Simvastatin alters human endothelial cell adhesion molecule expression and inhibits leukocyte adhesion under flow

Hypercholesterolaemia is implicated as an independent risk factor in the pathogenesis of atherosclerosis. HMG-CoA reductase inhibitors (statins) are prescribed for their lipid-lowering effects but recent evidence suggests they have pleiotropic effects independent of lipid balance regulation that may explain their role in dramatically decreasing cardiovascular mortality and morbidity. The mechanisms responsible are unclear but endothelial cell (EC) dysfunction is critical. To investigate potential anti-inflammatory properties of statins on EC, functional responses of human umbilical vein endothelial cells (HUVEC) and human neutrophils under physiological flow conditions were studied. These interactions were quantified in response to inflammatory mediators following pre-treatment with statin.Histamine stimulation resulted in significant (p < 0.001) increases in transient interactions between neutrophils and EC (tethering). These effects were significantly reduced (p < 0.001) on pre-treatment with statin. TNF-α stimulation resulted in significant (p < 0.001) increases in rolling interactions. These effects were significantly (p < 0.001) reduced following pre-treatment of EC with statin. Mevalonate pre-treatment of EC significantly reversed the effects of statin pre-treatment on both tethering and rolling (p < 0.001).Reductions in surface expression of P- and E-selectin were confirmed by ELISA. EC exposed to histamine demonstrated significantly increased (p < 0.01) levels of P-selectin, abrogated (p < 0.001) by pre-treatment with statin. EC exposed to TNF-α demonstrated a significant increase (p < 0.001) in levels of E-selectin, reduced (p < 0.05) by pre-treatment with statin.     

The endothelial cell cytoskeleton modulates extravascular polymorphonuclear leukocyte accumulations in vivo

Actin microfilaments, key elements in the endothelial cell (EC) cytoskeleton, have been noted in in vitro studies to play a modulating role in the diapedesis of polymorphonuclear leukocytes (PMN). The role of the cytoskeleton in PMN diapedesis in vivo was the subject of this study. Small skin abrasions were produced in rabbits. Cytoskeletal manipulation was accomplished by local application of phalloidin which promotes microfilament assembly or cytochalasin B which causes their disassembly, prior to addition of a chemotaxin. When the initial treatment to the abrasion site was saline, the secondary addition of more saline resulted in PMN accumulations, expressed as PMN/mm3 of 36 +/- 19, while 10(-4) M cytochalasin B led to 91 +/- 32 (P less than 0.05). Secondary addition of chemotaxins or histamine also led to significant PMN accumulations of 169 +/- 54 with 10(-8) M leukotriene (LT) B4, 318 +/- 85 with zymosan-activated plasma (ZAP), and 158 +/- 48 with 10(-4) M histamine. When the initial treatment was 10(-8) M phalloidin, PMN accumulations were reduced to 31 +/- 22 with cytochalasin B (P less than 0.05 relative to saline as initial treatment); 63 +/- 43 with LTB4 (P less than 0.05); 137 +/- 48 with ZAP (P less than 0.05); and 51 +/- 35 with histamine (P less than 0.05). In contrast, initial blister treatment with cytochalasin B rather than saline increased PMN accumulations in response to LTB4, 291 +/- 71 (P less than 0.05), and histamine, 270 +/- 56 (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions

Hemolytic uremic syndrome (HUS), which is the most common cause of acute renal failure in infants and small children, is caused by verotoxin (VT)-producing Escherichia coli infection. Endothelial injury determines microvascular thrombosis and evidence is available from recent studies that suggests that leukocyte activation participates in endothelial damage. We studied here the effect of VT-1 on leukocyte adhesion to vascular endothelium under physiologic flow conditions. Human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with VT-1 (0.1, 1, and 10 pmol/L) and then exposed to a total leukocyte suspension in a parallel plate flow chamber under laminar flow conditions (1.5 dynes/cm2). Adherent cells were counted by digital image processing. Results showed that VT-1 dose-dependently increased the number of adhering leukocytes to HUVECs as compared with unstimulated cells. The adhesive response elicited by VT-1 was comparable to that of interleukin-1 beta (IL-1 beta), one of the most potent inducers of endothelial cell adhesiveness. Exposure of HUVECs to VT-1 did not affect the number of rolling leukocytes, which was similar to that of control values. To examine the role of adhesion molecules in VT-1-induced leukocyte adhesion, HUVECs were incubated with mouse monoclonal antibodies against E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) before adhesion assay. Functional blocking of E-selectin, ICAM-1, and VCAM-1 on endothelial cells significantly inhibited VT-1-induced increase in leukocyte adhesion. In some experiments, before VT-1 incubation, HUVECs were pretreated for 24 hours with tumor necrosis factor alpha (TNF alpha; 100 U/mL), which is known to increase VT receptor expression on HUVECs. The number of adhering leukocytes on HUVECs exposed to TNF alpha and VT-1 significantly increased as compared with HUVECs incubated with VT-1 and TNF alpha alone. These results suggest that VT-1 modulates leukocyte-endothelium interaction, thus increasing leukocyte adhesion and upregulating adhesive proteins on endothelial surface membrane.     

The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). Using a computational method called "Adhesive Dynamics," we have simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor-ligand functional properties and the dynamics of adhesion. We express this relationship in a state diagram, a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors. Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. We varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate, k(r)(o), and the bond interaction length, gamma, are the most important molecular properties controlling the dynamics of adhesion. Experimental k(r)(o) and gamma values from the literature for molecules that are known to mediate rolling adhesion fall within the rolling region of the state diagram. We explain why L-selectin-mediated rolling, which has faster k(r)(o) than other selectins, is accompanied by a smaller value for gamma. We also show how changes in association rate, shear rate, and bond elasticity alter the dynamics of adhesion. The state diagram (which must be mapped for each receptor-ligand system) presents a concise and comprehensive means of understanding the relationship between bond functional properties and the dynamics of adhesion mediated by receptor-ligand bonds.     

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

During inflammation, E-selectin expressed on cytokine-activated endothelial cells mediates leukocyte rolling under flow. E-selectin undergoes endocytosis and may associate with lipid rafts. We asked whether distribution of E-selectin in membrane domains affects its functions. E-selectin was internalized in transfected CHO cells or cytokine-activated human umbilical vein endothelial cells (HUVECs). Confocal microscopy demonstrated colocalization of E-selectin with alpha-adaptin, a clathrin-associated protein. Deleting the cytoplasmic domain of E-selectin or disrupting clathrin-coated pits with hypertonic medium blocked internalization of E-selectin, reduced colocalization of E-selectin with alpha-adaptin, and inhibited E-selectin-mediated neutrophil rolling under flow. Unlike CHO cells, HUVECs expressed a small percentage of E-selectin in lipid rafts. Even fewer neutrophils rolled on E-selectin in HUVECs treated with hypertonic medium and with methyl-beta-cyclodextrin, which disrupts lipid rafts. These data demonstrate that E-selectin clusters in both clathrin-coated pits and lipid rafts of endothelial cells but is internalized in clathrin-coated pits. Distribution in both domains markedly enhances E-selectin's ability to mediate leukocyte rolling under flow.     

Effects of diet and of dietary components on endothelial leukocyte adhesion molecules

Endothelial-leukocyte adhesion molecules are involved in processes regulating the selective attachment of leukocytes to the vessel wall, which participate in tissue inflammation, atherogenesis, and immunity. There has been recent appreciation that diet or specific dietary components may modulate such processes. Highly unsaturated— particularly omega-3 — fatty acids and antioxidants are receiving increasing attention in this regard as potential antiatherogenic and anti-inflammatory agents. The vascular surface expression of endothelial leukocyte molecules can also be reflected by plasma levels of “soluble” adhesion molecules, thus allowing the assessment of the effects of diet and selected dietary components on these processes in vivo.     

Extracorporal-circulation-induced leukocyte/endothelial cell interaction is inhibited by dextran

Clinical complications of Extracorporeal Circulation (ECC) are the consequence of a systemic activation of cellular and humoral factors resulting in a dysregulation of the microcirculation. Previously we developed an animal model to study effects of ECC on the microcirculation in vivo. To prevent the systemic inflammatory reaction (SIRS) seen after ECC, colloids were used as priming solution. Intravital fluorescence microscopy was used on the hamster skinfold chamber model. ECC was introduced and the ECC-tube system was flushed with Ringer solution (control), with Dextran 60 (group I) or HES 10% (group II). ECC for 30 minutes resulted in an increase in rolling and adherent leukocytes in postcapillary venules (Roller: 11 +/- 3% to 38% +/- 20% 4 h after ECC, p < 0.05, Sticker: 19 +/- 16 cells/mm2 to 215 +/- 145 cells/mm2 4 h after ECC, p < 0.05; n = 7). Use of Dextran prevented L/E cell interaction (10 +/- 5%; 63 +/- 40 cells/mm2 at 4 h), whereas HES affected only adherent white cells. L/E cell interaction in the microcirculation is an indicator of the systemic activation induced by ECC. Dextran 60 prevented L/E cell interaction without side effects and may be preferable for ECC in regard to inhibition of SIRS.     

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.     

A novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow

Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective beta2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte beta1 and beta2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein-coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of beta2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.     

Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro

Bovine aortic endothelial cells were cultured on the basement membrane surface of amnionic membrane and used as a substrate for polymorphonuclear leukocyte (PMN) diapedesis in vitro. Norepinephrine (NE), serotonin (5HT), or phalloidin treatment of the endothelial cells (ECs) reduces, whereas histamine or cytochalasin B increases, the number of PMNs migrating across the ECs and amnionic membrane. In contrast, amine treatment of PMNs or acellular amnionic membrane does not alter PMN diapedesis or chemotaxis. The NE and histamine effects are blocked by appropriate receptor antagonists, but the 5HT effect is not. All the agents' effects are also reversible. Qualitatively similar effects on EC permeability to Evan's blue-labeled albumin occur with all agents; however, PMN adhesion to ECs is not affected. Previously, we reported that NE and 5HT increase stress fiber numbers and decrease EC permeability to macromolecules in vitro, whereas histamine has the opposite effects, and that NE and 5HT eliminate the erythrocyte extravasation associated with thrombocytopenia in vivo. In this study, we propose that these vasoactive amines also alter PMN diapedesis in vitro through a direct effect on the EC, in part due to alterations in the EC cytoskeleton.     

Direct cytotoxicity of polymorphonuclear leukocyte granule proteins to human lung-derived cells and endothelial cells

Neutrophils, in the course of defending the host against microbial invasion, release a potent arsenal of proteins that can potentially damage host tissues. Defensins are major peptides of human polymorphonuclear leukocyte (PMN) granules and are both broadly microbicidal and cytotoxic to several tumor cell lines. To determine whether these peptides could play a role in neutrophil-mediated lung injury, we examined the cytotoxicity of defensins and other PMN granule proteins in a chromium release assay with human lung-derived cell lines MRC-5 (lung fetal fibroblast), A549 (lung adenocarcinoma with features of alveolar epithelium), and primary cultures of human umbilical vein endothelial cells (HUVEC). Crude fractionation of an acid extract of human PMN granules yielded four fractions A-D. Only fraction D (containing mostly defensins) was significantly cytotoxic to all three target cells. In contrast, fraction A (containing myeloperoxidase and lactoferrin) and fraction C (containing lysozyme) had little effect, and fraction B (containing chiefly cathepsin G and elastase) was only injurious to endothelial cells. The cytotoxicity of whole PMN granule extracts on pulmonary epithelial and fibroblast targets could be completely accounted for by their defensin content. Fraction D- and defensin-mediated cytotoxicity was concentration dependent, required at least 10 to 12 h to become manifest, and was inhibited by serum. The role of these peptides in lung damage during acute and chronic inflammation deserves further study.     

氧化低密度脂蛋白对人脐静脉内皮细胞PECAM-1基因转录的影响

目的探讨氧化低密度脂蛋白（Ox-LDL）对人血管内皮细胞血小板内皮细胞黏附分子-1（PECAM-1）mRNA表达的影响。方法体外培养人脐静脉内皮细胞（HUVEC）,采用铜离子氧化24h制备Ox-LDL。取HUVEC以100mg／LOx-LDL分别孵育0．5、1、1．5、2h,另取HUVEC分别用100 mg／LLDL和0、50、100、200mg／LOx-LDL孵育1．5h．均采用RT-PCR技术检测细胞中PECAM-1基因的转录水平。结果Ox-LDL除正常LDL所含成分外．还含有胆固醇亚油酸酯的过氧化特异斑点X和甲基脂肪酸。Ox-LDL作用0．5h时PECAM-1 mRNA表达水平最低;随时间延长,表达水平逐渐升高,与0．5h时比较,P〈0．05;至2h时．PECAM-1表达水平有所回落,但与0．5h时比较,P-〈0．05。0、50、100、200mg／LOx-LDL作用1．5h,PECAM-1表达水平逐渐升高,以100mg／L时表达水平最高。100mg／LLDL作用1.5h时,PECAM-1表达水平无明显变化。结论Ox-LDL可在转录水平上调血管内皮细胞PECAM-1基因表达．从而促进血管内皮细胞高水平表达PECAM-1。     

The endothelial leukocyte adhesion molecule. Role in coronary artery disease

OBJECTIVES: It is hypothesized that adhesion molecules could be an early predictor of coronary artery disease. The endothelial leucocyte adhesion molecule (ELAM) is expressed on activated endothelial cells only and it has been found to exist in a soluble form. This soluble form (sELAM) may be an important marker for endothelial cell damage. The aim of the present study was to compare the sELAM levels in coronary artery disease (CAD) subjects and healthy controls and to evaluate their clinical usefulness. METHODS AND RESULTS: sELAM were measured using enzyme immunoassay methods in 145 subjects having angiographically determined CAD and compared with 70 healthy, normotensive controls having a normal stress test/angiogram. Significantly higher values (p < 0.0001 ) were observed in CAD subjects as compared to controls. Also, subjects who underwent angioplasty and were later on readmitted with restenosis within 1 year had significantly higher levels of sELAM as compared to those who did not get restenosis within a year. CONCLUSIONS: These findings show that sELAM concentration is elevated in the presence of CAD and is useful for determining the presence of coronary atherosclerosis. An increased level of sELAM in patients susceptible to restenosis supports a role for white blood cell/endothelial interaction in restenosis after angioplasty.     

Morphine attenuates leukocyte/endothelial interactions

Gram-negative sepsis and subsequent endotoxic shock after surgery remain problematic in the United States and throughout the world. While morphine is widely prescribed for postoperative trauma pain management, there are reports that morphine may compromise the immune system and contribute to postoperative sepsis. The current study tested the hypothesis that morphine attenuates leukocyte rolling and sticking in both arterioles and venules via nitric oxide production. Nude mice implanted with slow-release morphine pellets were used in this study. The dorsal skinfold chamber model for intravital fluorescence microscopy on awake mice was used. Leukocyte/endothelial interactions were evaluated after bolus injection of oxidized low density lipoprotein. Morphine was found to significantly attenuate leukocyte rolling and sticking in both the arterial and venular side of the microcirculation. This attenuation was reversed by simultaneous implantation of naloxone pellets. The mechanisms of this attenuation were further investigated by administration of the nitric oxide synthase inhibitors NG-nitro-l-arginine (NOLA) and aminoguanidine (AG) in drinking water. NOLA was found to significantly reverse this morphine-induced attenuation of leukocyte rolling and sticking in both arterioles and venules. However, AG did not have the same effect. The results indicate that morphine interferes with leukocyte/endothelial cell interactions via stimulation of nitric oxide production.     

RNAi-mediated silencing of CD40 prevents leukocyte adhesion on CD154-activated endothelial cells

The CD40-CD154 dyad has a central role in the development of immune-inflammatory processes. Therefore, disruption of CD40 signaling has the potential to be therapeutically useful in a number of disease indications, including autoimmune syndromes, atherosclerosis, and allograft rejection. Blocking antibodies to CD154 have been successfully employed in experimental animal models, and recently in clinical trials, to prevent or treat these immunologically induced diseases. However, the thrombotic events observed in some of these studies raise important issues regarding future use of anti-CD154 antibodies in humans. In this study, we demonstrate that a small interfering RNA (siRNA) can effectively reduce the surface expression of the human CD40 costimulatory receptor. Moreover, by rendering endothelial cells unresponsive to CD154(+) Jurkat cell-mediated activation through RNA interference, induction of endothelial cell-adhesion molecule expression and leukocyte adhesion is prevented in vitro. Thus, anti-CD40 siRNA may become a safe and effective therapeutic option for interfering with CD40-CD154-mediated acute or chronic immune-inflammatory conditions.     

Butyrate inhibits leukocyte adhesion to endothelial cells via modulation of VCAM-1

BACKGROUND: Leukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-kappaB)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. METHODS: VCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-alpha (TNF-alpha) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-kappaB activation. RESULTS: The observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-kappaB activation in human endothelial cells. In this context, the observed suppression of the TNF-alpha induced VCAM-1 expression is likely to play an essential role. CONCLUSIONS: Butyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis.     

Urokinase in conditioned medium from phorbol ester-pretreated endothelial cells promotes polymorphonuclear leukocyte migration.

Serum-free conditioned medium (CM) generated by human umbilical vein endothelial cell monolayers following pretreatment with 100 ng/ml of phorbol myristate acetate (PMA) promoted human polymorphonuclear leukocyte (PMNL) migration as assayed in blindwell chambers. Stimulation of PMNL migration in response to CM was dependent on the dose of PMA used to pretreat the endothelial cells as well as the duration of incubation time to generate CM. Phorbol esters have been previously shown to release plasminogen activators from vascular endothelial cells. In the present study, pretreatment of endothelial cells with PMA also increased plasminogen activator activity in CM at a time course similar to the generation of PMNL chemoattractant activity. Treatment of CM with a polyclonal antibody against human urokinase-type plasminogen activator (uPA) not only inhibited uPA activity, but also significantly reduced PMNL chemoattractant activity when compared with untreated CM. In contrast, treatment of CM with an antibody directed against tissue-type plasminogen activator (tPA) did not affect PMNL migratory activity. Furthermore, when CM was passed over an anti-uPA immunoaffinity column, plasminogen activator activity was reduced 90% and chemoattractant activities was reduced 68%. Both plasminogen activator and chemoattractant activities were reconstituted in the eluate from the anti-uPA column. These data demonstrate that uPA present in the CM from PMA-pretreated endothelial cells stimulates PMNL chemoattractant activity and suggests a possible role for endothelial cell-derived uPA in stimulating migration of peripheral blood leukocytes at an inflammatory locus.