Double-stranded RNA induces galectin-9 in vascular endothelial cells: involvement of TLR3, PI3K, and IRF3 pathway

Galectin-9 is a member of the galectin family, which induces various biological reactions such as chemotaxis of eosinophils and apoptosis of T cells. We previously reported that polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA), induces the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs). In the present study, we addressed the possible involvement of two potential receptors for dsRNA, Toll-like receptor (TLR) 3 and retinoic acid-inducible gene-I (RIG-I), in the expression of galectin-9 in HUVECs. Poly IC-induced galectin-9 expression was almost completely suppressed by RNA interference (RNAi) against TLR3, but not against RIG-I. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited the induction of galectin-9 by poly IC. RNAi against interferon regulatory factor 3 (IRF3) also inhibited poly IC-induced galectin-9 expression. We conclude that TLR3, PI3K, and IRF3 are involved in the poly IC-induced galectin-9 expression in HUVECs.     

Regulation of TLR2 Expression and Function in Human Airway Epithelial Cells

Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM₃CSK₄. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM₃CSK₄-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM₃CSK₄), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.     

Expression of interferon-stimulated gene 20 in vascular endothelial cells

ISG20 is an ribonuclease specific for single-stranded RNA and considered to play a role in innate immunity against virus infections. We herein show that both poly IC, an authentic double-stranded RNA, and IFN-gamma induced ISG20 expression in cultured HUVEC. Poly IC-induced ISG20 expression was inhibited by LY294002, an inhibitor of PI3K, or by RNA interference against IFN regulatory factor three. ISG20 expression was not induced by IFN-beta, loxoribine or CpG oligonucleotide. These results suggest that ISG20 induction by poly IC may not be dependent on the IRF-3-mediated type I IFN induction pathway in HUVEC. ISG20 may be involved in innate immunity against viral infection in vascular endothelial cells.     

Hepatitis C Virus Induced Endothelial Inflammatory Response Depends on the Functional Expression of TNFα Receptor Subtype 2

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.     

Endothelial LGALS9 splice variant expression in endothelial cell biology and angiogenesis

Galectins are carbohydrate binding proteins with versatile functions in tumor progression. Galectin-9, encoded by LGALS9, has been associated with metastasis and immunosuppression. We previously reported on regulation of LGALS9 expression during endothelial cell activation. Here, we show increased galectin-9 protein levels in the endothelium of different tumors, including carcinomas of the lung, liver, breast and kidney. Endothelial cells were found to express five LGALS9 splice variants, two of which have not been reported before. Splicing was found to be confined to exons 5, 6 and 10. Transfection of human microvascular endothelial cells (HMEC) with galectin-9∆5, a specific LGALS9 splice variant, induced a small but significant increase of proliferation, while migration was not affected by any LGALS9 splice variant. Application of recombinant galectin-9∆5 protein dose-dependently reduced proliferation and migration of HMEC as well as human umbilical vein endothelial cells in vitro. Enhanced sprouting and migration of human umbilical vein endothelial cell (HUVEC) towards a galectin-9∆5 gradient were observed. Interestingly, galectin-9∆5 was found to induce a small inhibitory effect on angiogenesis in vivo. Collectively, these data show that endothelial cells regulate the expression and splicing of LGALS9 during angiogenesis. The function of the dominant splice variant, i.e. galectin-9∆5, in endothelial cell biology depends on the concentration and environmental context in which it is presented to the cells.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.     

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide

Viral infection has been implicated as a triggering event that may initiate beta-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-gamma (IFN-gamma) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-gamma correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-gamma activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-alpha (TNF-alpha) + lipopolysaccharide, activated resident macrophages mediate beta-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by beta-cells. The inhibitory and destructive effects of poly(IC) + IFN-gamma, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-gamma results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-gamma on islets appear to be mediated by a direct interaction with beta-cells. Poly IC + IFN-gamma stimulates iNOS expression and inhibits insulin secretion by primary beta-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-gamma on glucose-stimulated insulin secretion by beta-cells. These results indicate that dsRNA + IFN-gamma interacts directly with beta-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of beta-cells during the development of autoimmune diabetes.     

Simvastatin modulates TNFalpha-induced adhesion molecules expression in human endothelial cells

Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDL-cholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor alpha (TNFalpha). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFalpha-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFalpha-induced activation of nuclear factor kappaB (NF-kappaB) and enhanced expression of peroxisome proliferator-activated receptor alpha (PPARalpha). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-kappaB.     

Zinc finger protein A20 overexpression inhibits monocyte homing and protects endothelial cells from injury induced by high glucose

Diabetes mellitus causes vascular lesions and may ultimately lead to atherosclerosis. One of the earliest steps in the development of atherosclerotic lesions is the adhesion of monocytes to endothelial cells of the vessel wall. It is currently unknown whether zinc finger protein A20 is able to protect endothelial cells from injury caused by high levels of glucose and monocyte homing. In our study, adhesion of monocytes to the vessel wall endothelium was detected by measuring the rolling velocity of monocytes along human umbilical vein endothelial cells (HUVECs). Activation of NF-κB was analyzed through Western blot. HUVEC apoptosis was monitored by TUNEL in situ end-labeling and flow cytometry. High glucose concentrations (25 mM) stimulated monocytes, reducing the velocity at which they roll along HUVECs. Stimulation of monocytes with high levels of glucose also induced HUVEC apoptosis. Overexpression of the zinc finger protein A20 inhibited monocyte recruitment, NF-κB activation, P-selectin expression, and HUVEC apoptosis induced by high glucose levels. We conclude that zinc finger protein A20 can protect HUVECs from injury induced by high levels of glucose and potentially could be used to develop treatments against diabetic vascular lesions.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Langerhans cells exhibit low responsiveness to double-stranded RNA

Double-stranded RNA (dsRNA) is a viral product recognized by Toll-like receptor 3 (TLR3), and it is a potent activator of dendritic cells (DC). We compared Langerhans cells (LC) and splenic CD11c(+) DC and investigated the responsiveness to dsRNA. We prepared highly purified LC (> 95%) using the panning method. TLR3 mRNA was expressed in LC, splenic DC, and keratinocytes (KC). The expression of IFN-beta mRNA was enhanced in LC and splenic DC by Poly(I:C) stimulation. However, cytokine/chemokine production in response to Poly(I:C) by LC was much lower than that by splenic DC. In addition, Poly(I:C) induced further maturation in splenic DC, but not in LC. Finally, we found that the mouse KC cell line, PAM212, produced a great amount of IL-1alpha by Poly(I:C) stimulation, and that IL-1alpha promoted the maturation of LC. These data altogether indicate that LC exhibit low responsiveness to dsRNA. It is possible that KC may primarily trigger anti-viral immune responses in the skin via cytokine production such as IL-1alpha.     

Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Both noncytopathic (ncp) and cytopathic (cp) biotypes of BVDV can be isolated from persistently infected cattle suffering from the lethal mucosal disease. The cp biotype correlates with the production of the NS3 nonstructural protein, which in the corresponding ncp biotype is present in its uncleaved form, NS23. Previously, we have shown that cp but not ncp BVDV induces the formation of alpha/beta interferons in bovine macrophages. In this study, we demonstrate that ncp BVDV inhibits the induction of apoptosis and the expression of interferon alpha/beta by poly(IC), a synthetic double-stranded RNA (dsRNA). Inhibition was observed only in cells which had been infected with ncp BVDV at least 12 h prior to the addition of dsRNA, which indicates that expression of viral proteins is necessary for the ncp virus to inhibit the effects of poly(IC). Additional experiments using transfected poly(IC) showed that ncp BVDV interfered with the intracellular action of dsRNA rather than with its uptake into the cells. Infected cells were not resistant to induction of apoptosis by actinomycin D or staurosporine, which suggests that ncp BVDV may specifically interfere with signaling through dsRNA. Interference with the innate antiviral host responses may explain the successful establishment of persistent infection by ncp BVDV in fetuses early in their development.     

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performedin vivoandin vitrodemonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.