Adhesion Of Monocytic Cells Research Articles

AMP-Activated Protein Kinase Suppresses Endothelial Cell Inflammation Through Phosphorylation of Transcriptional Coactivator p300

Considerable evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. AMP-activated protein kinase (AMPK) is highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related mechanisms are not fully understood. The present study was designed to evaluate the impact of and gain mechanistic insight into the signaling coupling AMPK function to the antiinflammatory response. 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment or overexpression of constitutively active AMPK markedly reduced human monocytic human acute monocytic leukemia cell line-1 cell adhesion and the expression of vascular cell adhesion molecule-1 in tumor necrosis factor-α-activated human aortic endothelial cells. Furthermore, AICAR or constitutively active AMPK overexpression strongly inhibited the histone acetyltransferase activity of the transcriptional coactivator p300 by phosphorylation of Ser89, which in turn decreased tumor necrosis factor-α-activated p300-mediated acetylation of nuclear factor-κB p65 on Lys221 and reduced the DNA binding activity of nuclear factor-κB by inhibiting its recruitment to its target gene promoters. AMPK phosphorylates the transcriptional coactivator p300 via the atypical protein kinase Cι/λ. Our findings demonstrate that transcriptional coactivator p300 phosphorylation at Ser89 by AMPK is critical for the therapeutic effect of AMPK and may be a potential target for pharmaceutical intervention in inflammatory diseases such as atherosclerosis.

A novel mechanism of coenzyme Q10 protects against human endothelial cells from oxidative stress-induced injury by modulating NO-related pathways

BackgroundAtherosclerosis is a chronic inflammatory disease of the vessel wall associated with oxidized low-density lipoprotein (oxLDL)-induced apoptosis of endothelial cells. Coenzyme Q10 (CoQ10), a potent antioxidant and a critical intermediate of the electron transport chain, has been reported to inhibit LDL oxidation and thus the progression of atherosclerosis. However, its molecular mechanisms on endothelial cells remain still unclarified. MethodsIn this study, primary human umbilical vein endothelial cell cultures treated with oxLDL were used to explore the protective effects of CoQ10. ResultsOur results showed that CoQ10 attenuated the oxLDL-induced generation of reactive oxygen species and improved the antioxidant capacity. CoQ10 also attenuated the oxLDL-mediated down-regulation of endothelial nitric oxide synthase (eNOS) and up-regulation of inducible nitric oxide synthase (iNOS). In addition, CoQ10 suppressed oxLDL-activated NF-κB and downstream inflammatory mediators, including expression of adhesion molecules, release of proinflammatory cytokines and the adherence of monocytic THP-1 cells. Moreover, CoQ10 attenuated oxLDL-altered proapoptotic responses. The inhibitor of eNOS (l-NIO 10 μM) and iNOS (1400W 10 μM) as well as NO enhancer (SNP 10 μM) were used to clean up the mechanism. ConclusionThese results provide new insight into the possible molecular mechanisms by which CoQ10 protects against atherogenesis by NO-related pathways.

Vascular Protective Role of Vitexicarpin Isolated from Vitex rotundifolia in Human Umbilical Vein Endothelial Cells

Pro-inflammatory cytokines induce injury of endothelial cells caused by increases of adhesion molecules, leading to vascular inflammation and the development of atherosclerosis. Recent pharmacological studies have demonstrated that vitexicarpin, a flavonoid isolated from Vitex rotundifolia, has anti-inflammatory, antitumor, and analgesic properties. In this study, we investigated whether vitexicarpin (5-100nM) prevented the TNF-α-induced vascular inflammation process in human umbilical vein endothelial cells (HUVEC). We found that pretreatment with vitexicarpin decreased TNF-α (10ng/ml)-induced expression of cell adhesion molecules such as vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin as well as matrix metalloproteinase-2 and -9 expression. Preincubation with vitexicarpin also dose-dependently inhibited TNF-α-induced adhesion of HL-60 monocytic cells. Vitexicarpin significantly decreased TNF-α-induced intracellular reactive oxygen species (ROS) production. Furthermore, vitexicarpin suppressed NF-κB nuclear translocation and transcriptional activity in TNF-α-treated HUVEC. In conclusion, vitexicarpin significantly reduced vascular inflammation, through inhibition of ROS-NF-κB pathway in vascular endothelial cells.

Effects of Prunella vulgaris in high glucose‐induced vascular dysfunction

Vascular inflammation is an important factor which can promote diabetic complications. In the present study, we designed to examine the inhibitory effects of high glucose (HG)-induced vascular dysfunction by aqueous extract from Prunella vulgaris (APV) in HUVECs and HASMCs. We found that pretreatment with APV decreased HG-induced expression of CAMs, MMPs, and adhesion of HL-60 monocytic cells on HUVECs. A further analysis indicated that APV attenuated HG-induced translocation and transcription activity in p65 NF-κB signaling pathway. Pretreatment with APV significantly inhibited the formation of intracellular ROS. Additionally, APV-induced Akt phosphorylation could obviously increase not only the induced activation of HO-1 and eNOS but also transcription factor Nrf2. Further, in this study, [3H]-thymidine incorporation was significantly increased compared with control and this increase was decreased by APV. Migration and invasion assay also suggested an anti-proliferative effect of APV. This data cast a new light on the actions of Prunella vulgaris and its potential benefits to diabetic patients for preventing diabetic vascular complications.

Chitosan oligosaccharides suppress LPS-induced IL-8 expression in human umbilical vein endothelial cells through blockade of p38 and Akt protein kinases

To investigate whether and how COS inhibited IL-8 production in LPS-induced human umbilical vein endothelial cells (HUVECs). RT-PCR, enzyme-linked immunosorbent assays (ELISA) and Western blotting were used to study IL-8 expression and related signaling pathway. Wound healing migration assays and monocytic cell adhesion analysis were used to explore the chemotactic and adhesive activities of HUVECs. COS 50-200 μg/mL exerted a significant inhibitory effect on LPS 100 ng/mL-induced IL-8 expression in HUVECs at both the transcriptional and translational levels. In addition, COS 50-200 μg/mL inhibited LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs in a concentration-dependent manner. Signal transduction studies suggest that COS blocked LPS-induced activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) as well as phosphorylation of p38 mitogen-activated protein kinase (MAPK) and phosphokinase Akt. Further, the over-expression of LPS-induced IL-8 mRNA in HUVECs was suppressed by a p38 MAPK inhibitor (SB203580, 25 μmol/L) or a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002, 50 μmol/L). COS inhibited LPS-induced IL-8 expression in HUVECs through the blockade of the p38 MAPK and PI3K/Akt signaling pathways.

Inhibitory Effects ofTerminalia chebulaExtract on Glycation and Endothelial Cell Adhesion

Terminalia chebula Retz. has been used in India for a long time to treat many diseases, and its extract was reported to have antidiabetic activity in vivo. In this study, T. chebula methanolic extract (TCE) containing 2.7 % chebulic acid was evaluated for its preventive effects against the formation of advanced glycation end products (AGEs) and endothelial cell dysfunction. When the effects of TCE on AGE formation and on protein crossing-linking by glycation with D-threose and lens crystallines were examined, TCE showed inhibitory activity in a dose-dependent manner, and the concentration of 1000 µg/mL presented an activity similar to that of 5 mM aminoguanidine as a positive control. Upon investigating the protective activity of TCE against AGE-induced vascular endothelium dysfunction, human umbilical vein endothelial cells (HUVEC) incubated with 100 µg/mL of AGEs had significantly enhanced reactive oxygen species (ROS) formation, whereas the treatment of T. chebula reduced AGE-induced ROS generation. The incubation of HUVEC with 100 µg/mL of AGEs caused a considerable increase in THP-1 monocytic cell adhesion, but this adhesion was reduced by the treatment of TCE. These results suggest that TCE is a potential agent for alleviating diabetic complications.

5‐Hydroxymethylfurfural from Black Garlic Extract Prevents TNFα‐induced Monocytic Cell Adhesion to HUVECs by Suppression of Vascular Cell Adhesion Molecule‐1 Expression, Reactive Oxygen Species Generation and NF‐κB Activation

5-Hydroxymethylfurfural (5-HMF) is a common Maillard reaction product; the reaction occurs during heat-processing and the preparation of many types of foods and beverages. Although 5-HMF has been proposed to have harmful effects, recently, its beneficial effects, including antioxidant, cytoprotective and antitumor effects have become increasingly apparent. It was found recently that a chloroform extract of aged black garlic shows antiinflammatory properties when administered to human umbilical vein endothelial cells (HUVECs). This study investigated the antiinflammatory potential of 5-HMF purified from the chloroform extract of aged black garlic in tumor necrosis factor-α (TNF-α)-stimulated HUVECs. Treatment of HUVECs with 5-HMF strongly suppressed TNF-α-induced cell surface and total protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) as well as their mRNA expression. In addition, 5-HMF significantly inhibited TNF-α-induced reactive oxygen species formation, and markedly reduced THP-1 monocyte adhesion to TNF-α-stimulated HUVECs. Furthermore, 5-HMF significantly inhibited NF-κB transcription factor activation in TNF-α-stimulated HUVECs. The data provide new evidence of the antiinflammatory properties of 5-HMF in support of its potential therapeutic use for the prevention and management of vascular diseases such as atherosclerosis through mechanisms involving the inhibition of VCAM-1 expression and NF-κB activation in vascular endothelial cells.

Telmisartan prevents VCAM-1 induction and monocytic cell adhesion to endothelium exposed to non-uniform shear stress and TNF-α

Atherosclerotic plaques develop at arterial regions subjected to non-uniform shear stress, and are initiated by increased leukocyte-endothelial interactions. Here we applied the in vitro model of arterial bifurcations to investigate whether telmisartan, an anti-inflammatory angiotensin II receptor blocker with PPAR-gamma activating ability, prevents monocyte recruitment by endothelium. Human umbilical vein endothelial cells (ECs) were exposed to 18 h non-uniform shear stress in bifurcating flow-through slides, followed by 2 h stimulation with 2.5 ng/mL TNF-alpha. During flow, cells were treated with telmisartan. To study cell adhesion, ECs were perfused with medium containing THP-1 monocytic cells. Adherent THP-1 monocytes were quantified by light microscopy. Endothelial protein expression was determined by immunofluorescence. Non-uniform shear stress in combination with TNF-alpha dramatically induced monocytic cell recruitment by endothelial cells. In cells treated with telmisartan (0.5-2.5 μmol/L) during exposure to non-uniform shear stress, dose-dependent inhibition of monocytic cell adhesion was observed, with about 45% reduction at 1 μmol/L. This effect was mediated by a significant reduction of endothelial VCAM-1 expression. On the contrary, the induction of E-selectin by TNF-alpha in ECs exposed to non-uniform shear stress was not affected by telmisartan. The inhibitory effect of telmisartan on monocytic cell recruitment and VCAM-1 induction was prevented in the presence of the PPAR-gamma antagonist GW9662. Treatment with telmisartan decreases the TNF-α-induced recruitment of monocytic cells and endothelial expression of VCAM-1 in regions of non-uniform shear stress in vitro. This mechanism can contribute to the beneficial pleiotropic effects of telmisartan in atherosclerosis-prone arterial regions.

Genipin Selectively Inhibits TNF-α-activated VCAM-1 But Not ICAM-1 Expression by Upregulation of PPAR-γ in Human Endothelial Cells

Vascular inflammation process has been suggested to be an important risk factor in the development of atherosclerosis. Recently we reported that induction of peroxisome proliferator-activated receptor-γ (PPAR-γ) selectively inhibits vascular cell adhesion molecule-1 (VCAM-1) but not intercellular cell adhesion molecule-1 (ICAM-1) in tumor necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVEC). In this study, we investigated whether genipin inhibits expression of cellular adhesion molecules, which is relevant to inflammation. Pretreatment with genipin reduced reactive oxygen species (ROS) production and expression of VCAM-1, but not ICAM-1 in TNF-α-activated HUVEC. Genipin dose- and time-dependently increased PPAR-γ expression and inhibited TNF-α-induced phosphorylation of Akt and PKC with different degrees. Finally, genipin prevented TNF-α-induced adhesion of U937 monocytic cells to HUVEC. Taken together, these results indicate that upregualtion of PPAR-γ by genipin selectively inhibits TNF-α-induced expression of VCAM-1, in which regulation of Akt and/or PKC play a key role. We concluded that genipin can be used for the treatment of cardiovascular disorders such as atherosclerosis.

αVβ3-integrin expression through ERK activation mediates cell attachment and is necessary for production of tumor necrosis factor alpha in monocytic THP-1 cells stimulated by phorbol myristate acetate

Macrophages play a key role in inflammation. Activated macrophages express adhesion molecules and produce tumor necrosis factor alpha (TNFα). Integrins are the main adhesion molecules that mediate binding to the extracellular matrix and they are involved in intracellular pathways. In the present study, human monocytic THP-1 cell adhesion to uncoated plastic plate was examined to investigate the regulatory mechanism of TNFα secretion. Addition of phorbol myristate acetate (PMA) for THP-1 cell activation induced cell adhesion in parallel with TNFα production. Among the mitogen-activated protein kinase pathways, the protein kinase C (PKC)–extracellular signal-regulated kinase (ERK) pathway was involved in αVβ3-integrin expression and PMA-induced cell adhesion. Flow cytometry and reverse transcription – quantitative polymerase chain reaction analysis revealed increased expression of matrix-binding integrins including integrin-αVβ3. Blockade of αVβ3-integrin by a specific antibody suppressed cell adhesion and TNFα production. These findings indicate that TNFα production from THP-1 cells is PKC–ERK, αVβ3-integrin and adhesion-dependent and its related pathway could be a target for TNFα-related diseases.

Indoxyl Sulfate Induces Leukocyte-Endothelial Interactions through Up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.

Human Alternatively Spliced Tissue Factor Promotes Monocyte-Endothelial Interactions Via Upregulation of Adhesion Molecules In Microvascular Endothelial Cells

AbstractAbstract 343 Background:Alternatively spliced Tissue Factor (asTF) is a soluble form of Tissue Factor (TF) that lacks the transmembrane domain and exhibits low pro-coagulant activity compared to decrypted full-length TF (Bogdanov et al, Nat Med 2003). Human asTF was recently shown to ligate integrins α6β1 and αVβ3 on endothelial cell surfaces, which triggered neovascularization in vitro, ex vivo, and in vivo in fVIIa- and/or PAR-2 independent fashion (van den Berg et al., PNAS 2009). This principally novel, non-proteolytic biologic activity of asTF was found to activate multiple integrin-linked kinases in human and murine macrovascular endothelial cells (EC), including those comprising the PI3K/Akt pathway. Engagement of PI3K/Akt signaling is known to activate NFkB – a transcription factor involved in upregulation of multiple leukocyte adhesion molecules that play a major role in various disease states, most notably atherogenesis and tumorigenesis. In this study, we sought to determine whether human asTF acts as an agonist on microvascular EC – the endothelial sub-type most relevant to monocyte egress from the circulation. Results:Primary cardiac and retinal human microvascular endothelial cells (micro-EC) expressed α6, β1, and β3 mRNA. Both micro-EC sub-types rapidly adhered to the recombinant N-terminally His-tagged human asTF: a 20+ fold increase in the number of adherent micro-EC was observed as early as 30 min after the cells were added (n=3,100 ng asTF vs BSA, p<0.0001). In both sub-types of micro-EC, anti-β1 integrin antibody completely blocked adhesion to asTF whereas anti-β3 and/or anti-αvβ3 antibody had no effect, likely due to the known scarcity of non-β1 integrins on the surface of human micro-EC (Wilson et al., IOVS 2003). We then examined the changes in gene expression elicited by asTF in micro-EC using Affymetrix Gene 1.0 ST microarrays. A marked upregulation of several cell adhesion molecules (CAMs) was observed in cardiac and retinal micro-EC including VCAM-1 and E-selectin, which was confirmed at the protein level by western blotting. We proceeded to investigate the functional significance of the upregulation of CAMs by performing adhesion assays using pre-labeled THP-1 cells. Under orbital shear stress conditions, a ∼76% increase in THP-1 adhesion was observed for cardiac micro-EC treated with asTF over control (n=5, p<0.001), and a ∼62% for retinal micro-EC (n=5, p<0.005). Addition of polymyxin B and/or non-charged agarose beads elicited no effect, whereas pre-treatment of asTF samples with Ni-charged beads and heat denaturation eliminated the effect, confirming that the observed findings are asTF-specific and not caused by LPS contamination. In the static assay and the laminar flow chamber assay performed under the flow rates found in postcapillary venules, the increase in THP-1 adhesion was 40% (n=3, p<0.001) and 250% (n=3, p<0.05), respectively, for cardiac micro-EC treated with asTF and 83% (n=3, p<0.001) and 290% (n=3, p<0.05), respectively, for retinal micro-EC. While performing laminar flow chamber assays, we observed relocation of some of the adhered THP-1 cells under the EC monolayer, which indicates that asTF may cause monocyte emigration in the absence of a chemokine gradient. To determine whether monocyte egress through asTF-stimulated micro-EC can also occur under a chemokine gradient, we performed transendothelial migration assays using transwell inserts with MCP-1 placed in the lower chamber. Here, we observed that asTF potentiated THP-1 migration through cardiac micro-EC by ∼206% (n=3, p<0.0001), and through retinal micro-EC – by ∼90% (n=3, p<0.005). Conclusions:We show for the first time that (i) human asTF ligates β1 integrin(s) on primary human micro-EC, (ii) this leads to transcriptional upregulation of CAMs, and (iii) this upregulation promotes adhesion and transendothelial migration of monocytic cells under physiologically relevant conditions. We note that the concentrations of asTF used here were in the range found in cervical cancer tissue (van den Berg et al., PNAS 2009). Our results expand the potential scope of asTF's contribution to atherogenesis and tumorigenesis. Evaluation of these findings in vivo is highly warranted as monocyte recruitment triggered by asTF may represent a novel nexus in atherosclerotic progression and/or tumor growth. Disclosures:No relevant conflicts of interest to declare.

Association of Gene-Environment Interactions with Venous Thromboembolism (VTE): A Pathway-Directed Candidate-Gene Case-Control Study

AbstractAbstract 480 Background:While interaction between Factor V Leiden and other VTE risk exposures (i.e., oral contraceptives, hormone therapy, pregnancy, cancer, minor trauma) compound VTE risk, whether other gene-environment interactions are associated with VTE is largely unknown. Objective:To test gene-environment interactions for an association with VTE. Methods:Cases (n=1488) were Mayo Clinic European-American patients of non-Hispanic ancestry with objectively-diagnosed VTE in the absence of active cancer, venous catheter or antiphospholipid antibodies. Controls (n=1439) were Mayo Clinic outpatients without VTE who were frequency-matched on case age, gender, race, MI/stroke status and state of residence. We selected candidate genes relevant to the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways, focusing on platelet, monocyte, neutrophil and endothelial cell agonists, receptors, ligands, signal transduction and adhesion molecules, granule contents and effectors; plasma proteases and inhibitors; matrix metalloproteases; inflammatory cytokines and receptors; estrogen, progesterone and androgen receptors; co-regulators and enzymes related to estrogen metabolism; important enzymes for catechol, homocysteine, thromboxane A2 and prostacyclin biosynthesis and metabolism; and HMG CoA reductase. For these genes (n=750), we selected all non-synonymous coding single nucleotide polymorphisms (SNPs) with minor allele frequency ≥0.5%; the remaining SNPs were selected using an LD tagging algorithm (Carlson et al. AJHG 2004); 479 ancestry-informative markers were also included (total n=13,241 SNPs). Leukocyte genomic DNA was genotyped using a custom Illumina Infinium iSelect platform, including appropriate controls. We tested all pairwise interactions between 12,483 SNPs (12296 autosomal, 187 chromosome X) that passed quality control and each of seven exposures (ever “stress”[defined as ever hospitalized, ever surgery, ever trauma, ever transfemoral procedure or ever leg paresis], ever surgery, ever neurosurgery, ever orthopedic surgery; and among women, ever pregnant, ≥3 pregnancies and ever oral contraceptives/hormone therapy), adjusted for age, gender, MI/stroke status and state of residence, using logistic regression. False discovery rates (q-value) were calculated to estimate the expected fraction of false positive associations due to multiple statistical tests. Results:The mean ± SD case and control ages were 55.3 ± 16.4 and 56.5 ± 15.9 years, respectively, and 51% were female. Analysis of ancestry-informative markers revealed no evidence of population stratification. Among the seven models, a significant interaction (q≤0.05) was found between ever surgery and two genes located on chromosome 1: USF1 (rs2516840, OR=2.1, p=2.0E-06; q=0.02), and F11R (rs790055 &rs790056; OR∼2.29, p∼4E-06; q=0.02); the two F11R SNPs are in complete linkage dysequilibrium (LD), and the USF1 SNP is in moderate LD with the F11R SNPs (r2∼0.6). USF1 encodes for upstream transcription factor 1 which regulates many genes involved in lipid and glucose homeostasis. F11R encodes for the platelet F11 receptor (JAM [junctional adhesion molecule], a cell adhesion molecule important for platelet adhesion to cytokine-stimulated endothelial cells. Conclusion:Interaction between surgery and SNPs within USF1 and F11R are associated with significantly increased risks for VTE. These potential associations require confirmation in future replication studies. Disclosures:No relevant conflicts of interest to declare.

Cobra CRISP Functions as an Inflammatory Modulator via a Novel Zn2+- and Heparan Sulfate-dependent Transcriptional Regulation of Endothelial Cell Adhesion Molecules

Cysteine-rich secretory proteins (CRISPs) have been identified as a toxin family in most animal venoms with biological functions mainly associated with the ion channel activity of cysteine-rich domain (CRD). CRISPs also bind to Zn(2+) at their N-terminal pathogenesis-related (PR-1) domain, but their function remains unknown. Interestingly, similar the Zn(2+)-binding site exists in all CRISP family, including those identified in a wide range of organisms. Here, we report that the CRISP from Naja atra (natrin) could induce expression of vascular endothelial cell adhesion molecules, i.e. intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin, to promote monocytic cell adhesion in a heparan sulfate (HS)- and Zn(2+)-dependent manner. Using specific inhibitors and small interfering RNAs, the activation mechanisms are shown to involve both mitogen-activated protein kinases and nuclear factor-κB. Biophysical characterization of natrin by using fluorescence, circular dichroism, and x-ray crystallographic methods further reveals the presence of two Zn(2+)-binding sites for natrin. The strong binding site is located near the putative Ser-His-Glu catalytic triad of the N-terminal domain. The weak binding site remains to be characterized, but it may modulate HS binding by enhancing its interaction with long chain HS. Our results strongly suggest that natrin may serve as an inflammatory modulator that could perturb the wound-healing process of the bitten victim by regulating adhesion molecule expression in endothelial cells. Our finding uncovers a new aspect of the biological role of CRISP family in immune response and is expected to facilitate future development of new therapeutic strategy for the envenomed victims.

Isoimperatorin, cimiside E and 23-O-acetylshengmanol-3-xyloside from Cimicifugae Rhizome inhibit TNF-α-induced VCAM-1 expression in human endothelial cells: Involvement of PPAR-γ upregulation and PI3K, ERK1/2, and PKC signal pathways

The methanol extract of Cimicifugae Rhizome has been traditionally used in various disorders including inflammation. The aim of the study is to explore whether anti-inflammatory action of 3 active compounds, two triterpenoid glycosides (cimiside E, 23-O-actylshengmanol-3-xyloside) and one furanocoumarin (isoimperatorin), isolated from Cimicifugae Rhizome is related with peroxisome proliferator-activated receptor-γ (PPAR-γ) expression in human umbilical endothelial cell line, EA.hy926 cells. Cell viability and production of reactive oxygen species were performed. In addition, adhesion of monocyte into endothelial cells and western blot for expression of adhesion molecules and signal proteins were investigated in tumor necrosis factor-α (TNF-α)-activated cells. Pretreatment of test compounds significantly reduced reactive oxygen species (ROS) production and expression of vascular cell adhesion molecule-1 (VCAM-1), but not intercellular cell adhesion molecule-1 (ICAM-1). Three compounds all dose-dependently increased not only PPAR-γ expression in EA.hy926 cells but inhibited TNF-α-induced phosphorylation of Akt, extracellular-signal-regulated kinase (ERK) and protein kinase C (PKC) with different specificity. Finally, they prevented TNF-α-induced adhesion of U937 monocytic cells to EA.hy926 cells. The present results show that cimiside E, 23-O-actylshengmanol-3-xyloside, isoimperatorin isolated from Cimicifugae Rhizome selectively inhibits TNF-α-induced expression of VCAM-1 at least by upregulation of PPAR-γ, and signals for ERK1/2, PI3K, and PKC are involved in this effect.

Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-κB signaling pathways

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2 h with diosgenin (0.1–10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.

Inhibition of TNF‐α‐mediated endothelial cell–monocyte cell adhesion and adhesion molecules expression by the resveratrol derivative, trans‐3,5,4′‐trimethoxystilbene

Resveratrol (RSV) has been shown to have anti-inflammatory activity and to have a protective role against atherosclerosis. Here it is shown, for the first time, that its derivative trans-3,5,4'-trimethoxystilbene (TMS) may be a more potent anti-inflammatory agent than resveratrol. A comparative analysis of the inhibitory activities of related stilbenes, resveratrol, TMS and polydatin (PD), on monocyte adhesion to TNF-α-activated endothelial cells showed TMS to be the most effective, with PD being the least effective. RSV and its analogues inhibited, albeit differentially, the protein and mRNA expression levels of inducible cell adhesion molecules, ICAM-1 and VCAM-1, in cultured endothelial cells. The mechanism of the inhibitory effects of these stilbenes on endothelial cell-monocyte cell adhesion can be attributed mainly to inhibition of NF-κB pathway activation. The results demonstrate that all three investigated stilbene compounds, especially TMS, exhibit a potent inhibitory effect on inflammation-induced cell-cell adhesion, expression of adhesion molecules and activation of the NF-κB pathway.

Time-dependent changes in the expression of lymphocyte and monocyte cell adhesion molecules after meals of different composition

The objective of the present study was to compare the acute effect of meals of different composition on the expression of adhesion molecules that play a key role in leucocyte trafficking. A total of twenty apparently healthy subjects randomly consumed three isoenergetic meals 1 week apart: enriched in carbohydrates (CHO), enriched in monounsaturated fat and enriched in saturated fat. Blood samples were obtained before the meals and at 2, 4, 6, 8 and 10 h after meal ingestion. Samples were analysed for LDL resistance to Cu-mediated oxidation and for the surface expression on peripheral blood mononuclear cells (PBMC) of CD62L, CD162, CD11a, CD11b, CD49d and CD54 by flow cytometry. The present results showed that there were no changes in LDL susceptibility to oxidation within and among the meals. After the CHO-enriched meal, there was a time-dependent increased expression of CD162, CD49d, CD11a and CD54 on PBMC that returned to basal values after 8-10 h. These changes were significantly greater than the ones observed after the consumption of the monounsaturated fat- and the saturated fat-enriched meals and were more evident in lymphocytes than in monocytes. In conclusion, acute ingestion of a CHO-enriched meal induces higher increases of lymphocyte activation markers than fat-enriched meals. These results suggest that long-term consumption of CHO-enriched diets may be associated with a sustained pro-inflammatory state.

Preventive effects of chebulic acid isolated from Terminalia chebula on advanced glycation endproduct-induced endothelial cell dysfunction

The aqueous extract of Terminalia chebular fruits was reported to have anti-hyperglycemia and anti-diabetic complication effects. The present study therefore investigated the protective mechanism of chebulic acid, a phenolcarboxylic acid compound isolated from the ripe fruits of Terminalia chebula against advanced glycation endproducts (AGEs)-induced endothelial cell dysfunction. To investigate the protective mechanism of chebulic acid against vascular endothelial dysfunction human umbilical vein endothelial cells (HUVEC) were treated with chebulic acid in the presence/absence of glyceraldehyde-related AGEs (glycer-AGEs). HUVEC incubated with 100 μg/ml of glycer-AGEs had significantly enhanced reactive oxygen species formation, whereas the treatment of chebulic acid dose-dependently reduced glycer-AGE-induced formation to 108.2 ± 1.9% for 25 μM versus 137.8 ± 1.1% for glycer-AGEs treated alone. The transendothelial electrical resistance (TER) value of the glycer-AGEs group was dramatically decreased to 76.9 ± 2.2% compared to the control, whereas chebulic acid treatment prevented glycer-AGE-induced TER change with a value of 91.3 ± 5.3%. The incubation of confluent HUVEC with 100 μg/ml of glycer-AGEs for 24h remarkably increased the adhesion of human monocytic THP-1 cells compared to non-stimulated HUVEC. These increases in HUVEC adhesiveness were dose-dependently reduced by chebulic acid. The present study shows the effects of chebulic acid against the progression of AGE-induced endothelial cell dysfunction suggesting that this compound may constitute a promising intervention agent against diabetic vascular complications.

Kallistatin Inhibits Vascular Inflammation by Antagonizing Tumor Necrosis Factor-α–Induced Nuclear Factor κB Activation

Kallistatin is a plasma protein with anti-inflammatory properties. In this study, we investigated the role and mechanisms of kallistatin in inhibiting endothelial inflammation through its heparin-binding domain. We showed that recombinant wild-type kallistatin dose-dependently competed with tumor necrosis factor (TNF)-alpha binding to TNF-alpha receptor in endothelial cells, whereas kallistatin mutant at the heparin-binding domain had no effect. Kallistatin, but not kallistatin mutant at the heparin-binding domain, abrogated TNF-alpha-induced endothelial cell activation, as evidenced by inhibition of TNF receptor 1-associated death domain protein activation, inhibitor of nuclear factor kappaB-alpha degradation, nuclear factor kappaB translocation, and p38 mitogen-activated protein kinase phosphorylation, as well as cell adhesion molecule and cytokine expression. Moreover, kallistatin, but not kallistatin mutant at the heparin-binding domain, inhibited TNF-alpha-induced human monocytic THP-1 cell adhesion to endothelial cells and prevented vascular endothelial growth factor-induced endothelial permeability. In mice, kallistatin gene delivery prevented vascular leakage provoked by complement factor C5a, whereas delivery of kallistatin heparin mutant gene had no effect. Similarly, gene transfer of kallistatin, but not the kallistatin heparin mutant, inhibited collagen/adjuvant-induced arthritis in rats. These results indicate that kallistatin's heparin-binding site plays an essential role in preventing TNF-alpha-mediated endothelial activation and reducing vascular endothelial growth factor-induced vascular permeability, resulting in attenuation of vascular inflammation in cultured endothelial cells and animal models. This study identifies a protective role of kallistatin in vascular injury, thereby implicating the therapeutic potential of kallistatin for vascular and inflammatory diseases.