Lack of inhibitory effect of HDL on TNFalpha-induced adhesion molecule expression in human aortic endothelial cells

Monocyte adhesion to and transmigration across the endothelium are initiating steps in atherogenesis. Cytokine-induced adhesion molecule expression in human umbilical vein endothelial cells (HUVEC) has been reported to be inhibited by either native HDL or reconstituted discoidal HDL (rHDL). In the present study we investigated these putative anti-atherosclerotic effects of HDL and rHDL in a more physiologically relevant cell type, i.e. human aortic endothelial cells (HAEC). HDL isolated by ultracentrifugation from eleven healthy subjects or rHDL made with apoA-I and either 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), or 1,2-dimyristoyl-sn-glycero-3-phosphocholine was incubated for 16 h with HAEC prior to stimulation with tumor necrosis factor-alpha (TNFalpha, 100 U/ml). Expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was measured by cell ELISA and Northern blot analysis. HDL (0.25, 0.5, 1.0 and 2.0 mgprotein/ml) failed to significantly inhibit TNFalpha-induced mRNA and protein expression of all three adhesion molecules. Furthermore, of the three rHDL preparations (16 micromol/l apoA-I) only that containing the polyunsaturated PLPC significantly reduced TNFalpha-induced VCAM-1 expression (by 29.9+/-9.1%). These data contrast with previously reported results using plasma HDL and HUVEC, and show that human HDL and rHDL, except for PLPC-rHDL, are ineffective inhibitors of TNFalpha-induced adhesion molecule expression in HAEC. The ability of polyunsaturated phospholipids in HDL to affect endothelial activation remains to be further investigated.     

Homocysteine increases monocyte and T-cell adhesion to human aortic endothelial cells

Although hyperhomocysteinemia has been recognized as an independent risk factor for atherosclerosis, its mechanism(s) are not well understood. Because chemotaxis and accumulation of leukocytes such as monocytes and T cells have been demonstrated to be critical events in the initiation and development of atherosclerosis, we investigated the effect of homocysteine (HCY) on U937 monocytic cells- and Jurkat T-cell-human aortic endothelial cell (HAEC) interactions under inflammatory cytokine-stimulated conditions. When HAEC were pretreated with HCY followed by stimulation with IL-1 beta, U937 and Jurkat T-cell adhesion to HAEC increased in a dose-dependent manner. The significant increase in U937 cell adhesion to HAEC was also observed when U937 cells were treated with HCY or when both cell types were treated with HCY. We also demonstrated that HCY increases endothelial surface expression and mRNA level of adhesion molecules, VCAM-1 and E-selectin. Attenuation of Jurkat T-cell and U937 cell adhesion to HAEC by monoclonal antibodies directed to specific adhesion molecules demonstrated that both VCAM-1 and E-selectin are involved in Jurkat T-cell adhesion, and VCAM-1 in U937 cell adhesion. Supplementation of HAEC with vitamin E was effective in preventing HCY-stimulated Jurkat T-cell adhesion and VCAM-1 and E-selectin expression in HAEC. These results indicate that HCY-mediated leukocyte-endothelial cell interaction is one potential mechanism by which homocysteinemia may lead to the development of atherosclerosis under inflammatory conditions. Dietary antioxidants such as vitamin E may attenuate HCY-stimulated activation of the endothelium and may help reduce the risk of vascular disease associated with hyperhomocysteinemia.     

Tibolone inhibits leukocyte adhesion molecule expression in human endothelial cells

Tibolone is a synthetic steroid with mixed estrogenic and progestogenic/androgenic activity used for post-menopausal hormone replacement therapy. Since its cardiovascular effects are still not clear, and no data have been published on possible direct actions on the vessel wall, we studied the effects of tibolone and its metabolites on lipopolysaccharide (LPS)-induced expression of leukocyte adhesion molecules on human endothelial cells. Tibolone and its two estrogenic 3alpha-OH and 3beta-OH metabolites, but not the progestogenic/androgenic Delta(4)-isomer, concentration-dependently decreased LPS-induced vascular cell adhesion molecule-1 protein expression. This effect was estrogen receptor dependent, since it was completely blocked by the pure estrogen receptor antagonist ICI 182780. Furthermore, only tibolone, the 3alpha-OH and the 3beta-OH metabolites decreased endothelial expression of E-selectin, while none of the compounds changed the levels of intercellular adhesion molecule-1. These findings were associated with parallel changes in mRNA levels for the three adhesion molecules. Our data show that tibolone and its estrogenic metabolites exert direct actions on the vascular wall, decreasing the expression of endothelial-leukocyte adhesion molecules, thus producing potentially important direct anti-atherogenic effects.     

Niacin inhibits vascular oxidative stress, redox-sensitive genes, and monocyte adhesion to human aortic endothelial cells

In pharmacological doses, nicotinic acid (niacin) reduces myocardial infarction, stroke and atherosclerosis. The beneficial effects of niacin on lipoproteins are thought to mediate these effects. We hypothesized that niacin inhibits oxidative stress and redox-sensitive inflammatory genes that play a critical role in early atherogenesis. In cultured human aortic endothelial cells (HAEC), niacin increased nicotinamide adenine dinucleotide phosphate (NAD(P)H) levels by 54% and reduced glutathione (GSH) by 98%. Niacin inhibited: (a) angiotensin II (ANG II)-induced reactive oxygen species (ROS) production by 24-86%, (b) low density lipoprotein (LDL) oxidation by 60%, (c) tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation by 46%, vascular cell adhesion molecule-1 (VCAM-1) by 77-93%, monocyte chemotactic protein-1 (MCP-1) secretion by 34-124%, and (d) in a functional assay TNF-alpha-induced monocyte adhesion to HAEC (41-54%). These findings indicate for the first time that niacin inhibits vascular inflammation by decreasing endothelial ROS production and subsequent LDL oxidation and inflammatory cytokine production, key events involved in atherogenesis. Initial data presented herein support the novel concept that niacin has vascular anti-inflammatory and potentially anti-atherosclerotic properties independent of its effects on lipid regulation.     

Comparative effect of dairy fatty acids on cell adhesion molecules,nitric oxide and relative gene expression in healthy and diabetic human aortic endothelial cells

Objective

Dairy intake, despite its high saturated fatty acid (SFA) content, is associated with a lower risk of cardiovascular disease and diabetes. This in vitro study determined the effect of individual fatty acids (FA) found in dairy, and FA mixtures representative of a high SFA and a low SFA dairy lipid on markers of endothelial function in healthy and type II diabetic aortic endothelial cells.

Methods

Cells were incubated for 24 h with FA mixtures (400 μM) and individual FA: oleic acid (OA; 150 μM); palmitic acid (PA; 150 μM); stearic acid (SA: 40 μM); trans-palmitelaidic acid (trans-PA; 20 μM); trans-vaccenic acid (trans-VA; 20 μM); α-linolenic acid (ALA; 20 μM) and linoleic acid (LA; 20 μM). Cellular adhesion molecules (sICAM-1, sVCAM-1 and sE-selectin) and nitric oxide (NO) were measured using ELISA and a chemiluminescent-based assay, respectively. Relative gene expression of these markers, including the insulin receptor, was performed using real-time PCR as well as FA compositions of cell pellets by gas chromatography.

Results

FA mixtures affected sE-selectin concentrations (P = 0.008), with concentrations lower following the high SFA compared to the low SFA mixture (P = 0.004), while NO concentrations were higher in diabetic compared to healthy cells (P = 0.029). Individual FA affected NO (P = 0.007) and sE-selectin (P = 0.040) concentrations with an increase following PA incubation relative to all other FA treatments (P < 0.05). PA increased sE-selectin compared with other FA treatments (P < 0.05). sE-selectin concentrations were also higher in healthy compared to diabetic cells (P = 0.023). Expression of ICAM-1 and insulin receptor was up-regulated in healthy compared to diabetic cells (P = 0.014 and P = 0.006 respectively).

Conclusions

Healthy and type II diabetic cells respond differently to incubation with FA treatments. Overall, physiological concentrations of dairy FA, but not dairy FA mixtures, substantially affected markers of endothelial function.     

Regulation of adhesion molecule expression by human synovial microvascular endothelial cells in vitro

Objective. To examine the in vitro expression of E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, vascular cell adhesion molecule 1 (VCAM-1), and platelet–endothelial cell adhesion molecule 1 (PECAM-1) by synovial microvascular endothelial cells (SMEC) in comparison with microvascular neonatal foreskin endothelial cells (FSE) and macrovascular human umbilical vein endothelial cells (HUVE). Methods. Cultured endothelial cells were treated for 4 hours with medium alone or tumor necrosis factor α (TNF α). The expression of endothelial adhesion molecules was evaluated by flow cytometry, cell enzyme-linked immunosorbent assay, and Northern blot analysis. Results. SMEC continuously expressed E-selectin under basal culture conditions, whereas FSE and HUVE did not. TNF α treatment of rheumatoid arthritis (RA) SMEC resulted in sustained peak expression of E-selectin for up to 24 hours, which subsequently declined but remained elevated even at 72 hours. In contrast, peak E-selectin expression in FSE and HUVE occurred between 4 hours and 16 hours after TNF α treatment and then declined to near basal levels by 24–48 hours. SMEC expressed significantly higher levels of ICAM-1 compared with HUVE under basal culture conditions. There was no difference between SMEC, FSE, and HUVE in the expression of P-selectin, VCAM-1, ICAM-2, or PECAM-1. Northern blot analysis demonstrated that the levels of E-selectin expression by TNF α-stimulated endothelial cells correlated with their respective messenger RNA levels. Conclusion. Regulation of E-selectin and ICAM-1 expression in RA synovial endothelium is different from that in neonatal foreskin and human umbilical vein endothelium. The augmented expression of adhesion molecules in RA synovial endothelium may facilitate the recruitment of leukocytes to this site.     

Effects of antioxidants and NO on TNF-alpha-induced adhesion molecule expression in human pulmonary microvascular endothelial cells

Pro-inflammatory cytokines initiate the vascular inflammatory response via upregulation of adhesion molecules on the endothelium. Recent observations suggest that reactive oxygen intermediates may play a pivotal role in TNF-alpha signaling and upregulate gene expression. We therefore evaluated the effects of pyrrolidine dithiocarbamate (PDTC; 0.1 mM) and spermine NONOate (Sper-NO; 1 mM) on adhesion molecule expression and nuclear factor kappa B (NF-kappaB) activation induced by TNF-alpha (10 ng/ml) in cultured human pulmonary microvascular endothelial cells (PMVEC). Treatment of cells with TNF-alpha for 4 h significantly induced the surface expression of E-selectin and ICAM-1. Treatment with TNF-alpha for 8 h significantly induced the surface expression of E-selectin, ICAM-1 and VCAM-1. The upregulation of these adhesion molecules was suppressed significantly by pretreatment with PDTC or Sper-NO for 1 h. 8-Bromo-cyclic GMP (1 mM) had no such effect, suggesting that the NO donor's effect was non-cGMP-dependent. The mRNA expression of E-selectin, ICAM-1 and VCAM-1, and activation of NF-kappaB induced by TNF-alpha for 2 h were decreased significantly by the above two pretreatments. N-acetylcysteine (10 mM) and S-nitroso-N-acetylpenicillamine (1 mM) had little inhibitory effects on the cell surface and mRNA expression of these adhesion molecules stimulated by TNF-alpha. Treatment with TNF-alpha for 4 h enhanced HL-60 leukocyte adhesion to human PMVEC, the effect of which was inhibited significantly by pretreatment with PDTC or Sper-NO. These findings indicate that both cell surface and mRNA expression of adhesion molecules in human PMVEC induced by TNF-alpha are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly in part through blocking the activation of NF-kappaB. Although our in vitro results cannot be directly extrapolated to the in vivo situation, they suggest a potential therapeutic approach for intervention in cytokine-mediated inflammatory processes in the human lung.     

Inhibitory effect of troglitazone on tumor necrosis factor alpha-induced expression of monocyte chemoattractant protein-1 in human mesangial cells

Insulin resistance is one of the risk factors for the progression of atherosclerosis and glomerulosclerosis. Recently, the new oral insulin-sensitizing agent troglitazone has been thought to offer potential in the treatment of diabetes. If adopted for this use, it might be helpful in protecting against the development of atherosclerosis and microvascular complications via its improvement of insulin resistance. However, it has not yet been clarified whether troglitazone acts directly on the vascular cells and inhibits the progression of atherosclerosis, including glomerulosclerosis. Meanwhile, monocyte chemoattractant protein-1 (MCP-1) is known to play an important role in the pathogenesis of atherosclerosis and glomerulosclerosis through the induction of monocyte migration. Therefore, we investigated the effect of troglitazone on the expression of MCP-1 in human mesangial cells (HMCs). HMCs were treated with or without troglitazone (1 or 10 micromol/L) in the presence or absence of tumor necrosis factor alpha (TNF-alpha) at various concentrations (50 or 500 ng/mL), and then MCP-1 secretion from the HMCs was measured. We found that TNF-alpha increased the secretion of MCP-1 by 55-fold versus the control and troglitazone significantly inhibited this TNF-alpha-induced increase in MCP-1 secretion (49.3%). Moreover, Northern blot analysis showed that troglitazone decreased the MCP-1 mRNA level in HMCs. We demonstrated that alpha-tocopherol also inhibited TNF-alpha-induced MCP-1 production in HMCs, although its effects were not as strong as troglitazone. The present study indicates that troglitazone may prevent the progression of atherosclerosis by inhibiting MCP-1 expression in mesangial cells.     

Low-density lipoprotein augments interleukin-1-induced vascular adhesion molecule expression in human endothelial cells.

In this study, the effect of low density lipoproteins (LDL) on the ability of the vascular endothelium to respond to vascular cell adhesion molecule 1 (VCAM-1) activation by a cytokine was investigated. After a 4-day pre-exposure to 240 mg/dl of LDL, human umbilical vein endothelial cells (HUVECs) were hyperresponsive to minute amounts of interleukin 1 alpha (IL-1 alpha) as demonstrated by an augmentation of VCAM-1 gene expression. Furthermore, in response to LDL exposure, endothelial recruitment of monocytes induced by minute amounts of IL-1 alpha was increased. This enhancing effect was blocked by an anti-VCAM antibody. The increased response appears not to be due to changes in IL-1 binding affinity or induction of endogenous IL-1 alpha. Transient transfection of HUVECs with a reporter driven by the VCAM promoter showed that LDL increased cellular response to IL-1 alpha by 46%. LDL itself does not increase NF-kappa B binding in endothelial cells (ECs). However, after a 2-day LDL incubation, NF-kappa B binding could be induced by over 63% with a very low dose of IL-1 alpha. IL-1 alpha at this dose (which activates NF-kappa B, but not AP-1) also enhanced LDL-activated AP-1 binding. This cross-enhanced effect may be an important intracellular signaling mechanism for EC activation. The results from this study provide new clues to understanding the mechanisms governing combined risk factors for atherosclerosis.     

Induction of IG9 monocyte adhesion molecule expression in smooth muscle and endothelial cells after balloon arterial injury in cholesterol-fed rabbits

The expression of monocyte-specific adhesion molecules and chemokines by cell types within the vessel wall plays an important role in foam cell accumulation during atherosclerotic plaque development. We previously identified IG9, a novel monocyte adhesion protein that is expressed on endothelial cells (ECs) overlying human and rabbit advanced atherosclerotic plaques. The present study was designed to determine the temporal and spatial expression of IG9 and the chemokine, monocyte chemoattractant protein-1 (MCP-1), after balloon injury with (double injury) or without (single injury) prior air desiccation EC injury in the femoral arteries of rabbits fed a high-cholesterol diet. By immunohistochemical analyses, intense reactivity with monoclonal antibodies to IG9 and MCP-1 was detected 24 hours after single injury in medial smooth muscle cells (SMCs) and in SMCs of adventitial microvessels. However, monocyte infiltration of the tunica media was minimal or not detected in these sections. IG9 and MCP-1 antibody reactivity in vessel sections 28 days after single injury and 24 hours, 7 days, and 28 days after double injury was localized to medial and neointimal SMCs, foam cells, and luminal ECs overlying the plaques. Uninjured rabbit (cholesterol or normal diet) vessel sections exhibited minimal IG9 and MCP-1 immunostaining. In vitro studies using human aortic SMCs demonstrated IG9 protein induction after 24 hours of treatment with platelet-derived growth factor-BB and interferon-gamma or epidermal growth factor. IG9 expression was further increased by pretreatment of SMCs with the proatherogenic lipid, minimally oxidized low density lipoprotein. After balloon injury (24 hours), IG9 is induced in vascular SMCs before the detectable accumulation of monocytes within the vessel wall. Thus, the expression of IG9 by SMCs as well as by ECs may be an important factor in the accumulation of foam cells in atherosclerotic plaque development after arterial injury.     

The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic endothelial cells

Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease (CVD). One proposed mechanism for the beneficial effect of carotenoids is through functional modulation of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell (HAEC) cultures (passages 4–8) for 24 h with each of the five most prevalent carotenoids in human plasma, which are -carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 μmol/l. Carotenoids were solubilized in 0.7% (v/v) tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate solubilities in aqueous medium, final concentrations of -carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 μmol/l and monolayers accumulated 647, 158, 7, 113, and 9 pmol/mg protein, respectively. Monolayers were then stimulated with IL-1β (5 ng/ml) for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay (ELISA). To assess endothelial cell adhesion to monocytes, IL-1β-stimulated monolayers were incubated for 10 min with ⁵¹Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with β-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13%, respectively. Pre-incubation with β-carotene and lutein significantly reduced E-selectin expression by 38 and 34%, respectively. Pre-treatment with β-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18%, respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1β-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25%, respectively. Thus, among the carotenoids, lycopene appears to be most effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface.     

Cytokine release and adhesion molecule expression by stimulated human bronchial epithelial cells are downregulated by salmeterol

Summary beta2-adrenoreceptor agonists are able to modulate various aspects of airway cell functions involved in the inflammatory and repair processes characterizing a variety of respiratory disorders. Human bronchial epithelial cells (HBECs), which can act as immune effector cells and express beta2-adrenoreceptors, were used to test the effects of different concentrations (0.1-100.0 nM) of salmeterol (Salm) on adhesion molecule expression and chemokine/cytokine release. HBECs, freshly isolated from resected bronchi at the time of surgery in ex-smokers with lung cancer, constitutively expressed over 3 times more ICAM-1 than VCAM-1 (P<0.05) and secreted greater amounts of IL-8 than of GM-CSF or RANTES (P<0.001). Stimulation of HBECs with IL-4, TNF-alpha or IL-4 plus TNF-alpha-upregulated ICAM-1 expression (P<0.05) and increased GM-CSF and IL-8 secretion (P<0.05). Similarly, VCAM-1 expression was significantly increased by IL-4 plus TNF-alpha, while RANTES release was significantly enhanced by IL-4 or by IL-4 plus TNF-alpha (P<0.05), but not by TNF-alpha alone (P>0.05). Dose-response curves showed that Salm, at concentration >1.0 nM, was effective in inhibiting adhesion molecule expression and cytokine release by HBECs (P<0.05). At a Salm concentration of 10 nM the degree of inhibition observed was similar for ICAM-1 and VCAM-1 expression (37.2 +/- 9.3% and 32.9 +/- 9.6%, respectively; P>0.05), but higher for RANTES (88.4 +/- 4.4%), as compared to IL-8 (21.8 +/- 7.0%) or GM-CSF (30.1 +/- 6.6%; P<0.05, each comparison). Thus, adhesion molecules and cytokines may be expressed/released at very different levels by unstimulated or stimulated HBECs and those activities appear to be modulated by Salm.