Intercellular Adhesion Molecule-1 Protein Expression Research Articles

17β-Estradiol protects against lung injuries after brain death in male rats.

Brain death elicits microvascular dysfunction and inflammation, and thereby compromises lung viability for transplantation. As 17β-estradiol was shown to be anti-inflammatory and vascular protective, we investigated its effects on lung injury after brain death in male rats. Wistar rats were assigned to: sham-operation by trepanation only (SH, n = 7); brain death (BD, n = 7); administration of 17β-estradiol (280 μg/kg, iv) at 60 minutes after brain death (BD-E2, n = 7). Experiments were performed 180 minutes thereafter. Histopathological changes in the lung were evaluated by histomorphometry. Gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and endothelin-1 was measured by real-time polymerase chain reaction. Protein expression of NO synthases, endothelin-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), BCL-2, and caspase 3 was assessed by immunohistochemistry. Cytokines were quantified by enzyme-linked immunosorbent assay. Treatment with 17β-estradiol after brain death decreased lung edema and hemorrhage (p < 0.0001), and serum levels of cytokine-induced neutrophil chemoattractant-1 (CINC-1; p = 0.0020). iNOS (p < 0.0001) and VCAM-1 (p < 0.0001) also diminished at protein levels, while eNOS accumulated (p = 0.0002). However, gene expression of iNOS, eNOS, and endothelin-1 was comparable among groups, as was protein expression of endothelin-1, ICAM-1, BCL-2, and caspase 3. 17β-Estradiol effectively reduces lung injury in brain-dead rats mainly due to its ability to regulate NO synthases. Thus, the drug may improve lung viability for transplantation.

Dose and timing of normal saline resuscitation on endothelial glycocalyx in early septic shock

To observe the effect of different doses and timing of normal saline (NS) resuscitation combined with norepinephrine (NE) on endothelial glycocalyx in rabbits with early septic shock. Thirty New Zealand male rabbits were randomly divided into sham group, model group, 30 mL and 60 mL timely resuscitation groups (30 mL and 60 mL timely group), and 30 mL delayed resuscitation group (30 mL delayed group) with 6 rabbits in each group. The rabbit model of septic shock was reproduced by cecal ligation and puncture (CLP). The rabbits in sham group were only received abdominal cavity open without cecal and ligation. The rabbits in 30 mL and 60 mL timely groups and 30 mL delayed group were intravenous infused with 30 mL/kg or 60 mL/kg NS immediately or 1 hour after model reproduction for 1 hour, and the mean arterial pressure (MAP) was maintained over 75 mmHg (1 mmHg = 0.133 kPa) compared with intravenous pumping of 0.02-0.05 μg×kg-1×min-1 NE followed by 5 mL/h NS infusion till the end of the experiment. The rabbits in sham and model groups were only given 5 mL/h NS. The changes in arterial blood gas before and immediately after resuscitation were observed in three fluid resuscitation groups. The internal jugular vein blood was collected at 0, 3, 6 hours after model reproduction. The levels of syndecan-1 (polysaccharide envelope marker) in plasma were determined by enzyme linked immunosorbent assay (ELISA). The rabbits were sacrificed at 6 hours after model reproduction, and the lung tissue was harvested. Western Blot was used to determine the protein expressions of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase 2 (MMP-2) and syndecan-1. The positive expression of syndecan-1 in lung tissue was observed by immunohistochemical method. (1) Blood gas analysis: compared with the results before resuscitation, the levels of lactic acid (Lac) after resuscitation in three fluid resuscitation groups were significantly decreased, especially in 30 mL timely group; the central venous blood oxygen saturation (ScvO2) was significantly increased, especially in 30 mL delayed group. Oxygenation index (PaO2/FiO2) was improved in 30 mL timely and 30 mL delayed resuscitation groups, which was decreased in 60 mL delayed group. (2) Plasma marker: compared with sham group, plasma syndecan-1 level in model group was significantly increased with a time-dependent manner. Plasma syndecan-1 levels at 3 hours in 30 mL timely and 30 mL delayed groups were significantly decreased as compared with those of model group (ng/L: 138.0±2.4, 139.7±15.7 vs. 161.5±4.1, both P < 0.05), but it was significantly increased at 6 hours in 30 mL delayed group (ng/L: 213.1±19.4 vs. 206.4±15.5, P < 0.05). The plasma syndecan-1 levels at 3 hours and 6 hours in 60 mL timely group were significantly higher than those in model group (ng/L: 233.0±28.9 vs. 161.5±4.1, 252.3±27.2 vs. 206.4±15.5, both P < 0.05). (3) Protein expression in lung tissue: compared with sham group, the protein expressions of ICAM-1 and MMP-2 in lung tissue of model group were significantly increased, and syndecan-1 protein expression was significantly decreased. After 30 mL timely or 30 mL delayed resuscitation, the protein expressions of ICAM-1 and MMP-2 in lung tissue were significantly decreased, and syndecan-1 protein expression was significantly increased, especially in 30 mL timely group, which showed statistical differences as compared with those of model group (ICAM-1 protein: 0.56±0.09 vs. 1.04±0.05, MMP-2 protein: 0.83±0.15 vs. 1.06±0.06, syndecan-1 protein: 2.09±0.08 vs. 0.99±0.03, all P < 0.05). The change tendency of protein expressions in 60 mL timely group was opposite to the other two resuscitation groups. (4) Immunohistochemistry: the positive expression of syndecan-1 in lung tissues was significant in the sham group, and it was lowered in model group. The positive expression of syndecan-1 was increased after 30 mL timely or 30 mL delayed resuscitation, but further weakened in 60 mL timely group. The dose and timing of resuscitation with NS in septic shock can affect pulmonary vascular endothelial glycocalyx function. The timely resuscitation with 30 mL NS in combination with NE plays a protective effect on endothelial cell and glycocalyx. However, NS resuscitation which was not timely or excessive infusion can make the glycocalyx degradation more obvious, resulting in increased endothelial permeability, microcirculation damaged, thus aggravate lung injury.

Mangiferin alleviates hypertension induced by hyperuricemia via increasing nitric oxide releases

Mangiferin, a natural glucosyl xanthone, was confirmed to be an effective uric acid (UA)- lowering agent with dual action of inhibiting production and promoting excretion of UA. In this study, we aimed to evaluate the effect of mangiferin on alleviating hypertension induced by hyperuricemia. Mangiferin (30, 60, 120 mg/kg) was administered intragastrically to hyperuricemic rats induced by gavage with potassium oxonate (750 mg/kg). Systolic blood pressure (SBP), serum levels of UA, nitric oxide (NO), C-reactionprotein (CRP) and ONOO− were measured. The mRNA and protein levels of endothelial nitric oxide synthase (eNOS), intercellular adhesion molecule-1 (ICAM-1), CRP were also analyzed. Human umbilical vein endothelial cells (HUVECs) were used in vitro studies. Administration of mangiferin significantly decreased the serum urate level and SBP at 8 weeks and last to 12 weeks. Further more, mangiferin could increase the release of NO and decrease the level of CRP in blood. In addition, mangiferin reversed the protein expression of eNOS, CRP, ICAM-1 and ONOO− in aortic segments in hyperuricemic rats. The results in vitro were consistent with the observed results in vivo. Taken together, these data suggested that mangiferin has played an important part in alleviating hypertension induced by hyperuricemia via increasing NO secretion and improving endothelial function.

Piper sarmentosum attenuates TNF-α-induced VCAM-1 and ICAM-1 expression in human umbilical vein endothelial cells.

ObjectivesInflammation plays a key role in the pathogenesis of atherosclerosis. Piper sarmentosum is an herb with antioxidant and anti-atherosclerotic activities. The aim of this study was to evaluate the anti-inflammatory properties of an aqueous extract of P. sarmentosum (AEPS) in human umbilical vein endothelial cells (HUVECs).MethodsHUVECs were divided into six groups: control, treatment with 10 ng/ml TNF-α, and co-treatment of 10 ng/ml TNF-α with four different concentrations of AEPS (100, 150, 250, and 300 μg/ml) for 24 h. Subsequently, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression, U937 monocyte cells adhesion, and nuclear factor-kappaB (NF-κB) p65 expression in HUVECs were measured.ResultsTreatment of TNF-α-stimulated HUVECs with AEPS at different concentrations resulted in decreased VCAM-1 and ICAM-1 protein expression in a dose-dependent manner. Furthermore, AEPS also inhibited TNF-α-stimulated U937 monocyte cells adhesion to HUVECs. In addition, AEPS reduced TNF-α-induced NF-κB p65 expression in a dose-dependent manner.ConclusionsThe results indicated that AEPS suppressed TNF-α-induced VCAM-1 and ICAM-1 expression NF-κB signaling.

Effects of thyroid-stimulating hormone on adhesion molecules and pro-inflammatory cytokines secretion in human umbilical vein endothelial cells.

Atherosclerosis is a multifactorial disorder, which affects the arterial wall. It has been reported that, hypothyroidism and thyroid hormone deficiency are related to cardiovascular disorders. Also, endothelial dysfunction plays an essential role in the development of atherosclerosis. We aimed to evaluate the effects of thyroid-stimulating hormone (TSH) on pro-inflammatory tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), angiogenic vascular endothelial growth factor (VEGF) and leukocyte adhesion, intercellular adhesion molecule 1 (ICAM-1) and E-selectin in human umbilical vein endothelial cells (HUVECs). In this study, HUVEC cells were treated with 1 and 2 μM of TSH in different treatment times. The gene and protein expression of ICAM-1, VEGF, and E-selectin were performed by real-time polymerase chain reaction and western blotting, respectively. Likewise, TNF-α and IL-6 protein levels were determined by the ELISA method. VEGF, ICAM-1, and E-selectin as endothelial dysfunction markers and also, TNF-α and IL-6 as pro-inflammatory cytokines were detectable in HUVEC. Besides, the results of this study revealed that TSH treatment down-regulates TNF-α and IL-6. Evaluating the gene and protein expression data revealed the upregulation of ICAM-1, E-selectin, and VEGF in TSH treated cases in different periods of exposure. Considering the multiple actions of TSH, it could be concluded that TSH plays a controversial role in atherogenesis by anti-inflammatory effects and on the other side, angiogenesis and leukocyte adhesion induction which is related to vascular cell proliferation.

CK2α promotes advanced glycation end products-induced expressions of fibronectin and intercellular adhesion molecule-1 via activating MRTF-A in glomerular mesangial cells

Advanced glycation end products’ (AGEs) modification of extracellular matrix proteins induces crosslinking, which results in thickening of the basement membrane and activating several intracellular signaling cascades, eventually promoting the pathological progression of diabetic nephropathy (DN). We have previously confirmed that casein kinase 2α (CK2α) activates the nuclear factor of kappaB (NF-κB) signaling pathway to enhance high glucose-induced expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs). However, to date, the mechanism by which CK2α regulates diabetic renal fibrosis is not fully understood. In view of the regulation of inflammation and fibrosis by myocardin-related transcription factor A (MRTF-A), we are highly concerned whether CK2α promotes AGEs-induced expressions of FN and ICAM-1 in glomerular mesangial cells via activation of MRTF-A, thus affecting the pathogenesis of DN. We found that CK2α and MRTF-A proteins were overexpressed in AGEs-induced diabetic kidneys. Inhibition of CK2α kinase activity or knockdown of CK2α protein expression suppressed the upregulation of FN and ICAM-1 expressions in GMCs induced by AGEs. MRTF-A knockdown compromised the expressions of FN and ICAM-1 in GMCs induced by AGEs. Moreover, inhibition of CK2α kinase activity or knockdown of CK2α protein expression restrained the protein expression and nuclear aggregation of MRTF-A. CK2α interacted with MRTF-A. Furthermore, knockdown of MRTF-A while overexpression of CK2α blocked the upregulation effect of CK2α on the protein expressions of FN and ICAM-1. These findings suggest that CK2α promotes diabetic renal fibrosis via activation of MRTF-A and upregulation of inflammatory genes.

Advanced glycation end-products increase IL-6 and ICAM-1 expression via RAGE, MAPK and NF-κB pathways in human gingival fibroblasts.

Diabetes mellitus (DM) is a risk factor for periodontal diseases and may exacerbate the progression of the pathogenesis of periodontitis. Advanced glycation end-products (AGEs) cause DM complications relative to levels of glycemic control and larger amounts accumulate in the periodontal tissues of patients with periodontitis and DM. In the present study, we investigated the effects of AGEs on the expression of inflammation-related factors in human gingival fibroblasts (HGFs) to elucidate the impact of AGEs on DM-associated periodontitis. HGFs were cultured with or without AGEs. Cell viability was examined, and RNA and protein fractions were isolated from AGE-treated cells. The expression of interleukin (IL)-6, intercellular adhesion molecule-1 (ICAM-1), and the receptor for AGE (RAGE) was investigated using reverse transcription-polymerase chain reaction, quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and reactive oxygen species activity was measured using a kit with 2',7'-dichlorofluorescin diacetate. Human monocytic cells (THP-1) labeled with a fluorescent reagent were co-cultured with HGFs treated with AGEs and IL-6 siRNA, and the adhesive activity of THP-1 cells to HGFs was assessed. The expression of IL-6 and ICAM-1 was examined when HGFs were pretreated with recombinant human IL-6, the siRNAs of RAGE and IL-6, and inhibitors of MAPK and NF-κB, and then cultured with and without AGEs. The phosphorylation of MAPK and NF-κB was assessed using western blotting. AGEs increased the mRNA and protein expressions of RAGE, IL-6, ICAM-1 and reactive oxygen species activity in HGFs, and promoted the adhesion of THP-1 cells to HGFs, but had no effect on cell viability until 72hours. Recombinant human IL-6 increased ICAM-1 expression in HGFs, while the siRNAs of RAGE and IL-6 inhibited AGE-induced IL6 and ICAM1 mRNA expression, and IL-6 siRNA depressed AGE-induced THP-1 cell adhesion. AGEs increased the phosphorylation of p38 and ERK MAPKs, p65 NF-κB and IκBα, while inhibitors of p38, ERK MAPKs and NF-κB significantly decreased AGE-induced IL-6 and ICAM-1 expression. AGEs increase IL-6 and ICAM-1 expression via the RAGE, MAPK and NF-κB pathways in HGFs and may exacerbate the progression of the pathogenesis of periodontal diseases.

Raf Kinase Inhibitor Protein (RKIP) Inhibits Tumor Necrosis Factor-α (TNF-α) Induced Adhesion Molecules Expression in Vascular Smooth Muscle Bells by Suppressing (Nuclear Transcription Factor-κB (NF-kappaB) Pathway.

BackgroundRaf kinase inhibitor protein (RKIP) regulates growth and differentiation and plays a role in key signal transduction cascades in mammalian cells. Nevertheless, the underlying mechanism for which RKIP regulates cell-cell adhesion remains unknown. Our study investigated the function of the RKIP overexpression on adhesion molecules expression induced by tumor necrosis factor (TNF)-α in cultured mouse vascular smooth muscle cells (MOVACs).Material/MethodsThe expression levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were detected by ELISA kit, reverse transcription-PCR, and western blot assays. The protein expression of RKIP, p65, and inhibitor of nuclear factor (NF)-κBα (IκBα) were detected by western blot analysis. The activity of NF-kappaB was determined using a Dual-Luciferase Reporter assay.ResultsThe results showed that MOVACs transfected with pCMV5-HA-RKIP significantly inhibited TNF-α induced mRNA and protein expression of ICAM-1 and VCAM-1. The adhesion of THP-1 cells was also detected and inhibited by pCMV5-HA-RKIP in TNF-α-treated MOVACs. RKIP also suppressed the TNF-α-induced activation of NF-kappaB and the protein expression of phosphorylated IκB-α, and promoted the protein expression of IκB-α and nuclear translocation of p65 NF-kappaB. Furthermore, RKIP and the inhibitor of NF-kappaB (BAY11-7082) reduced the upregulation of ICAM-1 and VACM-1 induced by TNF-α.ConclusionsTaken together, these results suggested that RKIP may inhibit the TNF-α-induced expression of adhesion molecules in MOVACs through inactivation of the NF-kappaB pathway.

Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress

Low shear stress (LSS)-induced endothelial inflammation is the basis for the development of atherosclerosis. However, the mechanism underlying LSS-induced inflammation is not well understood. The angiotensin II type 1 receptor (AT1R), a component of the renin-angiotensin system, participates in atherosclerotic plaque progression. The aim of this study was to investigate the role of AT1R in LSS-induced endothelial activation. Using immunohistochemistry, we noted significant increases in AT1R, vascular endothelial adhesion cell-1 (VCAM1), and intercellular adhesion molecule-1 (ICAM1) expression in the inner curvature of the aortic arch in C57BL/6 mice compared to the descending aorta in these mice. Moreover, western blotting revealed that these LSS-induced increases in AT1R, ICAM1 and VCAM1 expression were time dependent. However, the expression of these proteins was significantly abolished by treatment with the AT1R antagonist Losartan (1μM) or AT1R small interfering RNA (siRNA). AT1R inhibition significantly suppressed extracellular signal-regulated kinase 1/2 (ERK) upregulation, which also resulted in decreases in ICAM1 and VCAM1 protein expression. These findings demonstrate that LSS induces endothelial inflammation via AT1R/ERK signaling and that Losartan has beneficial effects on endothelial inflammation.

Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells.

The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest that huiPSCs exosomes could be used as a natural gene delivery vector to transport therapeutic siRNAs for alleviating inflammatory responses in recipient cells.

Effect of Heparin on the Expressions of Serum Intercellular Adhesion Molecule-1 and Hippocampus S100β Protein after Cardiopulmonary Resuscitation in Rabbits

Objective Use heparin during cardiac arrest (CA) in rabbits and observe the serum intercellular adhesion molecule-1 (ICAM-1) and hippocampal S100β protein expressions after cardiopulmonary resuscitation (CPR). Methods Thirty-two New Zealand rabbits were randomly divided into, Group I, control group; Group II, saline group; Group III, heparin group. Each animal underwent continuous hemodynamic monitoring including mean arterial pressure (MBP), heart rate (HR), and the end-tidal carbon dioxide partial pressure (PetCO2). Twenty-four hours after resuscitation, serum and hippocampal neurons were collected from all animals. Enzyme linked immunosorbent assay was used to detect serum ICAM-1 and immunohistochemistry to detect the S100β protein in hippocampal neurons. According to the rate of positive cells, each hippocampal specimen was categorized into four expression levels. Results The differences in the serum ICAM-1 concentration in the three groups were statistically significant. The expression of S100β protein in the hippocampus showed eight cases in group I at level 1 and none in groups II and III. There was 1 case in group II and 7 cases in group III at level 2; five cases in group II and 2 cases in group III at level 3; 2 cases in group II and 1 case in group III at level 4. The expression strength of S100β protein in the three groups differed significantly (P &lt; 0.05). Conclusions Heparin therapy can reduce the expression of serum ICAM-1 and S100β protein in hippocampal neurons during CPR. It is possible that heparin can have a positive effect on brain protection during CPR.

Quercetin inhibits LPS-induced adhesion molecule expression and oxidant production in human aortic endothelial cells by p38-mediated Nrf2 activation and antioxidant enzyme induction

Atherosclerosis, the underlying cause of ischemic heart disease and stroke, is an inflammatory disease of arteries in a hyperlipidemic milieu. Endothelial expression of cellular adhesion molecules, such as endothelial-leukocyte adhesion molecule-1 (E-selectin) and intercellular adhesion molecule-1 (ICAM-1), plays a critical role in the initiation and progression of atherosclerosis. The dietary flavonoid, quercetin, has been reported to inhibit expression of cellular adhesion molecules, but the underlying mechanisms are incompletely understood. In this study, we found that quercetin dose-dependently (5–20µM) inhibits lipopolysaccharide (LPS)-induced mRNA and protein expression of E-selectin and ICAM-1 in human aortic endothelial cells (HAEC). Incubation of HAEC with quercetin also significantly reduced LPS-induced oxidant production, but did not inhibit activation of the nuclear factor-kappaB (NF-κB). Furthermore, quercetin induced activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) and subsequent mRNA and protein expression of the antioxidant enzymes, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1, and glutamate-cysteine ligase. The induction of Nrf2 and antioxidant enzymes was partly inhibited by the p38 mitogen-activated protein kinase (p38) inhibitor, SB203580. Our results suggest that quercetin suppresses LPS-induced oxidant production and adhesion molecule expression by inducing Nrf2 activation and antioxidant enzyme expression, which is partially mediated by p38; and the inhibitory effect of quercetin on adhesion molecule expression is not due to inhibition of NF-κB activation, but instead due to antioxidant-independent effects of HO-1.

The role of N-glycosylation in high glucose-induced upregulation of intercellular adhesion molecule-1 on bovine retinal endothelial cells.

The development of diabetic retinopathy has been implicated as a consequence of chronic inflammation. Given the role of the intercellular adhesion molecule-1 (ICAM-1) in inflammation, the potential effect of N-glycosylation on the upregulated expression of ICAM-1 at the surface of bovine retinal endothelial cells (BRECs) induced by high glucose concentrations was investigated. Gene and protein expression of ICAM-1 in primary BRECs cultured in medium containing increasing concentrations of mannose or glucose in the presence or absence of tunicamycin were studied with reverse transcription-polymerase chain reaction and Western blot analysis, and the expression level of ICAM-1 at the surface of BRECs was examined with an immunofluorescence analysis. A lectin blot assay with PHA-L was performed to explore the level of N-glycans on cell total proteins or immunoprecipitated ICAM-1 from cells treated or untreated with high glucose. Both the mRNA and protein levels of ICAM-1, as well as the level of ICAM-1 on the cell surface, were significantly upregulated by increasing the concentration of glucose in the culture medium, with a peak concentration of 20mm. Consistent with these results, a dramatic increase in the N-glycosylation of ICAM-1 in BRECs cultured with a high concentration of glucose was observed, which could be partially attenuated by tunicamycin treatment. High glucose-induced upregulation of ICAM-1 on the surface of BRECs could be ascribed to the alterations in its N-glycosylation at least in part, indicating that interference with the glycosylation of ICAM-1 may contribute to improving the efficiency of current therapies with diabetic retinopathy.

Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressionsin rat vascular endothelial cells

Objective The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor- α(TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs).Methods We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF- α+ rosuvastatin (10 μmol/L), (4) TNF- α+ ethanol 0.5%, (5) TNF- α+ yellow wine 0.5%, (6) TNF- α+ ethanol 1.0%, (7) TNF- α+ yellow wine 1.0%, (8) TNF- α+ ethanol 1.5%, and (9) TNF- α+ yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting.Results Compared with the TNF-á group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different.Conclusion Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

Enhancement of ICAM-1 via the JAK2/STAT3 signaling pathway in a rat model of severe acute pancreatitis-associated lung injury.

Acute lung injury (ALI), which is associated with severe acute pancreatitis (SAP), results from damage to the pulmonary microvascular endothelial cells (PMVECs), which in turn leads to high levels of inflammatory cytokines that destroy PMVECs. However, the molecular mechanisms underlying SAP-associated ALI (SAP-ALI) are currently not well understood. Intercellular adhesion molecule-1 (ICAM-1) has been implicated in the persistent migration and accumulation of neutrophils and macrophages, which in turn has been associated with the increased permeability of microvascular endothelial cells. Signal transduction via the Janus kinase-2 (JAK2)/signal transducer and activator of transcription-3 (STAT3) transcription factors has been shown to be involved in inflammation. The present study aimed to investigate the expression levels of ICAM-1 and JAK2/STAT3 signaling components in a rat model of SAP-ALI. SAP was induced in the rat model, and dexamethasone (DEX) was administered to the treatment group. Subsequently, ICAM-1, interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, JAK2, STAT3 and nuclear factor (NF)-κB mRNA expression levels were determined using reverse transcription-polymerase chain reaction; ICAM-1 protein expression levels were determined using western blotting; and IL-6, IL-8 and TNF-α levels were measured via an enzyme-linked immunosorbent assay. In addition, an immunohistochemical analysis of ICAM-1, NF-κB, JAK2 and STAT3 was conducted, and the protein expression and cell morphology of the lungs in all rats was analyzed. ICAM-1 mRNA and protein expression levels were significantly increased following induction of SAP, and were significantly decreased in the DEX-treated group. Furthermore, treatment with DEX significantly reduced serum expression levels of IL-6, IL-8 and TNF-α and decreased expression levels of NF-κB, JAK2 and STAT3 in the lung tissue, as compared with the untreated SAP group. The present study demonstrated that DEX treatment was able to suppress ICAM-1 mRNA and protein expression in a rat model of SAP-ALI via the inhibition of IL-6 and TNF-α-induced JAK2/STAT3 activation; thus suggesting that DEX treatment may be considered a potential strategy in the treatment of patients with SAP-ALI.

Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice.

Matrine is isolated from Sophora flavescens and shows anti-inflammatory effects in macrophages. Here we evaluated matrine's suppressive effects on cyclooxygenase 2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expressions in lipopolysaccharide- (LPS-) stimulated human lung epithelial A549 cells. Additionally, BALB/c mice were given various matrine doses by intraperitoneal injection, and then lung injury was induced via intratracheal instillation of LPS. In LPS-stimulated A549 cells, matrine inhibited the productions of interleukin-8 (IL-8), monocyte chemotactic protein-1, and IL-6 and decreased COX-2 expression. Matrine treatment also decreased ICAM-1 protein expression and suppressed the adhesion of neutrophil-like cells to inflammatory A549 cells. In vitro results demonstrated that matrine significantly inhibited mitogen-activated protein kinase phosphorylation and decreased nuclear transcription factor kappa-B subunit p65 protein translocation into the nucleus. In vivo data indicated that matrine significantly inhibited neutrophil infiltration and suppressed productions of tumor necrosis factor-α and IL-6 in mouse bronchoalveolar lavage fluid and serum. Analysis of lung tissue showed that matrine decreased the gene expression of proinflammatory cytokines, chemokines, COX-2, and ICAM-1. Our findings suggest that matrine improved lung injury in mice and decreased the inflammatory response in human lung epithelial cells.

Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study.

Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1) mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1), in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α), and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (si)RNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

Extracellular Vesicles Derived from HT-29 Colorectal Cancer Cells Induce Endothelial Cell Activation and Procoagulant Activity

Introduction: Hypercoagulability leading to thromboembolic complications is associated with significant morbidity and mortality in patients with colorectal cancer, however the underlying mechanisms are not well understood. Elevated levels of extracellular vesicles (EV), both microparticles (> 100 nm) and exosomes (< 100 nm), circulate in patients with colorectal cancer. While some studies suggest that EV-expressed tissue factor is associated with thrombosis in patients with cancer, overall results are inconsistent. However, EV contain many other mediators such as inflammatory cytokines and microRNAs (miRNA) that may be transferred to other cells and induce phenotypic changes. We hypothesized that EV derived from colorectal cancer cells in vitro would induce endothelial cell activation and promote endothelial cell procoagulant activity.Methods: Human colorectal adenocarcinoma cells (HT-29) were cultured at a density of 1.0×107 cells per T75 flask in EV-free media containing 2% FBS, and after removal of floating cells and cell debris, microparticles were collected by centrifugation at 20,000 x G for 15 minutes; each flask contained approximately 4.0 x 106 annexin V+ microparticles. Exosomes were collected by additional centrifugation of the supernatant at 100,000 x G for 90 minutes. Microparticles and exosomes, at different dilutions, were separately co-cultured with confluent endothelial cell monolayers in 6 or 96-well plates, for 6 hours, using TNF-α (4 ng/ml) as a positive control. Endothelial cell activation was assessed by measuring expression of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), Il-1β and tissue factor by quantitative RT-PCR (qPCR) and immunoblotting. We also analyzed the effects of microparticles and exosomes on endothelial cell procoagulant activity by layering plasma over confluent endothelial cell monolayers. Briefly, after incubating endothelial cell monolayers with microparticles or exosomes (or 4 ng/ml TNFα as a positive control) for 6 hours, monolayers were washed three times with 20mM HEPES buffer (pH 7.4) containing 5mM calcium chloride and then overlaid with 100 µl per well of pooled normal human plasma. Plasma was re-calcified by the addition of 11 ul 200 mM calcium chloride, and plates were then placed in a kinetic microtiter-plate reader (Synergy HT) maintained at 37°C, and fibrin formation measured as an increase in the optical density at 405 nm.Results: Microparticles and exosomes constitutively released by HT-29 cells induced activation of endothelial cells in a concentration-dependent manner. Significant increases in the expression of E-selectin, ICAM-1, VCAM-1, tissue factor and IL-1β mRNA and protein were observed (Figure 1). In general, microparticles were more potent in inducing endothelial cell activation than exosomes when the EV were derived from the same number of HT-29 cells, though quantification of small exosomes is imprecise. Interestingly, while the response of endothelial cells to microparticles and exosomes was generally similar to that of 4 ng/ml TNFα, both types of EV appeared more potent in the induction of endothelial cell tissue factor, particularly at the mRNA level. HT-29 cell-derived EV, both microparticles and exosomes, also significantly shortened the clotting time of recalcified plasma added to endothelial cell monolayers (Figure 2).Conclusions: EV constitutively released from HT-29 cells, both microparticles and exosomes, directly activate cultured endothelial cells leading to increased expression of cell adhesion molecules, elaboration of IL-1β, and expression of tissue factor. The change in endothelial phenotype is not due to simple transfer of biomolecules since EV stimulated changes at the mRNA and protein level. While responses to microparticles and exosomes were similar, some differences were observed, suggesting that different underlying mechanisms may contribute. Endothelial cell activation in response to EV leads to accelerated clotting of plasma on the endothelial cell monolayer. These findings suggest a novel effect of cancer cell-derived EV that may contribute to the hypercoagulability present in patients with malignancy. [Display omitted] [Display omitted] DisclosuresKhorana:Daiichi Sankyo: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Leo Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; sanofi: Consultancy, Honoraria. McCrae:Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy.

Macrophages Are Involved in Gut Bacterial Translocation and Reversed by Lactobacillus in Experimental Uremia.

Uremia causes gut microbiome dysbiosis, which is characterized by a reduction in beneficial bacteria. Intestinal bacterial translocation (BT) contributes to microinflammation in uremia, which is associated with adverse outcomes. Whether macrophages are involved in BT remains unclear. We investigated the involvement of macrophages in BT and microinflammation in uremic rats and whether Lactobacillus LB can influence macrophage activity. Male Sprague-Dawley rats were randomly divided into three groups: sham, uremia, and uremia+probiotic. Macrophages and GFP-labeled tracer bacteria in intestinal and extraintestinal tissues were observed by fluorescence microscopy. The macrophage ultrastructure was examined by transmission electron microscopy. Immunochemistry was used to analyze the expression of cluster of differentiation 11a (CD11a), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1). RT-PCR and Western blot were employed to assess the mRNA and protein expression of early growth response gene 1 (EGR1) and toll-like receptor 4 (TLR4). In uremic rats, the colocalization of GFP-labeled tracer bacteria and macrophages was visible in intestinal and extraintestinal tissues. Compared with the sham group, the uremic macrophages showed fewer cytoplasmic protrusions and pseudopodia. Administration of Lactobacillus LB restored the protrusions and pseudopodia. Compared with the sham group, the uremia group exhibited macrophages with higher staining intensities for CD11a, iNOS, and ICAM-1, and higher mRNA and protein expression of TLR4 and EGR1. Intestinal macrophages in the uremic rats are polarized toward a proinflammatory phenotype, resulting in microinflammation. Macrophages with impaired phagocytic function are associated with BT. Lactobacillus LB reduces BT by enhancing macrophage phagocytosis.

TNF-α mediates PKCδ/JNK1/2/c-Jun-dependent monocyte adhesion via ICAM-1 induction in human retinal pigment epithelial cells.

Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases.