IL-8 In Endothelial Cells Research Articles

Abstract 2786: Seizure related 6 homolog (SEZ6), a cancer stem cell-specific secreted protein, is a novel GBM therapeutic target

Abstract Cancer Stem Cells (CSCs) are known to initiate a tumor, resist chemotherapy and radiotherapy, and also cause tumor recurrence. A deeper understanding of CSC biology is needed for a more effective therapeutic response. An integrative study of regulated transcriptome of human patient-derived Glioma Stem-like Cells (GSCs) using Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) identified a potential role for GSC in tumor angiogenesis. We chose Seizure Related 6 Homolog (SEZ6), one of the Wnt target genes, for further study, as its downregulation inhibited the potential of GSCs to induce angiogenesis without impacting GSC growth. SEZ6, a transmembrane protein, is shed upon cleavage by Beta-site APP cleaving enzyme 1 (BACE 1) and is known to be involved in normal neuronal functions; however, its role in cancer has not been studied yet. SEZ6 silencing inhibited GSC-initiated tumor growth in an intracranial orthotopic mouse model due to reduced tumor angiogenesis and increased the mouse survival. Pre-treatment with a pharmacological inhibitor of BACE1 (BACE1i) prevented SEZ6 shedding from GSCs and inhibited the ability of the GSC-secretome to induce angiogenesis. Further investigations are ongoing to establish the use of BACE1i or BACE1-silencing as a novel therapeutic strategy to inhibit SEZ6-mediated tumor angiogenesis. We also carried out a protein array-based investigation, which revealed that SEZ6 induced IL8 in endothelial cells to induce angiogenesis. Additional investigation showed that SEZ6 activated the TGFβ pathway in endothelial cells to induce IL8. SEZ6 interacted with TGFβRII as seen by confocal microscopy and co-immunoprecipitation. Generation of various deletion mutants of SEZ6 revealed that three Sushi [also known as complement control protein (CCP) module or the short consensus repeat (SCR)] domains, 3, 4, and 5, interacted with TGFβRII. Molecular docking and dynamic simulation studies identified Sushi-3 domain as the mediator of the interaction between SEZ6 and TGFβRII extracellular domain. Based on the above results, experiments are underway to design specific inhibitors of this interaction. We are also developing an improved BACE1 inhibitor that would inhibit the shedding of SEZ6 into the extracellular milieu to inhibit SEZ6 functions. Thus, this study establishes that GSC-secreted SEZ6 activates the TGFβ pathway in endothelial cells to induce tumor angiogenesis and proposes a novel therapeutic target. Citation Format: Arani Mukherjee, Shreoshi Sengupta, Abhishek Chowdhury, Anand Srivastava, Kumar Somasundaram. Seizure related 6 homolog (SEZ6), a cancer stem cell-specific secreted protein, is a novel GBM therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2786.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulatedin calcific areas. We further demonstrated TCF21 expression wasincreased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1β and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

Small Molecule BRD4 Inhibitors Apabetalone and JQ1 Rescues Endothelial Cells Dysfunction, Protects Monolayer Integrity and Reduces Midkine Expression.

NF-κB signaling is a key regulator of inflammation and atherosclerosis. NF-κB cooperates with bromodomain-containing protein 4 (BRD4), a transcriptional and epigenetic regulator, in endothelial inflammation. This study aimed to investigate whether BRD4 inhibition would prevent the proinflammatory response towards TNF-α in endothelial cells. We used TNF-α treatment of human umbilical cord-derived vascular endothelial cells to create an in vitro inflammatory model system. Two small molecule inhibitors of BRD4-namely, RVX208 (Apabetalone), which is in clinical trials for the treatment of atherosclerosis, and JQ1-were used to analyze the effect of BRD4 inhibition on endothelial inflammation and barrier integrity. BRD4 inhibition reduced the expression of proinflammatory markers such as SELE, VCAM-I, and IL6 in endothelial cells and prevented TNF-α-induced endothelial tight junction hyperpermeability. Endothelial inflammation was associated with increased expression of the heparin-binding growth factor midkine. BRD4 inhibition reduced midkine expression and normalized endothelial permeability upon TNF-α treatment. In conclusion, we identified that TNF-α increased midkine expression and compromised tight junction integrity in endothelial cells, which was preventable by pharmacological BRD4 inhibition.

TRIM47 is a novel endothelial activation factor that aggravates lipopolysaccharide-induced acute lung injury in mice via K63-linked ubiquitination of TRAF2.

Endothelial activation plays an essential role in the pathogenesis of sepsis-induced acute lung injury, however, the detailed regulatory mechanisms remain largely unknown. Here, we reported that TRIM47, an E3 ubiquitin ligase of the tripartite motif-containing protein family, was highly expressed in vascular endothelial cells. TRIM47-deficient mice were effectively resistant to lipopolysaccharide (LPS)-induced acute lung injury and death by attenuating pulmonary inflammation. TRIM47 was upregulated during TNFα-induced endothelial activation in vitro. Knockdown of TRIM47 in endothelial cells inhibited the transcription of multiple pro-inflammatory cytokines, reduced monocyte adhesion and the expression of adhesion molecules, and suppressed the secretion of IL-1β and IL-6 in endothelial cells. By contrast, overexpression of TRIM47 promoted inflammatory response and monocyte adhesion upon TNFα stimulation. In addition, TRIM47 was able to activate the NF-κB and MAPK signaling pathways during endothelial activation. Furthermore, our experiments revealed that TRIM47 resulted in endothelial activation by promoting the K63-linked ubiquitination of TRAF2, a key component of the TNFα signaling pathway. Taken together, our studies demonstrated that TRIM47 as a novel activator of endothelial cells, promoted LPS-induced pulmonary inflammation and acute lung injury through potentiating the K63-linked ubiquitination of TRAF2, which in turn activates NF-κB and MAPK signaling pathways to trigger an inflammatory response in endothelial cells.

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells.

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

Synthetic chalcone derivatives inhibit cytokine secretion via inhibition of ERK and JNK pathways in human U937 macrophage

Purpose: To investigate the inhibitory effects of a chalcone derivative on lipopolysaccharide (LPS)- induced interleukin (IL)-6 and IL-8 secretions and on LPS-induced mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation in human U937 macrophage-like cell line.
 Methods: The effects of chalcone derivative on LPS-induced secretion of IL-6 and IL-8 in endothelial cells were determined by enzyme-linked immunosorbent assay while the effects of chalcone on the activation of MAPK and NF-kB pathway were determined by Western blotting.
 Results: The results showed that 3-(5-methyl-furan-2-yl)-naphthalen-1-yl-propenone (compound 1) significantly inhibited the secretion of LPS-induced IL-6 and IL-8 in U937 macrophages. This compound also demonstrated significant suppression of c-Jun N-terminal kinases (JNK) and extracellular signalregulated kinases (ERK) phosphorylation. However, the compound did not reverse the degradation of inhibitor kappa B alpha (IκBα) and did not inhibit the phosphorylation of NF-κB subunit and P-38 MAPK.
 Conclusion: Compound 1 inhibits the secretion of cytokines via the inhibition of ERK and JNK pathways. These results suggest that chalcone derivative could act as an antiinflammatory agent by altering cytokine secretion and inflammatory pathways.

Increased Release of Soluble MD-2 in Sickle Cell Disease and Its Role in Pro-Inflammatory Signaling in Endothelial Cells

Sickle cell disease (SCD) is the most common hemoglobinopathy worldwide, resulting from a mutation in the beta globin gene. SCD has significant pathophysiological consequences -- hemolysis, inflammation, oxidative stress, hypercoagulability, endothelial dysfunction and painful vaso-occlusive crises. The latter can be precipitated by infection or other metabolic stressors. Hemolysis chronically exposes endothelial cells, leukocytes, and platelets to hemoglobin and heme that promote pro-inflammatory and prothrombotic phenotypes. We previously demonstrated that toll-like receptor 4 (TLR4) signaling is required for microvascular stasis induced by hemoglobin, heme, or lipopolysaccharide (LPS) in sickle mice. MD-2 is a glycoprotein, co-expressed with TLR4 at the surface of various cell types, principally myeloid and endothelial lineages. MD-2 also exists as a soluble plasma protein (sMD-2), mainly as a large disulfide-bound multimeric glycoprotein, as well as oligomers and monomers. sMD-2 binds LPS and confers TLR4 sensitivity to LPS . A marked increase in sMD-2 has been reported in plasma from patients with sepsis and rheumatoid arthritis. sMD-2 in SCD plasma has not been studied. Since SCD has a pro-inflammatory phenotype, we hypothesized that sMD-2 is increased in SCD plasma and promotes pro-inflammatory signaling of endothelial cells. We assessed plasma levels of sMD-2 by Western blot and found that sMD-2 was increased 1.7-fold in SS human plasma (n=8) compared to healthy AA plasma (p<0.05, n=7). In mice, plasma sMD-2 was increased 7.6-fold in Townes-SS sickle mice (n=9) compared to control Townes-AA mice (p<0.0002, n=7). In contrast, plasma CD14, another required component of LPS-TLR4 signaling, was not significantly different in SS humans (n=8) and SS mice (n=9) compared to AA controls (p<0.05). The liver is one potential source of sMD-2 in plasma. In mice, hepatic MD-2 mRNA was increased 2.1-fold in SS compared to AA (p<0.05, n=6). Activated vascular endothelium is another potential source and target of sMD-2 in plasma. It has been reported by other groups and confirmed by us that LPS induces sMD-2 secretion by human umbilical vein endothelial cells (HUVEC). To determine whether heme can induce sMD-2 secretion from endothelial cells, we treated HUVEC with heme (0-30 μM) for 18 hours and found heme increased sMD-2 in media in a dose-responsive manner. To determine if sMD-2 in plasma could activate TLR4 signaling in endothelial cells, we incubated HUVEC with 2% SS or AA human plasma for 18 hours and measured IL-8 in the media by ELISA. Media IL-8 concentration was 2.6-fold higher in HUVEC incubated with SS plasma compared to AA plasma (p<0.02, n=4). Tak242, a TLR4 signaling inhibitor, blocked IL-8 secretion by HUVEC + SS plasma. Since heme has been shown to activate TLR4 signaling, we examined whether heme could bind to sMD-2 in plasma using a heme-agarose pull-down assay. Human plasma was incubated with heme-agarose to pull down heme binding proteins, followed by Western blot for sMD-2 protein in the pellet. The blot confirmed that sMD-2 in plasma bound specifically to heme. When sMD-2 was removed from SS plasma using an anti-MD-2 affinity column, the sMD-2-depleted plasma reduced IL-8 secretion by HUVEC by 34.3% (p<0.002, n=4). Furthermore, when the high-affinity heme-binding protein hemopexin (10 μM) was added to SS plasma, IL-8 secretion by HUVEC was reduced by 31.6% (p<0.01, n=7). Next, we made recombinant human sMD-2 in CHO cells with protein-free ProCHO medium. UV/Vis absorption spectra (250-600 nm) and heme-agarose pull-down assays found there was heme bound to recombinant sMD-2 in the ProCHO medium. When recombinant sMD-2-heme was added to human AA plasma and incubated with HUVEC, IL-8 secretion increased 2.2-fold (p<0.004, n=3). TLR4 inhibitor Tak242 blocked this increase in IL-8 secretion. When hemopexin was added to the recombinant sMD-2-heme before adding it to AA plasma, IL-8 production was reduced 38% compared to non-hemopexin treated (p<0.01, n=7). In conclusion, these data indicate that sMD-2 is increased in SCD plasma, binds heme, and can stimulate endothelial cell IL-8 production through a TLR4-dependent mechanism. We speculate that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Factor VII activating protease (FSAP) regulates the expression of inflammatory genes in vascular smooth muscle and endothelial cells

Background and aimsThe factor VII activating protease (FSAP) knockout mice have a bigger neointima after vascular injury and a larger infarct volume after stroke. The Marburg I (MI) single nucleotide polymorphism (SNP) in the FSAP-encoding gene is associated with an increased risk of stroke and carotid stenosis in humans. We hypothesize that the regulation of gene expression by FSAP in vascular cells accounts for its vasculo-regulatory properties. MethodsVascular smooth muscle cells (VSMC) and endothelial cells (EC) were stimulated with FSAP and a microarray-based expression analysis was performed. Selected genes were further investigated by qPCR. Receptor- and pathway-inhibitors were used to elucidate the mechanisms involved. ResultsPathways significantly activated by FSAP include those related to inflammation, apoptosis and cell growth in VSMC and inflammation in EC. The key upregulated genes in VSMC were AREG, PTGS2 and IL6; and in EC these were SELE, VCAM1, and IL8. Secretion of IL6 in VSMC and IL8 in EC was also stimulated by FSAP. Recombinant wild type protease domain of FSAP, but not the MI-isoform, could recapitulate most of these effects. In VSMC, but not EC, gene expression by FSAP was impaired by PAR1 (protease-activated receptor1) receptor antagonists. In VSMC, FSAP-induced expression of AREG and IL6 was blocked by cAMP and MAPK pathway inhibitors indicating that multiple signalling pathways are likely to be involved. ConclusionsThe stimulation of inflammation- and proliferative/apoptosis-related genes in VSMC and EC provides a comprehensive basis for understanding the role of FSAP in vascular diseases.

Off-Target Anti-Inflammatory Activity of the P2X7 Receptor Antagonist AZ11645373

We have found that a well-characterized P2X7 receptor antagonist AZ11645373 blocked production of pro-inflammatory chemokine IL-8 in endothelial cells treated with OxPAPC. The effect was not due to toxicity of AZ11645373 as documented by cellular metabolic activity assay. The mechanism of inhibition by AZ11645373 was apparently independent of the P2X7 receptor because this receptor was not involved in induction of IL-8 under our experimental conditions. In support of this notion, two P2X7 agonists ATP and BzATP did not upregulate IL-8. On the other hand, a chemically different P2X7 receptor antagonist A740003 did not inhibit OxPAPC-induced production of IL-8. The inhibitory action of AZ11645373 was observed at the level of IL-8 protein and messenger RNA (mRNA) induction. Furthermore, AZ11645373 inhibited induction of mRNA encoding for COX-2 (PTGS2) suggesting that its anti-inflammatory potential is not limited to suppression of IL-8 production. In addition to inhibiting stimulation by OxPAPC, AZ11645373 suppressed induction of IL-8 by TNFα and LPS. To summarize, AZ11645373 inhibits in a P2X7-independent manner action of chemically different inflammatory agonists such as OxPLs, LPS, and TNFα. Thus, AZ11645373 may be especially effective for treatment of inflammatory disorders due to a beneficial combination of P2X7 receptor-dependent effects (inhibition of inflammasome activation, antinociceptive effects) with P2X7-independent general anti-inflammatory action described in this paper.

7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells.

Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 μg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.

Endotoxins affect diverse biological activity of chitosans in matters of hemocompatibility and cytocompatibility.

Chitosan is used in several pharmaceutical and medical applications, owing to its good cytocompatibility and hemocompatibility. However, there are conflicting reports regarding the biological activities of chitosan with some studies reporting anti-inflammatory properties while others report pro-inflammatory properties. In this regards we analyzed the endotoxin content in five different chitosans and examined these chitosans with their different deacetylation degrees for their hemocompatibility and cytocompatibility. Therefore, we incubated primary human endothelial cells or whole blood with different chitosan concentrations and studied the protein and mRNA expression of different inflammatory markers or cytokines. Our data indicate a correlation of the endotoxin content and cytokine up-regulation in whole blood for Poly-Morpho-Nuclear (PMN)-Elastase, soluble terminal complement complex SC5b-9, complement component C5/C5a, granulocyte colony-stimulating factor, Interleukin-8 (IL), IL-10, IL-13, IL-17E, Il-32α and monocyte chemotactic protein-1. In contrast, the incubation of low endotoxin containing chitosans with primary endothelial cells resulted in increased expression of E-selectin, intercellular adhesion molecule-1, vascular cell adhesion protein-1, IL-1β, IL-6 and IL-8 in endothelial cells. We suggest that the endotoxin content in chitosan plays a major role in the biological activity of chitosan. Therefore, we strongly recommend analysis of the endotoxin concentration in chitosan, before further determining if it has pro- or anti-inflammatory properties or if it is applicable for pharmaceutical and medical fields.

The role of adiponectin in the production of IL-6, IL-8, VEGF and MMPs in human endothelial cells and osteoblasts: implications for arthritic joints

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 μg ml−1) or IL-1β (0.1 ng ml−1) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1β. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1β-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1β. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1β in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.

AB0048 The role of adiponectin in the production of il-6, il-8, vegf, and mmps in human endothelial cells and osteoblasts: implications for arthritic joints

BackgroundThe role of adiponectin in the pathogenesis of rheumatoid arthritis was previously studied in relation to the production of IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs)...

Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

Induction of TLR2 expression by inflammatory stimuli is required for endothelial cell responses to lipopeptides

Human endothelial cells (EC) express Toll-like receptor 4 (TLR4), a receptor for lipopolysaccharides (LPS), but little or no TLR2, a lipopeptide receptor. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by EC of TLR4 and TLR2, of the TLR2 co-receptors TLR1 and TLR6 and of the TLR2-accessory proteins CD14 and CD36. Stimulation of umbilical vein derived EC with TNF-α, LPS or IL-1β for 24 h induced a strong increase in TLR2 mRNA but not in TLR1, TLR4 and TLR6 mRNA. Inflammatory activation had little effect on CD14 mRNA, but decreased the expression of CD36 mRNA. TLR2 antigen was readily detected by flow cytometry on activated EC, but not on resting EC. A significant proportion of TLR2 was found to be located intracellularly. By using specific signalling pathway inhibitors we established that the induction of TLR2 by inflammatory stimuli was dependent on NF-κB, p38-MAP kinase and c-Jun kinase. IRAK-1 phosphorylation after treatment with 10 μg/ml of lipoteichoic acid (LTA), a TLR2 agonist, was only observed in TNF-α-stimulated EC and not in resting EC. Furthermore, LTA potentiated the increase of the inflammatory markers E-Selectin or IL-8 in EC pre-treated with TNF-α, LPS or IL-1β, but not in resting EC. These results imply that the up-regulated TLR2 is functionally active. Interestingly, LTA had no effect on TLR2 expression, nor maintained TLR2 expression, in activated EC. This suggests that lipopeptide responses of EC are dependent on the continued presence of inflammatory cytokines, provided by other cell types, or LPS. In conclusion, inflammatory stimuli induce a high TLR2 expression in EC, which in turn enables the cells to strongly respond to lipopeptides. The up-regulation of TLR2 may be of relevance for the vascular effects of Gram-positive bacteria.

Direct Interaction of G-CSF-Stimulated-Neutrophils and HUVECs Are Necessary for Production of IL-8 in Endothelial Cells.

Background: We previously reported that interleukin (IL)-8 increased up to 207 pg/ml in vivo after administration of granulocyte colony-stimulating factor (G-CSF) at a dose 5μg/kg for five days to mobilize peripheral blood stem cells (PBSC) in hematopoietic stem cell transplantation in cancer patients or healthy donors. Since pre-apheresis serum IL-8 levels correlated with the yield of CD34-positive cells, IL-8 production may be critical to G-CSF induced stem cell mobilization. IL-8 is known to be surged from granules of endothelial cells (EC) by stimulation of GM-CSF, however, precise mechanisms of increased production of IL-8 by G-CSF in vivo is unclear. First, in this study we examined the relationship between G-CSF and IL-8 in vitro.Method: Peripheral blood was drawn from healthy donor with consent. Neutrophils were separated by density centrifugation method. Human umbilical vein endothelial cells (HUVECs) and G-CSF were purchased. HUVECs were cultured under serum-containing medium and stimulated by G-CSF or HUVECs were co-cultured with neutrophils. IL-8 in culture media was measured by ELISA.Results: G-CSF alone does not increase IL-8 production in the culture medium either of neutrophils or HUVECs alone, in either short time (6 hours) or long time (up to 4 days) stimulation. However, when HUVECs were co-cultured with neutrophils, IL-8 started to increase 10 hours after starting co-culture and gradually increased up to 3000pg/ml after 4 days culture. IL-8 in co-culture media increased five fold compared to that in the single culture of HUVECs. This increased production of IL-8 was dependent on the number of neutrophils. Further, when neutrophils were pre-incubated by G-CSF at a dose 50ng/ml, IL-8 production in co-culture media increased 70 percent compared to pre-incubation without G-CSF. This increased production of IL-8 was dependent on G-CSF dose. IL-8 production remarkably decreased to 15% when HUVECs and neutrophils were cultured separately by mesh to avoid direct contact.Conclusion: These data indicates that direct contact between HUVECs and neutrophils is necessary for production of IL-8. Some molecule(s) induced by G-CSF might play an important role in IL-8 production. The effect of stimulation by G-CSF seems less important than that of direct interaction between neutrophils and HUVECs. This correlates well to the fact that high WBC count after G-CSF administration in vivo correlates with the yield of CD34-positive cells in peripheral blood stem cells mobilization. Molecular mechanisms of interaction between HUVECs and neutrophils need to be clarified further.

Metformin Inhibits Proinflammatory Responses and Nuclear Factor-κB in Human Vascular Wall Cells

Metformin may benefit the macrovascular complications of diabetes independently of its conventional hypoglycemic effects. Accumulating evidence suggests that inflammatory processes participate in type 2 diabetes and its atherothrombotic manifestations. Therefore, this study examined the potential action of metformin as an inhibitor of pro-inflammatory responses in human vascular smooth muscle cells (SMCs), macrophages (Mphis), and endothelial cells (ECs). Metformin dose-dependently inhibited IL-1beta-induced release of the pro-inflammatory cytokines IL-6 and IL-8 in ECs, SMCs, and Mphis. Investigation of potential signaling pathways demonstrated that metformin diminished IL-1beta-induced activation and nuclear translocation of nuclear factor-kappa B (NF-kappaB) in SMCs. Furthermore, metformin suppressed IL-1beta-induced activation of the pro-inflammatory phosphokinases Akt, p38, and Erk, but did not affect PI3 kinase (PI3K) activity. To address the significance of the anti-inflammatory effects of a therapeutically relevant plasma concentration of metformin (20 micromol/L), we conducted experiments in ECs treated with high glucose. Pretreatment with metformin also decreased phosphorylation of Akt and protein kinase C (PKC) in ECs under these conditions. These data suggest that metformin can exert a direct vascular anti-inflammatory effect by inhibiting NF-kappaB through blockade of the PI3K-Akt pathway. The novel anti-inflammatory actions of metformin may explain in part the apparent clinical reduction by metformin of cardiovascular events not fully attributable to its hypoglycemic action.

Endothelial Cell IL-8, a New Target for Adiponectin

See related article, pages 1245–1252 Adipose tissue is no longer considered an inert energy storage tissue, but an active participant contributing to physiological and pathological processes associated with inflammation, immunity, appetite, insulin sensitivity, endocrine and reproductive systems, bone metabolism, and endothelial function.1,2 Adipose tissue synthesizes and secretes proinflammatory and antiinflammatory metabolically- and hormonally-active substances, collectively called adipokines or adipocytokines and include leptin, adiponectin, resistin, and visfatin.3,4 Adipose tissue also produces cytokines and chemokines, such as tissue necrosis factor (TNF)-1α, interleukin (IL)-1β, IL-6, IL-8, IL-10, transforming growth factor (TGF)-β, nerve growth factor and the acute-phase response plasminogen activator inhibitor-1, haptoglobin, and serum amyloid.1,2,5 Although adipose tissue produces various polypeptide and non-protein factors, only leptin, adiponectin, resistin, adipsin, and visfatin are primarily synthesized by adipocytes.3,4 Whereas leptin plays a role mainly in appetite regulation,6 resistin induces insulin resistance and is proinflammatory7 whereas visfatin acts as an insulin-mimetic and is antiapoptotic.8 Of all the adipokines, adiponectin is found in highest concentrations in the circulation.9 It is secreted specifically from adipocytes and it regulates insulin sensitivity.10 Low serum levels are causally linked to insulin resistance, obesity, and type 2 diabetes and are predictive for development of diabetes and cardiovascular disease.11,12 Obesity is associated with decreased adiponectin levels, and adiponectin is now being considered a putative therapeutic agent in the management of obesity.13,14 Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice.15,16 Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes.17,18 This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase19 and peroxisome proliferator-activated receptor-γ (PPAR-γ).20 Emerging evidence indicates that adiponectin has functions beyond insulin modulation. Recent studies demonstrated that adiponectin is synthesized and secreted not only …

Mast Cell Tryptase in Mast Cell Granules Enhances MCP-1 and Interleukin-8 Production in Human Endothelial Cells

Recent studies have highlighted the pathogenetic importance of chronic inflammation in cardiovascular disorders such as congestive heart failure and atherosclerosis. Mast cells release a wide variety of immune mediators that may initiate inflammatory responses, whereas endothelial cells (ECs) play a prominent role in the pathogenesis of cardiovascular diseases by secreting cytokines. The purpose of this study was to clarify the role of mast cells as an activator of ECs. ECs harvested from human umbilical cord veins were stimulated with mast cell granules (MCGs) prepared from sonicated human leukemic mast cells. The supernatants and total RNA from cells were collected. Levels of interleukin (IL)-1beta, tumor necrosis factor-alpha, and granulocyte colony-stimulating factor remained unchanged up to 24 hours. In contrast, levels of monocyte chemoattractant protein-1 (MCP-1) and IL-8 increased significantly within 6 hours. Northern blot analysis revealed an increase in MCP-1 and IL-8 mRNA expression in MCG-treated ECs. Induction of these chemokines was attenuated by antitryptase neutralizing antibody. Furthermore, MCP-1 and IL-8 were induced in ECs by incubation with human mast cell tryptase, but not with chymase. These results indicate that the production of MCP-1 and IL-8 in ECs was induced by MCG and amplified by tryptase.

Decoy Receptor 3 Increases Monocyte Adhesion to Endothelial Cells via NF-κB-Dependent Up-Regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 Expression

Decoy receptor 3 (DcR3), a soluble receptor for FasL, LIGHT and TL1A, is highly expressed in cancer cells. We show that pretreatment of HUVECs with DcR3 enhances the adhesion of THP-1 and U937 cells and primary monocytes. A similar stimulatory effect of DcR3 on THP-1 adhesion was also observed in human microvascular endothelial cells (HMVECs). Flow cytometry and ELISA showed that DcR3-treated HUVECs exhibited significant increases in ICAM-1 and VCAM-1 expression. We also demonstrate the ability of DcR3 to stimulate the secretion of IL-8 by HUVECs. RT-PCR and reporter assays revealed that the expression of adhesion molecules and IL-8 are regulated at the level of gene transcription. Experiments with pyrrolidine dithiocarbamate indicated the involvement of an NF-kappaB signaling pathway. DcR3 was found to induce IkappaB kinase activation, IkappaB degradation, p65 nuclear translocation, and NF-kappaB DNA-binding activity. The enhancement by DcR3 of cell adhesion to HUVECs was not mimicked by the TL1A-Ab, which has been shown in our previous work to be a neutralizing Ab against TL1A, thereby inducing HUVECs angiogenesis. Moreover, DcR3-induced cell adhesion could be detected in human aortic endothelial cells (ECs) in which TL1A expression is lacking. Together, our data demonstrate that DcR3 increases monocyte adhesion to ECs via NF-kappaB activation, leading to the transcriptional up-regulation of adhesion molecules and IL-8 in ECs. This novel action appears not to be due to TL1A neutralization, but occurs through an as yet undefined target(s). This study implicates DcR3 in the relationship between inflammation and cancer development.