Fusion with human lung cancer cells elongates the life span of human umbilical endothelial cells and enhances the anti-tumor immunity.

PURPOSE: Human umbilical endothelial cells (HUVECs) have been proved as an effective whole-cell vaccine inhibiting tumor angiogenesis. However, HUVECs divide a very limited number of passages before entering replicative senescence, which limits its application for clinical situation. Here, we fused HUVECs with human pulmonary adenocarcinoma cell line A549s and investigated the anti-tumor immunity of the hybrids against mice Lewis lung cancer. METHODS: HUVECs were fused with A549s using polyethylene glycol and were sorted by flow cytometry. The fusion cells (HUVEC-A549s) were confirmed by testing the expression of telomerase and VE-cadherin, the senescence-associated ß-galactosidase activity, and tube formation ability. HUVEC-A549s were then irradiated and injected into the C57BL/6 mice of protective, therapeutic, and metastatic models. The mechanism of the anti-tumor immunity was explored by analyzing mice sera, spleen T lymphocytes, tumor microenvironment, and histological changes. RESULTS: HUVEC-A549s coexpressed tumor and endothelial markers and maintained the vascular function of tube forming at passage 30 without showing signs of senescence. HUVEC-A549s could induce protective and therapeutic anti-tumor activity for LL(2) model and presented stronger activity against metastasis than HUVECs. Both humoral and cellular immunity were participated in the anti-angiogenic activity, as HUVECs-neutralizing IgG and HUVECs-toxic lymphocytes were increased. Angiogenic mediators (VEGF and TGF-ß) and tumor microenvironment cells MDSCs and Tregs were also diminished. CONCLUSIONS: Our findings might provide a novel strategy for HUVECs-related immunotherapy, and this vaccine requires lower culture condition than primary HUVECs while enhancing the anti-tumor immunity.

Male gender promotes an increased inflammatory response to lipopolysaccharide in umbilical vein blood.

OBJECTIVES: To establish gender-specific differences in maternal and fetal immune response in healthy human fetuses at term. METHODS: Forty-five women with elective caesarean sections for uncomplicated singleton pregnancies were recruited for two studies. Using a multiplex biomarker immunoassay system, unstimulated maternal and fetal plasma concentrations of interleukin (IL)-1ß, IL-1ra, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, and tumor necrosis factor (TNF)-α were measured from one study population. Lipopolysaccharide (LPS)-stimulated cytokine response was measured in a second study. RESULTS: There were no significant gender differences in either maternal or fetal unstimulated plasma cytokine concentrations, but concentrations of the proinflammatory cytokines IL-1ß and IL-6 were significantly greater in male fetal LPS-stimulated samples than in female fetal samples. CONCLUSIONS: Blood of male fetuses mounts a larger pro-inflammatory response to lipopolysaccharide (LPS). This heightened response could be a critical pathway in promoting premature rupture of membranes (PPROM) and may be associated with life long differential gender response to infection.

Polyphenolics from açaí ( Euterpe oleracea Mart.) and red muscadine grape (Vitis rotundifolia ) protect human umbilical vascular Endothelial cells (HUVEC) from glucose- and lipopolysaccharide (LPS)-induced inflammation and target microRNA-126.

Endothelial anti-inflammatory effects of açaí (Ac) and red muscadine grape (Gp) polyphenolics have not been extensively investigated. It was hypothesized that polyphenolics from Ac and Gp exert comparable protective effects in human vascular endothelial cells (HUVEC) upon inflammatory stress. Furthermore, this study investigated whether microRNAs relevant to endothelial function might be regulated by Ac and Gp. Results showed that Ac and Gp (5-20 mg gallic acid equivalent/L) protected HUVEC against glucose-induced oxidative stress and inflammation. Glucose-induced expression of interleukin-6 and -8 was down-regulated by Ac and Gp at mRNA and protein levels. Upon lipopolysaccharide (LPS; 1 µg/L)-induced inflammation, Ac and Gp inhibited gene expression of adhesion molecules and NF-κB activation to similar extents, although Gp was more effective in decreasing PECAM-1 and ICAM-1 protein. Of the screened microRNAs, only microRNA-126 expression was found to be modulated by Ac and Gp as the underlying mechanism to inhibit gene and protein expression of VCAM-1.

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells.

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells' supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB-DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

Sphingosylphosphorylcholine stimulates CCL2 production from human umbilical vein endothelial cells.

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.

Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus.

The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness.

Chronic homocysteine exposure upregulates endothelial adhesion molecules and mediates leukocyte: endothelial cell interactions under flow conditions.

AIMS: Homocysteine upregulates expression of adhesion molecules on endothelial cells which recruits leukocytes and initiates atherosclerosis. Endothelial cells in hyperhomocysteinemic patients are continuously exposed to high levels of homocysteine. This study exposed adult endothelial cells and endothelial cells from immune naïve foetal tissue to homocysteine chronically and studied effects on cellular adhesion molecule expression under static and flow conditions. METHODS: Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) were cultured in medium containing 1 mM dl-homocysteine or l-cysteine for 5-9 days. Proliferation was assessed. Cells were subjected to flowing neutrophils and numbers of tethered, rolled fixed and transmigrated neutrophils on endothelial cells were counted and compared to controls. Immunofluorescence staining with antibodies against Intercellular adhesion molecule-1 (ICAM-1), E-selectin and P-selectin were used to quantify expression. RESULTS: Chronic treatment with 1 mM homocysteine inhibited proliferation of HUVEC and HSVEC. Homocysteine treated cells showed significantly increased expression of ICAM-1, E-selectin and to a lesser extent P-selectin. In both cell types, homocysteine significantly increased interactions between neutrophils and endothelial cells under flow conditions (p < 0.05) while cysteine had no effect. CONCLUSION: Endothelial cells from adult and immune naïve foetal tissue showed similar responses to chronic treatment with homocysteine.

Human placental pericytes poorly stimulate and actively regulate allogeneic CD4 T cell responses.

OBJECTIVE: Cell-mediated immune responses in peripheral tissues begin with T cell infiltration through endothelial cell (EC) microvessels and accumulation in the perivascular space occupied by pericytes (PC). Here, we investigate how human T cells interact with PC. METHODS AND RESULTS: We compared human placental PC with autologous umbilical vein EC. Cultured PC express lower levels of major histocompatibility complex (MHC) and positive costimulatory molecules but higher levels of negative costimulatory molecules than do EC. Unlike EC, interferon-γ-treated MHC class II-positive PC (PC(+)) cannot stimulate resting allogeneic CD4 T cell proliferation or cytokine production. Instead, coculture of resting CD4 T cells with PC(+) induces CD25 expression and renders T cells unresponsive to restimulation by EC(+) from the same donor. PC cultured across a semi-permeable membrane decrease alloreactive CD4 T cell proliferation to EC(+), an effect enhanced by pretreatment of PC with interferon-γ and partially reversed by interleukin-10 and transforming growth factor-ß neutralization, but do not induce anergy. CONCLUSIONS: Human placental PC are poorly immunogenic and negatively regulate CD4 T cell responses through contact-dependent and contact-independent mechanisms.

Expression of MUC1 mucin in human umbilical vein endothelial cells (HUVEC).

Mucin 1 (MUC1) is a membrane-bound glycoprotein that is expressed by various epithelial cell types. MUC1 functions include modulation of cell adhesion, signal transduction, lubrication and hydration of epithelial surfaces, and their protection from infection. In this study we demonstrated that MUC1 is expressed in human umbilical vein endothelial cells (HUVECs) and could be released/shed from cellular membrane. MUC1 presence in these cells was verified using three methods: Western blotting, flow cytometry and metabolic labeling. We also showed that mucin expression is stimulated by proinflammatory cytokines: about a 2-fold increase was observed after TNF-α treatment and lower after IFN-γ alone and in combination with TNF-α treatment. It can be assumed that the presence of MUC1 in endothelial cells may have an important role in the interactions with different cell types in physiological and pathological processes.

Mechanisms of leukocyte distribution during sepsis: an experimental study on the interdependence of cell activation, shear stress and endothelial injury.

INTRODUCTION: This study was carried out to determine whether interactions of cell activation, shear stress and platelets at sites of endothelial injury explain the paradoxical maldistribution of activated leukocytes during sepsis away from local sites of infection towards disseminated leukocyte accumulation at remote sites. METHODS: Human umbilical venous endothelial cells (HUVEC) and polymorphonuclear neutrophils (PMN) were activated with lipopolysaccharide at 100 and 10 ng/ml to achieve adhesion molecule patterns as have been reported from the hyper- and hypo-inflammatory stage of sepsis. To examine effects of leukocyte activation on leukocyte-endothelial interactions, activated HUVEC were perfused with activated and non-activated neutrophils in a parallel plate flow chamber. Adhesion molecule expression and function were assessed by flow cytometry and blocking antibodies. In a subset of experiments the sub-endothelial matrix was exposed and covered with platelets to account for the effects of endothelial injury. To investigate interactions of these effects with flow, all experiments were done at various shear stress levels (3 to 0.25 dyne/cm(2)). Leukocyte-endothelial interactions were analyzed by videomicroscopy and analysis of covariance. RESULTS: Activation of neutrophils rendered adhesion increasingly dependent on shear stress reduction. At normal shear stress, shedding of L-selectin decreased adhesion by 56%. Increased rolling fractions of activated PMN at low shear stress revealed impaired integrin affinity despite numerical up-regulation of CD11b. On sub-maximally activated, intact HUVEC shear stress became the prevailing determinant of adhesion. Presence of a platelet-covered injury with high surface density of P-selectin was the strongest variable for adhesion. When compared to maximally activated HUVEC, platelets increased neutrophil adhesion by 2.7-fold. At sub-maximal activation a 10-fold increase was observed (P < 0.05 for all). CONCLUSIONS: L-selectin shedding and integrin dysfunction render leukocyte adhesion increasingly susceptible to shear stress and alternative adhesion receptors. In combination, these effects inhibit recruitment to normally perfused sites with intact endothelium and favor maldistribution towards sites with compromised perfusion or endothelial injury.

CD4+CD25+Foxp3+ regulatory T cells protect the proinflammatory activation of human umbilical vein endothelial cells.

OBJECTIVE: To investigate the role of CD4(+)CD25(+)forkhead box P 3 (Foxp3)(+) T-regulatory cells (Tregs) in protecting the activation and function of human umbilical vein endothelial cells (HUVECs) induced by proinflammatory stimulus and the mechanisms of it. METHODS AND RESULTS: ECs play a major role in atherogenic initiation, changing their quiescence into activated phenotypes to support every phase of the inflammatory process. HUVECs were incubated alone, with Tregs or CD4(+)CD25(-) T cells in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without oxidized low-density lipoprotein/lipopolysaccharide for an additional 24 hours. Tregs are able to induce alternative expression of immune phenotypic markers of activated HUVECs by down modulating CD86 and to inhibit the adhesion molecule, such as vascular cell adhesion molecule-1 (VCAM-1) and proinflammatory cytokine (eg, monocyte chemoattractant protein-1 and interleukin 6), response of HUVECs to oxidized low-density lipoprotein/lipopolysaccharide. Moreover, Tregs downregulate proinflammatory factor nuclear factor-κB activation and induce resistance to suppression of anti-inflammatory factor Kruppellike factor 2 in HUVECs induced by a proinflammatory stimulus. Mechanism studies reveal that Treg-mediated suppression of HUVEC proinflammatory cytokines and adhesion molecule expression impaired by oxidized low-density lipoprotein/lipopolysaccharide require cell contact by cytotoxic T-lymphocyte antigen-4 and CD86 and by soluble factors (mainly interleukin 10 and transforming growth factor [TGF]-ß). CONCLUSIONS: Tregs may exert their protective effects against atherogenesis in part through inducing an immune-inhibitory phenotype of ECs involving cytotoxic T-lymphocyte antigen-4-dependent cell-to-cell contact and also requiring soluble factors (mainly interleukin 10 and TGF-ß).

Vascular smooth muscle contractility assays for inflammatory and immunological mediators.

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α(L) subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K(D)) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4(+) and CD8(+) T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.

Pathogenic role of CXCR7 in rheumatoid arthritis.

OBJECTIVE: The interaction between CXCL12 and its receptor, CXCR4, in the synovium of patients with rheumatoid arthritis (RA) is important for local inflammatory cell recruitment, angiogenesis, and cytokine production. CXCR7 was recently identified as an alternative receptor for CXCL12. We undertook this study to analyze the expression of CXCR7 in RA synovium and the pathogenic role of the CXCL12/CXCR7 pathway in RA. METHODS: CXCR7 expression in RA synovial tissue was analyzed using immunohistochemistry, while expression of CXCR4 and CXCR7 on human umbilical vein endothelial cells (HUVECs) was examined using quantitative reverse transcription-polymerase chain reaction, and CXCR7 expression was also analyzed by flow cytometry. Tube formation and rat aortic ring angiogenesis assays were used to assess the effects of CCX733 (a CXCR7 antagonist) and AMD3100 (a CXCR4 antagonist) on CXCL12-induced angiogenesis. The effect of anti-CXCR4 monoclonal antibody (mAb) was also analyzed using a tube formation assay. The effects of CCX733 in a murine model of collagen-induced arthritis (CIA) were also evaluated. RESULTS: CXCR7 was expressed on endothelial cells in RA synovium and also on unstimulated HUVECs. The expression of CXCR7 on HUVECs was markedly up-regulated by interleukin-1ß (IL-1ß) stimulation, and this overexpression was further enhanced by CXCL12 treatment. Incubation with CXCL12 also promoted angiogenic activity, with addition of IL-1ß again augmenting the effect. CXCL12-induced angiogenesis was inhibited by both CXCR4 and CXCR7 antagonists and by anti-CXCR4 mAb. Furthermore, treatment with CCX733 significantly reduced the clinical arthritis scores and the numbers of vessels in the inflamed synovial tissue in mice with CIA. CONCLUSION: CXCR7 and CXCR4 are both important for angiogenesis in RA synovium, making CXCR7 another potential target molecule for novel RA angiogenesis-blocking therapies.

MCP-induced protein 1 suppresses TNFalpha-induced VCAM-1 expression in human endothelial cells.

Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. We here report that treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor (TNF) alpha substantially increased the expression of MCP-induced protein 1 (MCPIP1). Overexpression of MCPIP1 protected ECs against TNFalpha-induced endothelial activation, as characterized by the attenuation in the expression of the adhesion molecule VCAM-1 and monocyte adherence to ECs. Conversely, small interfering RNA-mediated knock down of MCPIP1 increased the expression of VCAM-1 and monocytic adherence to ECs. These studies identified MCPIP1 as a feedback control of cytokines-induced endothelial inflammation.

Effects of ropivacaine on adhesion molecule CD11b expression and function in human neutrophils.

Local anesthetics possess a wide range of anti-inflammatory properties through their effects on neutrophils. However, limited information is available on the effects of ropivacaine (a new local anesthetic) on neutrophil function. The aim of this study was to investigate anti-inflammatory properties of ropivacaine with regard to its effects on the expression and function of CD11b in human neutrophils. CD11b expression was examined by flow cytometry and its function was determined by measuring adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs). Ropivacaine inhibited CD11b expression in formyl-methionyl-leucyl-phenylalanine (fMLP)-activated neutrophils in a concentration-dependent manner, but not in a time-dependent manner. The inhibitory potency of ropivacaine was similar to that of bupivacaine and levobupivacaine, but was more potent than that of lidocaine. The up-regulation of CD11b induced by platelet-activating factor (PAF) priming was also inhibited by ropivacaine. fMLP increased adhesion of neutrophils to HUVECs, which was inhibited by ropivacaine. In addition, ropivacaine more potently inhibited the fMLP-induced CD11b expression in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N ,N -tetraacetic acid (EGTA), a chelator of extracellular Ca(2+). However, ropivacaine showed no effect on the fMLP-induced CD11b expression in the presence of butan-1-ol, a blocker of phospholipase D (PLD) pathway, which completely inhibited the fMLP-induced CD11b expression in neutrophils. Our results suggest that ropivacaine exerts anti-inflammatory activity, and this appears to be mediated through inhibiting the expression and function of adhesion molecule CD11b in neutrophils.

Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors.

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.

The inflammatory mediator oncostatin M induces angiopoietin 2 expression in endothelial cells in vitro and in vivo.

OBJECTIVES: Members of the glycoprotein 130 (gp130) receptor-gp130 ligand family play a role in angiogenesis in different tissues. We tested the effect of this cytokine family on the angiopoietin (Ang)-Tie system, which is involved in blood vessel maturation, stabilization, and regression. RESULTS: Oncostatin M (OSM) increased Ang2 expression in human umbilical vein endothelial cells via Janus kinase/signal transducer and activator of transcription (JAK/STAT) and mitogen-activated protein (MAP) kinase activation. Furthermore, OSM induced Ang2 expression in macrovascular endothelial cells isolated from the human aorta and in microvascular endothelial cells isolated from human heart. Our in vivo experiments revealed that mRNA expression of Ang2 in hearts of mice injected with OSM increased significantly, and levels of OSM mRNA significantly correlated with mRNA levels of Ang2 in human hearts. In addition, OSM increased the expression of its own receptors, gp130 and OSM receptor, in endothelial cells in vitro and in mice in vivo, and levels of OSM mRNA significantly correlated with mRNA levels of gp130 and OSM receptor in human hearts. CONCLUSION: Our data, showing the effects of OSM on the Ang-Tie system in endothelial cells, in hearts of mice, and in human heart tissue, provide yet another link between inflammation and angiogenesis.

Variable cell responses to P. gingivalis lipopolysaccharide.

Porphyromonas gingivalis is a major etiological agent of chronic periodontal diseases, the virulence of which has been attributed to different factors, including lipopolysaccharide (LPS). We investigated the differential responses induced by P. gingivalis LPS stimulation of human umbilical vein endothelial cells and human oral epithelial cells. RT-PCR analysis showed that P. gingivalis LPS used Toll-like receptor 2 (TLR2) to activate epithelial cells and Toll-like receptor 4 (TLR4) to activate endothelial cells. Both cell types were stimulated by P. gingivalis LPS to produce pro-inflammatory cytokines. Cytokine Array assay showed that although patterns of cytokine expression were similar in both cell types, some cytokines were specifically secreted by the endothelial cells, and others were specific to epithelial cells. These results support the idea that the same LPS preparation can act as a TLR2 or TLR4 agonist, depending on TLR expression of the host cell, inducing cytokine profiles that differ according to the cell type.

Human leukocyte histocompatibility antigen class II-induced cytokines from human gingival fibroblasts promote proliferation of human umbilical vein endothelial cells: potential association with enhanced angiogenesis in chronic periodontal inflammation.

BACKGROUND AND OBJECTIVE: The role of human leukocyte histocompatibility antigen (HLA) class II molecules on non-antigen-presenting cells has been a matter of controversy. We previously reported that HLA-II molecules on human gingival fibroblasts (GF) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed and secreted (RANTES) and IL-8. However, the exact role of these cytokines, as well as other cytokines which are potentially secreted from GF, in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to observe the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cells (HUVEC). MATERIAL AND METHODS: Antibody-based cytokine-microarray analyses were performed to detect potential cytokines associated with angiogenesis. Next, cytokine productivity was confirmed by quantitative methods. Then, cell proliferation assay was performed to see whether these cytokines promoted the proliferation of HUVEC. RESULTS: Besides IL-6, MCP-1, RANTES and IL-8, growth-related gene product (GRO) was newly identified as an HLA-II-induced cytokine released from GF. This was confirmed by a quantitative method. Cell culture supernatant from HLA-II-stimulated GF cultures promoted the growth of HUVEC. Addition of anti-IL-8 neutralizing antibody, anti-CXC receptor (CXCR)1 antibody and anti-MCP-1 antibody inhibited the growth of HUVEC in a dose-dependent manner, while addition of anti-GROalpha antibody did not. CONCLUSION: The HLA-II-induced IL-8, via CXCR1, as well as MCP-1 from GF, promotes endothelial cell proliferation, which is possibly associated with enhanced angiogenesis in chronic periodontal lesions.