as a novel inhibitor for the adhision of monocytes to cytokine-stimulated endothelial cells (Umetani et al. BBRC 2000). Interstingly, K7174 restored anemia induced by inflammatory cytokines in mice (Imagawa et al. FASEB J 2003), implying that K7174 might modulate hepcidin level. In the present study, we assessed the impact of K7174 on hepcidin expression based on human hematoma cell line and in vivo mice. MethodThe HepG2 hematoma cells as well as K562 erythroid cells were used for the analyses. The cells were treated with K7174 at doses of 10 and 20 uM for 24 h. For transcription profiling, Human Oligo chip 25K (Toray) were used for K7174-treated HepG2 cells. Western blotting and quantitative chromatin immunoprecipitation (ChIP) analyses were performed with antibodies to GDF15 (abcam), C/EBPbeta (CEBPB) (C-19) (Santa Cruz), Smad1 and Phospho-Smad1-5-8 (CST). For GDF15 knockdown in HepG2 cells, anti-GDF15 siRNA (Thermo Scientific Dharmacon) and Lipofectamine RNAiMAX (Invitrogen) were used. GDF15 promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Human GDF15 concentration in the K7174-treated media was evaluated with ELISA (R&D systems). For in vivo analysis, ICR mice were injected intraperitoneally with PBS (control) or 30 mg/kg K-7174, respectively, days 0, 1, 2, 3, 5, 6, 7 and 8, and the samples were taken on day 9. Serum hepcidin1 concentration was determined with LC-MS/MS method (MCProt Biotechnology, Kanazawa, Japan). ResultsWe first demonstrated that K7174 treatment (20 uM) in HepG2 cells significantly decreased HAMP expression (> 2-fold). Thus, we next conducted microarray analysis to reveal the molecular mechanism by which K7174 inhibits the HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Interestingly, K7174 strongly induced GDF15 (10-fold), a negative regulator of HAMP expression (Tanno et al. Nat Med 2007). Quantitative RT-PCR, Western blotting as well as ELISA analyses confirmed the induction of GDF15 by K7174 treatment. Furthermore, siRNA-mediated GDF15 knockdown during K7174 treatment significantly re-activated HAMP expression, suggesting that the increase of GDF15 induced by K7174 was responsible for the HAMP downregulation. Noticeably, we also found that K7174 upregulates CEBPB (2.9-fold). Promoter assay and quantitative ChIP analysis demonstrated that K7174-mediated upregulation of CEBPB contributes to the transcriptional activation of GDF15. On the other hand, the microarray analysis and quantitative RT-PCR-based validation identified the significant K7174-mediated downregulation of BMP4 (2.6-fold), a positive regulator of HAMP expression (Babitt et al. Nat Genet 2006). However, the level of SMAD1-5-8 phosphorylation in K7174-treated cells remained unchanged, implying that BMP-SMAD signaling might not be essential in K7174-mediated HAMP suppression.Next, we assessed if K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver sample from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp (n = 8, p< 0.05). Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice (Average: 138.1 and 110.4 ng/mL for K7174-treated and control mice, respectively. n = 8, p< 0.05). Beyond the regulation of Gdf15 in hepatocytes, erythroid cells have also been suggested to be one of the main sources of GDF15. Thus, we treated K7174 with K562 erythroid cells, and confirmed significant increase of GDF15, suggesting that systemic administration of K7174 may act on hepatocytes as well as erythroid cells to stimulate GDF15 production. ConclusionOur in vitro and in vivo analyses suggested that K7174 suppresses HAMP expression through modulating GDF15 expression. K7174 may be considered as a potential therapeutic option to treat anemia of chronic disease. Disclosures:No relevant conflicts of interest to declare.

A low-molecular-weight compound K7174 represses hepcidin: possible therapeutic strategy against anemia of chronic disease.

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. The expression of the hepcidin gene, HAMP, is increased in patients with anemia of chronic disease. Previously, the synthetic compound K7174 was identified through chemical screening as a novel inhibitor of the adhesion of monocytes to cytokine-stimulated endothelial cells. K7174 also ameliorated anemia induced by inflammatory cytokines in mice, which suggests a possible involvement of hepcidin regulation. The present study was performed to assess the impact of K7174 on hepcidin expression in a human hematoma cell line and in mice in vivo. We first demonstrated that K7174 treatment in HepG2 cells significantly decreased HAMP expression. Then, we conducted microarray analysis to determine the molecular mechanism by which K7174 inhibits HAMP expression. Transcriptional profiling confirmed the downregulation of HAMP. Surprisingly, we found that K7174 strongly induced GDF15, known as a negative regulator of HAMP expression. Western blotting analysis as well as ELISA confirmed the induction of GDF15 by K7174 treatment. Furthermore, K7174-mediated HAMP suppression was rescued by the silencing of GDF15 expression. Interestingly, we found that K7174 also upregulates CEBPB. Promoter analysis and chromatin immunoprecipitation analysis revealed that CEBPB could contribute to K7174-mediated transcriptional activation of GDF15. Subsequently, we also examined whether K7174 inhibits hepcidin expression in mice. Quantitative RT-PCR analysis with liver samples from K7174-treated mice demonstrated significant upregulation of Gdf15 and downregulation of Hamp expression, as compared to control mice. Furthermore, serum hepcidin concentration was also significantly decreased in K7174-treated mice. In conclusion, K7174 inhibits hepcidin expression partly by inducing GDF15. K-7174 may be a potential therapeutic option to treat anemia of chronic disease.

ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins

ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.

Cytokine-Stimulated Human Dermal Microvascular Endothelial Cells and Fibroblasts Produce Thymus and Activation-Regulated Chemokine (TARC)

Cytokine-Stimulated Human Dermal Microvascular Endothelial Cells and Fibroblasts Produce Thymus and Activation-Regulated Chemokine (TARC)

57 Mesenchymal stem cells inhibit recruitment of flowing neutrophils and lymphocytes by endothelial cells: roles of interleukin-6 and transforming growth factor-β

Mesenchymal stem cells (MSC) have been suggested to have protective effects through modulation of immune responses. We examined the hypothesis that MSC can modify the ability of endothelial cells (EC)...

Endothelium-derived GM-CSF influences expression of oncostatin M

During and after transendothelial migration, neutrophils undergo a number of phenotypic changes resulting from encounters with endothelium-derived factors. This report uses an in vitro model with human umbilical vein endothelial cells and isolated human neutrophils to examine the effects of two locally derived cytokines, granulocyte (G)-macrophage (M) colony-stimulating factor (GM-CSF) and G-CSF, on oncostatin M (OSM) expression. Neutrophils contacting activated HUVEC expressed and released increased amounts of oncostatin M (OSM), a proinflammatory cytokine known to induce polymorphonuclear neutrophil adhesion and chemotaxis. Neutrophil transendothelial migration resulted in threefold higher OSM expression and protein levels compared with nontransmigrated cells. Addition of anti-GM-CSF neutralizing antibody reduced OSM expression level but anti-G-CSF was without effect. GM-CSF but not G-CSF protein addition to cultures of isolated neutrophils resulted in a significant increase in OSM protein secretion. However, inhibition of β(2) integrins by neutralizing antibody significantly reduced GM-CSF-induced OSM production indicating this phenomenon is adhesion dependent. Thus cytokine-stimulated endothelial cells can produce sufficient quantities of GM-CSF to influence in an adhesion-dependent manner, the phenotypic characteristics of neutrophils resulting in the latter's transmigration. Both transmigration and adhesion phenomenon lead to increased production of OSM by neutrophils that then play a major role in inflammatory response.

Corrigendum

CorrigendumCorrigendumPublished Online:01 Jul 2011https://doi.org/10.1152/ajplung.zh5-5834-corr.2011Original articleMoreSectionsPDF (54 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 300, April 2011Volume 44, April 2011An article in the April issue was published with an incorrect spelling for the first author's name. The correct citation should be as follows:Chicoine LG, Stenger MR, Cui H, Calvert A, Evans RJ, English BK, Liu Y, Nelin LD. Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 300: , 2011.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesNitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells 01 Apr 2011American Journal of Physiology-Lung Cellular and Molecular Physiology More from this issue > Volume 301Issue 1July 2011Pages L135-L135 Copyright & PermissionsCopyright © 2011 the American Physiological Societyhttps://doi.org/10.1152/ajplung.zh5-5834-corr.2011History Published online 1 July 2011 Published in print 1 July 2011 Metrics

Docosahexaenoic Acid Inhibits the Adhesion of Flowing Neutrophils to Cytokine Stimulated Human Umbilical Vein Endothelial Cells

The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFa, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

Cimetidine inhibits the adhesion of gastric cancer cells expressing high levels of sialyl Lewis x in human vascular endothelial cells by blocking E-selectin expression

Cimetidine has been shown to have anti-metastatic activity and improves the survival of patients with colorectal cancer. One hypothesis is its modulation of the expression of the cell adhesion molecule by target organ endothelial cells. Because of the inconclusive results in clinical trials of gastric cancer, we investigated the effects of cimetidine on the adhesion of gastric cancer cells to activated endothelial cells and on the expression of some cell adhesion molecules. Human endothelial cells were pre-incubated with cimetidine for 6 h, incubated with the cytokine tumor necrosis factor for 4h, and the endothelial surface expression of E-selectin was evaluated by flow cytometry, immunostaining and ELISA. Further, we investigated E-selectin mRNA expression by RT-PCR. Three gastric cancer cell lines (SGC-7901, MGC-803, BGC-823) and a normal gastric epithelial cell line, GES-1, were studied for the surface expression of sialyl Lewis x by flow cytometry and immunostaining. Adherence of CFSE-labeled gastric cancer cells and GES-1 cells to endothelial cell monolayers was determined. Cimetidine significantly reduced E-selectin expression of activated endothelial cells, but did not influence E-selectin expression at the mRNA level. Three gastric cancer cell lines expressed high levels of sialyl Lewis x, whereas GES-1 did not. Cimetidine also significantly decreased gastric cancer cell adherence to stimulated endothelial cells. The inhibition of E-selectin expression corresponded to the reduction of tumor cell adherence. The effects of cimetidine on tumor adhesion were almost nullified by pre-incubation with E-selectin and sialyl Lewis x antibody. Furthermore, there was no significant change of GES-1 adherence to endothelial cells by TNF-α, cimetidine, E-selectin and sialyl Lewis x antibody. The inhibiton of gastric cancer cell adherence to cytokine-stimulated endothelial cells treated with cimetidine appears to result from blocking endothelial E-selectin expression. These data support the hypothesis that cimetidine may exert its anti-metastatic effects in gastric cancer, in part, by inhibiting E-selectin/sialyl Lewis x-mediated adherence of gastric cancer cells to endothelial cells in the metastasis target organs.