The chemokine and scavenger receptor CXCL16/SR-PSOX is expressed in human vascular smooth muscle cells and is induced by interferon gamma

Atherosclerosis is an inflammatory disease that is characterised by the involvement of chemokines that are important for the recruitment of leukocytes and scavenger receptors that mediate foam cell formation. Several cytokines are involved in the regulation of chemokines and scavenger receptors in atherosclerosis. CXCL16 is a chemokine and scavenger receptor and found in macrophages in human atherosclerotic lesions. Using double-labelled immunohistochemistry, we identified that smooth muscle cells in human lesions express CXCL16. We then analysed the effects of IFN-gamma, TNF-alpha, IL-12, IL-15, IL-18, and LPS on CXCL16 expression in cultured aortic smooth muscle cells. IFN-gamma was the most potent CXCL16 inducer and increased mRNA, soluble form, membrane form, and total cellular levels of CXCL16. The IFN-gamma induction of CXCL16 was also associated with increased uptake of oxLDL into these cells. Taken together, smooth muscle cells express CXCL16 in atherosclerotic lesions, which may play a role in the attraction of T cells to atherosclerotic lesions and contribute to the cellular internalisation of modified LDL.     

Leukocyte adhesion to endothelial cells

The adhesion of leukocytes to endothelium is a physiological phenomenon which is the first step for leukocyte emigration. The adhesion can be dramatically increased in pathological situations such as inflammation and vascular diseases. The molecular basis of leukocyte-endothelium interaction has been largely investigated in the last ten years. Using monoclonal antibodies it is possible to characterize the leukocyte adhesion molecule (LeuCAM) also named CD11/CD18 complex. These molecules responsible for leukocyte adhesion are heterodimers consisting of a common beta subunit and different subunit CD11a/CD18 corresponding to LFA-1; CD11b/CD18 to Mac1/Mol; CD11c/CD18 to GP150-95. Beside these receptors, other leukocyte structures such as the fibronectin receptors are involved in the adhesive process. On the endothelial cell side specialized structures implicated in leukocyte adhesion have been identified. Structures like Intercellular Adhesion Molecule (ICAM) are expressed on endothelial cells in the absence of stimulation, while other receptors Endothelial Leukocyte Adhesion Molecule (ELAM) are only detectable on activated endothelial cells. Cytokines such as IL-1 induced the expression of ELAM, increased the number of ICAM and Human Leukocyte Antigens (HLA) DR, DP, DQ. In various pathological circumstances, namely extracorporeal circulation, Acute Respiratory Distress Syndrome (ARDS), hypercholesterolemia and diabetes mellitus increased leukocyte adhesion has been reported and is potentially responsible for vascular damage. Therefore, the modulation of leukocyte-endothelial cell interactions is a possible target for antithrombotic and antiatherosclerotic therapy.     

CXCL16 is a novel angiogenic factor for human umbilical vein endothelial cells

CXCL16 is a unique chemokine with characteristics as a receptor for phosphatidylserine and oxidized low density lipoproteins in macrophages, and is involved in the accumulation of cellular cholesterol during atherosclerotic lesion development. In this study, we report a new function of CXCL16 as a novel angiogenic factor in human umbilical vein endothelial cells (HUVEC). CXCL16 stimulated proliferation and chemotaxis of HUVEC in a dose-dependent manner, reaching a maximum at 1 nM. CXCL16 also significantly induced tube formation of HUVEC on Matrigel. Further, exposure of HUVEC to CXCL16 led to a time- and dose-dependent activation of mitogen-activated protein kinase (ERK1/2), which was completely inhibited by a mitogen-activated protein kinase kinase inhibitor, PD98059. Proliferation and tube formation in response to CXCL16 were also blocked by the pretreatment with PD98059, but not CXCL16-induced chemotaxis. Thus, our data indicate that CXCL16 may act as a novel angiogenic factor for HUVEC and that ERK is involved as an important signaling molecule to mediate its angiogenic effects.     

Dynamic adhesion of T lymphocytes to endothelial cells revealed by atomic force microscopy

The recruitment of T lymphocytes to lymphoid organs or sites of inflammation is a crucial step in adaptive immunity. These processes require endothelial activation and expression of adhesion molecules, including E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, the complete characterization of the adhesion strength and dynamics between lymphocytes and endothelial cells has been hampered by the lack of sensitive quantitative techniques. Here we report on the application of atomic force microscopy to characterize the interaction between individual pairs of living T lymphocytes (i.e., Jurkat cells) and human umbilical vein endothelial cells (HUVECs). The detachment of individual cell-cell conjugates was a complex process involving several step-like rupture events and the viscoelastic deformation of cells on the scale of several microns. Adhesion between Jurkat cells and activated endothelial cells increased with compression force and contact time, with the most dramatic changes occurring within the first half second of contact. After 0.25 sec of contact, E-selectin, ICAM-1, and VCAM-1 contributed to 18%, 39%, and 41% of total adhesion strength, respectively, suggesting that ICAM-1 and VCAM-1 contributed more than the selectins in supporting cell attachment.     

Phosphatidylserine-dependent adhesion of T cells to endothelial cells

Phosphatidylserine (PS) was exposed at the surface of human umbilical vein endothelial cells (HUVECs) and cultured cell lines by agonists that increase cytosolic Ca(2+), and factors governing the adhesion of T cells to the treated cells were investigated. Thrombin, ionophore A23187 and the Ca(2+)-ATPase inhibitor 2, 5-di-tert-butyl-1,4-benzohydroquinone each induced a PS-dependent adhesion of Jurkat T cells. A23187, which was the most effective agonist in releasing PS-bearing microvesicles, was the least effective in inducing the PS-dependent adhesion of Jurkat cells. Treatment of ECV304 and EA.hy926 cells with EGTA, followed by a return to normal medium, resulted in an influx of Ca(2+) and an increase in adhering Jurkat cells. Oxidised low-density lipoprotein induced a procoagulant response in cultured ECV304 cells and increased the number of adhering Jurkat cells, but adhesion was not inhibited by pretreating ECV304 cells with annexin V. PS was not significantly exposed on untreated Jurkat cells, as determined by flow cytometry with annexin V-FITC. However, after adhesion to thrombin-treated ECV304 cells for 10 min followed by detachment in 1 mM EDTA, there was a marked exposure of PS on the Jurkat cells. Binding of annexin V-FITC to the detached cells was inhibited by pretreating them with unlabelled annexin V. Contact with thrombin-treated ECV304 cells thus induced the exposure of PS on Jurkat cells and, as Jurkat cells were unable to adhere to thrombin-treated ECV304 cells in the presence of EGTA, the adhesion of the two cell types may involve a Ca(2+) bridge between PS on both cell surfaces. The number of T cells from normal, human peripheral blood that adhered to ECV304 cells was not increased by treating the latter with thrombin. However, findings made with several T cell lines were generally, but not completely, consistent with the possibility that adhesion to surface PS on endothelial cells may be a feature of T cells that express both CD4(+) and CD8(+) antigens. Possible implications for PS-dependent adhesion of T cells to endothelial cells in metastasis, and early in atherogenesis, are discussed.     

C-reactive protein promotes adhesion of monocytes to endothelial cells via NADPH oxidase-mediated oxidative stress

Enhanced monocyte adhesion to endothelial cells is an early event in atherogenesis. It has been shown that C‐reactive protein (CRP) plays a key role in atherogenesis. Here, we investigated the effects of CRP on monocyte‐endothelial cell adhesion and tested the hypothesis that NADPH oxidase (NOX)‐mediated oxidative stress might play a key role in CRP‐induced monocyte‐endothelial cell adhesion. Firstly, 36 patients with carotid intima‐media thickness (IMT) incrassation and 34 controls were enrolled in this study. The levels of glucose, lipids, CRP, monocyte chemotractant protein (MCP‐1), malondialdehyde (MDA), and protein carbonylation were analyzed. The results showed that carotid IMT was associated with abnormal lipid metabolism, including elevated CRP, triglycerides (TG) (P < 0.01) and decreased high density lipoprotein (HDL) level (P < 0.05). The levels of CRP and MCP‐1 in patients with carotid IMT incrassation were increased compared with the controls (P < 0.01). Moreover, patients with carotid IMT incrassation displayed enhanced MDA and protein carbonylation levels (P < 0.01), accompanied by activation and up‐regulation of NOX in monocytes (P < 0.05) compared with the controls. The monocytes isolated from five healthy donors were used for in vitro experiments. Reactive oxygen species (ROS) production and NOX expression in monocytes were examined. The results also indicated that CRP could promote the adhesion of monocyte‐endothelial cell by up‐regulation of MCP‐1 expression (P < 0.05). Importantly, NFκ B and p38 MAPK signaling pathways, which were activated by NOX‐derived ROS, were involved in CRP‐induced monocyte‐endothelial cell adhesion and up‐regulation of MCP‐1 expression. These data suggested that CRP could promote the adhesion of monocytes to endothelial cells via NOX‐mediated oxidative stress. J. Cell. Biochem. 113: 857–867, 2012. © 2011 Wiley Periodicals, Inc.     

T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1.

The action of human rIL-1 beta on confluent, quiescent monolayers of human umbilical vein endothelial cells (HUVEC) has been studied for the induction of new membrane proteins. Two approaches have been taken. The first is a quantitative two-dimensional gel analysis of [35S]cysteine-labeled membrane proteins of HUVEC with and without cytokine treatment. This analysis indicates that there are a restricted number of new membrane proteins synthesized in the first 6 h of IL-1 treatment, on the order of 19 out of a total of over 600 detectable proteins. Second, we have prepared two mAb (1E7 and 2G7) to different epitopes of a major inducible sialoglycoprotein with molecular mass of 114 kDa and an isoelectric point of 4.6 to 4.8. These antibodies were compared with two additional antibodies, 3B7 and 7A9, which were shown to react with the endothelial leukocyte adhesion molecule-1 (ELAM-1) protein as expressed in COS cells. The 1E7/2G7 protein is distinct from ELAM-1, based upon biochemical comparisons as well as the inability of the 1E7 and 2G7 antibodies to react with ELAM-1-transfected COS cells. The protein defined as 1E7/2G7 is neither expressed constitutively nor in an inducible manner on PBMC, granulocytes, platelets, fibroblasts, or keratinocytes. The 7A9 and 3B7 antibodies are shown to block granulocyte binding to IL-1-activated HUVEC. The 2G7 antibody is effective at inhibiting the binding of T cells but not granulocytes to IL-1-activated endothelium, suggesting this new protein is an adhesion protein that may be active in vivo in T cell-endothelial cell adhesion-related events such as inflammation or lymphocyte recirculation. In addition, T cells were shown to utilize the ELAM-1 protein in binding to cytokine-activated HUVEC. Antibodies directed to both proteins had additive effects on inhibition of T cell adhesion.     

Selection of Babesia bovis-infected erythrocytes for adhesion to endothelial cells coselects for altered variant erythrocyte surface antigen isoforms

Sequestration of Babesia bovis-infected erythrocytes (IRBCs) in the host microvasculature is thought to constitute an important mechanism of immune evasion. Since Ig is considered to be important for protection from disease, an in vitro assay of B. bovis sequestration was used to explore the ability of anti-B. bovis Ig to interfere with IRBC cytoadhesion, and to identify IRBC surface Ags acting as endothelial cell receptors. Bovine infection sera reactive with the IRBC surface inhibited and even reversed the binding of IRBCs to bovine brain capillary endothelial cells (BBECs). This activity is at least partially attributable to serum IgG. IgG isolated from inhibitory serum captured the variant erythrocyte surface ag 1 (VESA1) in surface-specific immunoprecipitations of B. bovis-IRBCs. Selection for the cytoadhesive phenotype concurrently selected for antigenic and structural changes in the VESA1 Ag. In addition, the anti-VESA1 mAb, 4D9.1G1, proved capable of effectively inhibiting and reversing binding of adhesive, mAb-reactive parasites to BBECs, and by immunoelectron microscopy localized VESA1 to the external tips of the IRBC membrane knobs. These data are consistent with a link between antigenic variation and cytoadherence in B. bovis and suggest that the VESA1 Ag acts as an endothelial cell ligand on the B. bovis-IRBC.     

CXCL16 Promotes Gastric Cancer Tumorigenesis via ADAM10-Dependent CXCL16/CXCR6 Axis and Activates Akt and MAPK Signaling Pathways

Abnormal expression of CXC motif chemokine ligand 16 (CXCL16) has been demonstrated to be associated with tumor progression and metastasis, served as a prognostic factor in many cancers, with higher relative expression behaving as a marker of tumor progression. However, its role and mechanisms underlying progression and metastasis of gastric cancer (GC) are yet to be elucidated. In our investigation, public datasets and human GC tissue samples were used to determine the CXCL16 expression levels. Our results revealed that CXCL16 was upregulated in GC. The high expression CXCL16 in GC was significantly associated with histologic poor differentiation and pTNM staging. And high CXCL16 was positively correlated with the poor survival of GC patients. Gain-and loss-of-function experiments were employed to investigate the biological role of CXCL16 in proliferation and migration both in vitro and in vivo. Mechanically, Gene set enrichment analysis (GSEA) revealed that the epithelial‑mesenchymal transition (EMT), Akt and MAPK signal pathway related genes were significantly enriched in the high CXCL16 group, which was confirmed by western blot. Moreover, overexpression CXCL16 promoted the disintegrin and metalloproteases (ADAM10) and the CXC motif chemokine receptor 6 (CXCR6) expression, which mediated the CXCL16/CXCR6 positive feedback loop in GC, with activating Akt and MAPK signaling pathways. Knocking down ADAM10 would interrupted the CXCL16/CXCR6 axis in the carcinogenesis and progression of GC. In conclusion, our findings offered insights into that CXCL16 promoted GC tumorigenesis by enhancing ADAM10-dependent CXCL16/CXCR6 axis activation.     

Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway.

Accumulating evidence suggests that enhanced peroxynitrite (ONOO-) formation occurs during inflammation. We have studied the impact and the mechanisms of ONOO- action on expression of adhesion molecules on human neutrophils and coronary artery endothelial cells (HCAEC) and binding of neutrophils to HCAEC. Addition of ONOO- (0.1 to 200 5M) to isolated neutrophils resulted in a concentration-dependent down-regulation of L-selectin expression, and up-regulation of CD11b/CD18 expression. ONOO- stimulation of Erk activity was accompanied by activation of Ras, Raf-1 and MEK (mitogen-activated protein kinase kinase), and was sensitive to the MEK inhibitor PD 98059. We have observed a tight association between Erk activation and changes in CD11b/CD18 expression. ONOO- also evoked activation of neutrophil p38 MAPK. Neither ONOO--induced up-regulation of CD11b/CD18 expression nor Erk activation was affected by SB 203580, a selective inhibitor of p38 MAPK. ONOO- by itself had little effect on expression of ICAM-1 and E-selectin on HCAEC, whereas it markedly enhanced attachment of neutrophils to lipopolysaccharide-activated HCAEC only when it was added together with neutrophils. Increases in neutrophil adhesion evoked by ONOO- were blocked by an anti-CD18 monoclonal antibody. These data suggest that ONOO- activates Erk in neutrophils via the Ras/Raf-1/MEK signal transduction pathway, leading to up-regulation of surface expression of CD11b/CD18 and consequently to increased neutrophil adhesion to endothelial cells.     

Oxygen tension regulates neutrophil adhesion to human endothelial cells via an LFA-1-dependent mechanism

Extravasation of leukocytes at the sites of ischemia-reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO₂ of 50 torr for 3 hours, increased 2.5-fold, 2-, and 1.5-fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell-associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia-induced adhesion was inhibited by LFA-1-specific (CD 11 a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM-1 ligand for the LFA-1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA-1 molecule on edothelial cells which can be affected by hypoxic environments.     

Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α₅β₁ integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α₅β₁ with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.     

Low shear stress regulates monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha dependent pathway

In regions of a vessel that experience low shear stress and reversing flow patterns, early features in the pathogenesis of atherosclerosis include the accumulation of oxidized LDL (OxLDL) and adhesion of monocytes to endothelial cells (EC). Here we investigated the hypothesis that low shear stress (2 dyn/cm2) and OxLDL are synergistic for enhanced expression of vascular cell adhesion molecule (VCAM-1) and human aortic endothelial cell (HAEC)-monocyte adhesion. This study shows low shear stress can significantly reduce IkappaBalpha levels, activate NF-kappaB, increase the expression of VCAM-1 in HAEC and binding of monocytes. OxLDL itself cannot significantly increase the expression of VCAM-1 in HAEC and binding of monocytes, but through activation of NF-kappaB and degradation of IkappaBalpha induced by low shear stress it can significantly enhance VCAM-1 expression and monocyte adhesion, over that in unmodified LDL or control. These results suggest that low shear stress can regulate monocyte adhesion to oxidized lipid-induced endothelial cells via an IkappaBalpha-dependent pathway, and that low shear stress together with OxLDL may likely play an important role in atherogenesis.     

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

Background

Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.

Methods

RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.

Results

Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.

Conclusion

These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.

General significance

Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.     

Thrombospondin inhibits adhesion of endothelial cells

Adsorption of thrombospondin to a substratum inhibits adhesion of endothelial cells to that substratum. Four hours after plating of cells on glass covered with thrombospondin, the number of cells bound per unit area was only 8% of that bound to fibronectin, and 20% of that which could bind to albumin. While on fibronectin cells assumed a well-spread configuration with time in culture, on thrombospondin they stayed completely round. On surfaces constructed by sequential incubation of glass with thrombospondin and fibronectin or other proteins, thrombospondin retained its inhibitory effect on cell adhesion. Fibronectin surfaces treated with thrombospondin lost 50% of their capacity to adhere endothelial cells. Cell spreading was also greatly impaired. These observations indicate that thrombospondin, which is a component of the extracellular matrix, can modulate adhesion of endothelial cells to the matrix.