Hyperketonemia increases monocyte adhesion to endothelial cells and is mediated by LFA-1 expression in monocytes and ICAM-1 expression in endothelial cells

Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. The objective of this study was to examine the hypothesis that hyperketonemia increases monocyte-endothelial cell (EC) adhesion and the development of vascular disease in diabetes. Human U937 and THP-1 monocyte cell lines and human umbilical vein endothelial cells (HUVECs) were cultured with acetoacetate (AA) (0-10 mM) or β-hydroxybutyrate (BHB) (0-10 mM) for 24 h prior to evaluating adhesion and adhesion molecule expression. The results demonstrate a significant (P < 0.01) increase in both U937 and THP-1 adhesion to HUVEC monolayers treated with 4 mM AA compared with control. Equal concentrations of BHB resulted in similar increases in monocyte-EC adhesion. Similarly, treatments of AA or BHB to isolated monocytes from human blood also show increases in adhesion to endothelial cells. intercellular adhesion molecule-1 (ICAM-1) was significantly increased on the surface of HUVECs and an increase in total protein expression with AA treatment compared with control. The expression level of lymphocyte function-associated antigen-1 (LFA-1) was increased in monocytes treated with AA, and LFA-1 affinity was altered from low to high affinity following treatment with both AA and BHB. Monocyte adhesion could be blocked when cells were preincubated with an antibody to ICAM-1 or LFA-1. Results also show a significant increase in IL-8 and MCP-1 secretion in monocytes and HUVECs treated with 0-10 mM AA. These results suggest that hyperketonemia can induce monocyte adhesion to endothelial cells and that it is mediated via increased ICAM-1 expression in endothelial cells and increased expression and affinity of LFA-1 in monocytes.     

Cardiotrophin-1 stimulates intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human aortic endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) play critical roles in mediating monocyte adhesion to the vascular endothelium and monocyte migration into the subendothelial regions of the vessels. Inasmuch as cardiotrophin-1 (CT-1), an IL-6-type cytokine, was expressed in human atherosclerotic plaque, we examined whether CT-1 induces monocyte adhesion and migration by stimulating gene and protein expressions of ICAM-1 and MCP-1 in human aortic endothelial cells (HAECs). Immunocytochemistry revealed that CT-1 increased intensity of ICAM-1 and MCP-1 immunoreactivity in HAECs. Adhesion assay and chemotaxis assay revealed that CT-1 increased human monocytic THP-1 cell adhesion to HAECs and promoted chemotaxis in THP-1 cells, which were attenuated by anti-ICAM-1 and anti-MCP-1 antibody, respectively. Western blot analysis showed that CT-1 increased phosphorylation of ERK1/2 MAP kinase, p38 MAP kinase, and Akt and that their inhibitors, PD-98059, SB-203580, and LY-294002, respectively, inhibited phosphorylation. RNase protection assay and ELISA demonstrated that CT-1 increased gene and protein expressions of ICAM-1 and MCP-1. EMSA revealed that CT-1 enhanced NF-kappaB DNA-binding activity. CT-1-mediated upregulation of ICAM-1 and MCP-1 was suppressed by PD-98059, SB-203580, LY-294002, and parthenolide. The present study demonstrates that CT-1 promotes monocyte adhesion and migration by stimulating ICAM-1 and MCP-1 through mechanisms that involve ERK1/2 MAP kinase, p38 MAP kinase, phosphatidylinositol 3-kinase, and NF-kappaB pathways and suggests that CT-1 plays an important role in the pathophysiology of vascular inflammation and atherosclerosis.     

Toxoplasma gondii regulates ICAM-1 mediated monocyte adhesion to trophoblasts

Materno-foetal transmission causes one of the most serious forms of infection with the intracellular protozoan parasite Toxoplasma gondii. In the placenta, trophoblast cells constitute the barrier between maternal circulation and foetal tissue. We looked at the factors that determine the extent of cell adhesion to human BeWo trophoblast cells during T. gondii infection. BeWo monolayers stimulated with the supernatant of T. gondii-infected PBMC showed a large increase in THP-1 cell adhesion and upregulation of the intercellular adhesion molecule (ICAM)-1. Neutralization of cytokines by corresponding antibodies demonstrated that anti-IFN-gamma, but not anti-TNF-alpha or anti-IL-1beta, led to a significant reduction of THP-1 adhesion to a BeWo monolayer. Treatment of BeWo cells with single cytokines failed to induce upregulation of adhesion. In contrast, simultaneous treatment with IFN-gamma and either TNF-alpha or IL-1beta mimicked strongly the effect of infected cell supernatant. The results suggest that IFN-gamma plays a pivotal role in the cell adhesion process through upregulation of ICAM-1 and in the process of congenital transmission of T. gondii.     

Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (omega-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-alpha) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that omega-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.     

Lipoprotein lipase-mediated lipolysis of very low density lipoproteins increases monocyte adhesion to aortic endothelial cells.

Lipoprotein lipase (LPL) bound to vascular endothelial cells hydrolyses triglycerides in plasma lipoproteins. To explore the role of LPL in atherogenesis, the effect of LPL-mediated lipolysis of very low density lipoproteins (VLDL) on monocyte adhesion to endothelial cells was examined. Adhesion of U937 monocytes to porcine aortic endothelial cells that were incubated with VLDL and purified bovine milk LPL was markedly higher than endothelial cells that were incubated with VLDL alone. The increase in monocyte adhesion obtained with VLDL was dependent on the concentration of the lipoprotein, monocyte dose and time of incubation. The increase in adhesion correlated with generation of free fatty acids from the hydrolysis of triglycerides in VLDL by LPL. Furthermore, direct addition of oleic acid to endothelial cells also increased adhesion of monocytes. We postulate that LPL-derived lipolytic products increase monocyte adhesion to vascular endothelium and thereby promote atherogenesis.     

Human soluble E-selectin immunoadhesin inhibits leukemic monocyte adhesion to endothelial cells

Immunoadhesins are immunoglobulin (Ig)-like chimeric proteins comprised of target-binding regions fused to the Fc-hinge region of Ig, and are designed to have a long half-life and antibody-like properties. In an effort to find a good candidate for therapeutic use for inflammatory responses, we constructed a soluble human E-selectin immunoadhesin containing the extracellular region of human E-selectin fused to the Fc-hinge region of human IgG, and determined its effects on leukocyte adhesion and rolling in vitro. Our results revealed that the adhesion of leukocytes to endothelial cells was efficiently inhibited in the presence of 50 nM E-selectin immunoadhesin. In addition, the E-selectin immunoadhesin significantly inhibited leukocyte rolling on endothelial cells in perfusion experiments performed at 1.0 dyne/cm(2) wall shear stress. These findings indicate that our E-selectin immunoadhesin decreases leukocyte attachment and rolling in vitro, suggesting that this immunoadhesin may be a promising candidate for therapeutic anti-inflammatory use.     

Non-radioactive and colorimetric quantification of monocyte adhesion to endothelial cells in early atherogenesis

Monocyte adhesion to vascular endothelium is an initial step in atherogenesis. To quantify this, we incubated monocytes with cultured endothelial cells, and quantified the adhered live monocytes using a colorimetric assay. Endothelium activated with lipopolysaccharide attracted monocytes in a dose-dependent manner and the adhesion was attenuated with post-treatments with l-ascorbic acid (53%), α- (40%) and γ-tocopherol (39%), resveratrol (39%), and Lithospermum erythrorhizon root extract (45%). This non-radioactive, colorimetric assay may be useful for screening anti-atherogenic compounds in early atherogenesis.     

A membranous form of ICAM-1 on exosomes efficiently blocks leukocyte adhesion to activated endothelial cells

While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellular ICAM-1 have been detected in cell culture supernatants as well as in the serum: a soluble form of ICAM-1 (sICAM-1) and a membranous form of ICAM-1 (mICAM-1) associated with exosomes. Previous observations have demonstrated that sICAM-1 cannot exert potent immune modulatory activity due to its low affinity for leukocyte function-associated antigen-1 (LFA-1) or membrane attack complex-1. In this report, we initially observed that human cancer cells shed mICAM-1(+)-exosomes but were devoid of vascular cell adhesion molecule-1 and E-selectin. We demonstrate that mICAM-1 on exosomes retained its topology similar to that of cell surface ICAM-1, and could bind to leukocytes. In addition, we show that exosomal mICAM-1 exhibits potent anti-leukocyte adhesion activity to tumor necrosis factor-α-activated endothelial cells compared to that of sICAM-1. Taken together with previous findings, our results indicate that mICAM-1 on exosomes exhibits potent immune modulatory activity.     

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.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.     

细胞间粘附分子-1在血管内皮细胞冻融损伤中的作用

目的:探讨血管内皮细胞(VEC)表面细胞间粘附分子-1(ICAM-1)在VEC冻融损伤中的作用,以阐明冻融损伤的发病机制。方法:以大鼠主动脉VEC和大鼠外周血嗜中性粒细胞(PMN)为材料,使用WKL-Ⅴ型速率冷冻仪冷冻VEC然后在水浴中复温,制备VEC冻融模型。采用免疫组化法测定VEC冻融后4、12和24 h其表面ICAM-1的表达;将冻融VEC与正常PMN共同孵育后,以rose bengal染色法测定冻融VEC与PMN粘附,测定培养液中LDL活性确定VEC损伤程度。结果:冻融后4 h,VEC表面ICAM-1表达阳性率由冻融前的13.2%±3.6%增加至22.3%±4.4%,冻后12h达高峰(37.9%±2.5%)。冻融VEC与PMN共同孵育后,VEC-PMN粘附由对照组的0.204±0.025增加至0.363±0.022(P<0.01),培养液中LDH活性由对照组的104.64±20.14U/L增加至162.33±27.88U/L(P<0.01);ICAM-1Mab可部分阻断冻融VEC-PMN粘附(0.270±0.021,P<0.01),且使培养液中LDH活性降低至125.39±22.26U/L(P<0.05)。结论:冻融可诱发VEC表面ICAM-1的表达,进而增强VEC-PMN粘附而导致VEC损伤。     

3-deazaadenosine inhibits leukocyte adhesion and ICAM-1 biosynthesis in tumor necrosis factor-stimulated human endothelial cells

Previous reports demonstrate that cultured human umbilical vein endothelial cells (HEC) treated with TNF and other inflammatory mediators show an increased capacity to adhere human neutrophils. This increase is associated with the up-regulation of intercellular adhesion molecule 1 (ICAM-1) and other adhesion molecules on the HEC surface. We have found that 200 microM 3-deazaadenosine (c3Ado) prevented this TNF-induced increase in HEC adhesiveness. This effect resulted from interactions of c3Ado with HEC and not with polymorphonuclear neutrophils. Transport of c3Ado into the HEC was required for its activity, as evidenced by antagonism with the nucleoside transport inhibitor, nitrobenzylthioinosine. Treatment of HEC with c3Ado led to the intracellular buildup of S-adenosylhomocysteine and to the metabolic formation of S-3-deazaadenosylhomocysteine and 3-deazaadenosine 5'-triphosphate, events that appeared not to contribute to c3Ado activity. Exogenous L-homocysteine potentiated c3Ado activity, and this potentiation was prevented by the S-adenosylhomocysteine hydrolase inhibitor, periodate-oxidized adenosine. By using the mAb RR1/1, we have determined that c3Ado also inhibited the TNF-induced expression of ICAM-1 on the surface of the HEC, as well as cytosol-associated ICAM-1. Northern blot and in vitro translation analyses of poly(A+) RNA from c3Ado-treated HEC revealed that this nucleoside analog selectively decreased steady-state levels of ICAM-1 mRNA. The capacity of c3Ado to selectively inhibit HEC adhesiveness, ICAM-1 production, and steady-state levels of ICAM-1 mRNA may contribute to the drug's activity as an anti-inflammatory agent.     

Linoleic acid increases monocyte chemotaxis and adhesion to human aortic endothelial cells through protein kinase C- and cyclooxygenase-2-dependent mechanisms

The effects of polyunsaturated n-6 linoleic acid on monocyte-endothelial interactions were investigated with particular emphasis on the expression of platelet/endothelial cell adhesion molecule (PECAM)-1 and the role of protein kinase C (PKC) and cyclooxygenase-2 (COX-2). As a diet rich in polyunsaturated fatty acids may favour atherosclerosis in hyperglycaemia, this study was performed in both normal and high-glucose media using human aortic endothelial cells (HAEC). The HAEC were preincubated with normal (5 mM) or high (25 mM) D-glucose for 3 days before addition of fatty acids (0.2 mM) for 3 days. Linoleic acid enhanced PECAM-1 expression independently of tumor necrosis factor (TNF)-α and significantly increased TNF-α-induced monocyte adhesion to HAEC in comparison to the monounsaturated n-9 oleic acid. Chronic glucose treatment (25 mM, 6 days) did not modify the TNF-α-induced or fatty acid-induced changes in monocyte binding. The increase in monocyte binding was accompanied by a significant increase in E-selectin and vascular cell adhesion molecule (VCAM)-1 expression and could be abrogated by an interleukin (IL)-8 neutralising antibody and by the PKC and COX inhibitors. Inhibition of PKC-δ reduced VCAM-1 expression regardless of experimental condition and was accompanied by a significant decrease in monocyte binding. Conditioned medium from linoleic acid-treated HAEC grown in normal glucose conditions significantly increased THP-1 chemotaxis. These results suggest that linoleic acid-induced changes in monocyte chemotaxis and subsequent binding are not solely mediated by changes in adhesion molecule expression but may be due to secreted factors such as IL-8, monocyte chemoattractant protein-1 or prostaglandins (PGs) such as PGE(2), as IL-8 neutralisation and COX-2 inhibition reduced monocyte binding without changes in adhesion molecule expression.     

Membrane-type 1-matrix metalloproteinase regulates intracellular adhesion molecule-1 (ICAM-1)-mediated monocyte transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.     

Hypoxia/reoxygenation stimulates Ca2+-dependent ICAM-1 mRNA expression in human aortic endothelial cells

Increased endothelial ICAM-1 expression is found in normal aging and in atherosclerosis and is related to the chronic effects of oxidative stress. We examined the Ca(2+)-dependence of ICAM-1 mRNA expression in human aortic endothelial cells (HAEC) exposed to hypoxia/reoxygenation (H/R) as a model of oxidative stress. HAEC were exposed to glucose-free hypoxia (95% N(2)/5% CO(2)) for 60 min and were then reoxygenated (21% O(2)/5% CO(2)) and observed for up to 6h. Reactive oxygen species (ROS) generation was measured by dichlorofluorescein fluorescence and ICAM-1 mRNA was assessed by Northern blot. Upon reoxygenation after hypoxia, ROS production occurred in HAEC and was inhibited by diphenyleneiodonium and by polyethylene glycol-catalase, suggesting the involvement of NADPH oxidase-derived hydrogen peroxide. Hypoxia alone did not increase either ROS production or ICAM-1 mRNA levels, but a 2.5-fold increase in ICAM-1 mRNA was noted by 30 min of reoxygenation. This was not observed in Ca(2+)-free buffer or in cells treated with diphenyleneiodonium. Thus, H/R upregulates ICAM-1 mRNA in HAEC by a Ca(2+)- and ROS-dependent mechanism. Characterizing the signaling pathways involved in H/R-induced adhesion molecule expression may result in a better understanding of the vascular biology of normal aging and the pathobiology of atherosclerosis.