Expression Of Endothelial Leukocyte Adhesion Molecule-1 Research Articles

Bai Hu Tang, Si Ni Tang, and Xue Bi Tang amplify pro-inflammatory activities and reduce apoptosis in endothelial cells in a cell culture model of sepsis

Ethnopharmacological relevanceSepsis is a systemic inflammatory response of the body to a severe infection or massive tissue injury. Despite intensive research, sepsis continues to have a high mortality rate and successful treatment options are strongly needed. Bai Hu Tang (BHT), Si Ni Tang (SNT), and Xue Bi Tang (XBT) are ancient traditional Chinese formulas derived from Chinese herbs that are used to treat Sepsis, but their mechanisms of activity are largely unknown. Aim of the studyWe aimed to examine dose-dependent effects of BHT, SNT, and XBT in a cell culture model of Sepsis, with special focus on endothelial cell apoptosis and the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)6, IL8, the surface adhesion molecule intercellular adhesion molecule-1 (ICAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). Material and methodsWe stimulated THP1 monocytic cells with lipopolysaccharide (LPS, Escherichia coli (E. coli)) for 4 h and used the resulting culture medium to stimulate human umbilical vein endothelial cells (HUVECs). HUVECs were also simultaneously treated with hydrophilic concentrates of BHT, SNT or XBT. We evaluated the mRNA and protein expression levels of IL6, IL8, MCP-1, ICAM-1, and ELAM-1 and the activity of caspase 3/7, a marker of cell apoptosis, after stimulation and treatment. In addition, we stimulated cannulated veins from human umbilical cords for 24 h and treated them with BHT, SNT or XBT. Immunohistochemistry visualized expression of ICAM-1 and ELAM-1. ResultsThe mRNA and protein levels of IL6, IL8, ICAM-1, and ELAM-1 were higher in stimulated HUVECs than in controls. Treating stimulated HUVECs with BHT, SNT or XBT induced an additional increase in IL6 (13- to 132-fold) and IL8 (17- to 32-fold) mRNA levels but did not influence their protein levels. In addition, BHT induced an additional increase in ICAM-1 mRNA (9-fold) expression, whereas XBT increased the mRNA and protein levels of ELAM-1 by 42-fold and 10-fold, respectively. Finally, caspase 3/7 levels, and therefore apoptosis, were up to 100% lower in cells treated with BHT than in the stimulated control (P < 0.001). ConclusionThe results of this study indicate that BHT, SNT, and XBT interfere in inflammatory pathways during septic processes by reducing the apoptotic effects of LPS and modifying the endothelial expression of pro-inflammatory cytokines and surface adhesion molecules.

Erythrocyte-Derived Microparticles Activate Pulmonary Endothelial Cells in a Murine Model of Transfusion.

Erythrocyte-derived microparticles (MPs) are sub-micrometer, biologically active vesicles shed by red blood cells as part of the biochemical changes that occur during storage. We hypothesized that MPs from stored red blood cells would activate endothelial cells. MPs from aged murine packed red blood cells (pRBCs) were isolated and used to treat confluent layers of cultured endothelial cells. Endothelial expression of leukocyte adhesion molecules, endothelial-leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1(ICAM-1), and inflammatory mediator, interleukin-6 (IL-6), was evaluated at 0.5, 6, 12, and 24 h of treatment. Healthy C57BL/6 mice were transfused with a MP suspension and lung sections were analyzed for adhesion molecules and sequestered interstitial leukocytes. Increased levels of ELAM-1 and ICAM-1 were found on cultured endothelial cells 6 h after MP stimulation (6.91 vs. 4.07 relative fluorescent intensity [RFI], P < 0.01, and 5.85 vs. 3.55 RFI, P = 0.01, respectively). IL-6 in cell culture supernatants was increased after 12 h of MP stimulation compared with controls (1.24 vs. 0.73 ng/mL, P = 0.03). In vivo experiments demonstrated that MP injection increased ELAM-1 and ICAM-1 expression at 1 h (18.56 vs. 7.08 RFI, P < 0.01, and 23.66 vs. 6.87 RFI, P < 0.01, respectively) and caused increased density of pulmonary interstitial leukocytes by 4 h of treatment (69.25 vs. 29.25 cells/high powered field, P < 0.01). This series of experiments supports our hypothesis that erythrocyte-derived MPs are able to activate pulmonary endothelium, leading to the pulmonary sequestration of leukocytes following the transfusion of stored pRBCs.

Amla (Emblica officinalisGaertn.) extract inhibits lipopolysaccharide-induced procoagulant and pro-inflammatory factors in cultured vascular endothelial cells

Amla (Emblica officinalis Gaertn.) has been used for many centuries in traditional Indian Ayurvedic formulations for the prevention and treatment of many inflammatory diseases. The present study evaluated the anti-inflammatory and anticoagulant properties of amla fruit extract. The amla fruit extract potentially and significantly reduced lipopolysaccharide (LPS)-induced tissue factor expression and von Willebrand factor release in human umbilical vein endothelial cells (HUVEC) in vitro at clinically relevant concentrations (1-100 μg/ml). In a leucocyte adhesion model of inflammation, it also significantly decreased LPS-induced adhesion of human monocytic cells (THP-1) to the HUVEC, as well as reduced the expression of endothelial-leucocyte adhesion molecule-1 (E-selectin) in the target cells. In addition, the in vivo anti-inflammatory effects were evaluated in a LPS-induced endotoxaemia rat model. Oral administration of the amla fruit extract (50 mg/kg body weight) significantly decreased the concentrations of pro-inflammatory cytokines, TNF-α and IL-6 in serum. These results suggest that amla fruit extract may be an effective anticoagulant and anti-inflammatory agent.

Sesamin attenuates intercellular cell adhesion molecule‐1 expression in vitro in TNF‐α‐treated human aortic endothelial cells and in vivo in apolipoprotein‐E‐deficient mice

Sesame lignans have antioxidative and anti-inflammatory properties. We focused on the effects of the lignans sesamin and sesamol on the expression of endothelial-leukocyte adhesion molecules in tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs). When HAECs were pretreated with sesamin (10 or 100 microM), the TNF-alpha-induced expression of intercellular cell adhesion molecule-1 (ICAM-1) was significantly reduced (35 or 70% decrease, respectively) by Western blotting. Sesamol was less effective at inhibiting ICAM-1 expression (30% decrease at 100 microM). Sesamin and sesamol reduced the marked TNF-alpha-induced increase in human antigen R (HuR) translocation and the interaction between HuR and the 3'UTR of ICAM-1 mRNA. Both significantly reduced the binding of monocytes to TNF-alpha-stimulated HAECs. Sesamin significantly attenuated TNF-alpha-induced ICAM-1 expression and cell adhesion by downregulation of extracellular signal-regulated kinase 1/2 and p38. Furthermore, in vivo, sesamin attenuated intimal thickening and ICAM-1 expression seen in aortas of apolipoprotein-E-deficient mice. Taken together, these data suggest that sesamin inhibits TNF-alpha-induced extracellular signal-regulated kinase/p38 phosphorylation, nuclear translocation of NF-kappaB p65, cytoplasmic translocalization of HuR and thereby suppresses ICAM-1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that sesamin may prevent the development of atherosclerosis and inflammatory responses.

Modulation of adhesion molecule expression on endothelial cells during the late asthmatic reaction: role of macrophage-derived tumour necrosis factor-alpha.

In a previous work we have demonstrated that in patients exhibiting a late allergic reaction (LAR), alveolar macrophages (AM) collected 18 h after bronchial allergen challenge produced high levels of IL-6 and tumour necrosis factor-alpha (TNF) which is known to up-regulate the endothelial cell expression of adhesion molecules participating in the development of the inflammatory reaction in bronchial asthma. For these reasons, we evaluated the effect of AM supernatants from asthmatic patients developing an LAR on intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) expression by human endothelial cells. The expression of adhesion molecules was assessed by an ELISA method and compared with the effect of an optimal dose of human recombinant (hr) TNF. Results showed that AM supernatants, from challenged asthmatics developing an LAR, increased significantly the ICAM-1 and ELAM-1 expression on endothelial cells to a level similar to that obtained in the presence of hrTNF (500 U/ml) (P < 0.001 in both cases, respectively 90.4% and 75.2% of the level obtained with hrTNF). In contrast, AM supernatants from asthmatics at baseline or exhibiting, after challenge, a single early reaction had no significant effect on these parameters (P = NS in both cases, respectively 23.5% and 24.7% of the ICAM-1 expression, 22.7% and 15.3% of the ELAM-1 expression obtained with hrTNF). AM-derived TNF present in these supernatants was thought to play a key role in endothelial cell stimulation, since: (i) TNF concentration in AM supernatants correlated with its effect on ICAM-1 (r = 0.80, P < 10(-4)) and ELAM-1 expression (r = 0.88, P < 10(-5)); and (ii) a neutralizing anti-TNF antibody decreased their effect (68% and 80% respectively on ICAM-1 and ELAM-1 expression). Moreover, the role of IL-6 was excluded on the basis both of the hrIL-6 inefficiency to induce ICAM-1 and ELAM-1 synthesis, even in costimulation with hrTNF, and of anti-IL-6 antibody to neutralize the effect of AM supernatants. Our results suggest that, beside mast cells and lymphocytes, macrophages might participate in the induction of the local inflammatory reaction observed in bronchial asthma. During the LAR, cytokines and especially TNF are able, through an enhanced adhesion molecule expression on endothelial cells, to facilitate the bronchial cellular influx.

Antibodies to proteinase 3 increase adhesion of neutrophils to human endothelial cells.

The detection of anti-neutrophil cytoplasmic antibodies (ANCA), especially those with specificity for proteinase 3, is important in the diagnosis and in monitoring disease activity of Wegener's granulomatosis and related vasculitides. An ubiquitous feature of all ANCA-associated acute vascular injury is lytic necrosis. Adhesion of neutrophils to endothelium is a fundamental early step of the inflammatory response. Recently we were able to show that ANCA recognize their target antigen (proteinase 3) translocated into the membrane of human endothelial cells. The aim of this study was to investigate the effect of ANCA on the adhesion of neutrophils to human endothelial cells. Incubation of endothelial cells with affinity-purified antibodies to proteinase 3 (IgG- and F(ab')2-fractions) led to a marked increase of neutrophil adhesion, with a peak after 4 h and a rapid decrease after 8 h. This effect could be inhibited by preincubation of the endothelial cells with an antibody to endothelial-leucocyte adhesion molecule-1 (ELAM-1). Incubation with antibodies to proteinase 3 also led to an increase of endothelial ELAM-1 expression as measured in a cyto-ELISA and by flow cytometry. Our data demonstrate a direct effect of ANCA on neutrophil-endothelial interactions. The enhanced adhesion of neutrophils occurs time-dependently via induction of ELAM-1 expression on the surface of endothelial cells. Our data give a hint of an ANCA-mediated mechanism of endothelial injury via induction of neutrophil adhesion to vascular endothelium in Wegener's granulomatosis and other ANCA-related vasculitides.

The effect of endothelial leukocyte-adhesion molecule-1 on the shape and cytoskeleton of porcine trabecular meshwork cells

To study the effects of endothelial leukocyte-adhesion molecule-1 (ELAM-1) on the shape and actin cytoskeleton of cultured porcine trabecular meshwork cells (PTCs). The cultured PTCs were stimulated by interleukin-1 (IL-1) and conjugated by ELAM-1 antibody or further cross-linked by IgG. Immunocytochemical procedures were used to study the expression of ELAM-1 and FITC-phalloidin was used to stain the actin filaments on PTCs; phase contrast microscope was employed to observe the dynamic changes of cellular shape and the intercellular conjunction and confocal microscope was used to observe the changes of actin and cellular thickness. IL-1 treatment increased the expression of ELAM-1 in PTCs which resulted in enlarged intercellular space in PTCs with more rounded and stereoscopic morphology after specific antibody conjugation or IgG cross linking. In addition, actin filaments became sparse, cellular rigidity decreased, and the cellular thickness increased after these treatments. Our results demonstrated that IL-1 activated PTCs can express ELAM-1 and interaction of ELAM-1 with specific antibodies and cross linking with IgG, induced a rounding-up of PTCs. This change in cell shape may be due to its action on the actin cytoskeleton.

Effect of High Glucose Concentrations on Expression of ELAM 1, VCAM-1 and ICAM-1 in HUVEC with and without Cytokine Activation.

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.

Elevated cyclic adenosine monophosphate ameliorates ischemia–reperfusion injury in rat cardiac allografts

Background: Oxidative stress after ischemia and reperfusion leads to leukocyte activation, the production of injurious cytokines, and increased expression of inflammatory adhesion molecules. This initial event is one of the most important alloantigen-independent factors associated with graft coronary artery disease (GCAD). Cyclic adenosine monophosphate (cAMP) is an important second messenger that inhibits the expression of tumor necrosis factor α (TNF-α), vascular cell adhesion molecule 1 (VCAM-1), and endothelial leukocyte adhesion molecule 1 (ELAM-1) in vitro. Its levels decrease during organ preservation. We hypothesized that augmenting allograft cAMP levels with the water-soluble adenylate cyclase activator, NKH477, could decrease ischemia–reperfusion injury and inhibit the progression of GCAD. Methods: PVG to ACI rat heterotopic cardiac allografts, treated with NKH477 solution or vehicle, were reperfused for 4 hours or 90 days after 60 minutes of ischemia. We analyzed grafts for intracellular adhesion molecule 1 (ICAM-1), VCAM-1, and ELAM-1 mRNA expression; TNF-α and interleukin-6 (IL-6) protein expression; and myeloperoxidase activity. We also performed immunohistochemical analysis for ICAM-1 and VCAM-1 protein expression. At post-operative Day 90, the progression of GCAD had increased morphometrically. Results: NKH477-treated grafts had significantly decreased levels of myeloperoxidase activity compared with controls. In this group, TNF-α, IL-6, and VCAM-1 protein expression was inhibited; however, ICAM-1 and ELAM-1 expression did not alter. We found no differences in the degree of development of GCAD between groups. Conclusion: Although augmented intracellular cAMP prevented acute reperfusion injury, it was insufficient to prevent the development of GCAD. Intracellular adhesion molecule 1 and ELAM-1, whose expression NKH477 does not inhibit, may play important roles in the development of GCAD.

Magnolol attenuates VCAM-1 expression in vitro in TNF-alpha-treated human aortic endothelial cells and in vivo in the aorta of cholesterol-fed rabbits.

1. In a previous study, we showed that magnolol, a potent antioxidant derived from a Chinese herb, attenuates monocyte chemotactic protein-1 (MCP-1) expression and intimal hyperplasia in the balloon-injured aorta of cholesterol-fed rabbits. Expression of cell adhesion molecules by the arterial endothelium and the attachment of leukocytes to the endothelium may play a major role in atherosclerosis. In the present study, the effects of magnolol on the expression of endothelial-leukocyte adhesion molecules and the activation of nuclear factor kappa B (NF-kappa B) in tumour necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. 2. Pretreatment of HAECs with magnolol (5 microM) significantly suppressed the TNF-alpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1) (64.8+/-1.9%), but had no effect on the expression of intercellular cell adhesion molecule-1 and endothelial cell selectin. 3. Magnolol (5 and 10 microM) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (58.4 and 56.4% inhibition, respectively). Gel shift assays using the (32)P-labelled NF-kappa B consensus sequence as probe showed that magnolol pretreatment reduced the density of the shifted bands seen after TNF-alpha-induced activation. Immunoblot analysis and immunofluorescence staining of nuclear extracts demonstrated a 58% reduction in the amount of NF-kappa B p65 in the nuclei in magnolol-treated HAECs. Magnolol also attenuated intracellular H(2)O(2) generation in both control and TNF-alpha treated HAECs. 4. Furthermore, in vivo, magnolol attenuates the intimal thickening and TNF-alpha and VCAM-1 protein expression seen in the thoracic aortas of cholesterol-fed rabbits. 5. Taken together, these data demonstrate that magnolol inhibits TNF-alpha-induced nuclear translocation of NF-kappa B p65 and thereby suppresses expression of VCAM-1, resulting in reduced adhesion of leukocytes. These results suggest that magnolol has anti-inflammatory properties and may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo.

Stimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1, VCAM-1 or ELAM-1 expression by human vascular endothelial cells.

A previous study reported that intercellular adhesion molecule-1 (ICAM-1) expression by human vascular endothelial cells (HUVEC) is augmented by intracellular signal transmission mainly through the protein kinase C (PKC) system stimulated by TXA2 receptors. In the present study, we show that a TXA2 receptor agonist, U46619, augments the expression of not only ICAM-1, but also vascular cell adhesion molecule-1 (VCAM-1) or endothelial leucocyte adhesion molecule-1 (ELAM-1) in HUVEC both at protein and mRNA levels. Pretreatment with SQ29,548 (a TXA2 receptor antagonist) or PKC inhibitors greatly diminished the extent of U46619-induced mRNA accumulation and surface expression of the adhesion molecules. An inhibitor of nuclear factor kappaB (NF-kappaB) activation, PDTC, diminishes U46619-induced VCAM-1 mRNA accumulation. NAC, which inhibits NF-kappaB and activation protein 1 (AP-1) binding activity, inhibits the expression of ICAM-1 or ELAM-1 at protein and mRNA levels. These findings suggest that ICAM-1 or ELAM-1 expression of HUVEC stimulated via TXA2 receptors is augmented by induction of NF-kappaB and AP-1 binding activity through the PKC system, and that VCAM-1 expression is augmented by induction of NF-kappaB binding activity.

Inhibition of dextran sulphate sodium (DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules (endothelial leucocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1)).

We examined the effect of intracolonic administration of anti-adhesion molecule antibodies on DSS-induced colitis in mice. Immunohistochemical staining in mice with colitis showed increased expression of ELAM-1 and ICAM-1 on endothelial cells of vessels in the lamina propria and submucosa at sites of inflamed lesions. Intracolonic administration of anti-ELAM-1 or anti-ICAM-1 antibody decreased bloody stools, anaemia, and histologically evident damage, as well as myeloperoxidase activity and IL-1beta content. We concluded that adhesion molecule expression is important in the development of DSS-induced colitis in mice and that intracolonic administration of anti-adhesion molecule antibodies, especially anti-ELAM-1 antibody, effectively inhibits the colonic inflammation. Intracolonic administration of anti-adhesion molecule antibodies may show therapeutic promise in ulcerative colitis.

Quantitative assessments of physiological and biological parameters in psoriatic lesions and its correlations to the clinical severity of psoriasis.

Twenty-nine psoriatics were examined using a model with clinical, physiological and pathological assessment parameters. The three parts in this assessment model include: (1) clinical assessment: psoriasis area and severity index (PASI); (2) assessment of skin physiology and microcirculation: water content of stratum corneum, water-holding capacity of stratum corneum, transepidermal water loss, intravital dynamic videocapillaroscopy-measuring the capillary diameters and blood cell velocity in proximal nailfold of ring finger, and fluorescence angiography-measuring transcapillary Na-fluorescein(NAF) diffusion; and (3) immunohistochemistry examination: markers of proliferation (Ki67Ag), differentiation (involucrin), and inflammation (neutrophil elastase, intercellular adhesion molecule-1(ICAM-1), endothelial leukocyte adhesion molecule-1(ELAM-1)). Our results showed both the transcapillary diffusion of NAF and the expression of cell markers-dermal neutrophil elastase, epidermal ELAM-1 and Ki67Ag--correlated significantly to PASI scores (P < 0.05, linear regression). According to our results, the increased capillary permeability and inflammation markers, and enhanced expression of Ki67Ag correlated very well with PASI score. These markers could serve as alternative methods for assessment of the clinical severity of psoriatic patients.

Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-alpha-treated human aortic endothelial cells.

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 microg/ml), the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2 +/- 3.2% and 80.0 +/- 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 microg/ml), 84.5 +/- 1.9%, 78.8 +/- 1.2%, 58.9 +/- 0.4%, 58.7 +/- 0.9%, and 57.4 +/- 0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 microg/ml) or Sal B (5 microg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (45.7 +/- 2.5% and 55.8 +/- 1.2%, respectively). SME or Sal B significantly inhibited TNF-alpha-induced activation of nuclear factor kappa B (NF-kappaB) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis.

Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype. Wang N, Chintala SK, Fini ME, Schuman JS. Nat Med 2001;7:304–309.

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-B, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye’s aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.—Author’s abstract.

Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype.

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-kappaB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eye's aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.

Human lactoferrin interacts with soluble CD14 and inhibits expression of endothelial adhesion molecules, E-selectin and ICAM-1, induced by the CD14-lipopolysaccharide complex.

Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (K(d) = 16+/-7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A-2-keto-3-deoxyoctonic acid-heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.

Enhanced expression of ELAM-1 on endothelium of renal cell carcinoma compared to the corresponding normal renal tissue

Renal cell carcinoma (RCC) has been shown to respond to an immunological therapy with tumor infiltrating lymphocytes (TIL), which accumulate in RCC at a higher density than in normal renal tissue, suggesting that there is selective tumor invasion. Since invasion of TIL into the malignant tissue is mediated by adhesion molecules, we examined the different expression of the adhesion molecule endothelial-leukocyte-adhesion-molecule-1 (ELAM-1) on endothelial cells of RCC versus normal renal tissue. For a specific quantification, the level of ELAM-1 mRNA was investigated by both semi-quantitative reverse transcription–polymerase chain reaction (RT–PCR) and Northern blot analysis and referred to the content of endothelial cells in the tissue, determined by endothelium specific staining. Quantification of mRNA was evaluated by computer-aided integration. We observed a significantly lower amount of endothelial cells in RCC compared to normal renal tissue. The specific transcription rate of ELAM-1 in RCC, determined by RT–PCR was about 5.2 times that of normal tissue, while Northern blot analysis indicated an approximately 11.8 times increase. Our investigations show a significantly increased expression of ELAM-1 in tumor tissue compared to normal renal tissue, presumably caused by a higher amount of cytokines in the tumor tissue. This enhanced expression may be responsible for the high concentration of TIL in renal tumors.

Gallates inhibit cytokine-induced nuclear translocation of NF-kappaB and expression of leukocyte adhesion molecules in vascular endothelial cells.

Gallates (gallic acid esters) belong to the class of phenolic compounds, which are abundant in red wine. In this study, we show that gallates can inhibit cytokine-induced activation of nuclear factor kappaB (NF-kappaB) and thereby reduce expression of endothelial-leukocyte adhesion molecules in cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of HUVECs with ethyl gallate (3 to 10 micromol/L) significantly suppressed interleukin-1alpha (IL-1alpha)- or tumor necrosis factor-alpha (TNF-alpha)- induced mRNA and cell-surface expression of vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin, which was associated with reduced adhesion of leukocytes to HUVECs. Gel shift assays with the NF-kappaB consensus sequence showed the decreased densities of the shifted bands in gallate-treated HUVECs. Furthermore, gallate pretreatment inhibited cytokine-induced transcription of a fusion gene, which consisted of 4 repeats of the NF-kappaB consensus sequence and the luciferase reporter gene. Immunoblot analysis of nuclear extracts and whole-cell lysates demonstrated the decreased amounts of NF-kappaB p65 in nuclei but equal amounts of inhibitor-kappaBalpha (I-kappaBalpha) in whole-cell lysates of ethyl gallate-treated HUVECs. Incubation of the nuclear extracts from cytokine-activated HUVECs with ethyl gallate did not affect the NF-kappaB shifted bands induced by cytokines in gel shift assays. Taken together, these data demonstrate that ethyl gallate can inhibit cytokine-induced nuclear translocation of NF-kappaB p65 by way of a mechanism independent of I-kappaBalpha degradation and thereby suppress expression of VCAM-1, ICAM-1, and E-selectin, which was associated with reduced adhesion of leukocytes. These results in vitro demonstrate that gallates can exhibit anti-inflammatory properties by blocking activation of NF-kappaB and suggest that these natural compounds, abundant in red wine, may play important roles in the prevention of atherosclerosis and inflammatory responses in vivo.

The inhibition of endothelial activation by unsaturated fatty acids

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-kappaB (NF-kappaB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-kappaB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.