1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Suppression of Tie-1 in endothelial cells in vitro induces a change in the genome-wide expression profile reflecting an inflammatory function

Tie-1 is an endothelial specific receptor tyrosine kinase that is upregulated in diseases such as atherosclerosis and rheumatoid arthritis. We recently demonstrated that Tie-1 induced a proinflammatory response when overexpressed in endothelial cells. Here, we used a complementary approach and suppressed endogenous Tie-1 expression in endothelial cells to examine its function by microarray analysis. Tie-1 appeared to govern expression of many genes involved in inflammation. Expression knockdown of Tie-1 significantly reduced endothelial conditioned medium ability to stimulate MCP-1 production in U937 cells. Collectively, our results support the notion that Tie-1 has an inflammatory function in endothelial cells.     

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.     

In vitro endothelial potential of human UC blood-derived mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (MSC) possess powerful ex vivo expansion and versatile differentiation potential, placing themselves at the forefront of the field of stem cell-based therapy and transplantation. Of high clinical relevance is the endothelial differentiation potential of MSC, which can be used to treat various forms of ischemic vascular disease. METHODS: We investigated whether human umbilical cord blood (UCB)-derived MSC are able to differentiate in vitro along an endothelial lineage, by using flow cytometry, RT-PCR and immunofluorescence analyzes, as well as an Ab array method. RESULTS: When the cells were incubated for up to 3 weeks in the presence of VEGF, EGF and hydrocortisone, they began to express a variety of endothelial lineage surface markers, such as Flk-1, Flt-1, VE-Cadherin, vWF, VCAM-1, Tie-1 and Tie-2, and to secrete a specific set of cytokines. Differentiated cells were also found to be able to uptake low-density lipoprotein and form a tubular network structure. DISCUSSION: These observations have led us to conclude that UCB-derived MSC retain endothelial potential that is suitable for basic and clinical studies aimed at the development of vasculature-directed regenerative medicine.     

A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

Insulin administration in the mild hyperglycaemia changes expression of proinflammatory adhesion molecules on human aortic endothelial cells

An overexpression of cell adhesion molecules (CAMs) on the surface of endothelial cells is one of the first steps in a high glucose-mediated endothelial dysfunction in diabetic patients. The effect of insulin administration in the condition of elevated glucose concentration on the E-selectin, intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) expression on human aortic endothelial cells (HAEC) was investigated. Cells were cultured for 4 h in a medium supplemented with homocysteine (7 pM) and different concentration of glucose (5.5, 8.0, 12.0 and 16.5 mM respectively) with or without insulin (1 mlU/mL) addition. Expression of CAMs was analysed by flow-cytometry using monoclonal antibodies. Controls were CAMs expression in the medium with a corresponding glucose concentration. Obtained results show that short-term exposure of HAECs to moderate high glucose concentrations results in increased expression of E-selectin (2-fold), VCAM-1 (3-fold) and ICAM-1 (47%). At the same time, HAEC grown with 12 mM glucose expressed lesser E-selectin and, more ICAM-1 (for 64%) and VCAM-1 (41%) molecules. 16.5 mM glucose decreased expression of all investigated adhesion molecules. Addition of insulin was not changed expression of CAMs in a medium with 5.5 mM glucose. In conditions of elevated glucose concentration (12 mM), addition of insulin significantly dropped E-selectin (27%) and increased VCAM-1 (23%) expression. In conclusion, moderate elevated glucose concentration increased expression of cell adhesion molecules on HAEC. Insulin administration in the mild hyperglycaemia reduces an expression of the proinflammatory adhesion molecule E-selectin which could contribute in deceleration of macrovascular complications development in diabetic patients.     

Development of a disease-specific model to evaluate endothelial dysfunction in patients with diabetes mellitus

Diabetic patients have an increased cardiovascular risk. We propose to characterize the endothelial dysfunction in a disease-specific in vitro model. Human saphenous vein endothelial cells (HSVEC) were isolated from coronary artery bypass patients without and with non-insulin-dependent diabetes mellitus. Growth kinetics and proinflammatory responses (expression of adhesion molecules, cytokines) were documented under non-stimulating conditions. Diabetic HSVEC showed delayed growth kinetics with reduced cell densities of about 40%. During exponential growth of diabetic EC, the surface expression of adhesion molecules was increased 10-fold (p< or =0.05). However, in a monolayer the expression adapted to low levels of non-diabetic EC. In addition, diabetic EC produced significantly more soluble E-selectin, VCAM-1, IL-6 and MCP-1. Our results suggest a link between the pathologically proinflammatory basic state of diabetic EC and the endothelial dysfunction in diabetic disease. Therefore, this in vitro model could be used for investigating early dysfunction and environmental effects on pathological endothelium.     

NF-kappaB activation for constitutive expression of VCAM-1 and ICAM-1 on B lymphocytes and plasma cells.

Cytokine stimulation can activate NF-kappaB that triggers inducible expression of E-selectin, VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intercellular Cell Adhesion Molecule-1) in endothelial cells. In the previous study, we have shown that B lymphocytes and plasma cells can express E-selectin by constitutive activation of NF-kappaB. Here we show that human B lymphocytes and ARH-77 plasma cells expressed VCAM-1 and ICAM-1 in a cytokine dispensable mechanism. NF-kappaB antagonists could inhibit their expressions in ARH-77 cells. The activities of NF-kappaB for VCAM-1 and ICAM-1 promoters prior to cytokine stimulation were detected in ARH-77 cells using electrophoretic mobility shift assays. Again, NF-kappaB antagonists could abrogate these promoter activities. Taken together, our results demonstrate that NF-kappaB activation is the underlying molecular mechanism for constitutive expression of E-selectin, VCAM-1, and ICAM-1 on human B lymphocytes and plasma cells.     

Expression of Tie-2 by human monocytes and their responses to angiopoietin-2

Angiopoietins 1 and 2 bind to Tie-2 expressed on endothelial cells and regulate vessel stabilization and angiogenesis. Tie-2(+) monocytes have been shown to be recruited to experimental tumors where they promote tumor angiogenesis. In this study, we show that 20% of CD14(+) human blood monocytes express Tie-2, and that these cells coexpress CD16 (FcgammaRIII) and are predominantly CD34 negative. Ang-2 is up-regulated by endothelial cells in malignant tumors and inflamed tissues, so our finding that Ang-2 is a chemoattractant for human Tie-2(+) monocytes and macrophages, suggests that it may help to recruit and regulate their distribution in such tissues. Ang-2 was also found to markedly inhibit release of the important proinflammatory cytokine, TNF-alpha, by monocytes in vitro. Following extravasation of monocytes, and their differentiation into macrophages, many accumulate in the hypoxic areas of inflamed and malignant tissues. Ang-2 is known to be up-regulated by hypoxia and we show that monocytes and macrophages up-regulate Tie-2 when exposed to hypoxia. Furthermore, hypoxia augmented the inhibitory effect of Ang-2 on the release of the anti-angiogenic cytokine, IL-12 by monocytes. In sum, our data indicate that Ang-2 may recruit Tie-2(+) monocytes to tumors and sites of inflammation, modulate their release of important cytokines and stimulate them to express a proangiogenic phenotype.     

The effect of metronidazole on stimulation of adhesion molecule expression on the surface of vascular endothelial cells by Bacteroides fragilis endotoxins and enterotoxin]

The influence of metronidazole on the level of expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of vascular endothelial cells activated with B. fragilis endotoxins and enterotoxin was examined. Three enterotoxigenic (ETBF) strains and one nonenterotoxigenic (NTBF) strain were used for lipopolysaccharide extraction. Enterotoxin was prepared from the culture supernatant of the reference B. fragilis ATCC 43858 strain. Expression of adhesion molecules on vascular endothelial cells (HMEC-1 cell line) was determined after their stimulation with bacterial compounds at the concentration of 10 micrograms/ml in the presence of metronidazole at the concentration of 4 micrograms/ml. Endothelial cells were activated for 4 hours (E-selectin expression) and for 24 hours (ICAM-1 and VCAM-1 expression). Adhesion molecules were detected in immunoenzymatic test (ELISA) with mouse, monoclonal antibodies against human ICAM-1, VCAM-1 and E-selectin. The results of experiments suggest, that metronidazole enhances the expression of examined adhesion molecules on endothelial cells. This antimicrobial agent causes some changes in the expression of endothelial ICAM-1, VCAM-1 and E-selectin stimulated by B. fragilis endotoxins and enterotoxin.     

Oxidized LDL further enhances expression of adhesion molecules in Chlamydophila pneumoniae-infected endothelial cells

Chlamydophila pneumoniae is a common respiratory pathogen that has been shown to be associated with coronary artery disease. Recent studies have shown that one of the possible mechanisms of the atherogenicity of C. pneumoniae is overexpression of cell adhesion molecules (CAMs) in infected endothelial cells. We investigated whether exposure of C. pneumoniae-infected endothelial cells to oxidized LDL (oxLDL) leads to further upregulation of CAMs. Flow cytometry and immunoblot analysis of human aortic endothelial cells (HAECs) was performed for intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. ICAM-1 was expressed in 78.7% of C. pneumoniae-infected HAECs. The addition of oxLDL (100 microg/ml) to infected HAECs increased the proportion of ICAM-1-positive cells to 92%. VCAM-1 was only observed in 9.3% of infected HAECs, and the addition of oxLDL had no further effect on the surface expression of VCAM-1. C. pneumoniae also upregulated the surface expression of E-selectin on 52.2% of the cells, and incubation with oxLDL further increased the proportion of positive cells to 63.64%. In conclusion, C. pneumoniae upregulated the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin on HAECs. The addition of oxLDL to the infected cells further enhanced the surface expression of ICAM-1 and E-selectin.     

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

E-selectin-inducing activity in plasma from type 2 diabetic patients with maculopathy

Diabetic maculopathy (DMa) is a leading cause of visual loss in the western world. We examined whether plasma from type 2 diabetic patients with DMa contains factor(s) capable of inducing expression of the adhesion molecules E-selectin and VCAM-1 or cellular proliferation in cultured endothelial cells. Four gender-, age-, and duration (diabetes groups)-matched groups of 20 subjects each participated: 1) subjects with normal glucose tolerance (NGT), 2) subjects with impaired glucose tolerance (IGT), 3) type 2 diabetic patients without retinopathy, and 4) type 2 diabetic patients with DMa. Fasting plasma was added to in vitro-grown human umbilical vein endothelial cells for 6 h, after which E-selectin and VCAM-1 expression was measured. Proliferation was evaluated by thymidine incorporation. The individuals were characterized by measurement of 24-h ambulatory blood pressure, urinary albumin excretion rate, Hb A(1c), and blood lipids. Plasma from type 2 diabetic patients with DMa induced a significantly higher expression of E-selectin in endothelial cells than did plasma from subjects with NGT (259 +/- 23 x 10(3) vs. 198 +/- 19 x 10(3); arbitrary absorbance units; P < 0.05). There were no significant differences in plasma stimulatory effects on VCAM-1 expression or on thymidine incorporation between groups. These findings suggest that plasma from type 2 diabetic patients with DMa contains factor(s) capable of inducing the expression of E-selectin in endothelial cells. Enhanced expression of E-selectin may contribute to the development of DMa in type 2 diabetes.     

Influence of nebivolol and metoprolol on inflammatory mediators in human coronary endothelial or smooth muscle cells. Effects on neointima formation after balloon denudation in carotid arteries of rats treated with nebivolol.

OBJECTIVE AND BACKGROUND: Inflammation plays a critical role in all stages of atherogenesis. Proliferating vascular smooth muscle cells (SMC) and endothelial cells (EC) enhancing the inflammatory response, both contribute to the progression of atherosclerosis. Anti-proliferative, anti-inflammatory and anti-oxidative therapy seems to be a promising therapeutic strategy. The aim of this study was to assess the anti-proliferative and anti-inflammatory effect of the beta-blocker nebivolol in comparison to metoprolol in vitro and to find out whether nebivolol inhibits neointima formation in vivo. METHODS AND RESULTS: Real-time-RT-PCR revealed a decrease in VCAM-1, ICAM-1, PDGF-B, E-selectin and P-selectin mRNA expression in human coronary artery EC and SMC incubated with nebivolol for 72 hours while metoprolol did not have this effect. Nebivolol reduced MCP-1 and PDGF-BB protein in the culture supernatant of SMC and EC, respectively. Sprague-Dawley rats were treated with nebivolol for 0 or 35 days before and 28 days after carotid balloon injury. Immunohistological analyses showed that pre-treatment with nebivolol was associated with a decreased number of SMC layers and macrophages and an increased lumen area at the site of the arterial injury. The intima area was reduced by 43% after pre-treatment. CONCLUSION: We found that nebivolol reduced the expression of proinflammatory genes in endothelial cells and vascular smooth muscle cells in vitro whereas metoprolol did not. In vivo, nebivolol inhibited neointima formation by reducing SMC proliferation and macrophage accumulation.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.     

Retinol-Binding Protein 4 Induces Inflammation in Human Endothelial Cells by an NADPH Oxidase- and Nuclear Factor Kappa B-Dependent and Retinol-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.