Challenge with LPS or TNF-α Demonstrates Unique Differences in Primary Arterial Endothelial Cells Pretreated with Recombinant Human Activated Protein C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1935-1935
Author(s):  
James A. Martin ◽  
David E. Joyce ◽  
Rashna Balsara ◽  
Victoria A. Ploplis ◽  
Francis J. Castellino
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Inflammatory Responses ◽  
Transcript Levels ◽  
Total Rna ◽  
Inflammatory Agent ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Arterial Endothelial Cells ◽  
Rna Transcript

Abstract A human recombinant form of the endogenous anticoagulant APC (rhAPC) has been approved for treatment of severe sepsis, a condition with 30-50% mortality and affecting 750,000 US patients per year. Clinical and in vitro studies show that rhAPC has pro-fibrinolytic, anti-inflammatory, and anti-apoptotic properties. In order to better understand the anti-inflammatory mechanism of rhAPC and its receptor EPCR on primary murine aortic endothelial cells (EC), responses were compared between wild type (WT) and low-expressing endothelial protein C receptor (EPCRδ/δ) EC by total RNA for specified endothelial inflammatory markers. The purpose was to determine the effect of rhAPC and low expression of EPCR on murine arterial EC responses to tumor necrosis factor alpha (TNF-α) or endotoxin (LPS). EC from C57BL/6 mice aorta, WT or EPCRδ/δ, were isolated, cultured, and positively selected for EC markers (CD105, CD106). EC in serum free media were pretreated with 5ug/mL rhAPC (Eli Lilly) for 16 hours followed by challenge with 100ng/mL TNF-α or 10ug/mL LPS for 8 hours. Total RNA was analyzed by Quantitative Real-time PCR (QRT-PCR) for CXC chemokines MIP-2 and KC, adhesion markers E-Selectin or ICAM-1, cytokines MCP-1 and IL-6, and NFκB-1. Mean +/− standard error of the mean for the time points (T0, 0.5hr, 1hr, 2hr, 4hr, and 8hr) after TNF-α or LPS were compared between treatment groups. Both TNF-α and LPS produced expected characteristic fold changes of RNA expression over the eight hour time period in the murine EC. Without rhAPC EPCRδ/δ EC showed a similar response compared to WT EC. When pretreated with rhAPC for 16 hours followed by LPS challenge, EC RNA transcript levels for CXC chemokines and adhesion markers were suppressed more in EPCRδ/δ compared to WT EC. When pretreated with rhAPC for 16 hours followed by TNF-α challenge, RNA transcript levels for CXC chemokines and adhesion markers were elevated or showed little change in WT EC and EPCRδ/δ EC compared to EC not given rhAPC. Nuclear factor NFκB-1 RNA was suppressed in both WT EC and EPCRδ/δ EC with rhAPC pretreatment and subsequent inflammatory agent (LPS or TNF-α). Most striking was the unexpected suppressed response of rhAPC pretreated EPCRδ/δ EC compared to WT EC after addition of either inflammatory agent. Further studies suggested that surface EPCR protein did not appear to be enhanced with any treatment combination, or with rhAPC alone. These results are consistent with previously reported endothelial cell specific rhAPC response of CXC chemokines and the ability of rhAPC to suppress other TNF-α mediated inflammatory responses (eg. MCP-1 and NFkB-1). In addition, rhAPC pretreatment appeared to suppress LPS mediated inflammatory responses, including CXC chemokines. The enhanced suppression of inflammatory responses seen in arterial EPCRδ/δ EC compared to WT EC remains unexplained. Results from this study also indicate primary murine arterial endothelial cells treated with rhAPC respond differently to challenge with TNF-α versus LPS.

scholarly journals PSVI-13 Anti-inflammatory effects of polyphenol-rich red osier dogwood extracts in Caco-2 mono- and Caco-2/EA.hy926 co-culture models

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 211-212
Author(s):  
Hua Zhang ◽  
Yuhuan Chen ◽  
Lili Mats ◽  
Qianru Hui ◽  
Rong Tsao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Phenolic Compounds ◽  
Inflammatory Responses ◽  
Cell Model ◽  
Circulation System ◽  
Systemic Diseases ◽  
Potential Health ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
The Impact

Abstract An impaired intestinal barrier function results in aggravating inflammatory response at a systemic scale, eventually leading to rising risk for systemic diseases (e.g., muscle myopathy and vascular disorders). In the present study, the impact of intake polyphenol-rich red osier dogwood extracts (RWE) on the inflammation of endothelial cells was exploited. A strong anti-inflammatory activity of RWE was found to suppress the expression of pro-inflammatory mediators (e.g., IL-8, TNF-α, IL-6, and ICAM) in the inflamed intestinal epithelial cell model. Furthermore, the intestinal transported RWE derived phenolic compounds was shown to protect the endothelial cells against both oxidative and inflammatory damages in a Caco-2/EA.hy926 co-culture cell model. Their protective activities in EA.hy926 was found to be strongly associated with intestinal absorption efficiency. The accumulation of transported rutin and unknown monoglyceride quercetin from RWE were identified across the Caco-2 BBe1 monolayer by HPLC up to 24 h. The highest concentration of transported rutin and monoglyceride quercetin derived from RWE were detected as 2.0 ± 0.22 µg/mL and 0.5 ± 0.08 µg/mL in the basolateral compartment after 12 h and 24 h of incubation, respectively. Profound anti-inflammatory effects of RWE derived polyphenols was observed to suppress pro-inflammatory mediator expression, including IL-8, TNF-α, IL-6, ICAM, VCAM and Cox2, in the TNF-α or oxidized low-density lipoprotein (oxLDL)-induced basolateral EA.hy926 cells (co-culture model). Moreover, we observed a significant inhibitory effect of the transported RWE on oxLDL-induced inflammation after 6 h incubation rather than 24 h, indicating the potential health benefits of RWE is determined by its bioavailability. Results of this study demonstrated that phenolic compounds derived from RWE could be delivered into the circulation system to mitigate inflammatory responses thereby being a promising dietary agent for preventing systemic diseases (e.g., cardiovascular diseases in humans and white stripping/woody meat in broiler chickens).

scholarly journals Insulin and IGF1 receptors in human cardiac microvascular endothelial cells: metabolic, mitogenic and anti-inflammatory effects

2012 ◽  
Vol 215 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Karolina Bäck ◽  
Rakibul Islam ◽  
Git S Johansson ◽  
Simona I Chisalita ◽  
Hans J Arnqvist
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Receptor Protein ◽  
Microvascular Endothelial Cells ◽  
Tnf Α ◽  
Glucose Accumulation ◽  
Anti Inflammatory ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10−8 mol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10−8 mol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10−8 mol/l and glucose accumulation at 10−7 mol/l in HMVEC-Cs. TNF-α dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.

scholarly journals Anti-Inflammatory Effect and Cellular Uptake Mechanism of Carbon Nanodots in in Human Microvascular Endothelial Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1247
Author(s):  
Sarah Belperain ◽  
Zi Yae Kang ◽  
Andrew Dunphy ◽  
Brandon Priebe ◽  
Norman H. L. Chiu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Interleukin 1 ◽  
Antioxidant Properties ◽  
Synthesis Method ◽  
Microvascular Endothelial Cells ◽  
Carbon Nanodots ◽  
Uptake Mechanism ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Microvascular Endothelial

Cardiovascular disease (CVD) has become an increasingly important topic in the field of medical research due to the steadily increasing rates of mortality caused by this disease. With recent advancements in nanotechnology, a push for new, novel treatments for CVD utilizing these new materials has begun. Carbon Nanodots (CNDs), are a new form of nanoparticles that have been coveted due to the green synthesis method, biocompatibility, fluorescent capabilities and potential anti-antioxidant properties. With much research pouring into CNDs being used as bioimaging and drug delivery tools, few studies have been completed on their anti-inflammatory potential, especially in the cardiovascular system. CVD begins initially by endothelial cell inflammation. The cause of this inflammation can come from many sources; one being tumor necrosis factor (TNF-α), which can not only trigger inflammation but prolong its existence by causing a storm of pro-inflammatory cytokines. This study investigated the ability of CNDs to attenuate TNF-α induced inflammation in human microvascular endothelial cells (HMEC-1). Results show that CNDs at non-cytotoxic concentrations reduce the expression of pro-inflammatory genes, mainly Interleukin-8 (IL-8), and interleukin 1 beta (IL-1β). The uptake of CNDs by HMEC-1s was examined. Results from the studies involving channel blockers and endocytosis disruptors suggest that uptake takes place by endocytosis. These findings provide insights on the interaction CNDs and endothelial cells undergoing TNF-α induced cellular inflammation.

scholarly journals The potential effects of Centella asiatica ethanolic extracts as an anti-inflammatory agent through decreasing TNF-α expression in indomethacin-induced gastric ulcer model rats

2021 ◽  
Author(s):  
Priscilla Maiselina Sriepindonnta ◽  
Fatimah Nur Fitriani ◽  
Savannah Quila Thirza ◽  
Made Dinda Pratiwi ◽  
Dwi Evan Prima Putra Noviardi ◽  
...  
Keyword(s):  
Gastric Ulcer ◽  
Centella Asiatica ◽  
Inflammatory Agent ◽  
Tnf Α ◽  
Ethanolic Extracts ◽  
Ulcer Model ◽  
Anti Inflammatory

scholarly journals In Vitro Evaluation of the Anti-Inflammatory Effect of KMUP-1 and in Vivo Analysis of Its Therapeutic Potential in Osteoarthritis

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 615
Author(s):  
Shang-En Huang ◽  
Erna Sulistyowati ◽  
Yu-Ying Chao ◽  
Bin-Nan Wu ◽  
Zen-Kong Dai ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Antioxidant Properties ◽  
Serum Levels ◽  
Gene Expressions ◽  
Tnf Α ◽  
Anti Inflammatory

Osteoarthritis is a degenerative arthropathy that is mainly characterized by dysregulation of inflammatory responses. KMUP-1, a derived chemical synthetic of xanthine, has been shown to have anti-inflammatory and antioxidant properties. Here, we aimed to investigate the in vitro anti-inflammatory and in vivo anti-osteoarthritis effects of KMUP-1. Protein and gene expressions of inflammation markers were determined by ELISA, Western blotting and microarray, respectively. RAW264.7 mouse macrophages were cultured and pretreated with KMUP-1 (1, 5, 10 μM). The productions of TNF-α, IL-6, MMP-2 and MMP- 9 were reduced by KMUP-1 pretreatment in LPS-induced inflammation of RAW264.7 cells. The expressions of iNOS, TNF-α, COX-2, MMP-2 and MMP-9 were also inhibited by KMUP-1 pretreatment. The gene expression levels of TNF and COX families were also downregulated. In addition, KMUP-1 suppressed the activations of ERK, JNK and p38 as well as phosphorylation of IκBα/NF-κB signaling pathways. Furthermore, SIRT1 inhibitor attenuated the inhibitory effect of KMUP-1 in LPS-induced NF-κB activation. In vivo study showed that KMUP-1 reduced mechanical hyperalgesia in monoiodoacetic acid (MIA)-induced rats OA. Additionally, KMUP-1 pretreatment reduced the serum levels of TNF-α and IL-6 in MIA-injected rats. Moreover, macroscopic and histological observation showed that KMUP-1 reduced articular cartilage erosion in rats. Our results demonstrated that KMUP-1 inhibited the inflammatory responses and restored SIRT1 in vitro, alleviated joint-related pain and cartilage destruction in vivo. Taken together, KMUP-1 has the potential to improve MIA-induced articular cartilage degradation by inhibiting the levels and expression of inflammatory mediators suggesting that KMUP-1 might be a potential therapeutic agent for OA.

Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Helong Zhao ◽  
Appakkudal Anand ◽  
Ramesh Ganju
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Model Studies ◽  
Endothelial Inflammation ◽  
Anti Inflammatory ◽  
Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

Abstract 553: Siglec-1 (CD169) on Monocytes/Macrophages: A New Receptor For Extracellular Self-RNA in Triggering Inflammatory Responses via the Sheddase TACE/ADAM17

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Hector A Cabrera-Fuentes ◽  
Klaus T Preissner ◽  
William A Boisvert
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Responses ◽  
Transmembrane Protein ◽  
Macrophage Polarization ◽  
Extracellular Rna ◽  
Tnf Α ◽  
M1 Phenotype ◽  
Phenotype Markers ◽  
Anti Inflammatory

As an important component of atherosclerosis, monocytes/macrophages respond to external stimuli with rapid changes in their expression of many inflammation-related genes to undergo polarization towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. Although sialoadhesin (Sn), also known as SIGLEC-1 or CD169, is a transmembrane protein receptor expressed on monocytes and macrophages whether it has a role in macrophage polarization and ultimately, macrophage-driven atherogenesis, has not been investigated. We have previously shown that, independently of Toll-like receptor signaling, extracellular RNA (eRNA) could exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system by inducing cytokine mobilization. In the current study, recombinant mouse macrophage CSF[[Unable to Display Character: –]]driven bone marrow-derived macrophage (BMDM) differentiation was found to be skewed towards the M1 phenotype by exposure of cells to eRNA. This resulted in up-regulation of inflammatory markers, whereas anti-inflammatory genes were significantly down-regulated by eRNA. Interestingly, eRNA was released from BMDM under hypoxia and induced TNF-α liberation by activating TNF-α converting enzyme (TACE) to provoke inflammation. Conversely, TNF-α promoted eRNA release, especially under hypoxia, feeding a vicious cycle of cell damage. Administration of RNase1 or TAPI (a TACE-inhibitor) prevented the production of inflammatory mediators. Murine BMDM isolated from mice deficient in sialoadhesin had the opposite reaction to eRNA treatment with a prominent down-regulation of pro-inflammatory cytokines/M1 phenotype markers, while anti-inflammatory cytokines/M2 phenotype markers were significantly raised. In keeping with the proposed role of eRNA as a pro-inflammatory “alarm signal”, these data further shed light on the role of eRNA in macrophage function in the context of chronic inflammatory diseases such as atherosclerosis. The identification of sialoadhesin as putative eRNA recognition site on macrophages may allow further investigation of the underlying mechanisms of eRNA-macrophage interaction and related signal transduction pathways. Siglec-1 thereby may provides a new target to treat eRNA-mediated vascular diseases.

Abstract 153: Bile Acid Receptor TGR5 Agonism Represents Anti-inflammatory Effects via Stimulating Nitric Oxide Production in Vascular Endothelial Cells

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Taiki Kida ◽  
Yoshiki Tsubosaka ◽  
Masatoshi Hori ◽  
Hiroshi Ozaki ◽  
Takahisa Murata
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Bile Acids ◽  
Inflammatory Responses ◽  
Vascular Endothelial Cells ◽  
Lithocholic Acid ◽  
Signal Pathways ◽  
No Production ◽  
Vascular Endothelial ◽  
Anti Inflammatory

Objective TGR5, a membrane-bound, G-protein-coupled receptor for bile acids, is known to be involved in regulation of energy homeostasis and inflammation. However, little is known about the function of TGR5 in vascular endothelial cells. In the present study, we examined whether TGR5 agonism represents anti-inflammatory effects in vascular endothelial cells focusing on nitric oxide (NO) production. Methods and Results In human umbilical vein endothelial cells (HUVECs), treatment with taurolithocholic acid (TLCA), which has the highest affinity to TGR5 among various bile acids, significantly reduced tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule (VCAM)-1 protein expression and adhesion of human monocytes, U937. These effects were abrogated by a NO synthase (NOS) inhibitor, N G -Monomethyl-L-arginine (L-NMMA). In bovine aortic endothelial cells (BAECs), treatment with TLCA as well as lithocholic acid, which also has high affinity to TGR5, significantly increased the NO production. In contrast, deoxycholic acid and chenodeoxycholic acid, which possess low affinity to TGR5, did not affect the NO production. Gene depletion of TGR5 by siRNA transfection abolished TLCA-induced NO production in BAECs. TLCA-induced NO production was also observed in HUVECs measured as intracellular cGMP accumulation. We next investigated the signal pathways responsible for the TLCA-induced NO production in endothelial cells. Treatment with TLCA increased endothelial NOS (eNOS) ser1177 phosphorylation in HUVECs. This response was accompanied by increased Akt ser473 phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ). Treatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or blockade of calcium channel with La 3+ , significantly decreased TLCA-induced eNOS ser1177 phosphorylation and subsequent NO production. Conclusion These results indicate that TGR5 agonism can mediate anti-inflammatory responses by suppressing VCAM-1 expression and monocytes adhesion to endothelial cells. This function is dependent on NO production via Akt activation and [Ca 2+ ] i increase.

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