Levels of soluble VCAM-1, soluble ICAM-1, and soluble E-selectin in patients with tuberculous pleuritis

Tuberculosis is characterized by the presence of activated mononuclear cells both in the peripheral circulation and in pleural fluid. Expression and up-regulation of adhesion molecules is the basis of cell-cell adhesion in granuloma formation and in leukocyte migration to the inflammatory site. Soluble isoforms of adhesion molecules have been described, and their expression at high levels indicated an activated state. The purpose of this study was to evaluate levels of soluble adhesion molecules in serum and pleural fluid from patients with tuberculous pleural effusions, compared with non-tuberculous pleural effusions. We analysed levels of soluble vascular cell adhesion molecule-1 (s.VCAM-1), soluble intercellular adhesion molecule-1 (s.ICAM-1), and soluble E-selectin (sE-selectin) in serum and pleural fluid from patients with tuberculous pleuritis, by sandwich ELISA. Serum levels of s.ICAM-1 and s.VCAM-1 in patients with tuberculosis were higher than those in healthy controls (p < 0.001). Levels of sE-selectin levels were in the normal range compared with control groups. In pleural fluid, levels of s.VCAM-1 and s.ICAM-1 were increased in pleural effusions. Patients with tuberculous pleural effusion exhibited high levels of s.ICAM-1 compared with patients with neoplastic pleural involvement. Up-regulation of s.VCAM-1 and s.ICAM-1 in serum, along with increased levels of sE-selectin in pleural effusions from tuberculous patients, may result in transmigration of activated inflammatory cells inducing pleural damage, which may contribute to the pathological processes involved.     

Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium

Resting membrane potential (RMP) and whole cell currents wererecorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),lipopolysaccharide (LPS)-treated HUVECs, immobilizedE-selectin, or vascular cell adhesion molecule 1 (VCAM-1)using the patch-clamp technique. RMP after 5 h on polystyrene was24.3 ± 1.7 mV (n = 42) with delayed rectifier K⁺(I_dr) andCl currents(I_Cl) presentin >75% of the cells. Inwardly rectifying K⁺ currents(I_ir) werepresent in only 14% of THP-1 cells. Adherence to unstimulated HUVECsor E-selectin for 5 h had no effect on I_ir orI_Cl but decreasedI_dr. Five hoursafter adherence to LPS-treated HUVECs, outward currents were unchanged,but I_ir waspresent in 81% of THP-1 cells. A twofold increase inI_ir and ahyperpolarization (41.3 ± 3.7 mV,n = 16) were abolished by pretreatmentof THP-1 cells with cycloheximide, a protein synthesis inhibitor, orherbimycin A, a tyrosine kinase inhibitor, or by pretreatment of theLPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interactionbetween THP-1 cells and LPS-treated HUVECs was required toinduce I_ir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similarconductances to cells adherent to LPS-treated HUVECs. Thus engagementof specific integrins results in selective modulation of differentK⁺ conductances.

     

Contrasting roles for domain 4 of VCAM-1 in the regulation of cell adhesion and soluble VCAM-1 binding to integrin alpha4beta1

Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 10(-9) M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to alpha4beta1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through alpha4beta1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.     

Interleukin-1 receptor antagonist, soluble tumor necrosis factor-alpha receptor type I and II, and soluble E-selectin serum levels in multiple sclerosis patients receiving weekly intramuscular injections of interferon-beta1a

BACKGROUND: interferon beta (IFN-beta) reduces relapse rate and disease progression in patients with the relapsing-remitting form of multiple sclerosis (RRMS). IFN-beta may act by upregulating the expression of anti-inflammatory components of the immune system. OBJECTIVES: To determine whether weekly intramuscular (i.m.) injection of IFN-beta1a had a short- or long-term effect on the expression of naturally occurring soluble factors that play an immunosuppressive role within the cytokine network. MATERIALS AND METHODS: serum levels of interleukin-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor alpha receptor type I and type II (sTNF-alphaRI and sTNF-alphaRII), and soluble E-selectin (sE-Sel) were followed over time in ten patients with RRMS who were treated with weekly i.m. injections of 30 mg (= 6 MU) of IFN-beta1a. Patient sera were sampled before, and 24, 48, 72, 96, and 168 hours after the first IFN-beta1a injection (short-term), and then at 1, 3, 6, 9 and 12 months after therapy initiation (long-term); highly sensitive, commercially available ELISA tests were used. RESULTS: serum levels of IL-1Ra, sTNF-alphaRI and sTNF-alphaRII, but not sE-Sel were significantly increased in both short- and long-term follow-up. Interestingly, IL-1Ra, sTNF-alphaRI and sTNF-alphaRII behaviors were completely different, suggesting that these naturally occurring immunoregulatory factors were differentially affected by IFN-beta1a. CONCLUSION: our study demonstrates that weekly i.m. injection of 30 mg of IFN-beta1a induces the expression of soluble mediators that may suppress the activities of pro-inflammatory cytokines such as IL-1 and TNF-alpha.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin,P-selectin,ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

Borrelia Burgdorferi Upregulates the Adhesion Molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on Mouse Endothelioma Cells in vitro

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (≈50 h), bEnd3 cells were found to bind cells of a VLA-4⁺ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.     

The activity of soluble VCAM-1 in angiogenesis stimulated by IL-4 and IL-13

IL-13 is a multifunctional lymphokine sharing a number of biological properties with IL-4. We previously observed that IL-4 shows angiogenic activities in vitro as well as in vivo. In this study we examined the effect of IL-13 on angiogenesis in vitro and in vivo and also the underlying mechanisms. Human IL-13 significantly stimulated the formation of tube-like structures in collagen gels by human microvascular endothelial cells and bovine aortic endothelial cells by about 3-fold over the controls in the absence of the cytokines. Administration of murine IL-13 led to neovascularization when implanted in the rat cornea. Coadministration of neutralizing mAb to the IL-4R inhibited both tubular morphogenesis in vitro and activation of STAT6 induced by IL-4 or IL-13. Both IL-4 and IL-13 markedly increased mRNA levels of VCAM-1 in vascular endothelial cells, and the production of the soluble form of VCAM-1 was also stimulated in response to IL-4 or IL-13. Administration of anti-VCAM-1 Ab in vitro blocked tubular morphogenesis induced by IL-4 and IL-13. Angiogenesis induced in vivo in rat cornea by IL-4 and IL-13 was also inhibited by Ab against the rat alpha4 integrin subunit. These findings suggest that angiogenesis dependent on IL-4 and IL-13 is mainly mediated through a soluble VCAM-1/alpha4 integrin pathway.     

Synergistic effect of TNF-alpha in soluble VCAM-1-induced angiogenesis through alpha 4 integrins

In our present study we focused on soluble VCAM-1 (sVCAM-1)/alpha(4) integrin-induced angiogenesis and found that this type of angiogenesis was mediated through p38 mitogen-activated protein kinase and focal adhesion kinase (FAK). HUVEC expressed both alpha(4) and beta(1) integrins, and it was reported that expression of alpha(4) integrin and its counterreceptor, sVCAM-1/VCAM-1, was enhanced in response to an inflammatory cytokine, TNF-alpha. In endothelial cells phosphorylation of p38 and FAK, but not that of extracellular signal-regulated kinase 1/2 was induced by sVCAM-1. Migration of endothelial cells was stimulated in response to sVCAM-1 at similar levels as those induced by vascular endothelial growth factor, and sVCAM-1-induced migration was almost completely blocked by neutralizing Ab against alpha(4) integrin, by either an inhibitor of p38 (SB203580), or by adenovirus containing FAK-related nonkinase. sVCAM-1 also induced the formation of blood vessels in Matrigel plug assay in vivo, and this neovascularization was blocked by SB203580 or neutralizing Ab against alpha(4) integrin. Moreover, we also confirmed that both TNF-alpha and sVCAM-1 could synergistically induce angiogenesis in the corneas of mice when each factor at used dose could not induce. This angiogenesis by TNF-alpha and sVCAM-1 was almost completely blocked by coadministration of SB203580 and also by neutralizing Ab against alpha(4) integrin. These results suggest that sVCAM-1/alpha(4) integrin induces angiogenesis through p38 and FAK signaling pathways.     

Induction of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in a co-culture of human endothelial and smooth muscle cells

Using immunofluorescence and flow cytometry, we studied the surface expression of cell adhesion molecules, E-selectin, VCAM-1 and ICAM-1, in human umbilical vein endothelial cells (HUVEC) co-cultured with human aortic intimal smooth muscle cells (SMC). It was found that inactivated HUVEC constitutively expressed only ICAM-1. After 3-4 h of co-culturing with SMC in the Transwell system we observed the appearance of E-selectin and VCAM-1, and the increase of ICAM-1 content on the cell surface. In all the cases, the maximum expression of these molecules (85-100% of positively stained cells) was detected within 18-24 h after co-culturing. Similar effect was exerted by SMC-conditioned culture medium, whose action well compared with that of a direct addition of interleukin-1 to EC at a concentration of 5-10 u/ml. The obtained data suggest that the cytokines secreted by SMC may participate in the regulation of endothelial cell adhesion molecule expression, and influence cell accumulation in sites of inflammation, immune disorders, etc.     

Potential of serum soluble CD93 as a biomarker for asthma in an ovalbumin-induced asthma murine model

Background: CD93 is a membrane-associated glycoprotein, which can be released in a soluble form (sCD93) into the serum. CD93 has received renewed attention as a candidate biomarker of inflammation in various inflammatory and immune-mediated diseases, including asthma.

Objective: We aimed to evaluate the effects of airway inflammation on CD93 levels in murine models.

Methods: We established an ovalbumin (OVA)-induced acute asthma murine model (OVA model) and a lipopolysaccharide (LPS)-induced airway inflammation murine model (LPS model). Dexamethasone was administered by gavage to attenuate the airway inflammation.

Results: The OVA model demonstrated typical allergic asthma features with increased airway hyper-responsiveness, inflammatory cell infiltration, increased Th2 cytokine levels, compared to the control group. CD93 levels were decreased in lung homogenates and, respiratory epithelial cells, whereas serum sCD93 levels were increased in the OVA model, as compared to the control group. Dexamethasone reversed these effects of OVA. In contrast, in the LPS model, CD93 levels were not affected in neither respiratory epithelial cells nor serum.

Conclusions: Our findings demonstrate the potential of using sCD93 as a biomarker for allergic asthma.     

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.     

Significance of soluble endothelial molecule E-selectin in patients with breast cancer

Increasing evidence suggests that endothelial cells are involved in tumor growth and metastasis. E-selectin, an adhesion molecule specifically expressed or secreted by activated endothelial cells, may enhance tumor angiogenesis and the adhesion of tumor cells to endothelial cells at distant sites. The aim of this study was to assess the relationship between concentrations of circulating soluble E-selectin and clinical, pathological and biological features in patients with breast cancer (BC). sE-selectin concentrations were analyzed by an ELISA method in sera from 113 patients with metastatic BC, 30 patients with primary inflammatory BC, 105 patients with primary non-inflammatory BC, 456 patients with node-negative BC, and 42 healthy controls. sE-selectin in the metastatic BC group was significantly higher than in the healthy control group. In metastatic BC, sE-selectin was significantly higher in patients with liver metastases than in patients without liver metastases. In patients with primary non-inflammatory BC, a negative correlation was found between sE-selectin concentrations and tumoral microvessel count. In overall and disease-free survival studies performed in the node-negative population (median follow-up duration 7.5 years), multivariate analyses demonstrated a prognostic value of sE-selectin and tumor size. This study suggests that endothelial activation might play a role in the development of BC. This role seems not to be related to angiogenesis.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

Activin,a grape seed-derived proanthocyanidin extract,reduces plasma levels of oxidative stress and adhesion molecules (ICAM-1, VCAM-1 and E-selectin) in systemic sclerosis

This study evaluated whether a new generation antioxidant Activin derived from the grape seed proanthocyanidins, could reduce the induction of the adhesion molecules as a result of inflammatory response in the plasma of systemic sclerosis (SSc) patients. SSc patients were divided into two groups: one group was treated with Activin, a grape seed-derived proanthocyanidins, while the other group served as control. Patients were given Activin 100 mg/day orally for one month after which the blood samples were withdrawn from both groups of the patients. Blood was also taken from normal human volunteers. Plasma was obtained in fasting state between 8 to 9 A.M. from two groups of SSc patients and controls. Soluble adhesion molecules including ICAM-1, VCAM-1, E-selectin and P-selectin as well as malonaldehyde, a marker for oxidative stress, were measured. The results of our study demonstrated up-regulation of these soluble adhesion molecules except for P-selectin, in the plasma of the SSc patients compared to those obtained from human volunteers. Activin significantly attenuated the increased expression of these adhesion molecules. In addition, there was a significant increase in the amount of malondialdehyde formation in the plasma of the SSc patients, which was also attenuated by Activin. The results of this study demonstrated that Activin could reduce the inflammatory response and the oxidative stress developed in SSc patients.     

Elevation of soluble interleukin-2 receptor levels in nasal allergy

To investigate soluble IL-2 receptor (sIL-2R) levels in nasal allergy, the sera and nasal secretions from patients with nasal allergy and from healthy subjects were subjected to a double-epitope enzyme-linked immunosorbent assay. Significant elevation of sIL-2R concentrations in the sera and nasal secretions was observed in the allergy patients (n = 26) compared with those of healthy subjects (n = 9). IL-2R-positive (CD25(+)) cells were observed in the crust formed in an allergic nasal mucosa. The concentration of sIL-2R in the sera correlated neither with the eosinophil count of the peripheral blood count nor with clinical severity. The concentration of sIL-2R in the nasal secretions was significantly higher compared with that in the sera from allergic patients (p < 0.01), whereas no significant difference was observed between sIL-2R levels in the sera and nasal sections from normal subjects. These findings indicate that sIL-2R plays an essential role in allergic processes by regulating IL-2R-positive cells recruited into the nasal mucosa.     

Activin,a Grape Seed-derived Proanthocyanidin Extract,Reduces Plasma Levels of Oxidative Stress and Adhesion Molecules (ICAM-1, VCAM-1 and E-selectin) in Systemic Sclerosis

This study evaluated whether a new generation antioxidant Activin derived from the grape seed proanthocyanidins, could reduce the induction of the adhesion molecules as a result of inflammatory response in the plasma of systemic sclerosis (SSc) patients. SSc patients were divided into two groups: one group was treated with Activin, a grape seed‐derived proanthocyanidins, while the other group served as control. Patients were given Activin 100 mg/day orally for one month after which the blood samples were withdrawn from both groups of the patients. Blood was also taken from normal human volunteers. Plasma was obtained in fasting state between 8 to 9 A.M. from two groups of SSc patients and controls. Soluble adhesion molecules including ICAM‐1, VCAM‐1, E‐selectin and P‐selectin as well as malonaldehyde, a marker for oxidative stress, were measured. The results of our study demonstrated up‐regulation of these soluble adhesion molecules except for P‐selectin, in the plasma of the SSc patients compared to those obtained from human volunteers. Activin significantly attenuated the increased expression of these adhesion molecules. In addition, there was a significant increase in the amount of malondialdehyde formation in the plasma of the SSc patients, which was also attenuated by Activin. The results of this study demonstrated that Activin could reduce the inflammatory response and the oxidative stress developed in SSc patients.     

Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.