Vascular Cell Adhesion Molecule-1 mRNA Expression Research Articles

Anti-Inflammatory Activity of Ferula assafoetida Oleo-Gum-Resin (Asafoetida) against TNF-α-Stimulated Human Umbilical Vein Endothelial Cells (HUVECs).

Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 μg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 μg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.

Paeoniflorin improves myocardial injury via inhibition of Src/VE-cadherin pathway in septic rats

Objective: To study the protective effect and mechanism of paeoniflorin (pae) on myocardial injury in septic rats. Methods: Sprague-Dawley (SD) rats were randomly divided into 4 groups with 10 rats in each group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml 5% dimethyl sulfoxide (DMSO)solution independently in control group and DMSO group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml pae independently, then with 0.1 ml lipopolysaccharide (LPS) 1 hour later in sepsis group and pae group. Enzyme linked immunosorbent assay (ELISA) was used to detect serum cardiac troponin I (cTnI) levels and myocardial tissue tumor necrosis factor alpha (TNFα), interleukin(IL)-6, IL-1β, chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), vascular cell adhesion molecule 1 (VCAM-1) levels. Evans blue (EB) method was used to detect the EB content of myocardial tissue. HE staining method was used to observe the pathological changes, real-time quantitative polymerase chain reaction (RT-qPCR) to detect mRNA expression levels of the above molecules, and Western-blot to detect vascular endothelium-cadherin (VE-cadherin), phosphorylated p38 mitogen-activated protein kinase (P-p38MAPK), phosphorylated Src protein (P-Src), Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) levels. Results: Compared with control group, cTnI level and the EB content in sepsis group increased significantly, and the myocardial inflammatory cell infiltration was obvious. The cTnI level and EB content in pae group were significantly reduced, and myocardial inflammatory cell infiltration was reduced [cTnI: (227.7±15.9)pg/ml vs. (312.9±17.9)pg/ml;EB: (13.2±2.3)μg/g vs. (23.8±2.9)μg/g; P<0.05]. Compared with control group, the levels of TNFα, IL-6, IL-1β, CXCL1, CXCL2, and VCAM-1 in sepsis group were increased. Compared with sepsis group, the above-mentioned molecular levels of pae group were significantly decreased [TNFα: (63.39±9.55)pg/ml vs. (126.54±19.17)pg/ml ;IL-6: (64.03±8.82)pg/ml vs. (85.60±9.52)pg/ml;IL-1β: (69.52±9.23)pg/ml vs. (130.45±15.10)pg/ml;CXCL1: (2 600.19±379.54)pg/ml vs. (4 903.89±533.42)pg/ml;CXCL2: (93.71±10.83)pg/ml vs. (127.24±13.92)pg/ml;VCAM-1: (112.22±13.49)pg/ml vs. (149.32±15.65)pg/ml, both P<0.05]. RT-qPCR results showed that the mRNA expressions of TNFα, IL-6, IL-1β, CXCL1, CXCL2 and VCAM-1 in the sepsis group were increased compared with the control group; Compared with sepsis group, the IL-6 mRNA (1.271±0.139 vs. 1.920±0.191, P<0.05), IL-1βmRNA (1.180±0.130 vs. 1.817±0.191, P<0.05), VCAM-1 mRNA (1.088±0.144 vs. 1.460±0.166, P<0.05) expression decreased significantly in the pae group. Compared with control group, the levels of P-p38MAPK and P-Src in sepsis group increased, and the level of VE-cadherin decreased. Compared with sepsis group, the levels of p38MAPK and P-p38MAPK in pae group were significantly decreased, and the level of VE-cadherin was increased (p38MAPK/β-actin: 1.125±0.078 vs. 1.520±0.164; P-p38MAPK protein: 1.639±0.133 vs. 2.112±0.222; both P<0.05). Conclusion: Paeoniflorin could improve the permeability of cardiac microvascular endothelium in sepsis rats and inhibit the secretion and expression of inflammation-related proteins and genes, which might be related to the inhibition of Src/VE-cadherin pathway by paeoniflorin.

WITHDRAWN: Differential Effects of Omega-3 Fatty Acids on HO-1, VCAM-1, and Cytotoxicity in Endothelial Cells

<h2>ABSTRACT</h2><h3>Introduction</h3> : Omega-3-fatty acids are considered to have beneficial effects on the initiation and progression of atherosclerosis beyond lowering blood lipids. Clinical trials demonstrated that treatment with high-dose eicosapentaenoic acid (EPA) reduced the incidence of cardiovascular events in at-risk patients, yet not with an EPA and docosahexaenoic acid (DHA) combination treatment. <h3>Objectives</h3> : This <i>in-vitro</i> experiment evaluates molecular mechanisms for the observed differential pleiotropic cardioprotective effects of DHA and EPA on endothelial cells. <h3>Materials and Methods</h3> : Human umbilical vein endothelial cells (HUVEC) were stimulated with 25 ng/ml tumor necrosis factor alpha (TNF-α) and (pre-)treated with 125 µmol/l DHA or EPA. Vascular cell adhesion molecule-1 (VCAM-1) and hemoxygenase-1 (HO-1) mRNA fold change were measured in HUVEC. HUVEC cell viability was determined by MTT assay. <h3>Results</h3> : Pre-treating HUVEC with 125 μmol/l EPA 24 hours before the TNF-α stimulus significantly reduced VCAM-1 mRNA expression by -17.5% (<i>p</i>=.011), yet not DHA (+7.4%, <i>p</i>=.060). Treating HUVEC at the time of the TNF-α stimulus lowered VCAM-1 expression by -22.8% for DHA and -56.7% for EPA (<i>p</i>=.002). Treating HUVEC simultaneous to the TNF-α stimulus increased HO-1 expression for DHA (+571.4%, <i>p</i>=.007) and EPA (+581.4%, <i>p</i>=.003). Repeated treatment of HUVEC with DHA or EPA further reduced VCAM-1 expression, whilst HO-1 expression surged by +3,133.4% for DHA and +919.1% for EPA (<i>p</i>=.002). MTT assays displayed DHA and EPA were cytotoxic on HUVEC beyond concentrations of 250 µmol/l. <h3>Conclusions</h3> : (Pre-)treatment of HUVEC with EPA reduces VCAM-1 expression, which is known to impact the initiation and progression of arteriosclerosis, to a greater extent than DHA. In contrast, DHA displays a greater effect on HO-1 expression than EPA. These results offer potential molecular mechanisms for the clinically observed different pleiotropic effects in cardiometabolic treatment of omega-3 fatty acids.

High-Density Lipoprotein Anti-Inflammatory Capacity and Incident Cardiovascular Events.

The role of high-density lipoprotein (HDL) function in cardiovascular disease represents an important emerging concept. The present study investigated whether HDL anti-inflammatory capacity is prospectively associated with first cardiovascular events in the general population. HDL anti-inflammatory capacity was determined as its ability to suppress TNFα (tumor necrosis factor α)-induced VCAM-1 (vascular cell adhesion molecule-1) mRNA expression in endothelial cells in vitro (results expressed as achieved percent reduction by individual HDL related to the maximum TNFα effect with no HDL present). In a nested case-control design of the PREVEND (Prevention of Renal and Vascular End Stage Disease) study, 369 cases experiencing a first cardiovascular event (combined end point of death from cardiovascular causes, ischemic heart disease, nonfatal myocardial infarction, and coronary revascularization) during a median of 10.5 years of follow-up were identified and individually matched to 369 controls with respect to age, sex, smoking status, and HDL cholesterol. Baseline samples were available in 340 cases and 340 matched controls. HDL anti-inflammatory capacity was not correlated with HDL cholesterol or hsCRP (high-sensitivity C-reactive protein). HDL anti-inflammatory capacity was significantly lower in cases compared with controls (31.6% [15.7-44.2] versus 27.0% [7.4-36.1]; P<0.001) and was inversely associated with incident CVD in a fully adjusted model (odds ratio [OR] per 1 SD, 0.74 [CI, 0.61-0.90]; P=0.002). Furthermore, this association was approximately similar with all individual components of the cardiovascular disease end point. The HDL anti-inflammatory was not correlated with cholesterol efflux capacity (r=-0.02; P>0.05). When combining these 2 HDL function metrics in 1 model, both were significantly and independently associated with incident cardiovascular disease in a fully adjusted model (efflux: OR per 1 SD, 0.74; P=0.002; anti-inflammatory capacity: OR per 1 SD, 0.66; P<0.001). Adding HDL anti-inflammatory capacity improved risk prediction by the Framingham risk score, with a model likelihood-ratio statistic increase from 10.50 to 20.40 (P=0.002). The HDL anti-inflammatory capacity, reflecting vascular protection against key steps in atherogenesis, was inversely associated with incident cardiovascular events in a general population cohort, independent of HDL cholesterol and HDL cholesterol efflux capacity. Adding HDL anti-inflammatory capacity to the Framingham risk score improves risk prediction.

Skeletal muscle-specific forkhead box protein-O1 overexpression suppresses atherosclerosis progression in apolipoprotein E-knockout mice

Calorie restriction (CR) reportedly prevents atherosclerotic diseases. Furthermore, CR induces forkhead box protein-O1 (FOXO-1) expression in the skeletal muscle, altering the character of the skeletal muscle. We previously reported that the change in skeletal muscle character, induced by the overexpression of peroxisome proliferator-activated receptor γ coactivator-1α, suppresses atherosclerotic progression in an atherosclerotic apolipoprotein E-knockout (ApoE-KO) mouse model. Thus, we hypothesized that skeletal muscle alternation induced by FOXO-1 may also have an anti-atherosclerotic effect in ApoE-KO mice. In this study, we investigated whether skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. We generated ApoE-KO/FOXO-1 mice, in which an ApoE-KO mouse was crossbred with a mouse presenting skeletal muscle-specific FOXO-1 overexpression (FOXO-1Tg). The mice were sacrificed at 20 weeks of age, and atherosclerotic plaque area and protein expression in the plaque were measured. Additionally, we measured the tumor necrosis factor α (TNFα)- induced mRNA expression in human umbilical vein endothelial cells (HUVECs), using serum collected from the FOXO-1Tg mice. Accordingly, ApoE-KO/FOXO-1 mice showed a 65% reduced atherosclerotic plaque area when compared with the ApoE-KO mice, with concomitantly reduced vascular cell adhesion molecule-1 (VCAM-1) and macrophage infiltration. As compared to serum from wild-type mice, the serum collected from the FOXO-1Tg mice significantly suppressed the mRNA expression of VCAM-1, an atherosclerosis initiation factor, in TNFα-treated HUVECs. Therefore, these data suggest that skeletal muscle-specific FOXO-1 overexpression suppresses the progression of atherosclerosis in ApoE-KO mice. In part, the CR-induced anti-atherosclerotic effect could be attributed to FOXO-1 upregulation in the skeletal muscle.

Cardio-protective and Anti-atherosclerosis Effect of Crocetin on Vitamin D3 and HFD-induced Atherosclerosis in Rats.

Cardiovascular disease (CVD) is a chronic disease and causes the highest rate of death globally. CVD-related deaths account for 80% of all deaths in low and middle-income countries, such as China. Crocetin (CT), a carotenoid phytoconstituent already confirm their anti-inflammatory and antioxidant effects in various diseases animal models. In the study, we make effort to access the cardio-protective effect of Crocetin against vitamin D3 and high fat induced atherosclerosis in rats and scrutinize the underlying mechanism. Sprague Dawley (SD) rats were used in this study and rats were divided into different groups and high fat diet and vitamin D was used for induction the atherosclerosis. The rats were received oral administration of crocetin (5, 10 and 15 mg/kg) and simvastatin (0.5 mg/kg) until 30 days. At the end of the experimental period, lipid, cardiac markers, anti-inflammatory, antioxidant, pro-inflammatory cytokines and atherogenic index were estimated. The mRNA expression of Intercellular adhesion molecule-1 (ICAM-1), Monocyte Chemoattractant Protein-1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) in aortic tissue of the atherosclerotic rats. Crocetin significantly reduced the aortic membrane thickness and platelet aggregation rates. Crocetin also dose-dependently reduced total cholesterol (TC), very low-density lipoprotein (VLDL), triacylglycerol (TG), low-density lipoprotein (LDL) and augmented the level of high-density lipoprotein (HDL) level. Additionally, Crocetin significantly (p < 0.001) abridged the level of malonaldehyde (MDA) and augmented the level of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione peroxidase (GPx). Furthermore, Crocetin significantly (p < 0.001) dose-dependently reduced the levels of pro-inflammatory cytokines and inflammatory mediators. Crocetin attenuated mRNA expression of VCAM-1, ICAM-1 and MCP-1. Crocetin had anti-atherosclerosis and cardio-protective effects on vitamin D3 and high fat induced atherosclerosis in rats through anti-inflammatory and antioxidant mechanisms.

SDPR/Cavin-2 loss inhibits monocyte adhesion to endothelial cells in abdominal aortic aneurysm via suppressing the expression of adhesion molecules

Abstract Background Abdominal aortic aneurysm (AAA) is a common and life-threatening vascular disease. The initial phase of AAA progression is vascular inflammation. Inflammation sites present adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1). These molecules play a crucial role in recruiting inflammatory cells to endothelial cells through NF-κB signaling. Endothelial cells express serum deprivation response (SDPR)/Cavin-2 localized in caveolae on the cell membrane. Although Cavin-2 is involved in such as cell proliferation, migration, and signal transduction, the role of Cavin-2 in vascular inflammation in the development of AAA is still unclear. Purpose To assess the influence of Cavin-2 deficiency in AAA development and clarify the role of Cavin-2 in the regulation of inflammatory cell adhesion in endothelial cells. Methods CaCl2-induced AAAs were induced by the periaortic application of 0.5 M CaCl2 in male SDPR-knockout (KO) and wild-type (WT) mice at 8–10 weeks of age. Angiotensin II (Ang II)-induced AAAs were created by 4-week-subcutaneous drug infusion in male ApoE-KO and ApoE/Cavin-2-double KO (DKO) mice at 24 weeks of age. Inflammatory response and cell adhesion were evaluated using human aortic endothelial cells (HAECs) and human monocytes (THP-1 cells). Results Six weeks after CaCl2 treatment, Cavin-2 deficiency significantly attenuated the development of AAAs. Elastin degradation was markedly suppressed and F4/80-positive macrophages infiltration in aortic walls were decreased in Cavin-2-KO mice. Although Ang II infusion for 4 weeks formed AAAs in ApoE KO mice and ApoE/Cavin-2-DKO mice, ApoE/Cavin-2-DKO mice exhibited the suppression of AAA formation independently of blood pressure. Immunohistochemical staining showed VCAM-1 expression on endothelial cells was suppressed in ApoE/Cavin-2-DKO mice. Further, in vitro co-culture experiment, the number of THP-1 cells adhered to TNF-treated SDPR-knockdown HAECs was decreased compared with that to control HAECs. Moreover, mRNA expression of VCAM-1 and ICAM-1 was decreased in TNFα-treated SDPR-knockdown HAECSs. Protein expression of VCAM-1 was also suppressed in TNFα-treated SDPR-knockdown HAECSs. The activity of NF-κB p65, an upstream regulator of VCAM-1 and ICAM-1,tended to be suppressed in TNFα-treated SDPR-knockdown HAECs. Conclusion In this study, we revealed that SDPR/Cavin-2 loss attenuated AAA development with the suppression of elastin degradation and macrophage infiltration. Our findings suggest that SDPR/Cavin-2 in the endothelial cells regulates the expression of adhesion molecules via NF-κB signaling and promotes the adhesion and infiltration of inflammatory cells to the aortic wall. Funding Acknowledgement Type of funding source: None

Black mulberry fruit extract alleviates streptozotocin-induced diabetic nephropathy in rats: targeting TNF-α inflammatory pathway.

This study was designed to investigate the effect of Morus nigra fruit extract in retarding the progression of diabetic nephropathy in streptozotocin (STZ)-induced diabetic rats. Diabetic male Wistar rats were injected with black mulberry fruit extract (BMFE) at doses of 150 and 300mg/kg body weight. After 4weeks, microalbuminuria was estimated in addition to serum concentrations of glucose, insulin, creatinine and albumin. The study revealed a significant amelioration of all the measured parameters in diabetic animals. In addition, MDA, lipid peroxide levels and catalase activity were also improved. The histopathological examination of kidney tissues revealed significant improvement of the pathological changes and glomerular sclerosis in diabetic rats treated with BMFE. Treated rats showed downregulation of TNF-α, vascular cell adhesion molecule-1 (VCAM-1) and fibronectin mRNA expression. The ameliorative effect of BMFE on diabetic nephropathy is not only through its potent antioxidant and hypoglycaemic effects but also through its downregulation of TNF-α, VCAM-1 and fibronectin mRNA expression in renal tissues of diabetic-treated rats. Therefore, BMFE as dietary supplement could be a promising agent in improving diabetic nephropathy.

Expression and pathogenesis of VCAM-1 and VLA-4 cytokines in multiple myeloma.

ObjectiveThe objective of this study is to investigate the expression of Vascular cell adhesion molecule-1 (VCAM-1) and very late appearing antigen-4 (VLA-4) cytokines in MM (multiple Myeloma).MethodForty patients with MM are selected as the experimental group and 30 healthy persons as the control group. Flow cytometry is used to detect the expression of VCAM-1 (CD106), VLA-4 (CD49d), CD38 and CD138 antigens in experimental group and control group. ELISA (Enzyme Linked Immunosorbent Assay) is used to detect the concentration of VCAM-1 in serum of experimental group and control group. RT-PCR is used to detect the expression of VCAM-1.ResultsThe positive rate and antigen expression rate of VACM-1 antigen in the experimental group were significantly higher than those in the control group (P < 0.05). There were statistical differences of VLA-4 and VCAM-1 antigens between the initial diagnosis group and the relapse/refractory group, and between the relapse/refractory group and the platform stage group (P < 0.05). There were significant differences between VLA-4 antigen and VACM-1 antigen, phase I and phase II, and between phase I and phase III (P < 0.05). The concentration of VCAM-1 and the expression of VCAM-1 mRNA in the experimental group were significantly higher than (P < 0.01). In the different stages of ISS (International Staging System) and different disease groups in the experimental group, the concentration of VCAM-1 and the expression level of VCAM-1 mRNA are significantly different among the three groups of stage I, II and III (P < 0.01). There is a significant difference between the initial diagnosis group, the relapse/refractory group and the platform group (P < 0.05).ConclusionThere are abnormal expressions of adhesion molecules VCAM-1 and VLA-4 in multiple myeloma patients, which are related to ISS staging.

Effects of in‐vitro exercise‐induced endothelial shear stress on adhesion molecule gene expression

BackgroundExercise‐Induced Shear Stress (ESS) is characterized as a mechanical stimulus effecting cellular morphology and physiology. Varying ESS intensity elicit different response mechanisms through cellular signaling pathways. Higher intensity ESS enhances the expression of genes involved in protection against atherosclerosis. Adhesion molecules expression is common in atherosclerosis. Therefore, the purpose of this study was to determine the effects of different intensities of pulsatile ESS on vascular cell adhesion molecule‐1 (VCAM1) and intercellular adhesion molecule (ICAM1) gene expression.MethodsCommercially available human carotid artery endothelial cells (Sigma Aldrich) were cultured until 95–100% confluence and then exposed to pulsatile resting ESS (18 dynes/cm2) for 5 hours, followed by 1 hour low‐intensity ESS (35 dynes/cm2), or high‐intensity ESS (80 dynes/cm2) on parallel fluidic units (Ibidi, USA). Ibidi Pump Control Software (version 1.5.4, Ibidi, USA) was used to control shear stress intensity. mRNA was extracted and expression of VCAM1 and ICAM1 was assessed using qRT‐PCR. Samples were run in Changes in gene expression were calculated using the ΔΔ‐CT method, using GAPDH as the normalizing control gene. One‐way ANOVA was used to asses statistically significant differences (p &lt; 0.05) using Prism version 8 (Graphpad).ResultsThe mRNA expression of VCAM1 and ICAM1 significantly increased (p &lt;0.01). 6 and 8 folds respectively after exposure to low‐intensity ESS. Furthermore, high‐intensity ESS did not significantly increased the expression of VCAM1 and ICAM1.ConclusionLow‐intensity pulsatile exercise‐induced shear stress improves endothelial function in‐vitro through the increase in adhesion molecule’s gene expression.

Effects of hydrogen sulphide on oxidative stress, inflammatory cytokines, and vascular remodelling in l-NAME-induced hypertension.

This study was designed to evaluate the protective effects of hydrogen sulphide (H2 S) against NG-Nitro l-Arginine Methyl Ester (l-NAME)-induced hypertension and its possible effects on the inflammatory process, oxidative stress, and vascular remodelling in rats. Forty male Wistar Albino rats were assigned to four equal groups: the control group, the H2 S control group, the hypertensive group, and the treated group, which received concomitant treatment with sodium hydrosulphide (NaHS) and l-NAME. Systolic blood pressure (SBP) was measured weekly. Serum levels of nitric oxide (NO), total peroxide, and total antioxidant capacity (TAC) were measured and the oxidative stress index (OSI) was calculated. Aortic weight and length were measured and the aortic weight/length ratio determined. Aortic fold expression of interferon-γ (IFN-γ) and vascular cell adhesion molecule-1 (VCAM-1) mRNA was measured using qPCR. Aortic media thickness and elastin content were measured morphometrically. l-NAME administration increased SBP, serum levels of total peroxide and OSI, but reduced serum levels of NO and TAC. Aortic fold expression of IFN-γ and VCAM-1 mRNA, aortic weight, aortic weight/length ratio, aortic media thickness, and elastin area percentage were increased in the hypertensive group. Concurrent administration of l-NAME and H2 S attenuated these changes. Thus, H2 S could attenuate the increase in ABP through restoration of the NO level, reduction in the oxidative state, and attenuation of the inflammatory process, thereby reduced vascular remodelling.

Liraglutide prevents high glucose-induced adhesion of endothelial cells to monocyte by targeting IKK/NF-κB

Objective To investigate the inhibition effect of liraglutide (lira) on high glucose-induced adhesion of endothelial cells to monocyte. Methods Human umbilical vascular endothelial cells (HUVECs) induced by high glucose (20 mmol/L) were incubated with different concentrations of liraglutide (0, 0.3, 3, 30 nmol/L) for different time. THP-1 cells were pre-labeled with Calcein-AM and then incubated with HUVECs for 1 h, M199 medium were used for washing the nonadherent cells for 3 times, and the adhesion of HUVECs to THP-1 cells were measured by Fluorence microplate reader; real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) were employed to detect the mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), respectively. Western blot was used to measure the content of IκBα, p-IκBα and the distribution of p65 in plasma and nuclear. GST-IκBα was introduced as substrate to test the activity of IκB-kinase (IKK). Results The adhesion of HUVECs to THP-1(P<0.01), the mRNA and protein expression of VCAM-1 and ICAM-1(P<0.05, P<0.01), the phosphorylation of κBα (P<0.05, P<0.01), the translocation of p65 subunit from plasma to nuclear and the activity of IKK (P<0.01) in HUVECs were all elevated by stimulation with 20 mmol/L glucose, and the content of IκBα was decreased accordingly (P<0.01). Pre-incubation with lira could reverse the above effect of high concentration of glucose. Conclusions Lira could reduce VCAM-1 and ICAM-1 expression through IKK/NF-κB pathways in 20 mmol/L glucose-induced HUVECs, which finally suppress THP-1-HUVECs adhesion. Key words: Liraglutide; Endothelial cells; Monocytes; Cell adhesion; In vitro

Effect of macrophages on the expression of vascular cell adhesion molecule 1 in ovarian carcinoma cells and its mechanism

Objective To explore the effect of macrophages on the expression of vascular cell adhesion molecule 1 (VCAM1) in ovarian carcinoma cells and its mechanism. Methods Phorbol ester and lipopolysaccharide were used to activate the monocyte THP-1 that would become macrophages . Enzyme linked immunosorbent assay (ELISA) was used to detect the cytokines level in the supernatant of macrophages. The effect of macrophages' supernatant on VCAM1 mRNA expression of ovarian HEY and IGROV1 carcinoma cells was detected by using quantitative real-time and polymerase chain reaction (qRT-PCR). Western blot was used to detect the effect of macrophages' supernatant on VCAM1 protein expression of ovarian carcinoma cells with VCAM1 over expression (HEY-VCAM1 and IGROV1-VCAM1). Dual-luciferase report gene assay was used to detect the effect of macrophages' supernatant and the cytokines on promoter transcriptional activity in different truncations of human embryonic kidney cells HEK293T VCAM1 gene. Results Compared with the supernatant of THP-1 cells, the release number of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-12 in supernatant from macrophages was increased (all P < 0.05), and IL-10 was decreased (t=3.841, P=0.019). The levels of VCAM1 mRNA in HEY and IGROV1 cells were upregulated by macrophages' supernatant and 1 ng/ml TNF-α, and macrophages' supernatant could promote the expression levels of VCAM1 protein in HEY-VCAM1 cells and IGROV1-VCAM1 cells. Compared with the empty vector (pGV354) control group [(8.6±0.2)×10-3 relative light unit (RLU)], the reporter gene luciferase activity of human embryonic kidney cells HEK293T VCAM1 gene promoter region were upregulated by supernatant from macrophages located at -1 641 bp to +12 bp including the transcription binding site of AP-1 [(109.4±3.4)×10-3 RLU], and there was a significant difference (t=29.42, P < 0.001). Compared with the negative control group untreated by cytokine [(21.0±0.5)×10-3 RLU], 100 ng/ml TNF-α could promote the transcriptional activity of -1641 bp to +12 bp promoter of VCAM1 in HEK293T cells [(23.4±0.4)×10-3 RLU; t=4.134, P=0.001]. 150 ng/ml IL-6 had no effect on the transcriptional activity of the promoter [(21.4±1.0)×10-3 RLU; t=0.328, P=0.708]. 5 ng/ml IL-12 inhibited the transcriptional activity of the promoter [(14.3±1.0)×10-3 RLU; t=6.390, P < 0.001]. Conclusion Macrophages can promote VCAM1 expression in ovarian carcinoma cells by secreting inflammatory factors like TNF-α to affect VCAM1 promoter region containing AP1 transcription binding site and can promote VCAM1 mRNA expression in ovarian cancer cells. Key words: Ovarian neoplasms; Macrophages; Cytokines; Vascular cell adhesion molecule 1

82. Disulfiram inhibits placental sFLT-1 secretion

Introduction Preeclampsia is associated with elevated placental secretion of the anti-angiogenic factor sFLT-1. Surprisingly, little is understood about its regulation, identifying such mechanisms may uncover therapeutic leads. Objectives Our objective was to determine the effects of disulfiram, a 20S subunit proteasome inhibitor, on placental secretion of sFLT-1 and endothelial dysfunction. Methods Primary trophoblast were isolated from normotensive term placenta, treated with disulfiram and mRNA expression of sFLT-1 variants and protein secretion assessed. To assess the effects of disulfiram on the 20S subunit of the proteasome, primary trophoblast were treated with disulfiram in the presence of Suc-LLVY-AMC fluorogenic substrate. Next, we investigated the effect of disulfiram on endothelial dysfunction using isolated primary human umbilical vein endothelial cells (HUVECs) treated with 5% preeclamptic serum +/− disulfiram. The effect of disulfiram on markers of endothelial dysfunction were measured via qRT-PCR for vascular cell adhesion molecule-1 (VCAM-1) and adhesion of fluorescently labelled peripheral blood mononuclear cells (PBMCs) to HUVECs. Results Disulfiram significantly reduced mRNA expression of the sFLT-1 variants, sFLT-1-i13 and sFLT-e15a. This translated into a significant and dose dependent reduction in the protein secretion of sFLT-1. In primary trophoblast disulfiram did not inhibit the 20S subunit of the proteasome, evidenced by no change in cellular fluorescence following addition of Suc-LLVY-AMC. Treating primary HUVECs with serum from preeclamptic women induced endothelial dysfunction with significantly increased mRNA expression of VCAM-1 as well as the adhesion of PBMCs to HUVECs. Disulfiram reduced VCAM-1, with expression reduced below control levels, however, did not alter the adhesion of PBMCs to primary HUVECs. Conclusions We have identified disulfiram to quench placental sFLT-1 secretion via a mechanism independent of inhibiting the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits sFLT-1 secretion may assist in identifying therapeutic targets. Interestingly, disulfiram also reduced markers of endothelial dysfunction.

Disulfiram inhibits placental soluble FMS-like tyrosine kinase-1 and soluble endoglin secretion independent of the proteasome.

Preeclampsia is associated with intermittent placental hypoxia, inflammation and the release of antiangiogenic factors, namely sFLT-1 and sEng. These factors cause maternal endothelial dysfunction and the manifestation of clinical disease. Disulfiram is a dehydrogenase inhibitor used to treat alcoholism and has been suggested as a proteasome inhibitor. Inhibiting the proteasome has been previously shown to reduce FLT-1 gene expression. Thus, we aim to investigate whether disulfiram alters the secretion of sFLT-1 and sEng and reduces endothelial dysfunction. METHODS AND RESULTS: We assessed the effects of disulfiram on primary cytotrophoblast and human umbilical vein endothelial cells (HUVECs). Disulfiram significantly reduced mRNA expression of membrane bound FLT-1 and sFLT-1 variants in primary cytotrophoblasts, which translated into a significant reduction in the protein secretion of sFLT-1. Additionally, sFLT-1 was reduced in primary HUVECs treated with disulfiram, whilst sEng was only reduced in primary cytotrophoblasts. Next, we investigated the effect of disulfiram on endothelial dysfunction using primary HUVECs treated with 5% preeclamptic serum ± disulfiram. Serum from preeclamptic women induced endothelial dysfunction evidenced by increased mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs. The addition of disulfiram reduced VCAM-1 mRNA expression, however did not affect the adhesion of PBMCs to endothelial cells. Lastly, we assessed the effects of disulfiram on the 20S subunit of the proteasome and found disulfiram did not inhibit this subunit in either primary cytotrophoblast or HUVECs. CONCLUSIONS: Disulfiram quenches sFLT-1 and sEng via mechanisms independent of the 20S subunit of the proteasome. Understanding of the mechanisms by which disulfiram inhibits antiangiogenic secretion may reveal insights into the pathogenesis and potential therapeutic targets for preeclampsia.

Alterations in intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in human endothelial cells.

Alterations of Endothelial cells (ECs) play a critical role in different pathogenesis of many serious human diseases, and dysfunction of the vascular endothelium is an indicator for human disorders. Endothelial dysfunction is considered to be an early indicator for atherosclerosis, which is characterised by overexpression of adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Hydrogen peroxide (H2O2) released via neutrophils is an important mediator of endothelial cell function. Ambient production of superoxide anion (O2-) and subsequently H2O2 at low levels is critical for regulating endothelial cell functions and proliferation. In this study, we investigated the effects of H2O2 on the expression of adhesion molecules VCAM-1 and ICAM-1 in cultured human umbilical vein endothelial cells (HUVECs). Intracellular superoxide anion production was detected by using p-Nitro Blue Tetrazolium (NBT) assay. Our results showed that administration of 100μM of H2O2 on HUVECs for 2, 6, 12 and 24 h induced a time-dependent increase in ICAM-1 and VCAM-1 mRNA and protein expression levels with a significant increase observed from 6 h. HUVECs exposed to H2O2 exhibit increased O2-, suggesting that H2O2 induced oxidative stress may be a reasonable for atherosclerosis. This increase can be reduced by the flavonoid, N-acetyl cysteine (NAC). The modulation of endothelial cell function through this mechanism may underlie the contribution of H2O2 to the development of vascular disease.

Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G

Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1β-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.

High-density lipoproteins (HDL) composition and function in preeclampsia.

To evaluate (a) the properties of high-density lipoproteins (HDL)/cholesterol, which include apolipoprotein A-1 (ApoA1) and paraoxonase1 (PON1), both are negative predictors of cardiovascular risk and (b) HDL function, among women with preeclampsia (PE). PE is a multi-system disorder, characterized by onset of hypertension and proteinuria or other end-organ dysfunction in the second half of pregnancy. Preeclampsia is associated with increased risk for later cardiovascular disease. The inverse association between HDL, cholesterol levels and the risk of developing atherosclerotic cardiovascular disease is well-established. Twenty-five pregnant women [19 with PE and 6 with normal pregnancy (NP)] were recruited during admission for delivery. HDL was isolated from blood samples. PON1 activity and HDL were analyzed. An in vitro model of endothelial cells was used to evaluate the effect of HDL on the transcription response of vascular cell adhesion molecule-1 (VCAM-1) and endothelial nitric oxide synthase (eNOS) mRNA expression. PON1 activity (units/ml serum) was lower in the PE group compared to normal pregnancy (NP) (6.51 ± 0.73 vs. 9.98 ± 0.54; P = 0.015). Increased ApoA1 was released from PE-HDL as compared to NP-HDL (3.54 ± 0.72 vs. 0.89 ± 0.35; P = 0.01). PE-HDL exhibited increased VCAM-1 mRNA expression and decreased eNOS mRNA expression on TNF-α stimulated endothelial cells as compared to NP-HDL. HDL from women with PE reduced PON1 activity and increased ApoA1 release from HDL particles. This process was associated with increased HDL diameter, suggesting impaired HDL anti-oxidant activity. These changes might contribute to higher long-term cardiovascular risks among women with PE.

VCAM1-targeted RNA interference inhibits the proliferation of human oral squamous carcinoma HN12 cells.

In the present study, RNA interference (RNAi) was used to investigate the effect of vascular cell adhesion molecule 1 (VCAM1) silencing on the proliferation of human oral squamous carcinoma HN12 cells. HN12 cells were divided into three groups: The untreated blank control cell group (CK), the negative control group transfected with non-homologous vector (NC) and the positive group transfected with the target sequence VCAM1 small hairpin RNA (KD). Reverse-transcription polymerase chain reaction and western blot analysis were used to examine the effects of VCAM1-knockdown on the mRNA expression of VCAM1 and subsequent protein expression. Furthermore, the HN12 cell growth inhibition rate was detected using the cell counting kit-8 method. The VCAM1-targeted lentiviral vector RNAi significantly inhibited VCAM1 mRNA, and subsequent protein, expression. Compared with the NC group, the VCAM1 gene knockdown efficiency was ~85%, while the expression level of VCAM1 protein was reduced by ~74% in KD group cells. In addition, cell growth was significantly inhibited in the KD group, with a growth inhibition rate of ~34%. Therefore, this targeted lentiviral vector RNAi effectively inhibited VCAM1 gene, and subsequent protein, expression, as well as the proliferation of oral squamous carcinoma cells. These results may provide an experimental reference for the diagnosis and treatment of oral squamous cell carcinoma.

MiR-142-5p in Bone Marrow-Derived Mesenchymal Stem Cells Promotes Osteoporosis Involving Targeting Adhesion Molecule VCAM-1 and Inhibiting Cell Migration.

Osteoporosis is a systemic bone metabolic disease that is highly prevalent in the elderly population, particularly in postmenopausal women, which results in enhanced bone fragility and an increased susceptibility to fractures. However, the underlying molecular pathogenesis mechanisms still remain to be further elucidated. In this study, in a rat ovariectomy- (OVX-) induced postmenopausal osteoporosis model, aberrant expression of a microRNA miR-142-5p and vascular cell adhesion molecule 1 (VCAM-1) was found by RNA sequencing analysis and qRT-PCR. Using a dual-luciferase reporter assay, we found that miR-142-5p can bind to and decrease expression of VCAM-1 mRNA. Such reduction was prohibited when the miR-142-5p binding site in VCAM-1 3′UTR was deleted, and Western blotting analyses validated the fact that miR-142-5p inhibited the expression of VCAM-1 protein. Bone marrow-derived mesenchymal stem cells (BMMSCs) transfected with miR-142-5p showed a significantly decreased migration ability in a Transwell migration assay. Collectively, these data indicated the important role of miR-142-5p in osteoporosis development involving targeting VCAM-1 and inhibiting BMMSC migration.