Human Umbilical Vein Endothelial Cells Membrane Research Articles

Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

Background: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. Methods: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. Results: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. Conclusions: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Arsenic trioxide induces procoagulant activity through phosphatidylserine exposure and microparticle generation in endothelial cells

Background Coagulopathy is a major cause of early death when arsenic trioxide (As 2O 3) therapy fails. In addition to the procoagulant properties of blast cells, the cytotoxic therapy may contribute to the coagulation disorders. The aim of the present study was to evaluate the possible impact of As 2O 3 on membrane alterations, including phosphatidylserine (PS) exposure and microparticle generation, and the consequent procoagulant properties of endothelial cells. Methods Procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVECs) was assessed by measuring clotting time and through purified coagulation complex assays. PS exposure on HUVEC membrane was observed by confocal microscopy and quantified with flow cytometry. In addition, counts and PCA of endothelial microparticles were determined by flow cytometry and plasma coagulation assay. Results As 2O 3 increased the ability of HUVECs to accelerate coagulation process and promote formation of coagulation complexes. Procoagulant activity corresponded to PS exposed on HUVECs. In coincidence with the PS externalization, As 2O 3 increased the production of PS-bearing microparticles, which then accelerated fibrin strand formation significantly. By blocking PS, lactadherin was able to inhibit over 90% of the intrinsic tenase/prothrombinase activity of As 2O 3-treated HUVECs, and restored coagulation times of As 2O 3–treated cells and microparticles to control levels. Conclusions As 2O 3 increases PCA of HUVECs through PS exposure and PS-bearing microparticle generation, which might cause thrombosis and act as a contributing factor in As 2O 3 therapy-related coagulopathy.

Effect of lipoprotein (a) on annexin A5 binding to cell membrane

Background High blood lipoprotein (a) [Lp(a)] concentration is a risk factor for a thrombotic event. Annexin A5 is involved in anticoagulation on the endothelial surface. How Lp(a) affects the annexin A5 function is not clear. This study investigates annexin A5 binding on the cell membrane in the presence of Lp(a). Methods Lp(a) was isolated from human blood plasma by ultracentrifugation and annexin A5 protein was purchased commercially. The cell membrane was prepared from primary human umbilical vein endothelial cells (HUVEC) and cultured cell line HepG2 by sucrose density gradient centrifugation. Enzyme-linked immunosorbent assays (ELISA) were used to examine annexin A5 binding to the cell membrane in the presence of Lp(a). Flow cytometry was used to analyze the binding of fluorescence-labeled annexin A5 to phosphatidylserine (PS)-translocated intact cells in the presence of Lp(a). Results Annexin A5 binding to the cell membrane was attenuated by a high concentration of Lp(a) in both HUVEC and HepG2 membrane surfaces. The phenomenon was also observed with annexin A5 surface labeling of HepG2 cells and flow cytometry analysis. Conclusions The results imply that Lp(a) interferes with annexin A5 binding to the procoagulant PS which translocates to the membrane surface under stress condition and therefore may increase the risk for thrombosis.

Subcellular localization of leukotriene receptors in human endothelial cells

Leukotriene B 4 (LTB 4), a potent chemotactic and immune-modulating mediator, signals via two receptors, BLT 1 and BLT 2. Recently, we reported that BLT 1 is the predominating BLT expressed on human umbilical vein endothelial cells (HUVEC), and that BLT 1 mediated functions are enhanced by LTB 4 and lipopolysaccharide (LPS), but not by TNFα. Here, we demonstrate that BLT 1 is found on the outer cell membrane of HUVECs but also in intracellular granules, co-localized with monocyte chemotactic protein-1 and P-selectin, but not with interleukin-8 and von Willebrand factor. Upon stimulation with LTB 4 or LPS, more BLT 1 protein is found, now evenly distributed over the cytoplasm and in the cell nucleus, but less on the cell surface. An MAP kinase inhibitor prevented this enhancement and translocation, suggesting this signaling pathway to be crucial. Thus, BLT 1, a G-protein-coupled 7-transmembrane receptor, is located in various subcellular compartments in endothelial cells, which may have implications for cellular LT dependent responses and target accessibility for BLT 1 antagonists.

Pravastatin Counteracts Angiotensin II-Induced Upregulation and Activation of NADPH Oxidase at Plasma Membrane of Human Endothelial Cells

Endothelial dysfunction has been linked to reactive oxygen species (ROS) production by nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase. Angiotensin II (ANG), which levels are elevated in some cardiovascular diseases, can stimulate this enzyme, whereas statins have been demonstrated pleiotropic effects related with the restoration of endothelial function. Therefore, our purpose was to study the mechanism of the possible beneficial effects of pravastatin on ANG-activated human umbilical vein endothelial cells (HUVEC). ANG induced an increase in the extracellular superoxide anion produced by NADPH oxidase but had no effect in the intracellular ROS production. Pravastatin, which alone did not have any effect on ROS production, totally blocked the stimulating effects of ANG when combined with it. These effects were not due to a direct action of ANG or pravastatin on the activity of NADPH oxidase measured in HUVEC lisates. On the contrary, the results correlated well with other effects of ANG: a Nox4 and p22phox upregulated expression and an enhanced Nox4 translocation to the cell membrane. All these effects were inhibited by pravastatin, which had no effect when incubated alone. These data reveal for the first time that pravastatin interrupts the signaling pathway activated by ANG that leads to an enhanced NADPH oxidase activity at the cell membrane of HUVEC. For that, pravastatin inhibits ANG-induced upregulation of catalytic NADPH oxidase subunits and blocks the migration of them to the endothelial cell membrane.

Characterization and specificity of anti-endothelial cell membrane antibodies and their relationship to thrombosis in primary antiphospholipid syndrome (APS).

Immunoblotting was used to detect antibodies reacting with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), fibroblasts and a T lymphoma line HUT78 in 18 patients with anticardiolipin antibodies (ACA) (14 of whom had had a thrombotic event), 11 patients with a recent myocardial infarction and 17 controls. Multiple membrane-specific antibodies to HUVEC were found in 10 of the patients with ACA (28 bands) and in nine of the patients with thromboses (27 bands) in contrast to only three of the patients with myocardial infarction (four bands) and one control (one band). The most frequently recognized HUVEC membrane epitopes were at 33 kD (four sera), 61-63 kD (five sera) and 76-79 kD (four sera). Although cross-reactivity with fibroblast and/or HUT78 membranes was seen at 33 kD, binding at 61-63 kD and 76-79 kD was specific for endothelial membranes. Although no correlations with the presence and titre of ACA were seen, HUVEC membrane-specific antibodies showed a correlation with venous thrombotic events.

Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Thematic Review Series: Sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.

Rapid Activation of Unstimulated Endothelial Cells Containing Tissue Factor Following Laser-Induced Injury as Monitored Via Calcium Mobilization.

The induction of thrombus formation in vivo through a laser-induced injury to the vessel wall in the living mouse has demonstrated the rapid expression of tissue factor antigen, thrombin generation and fibrin formation during thrombus formation. This thrombosis model is dominated by a tissue factor-dependent mechanism of platelet activation and fibrin formation. Specifically, this model does not lead to exposure of detectable sub-endothelial collagen, and the growth of the resulting thrombus is not dependent upon collagen-induced signaling. In light of prior in vivo results suggesting the rapid expression of active tissue factor on the vessel wall, we have determined if endothelial cells are activated in response to the injury induced by a pulsed laser. Laser-induced activation of individual cultured endothelial cells and cell populations were monitored through elevation of intracellular calcium and fluorescence microscopy. Cultured human umbilical vein endothelial cells (HUVECs) were loaded with the calcium-sensitive dye Fluo-4 and subjected to a single pulse of the nitrogen dye-tuned laser. HUVEC activation, was characterized by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. Comparable calcium elevation could be achieved by exogenous addition of either ADP (10 μM) or thrombin (1 U/ml). Although reduced, significant laser-induced elevation of intracellular calcium remained when cells were bathed in calcium-free media, thus suggesting that both calcium influx and calcium mobilization play a part in the total calcium elevation observed. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation of the targeted cell and was followed by a wave of calcium elevation in surrounding cells. These results imply either a release of secondary mediators or cell-cell communication. Using both isolated and confluent cultured HUVECs, we performed widefield immunofluorescence and differential interference contrast microscopy to detect tissue factor (TF) in unstimulated HUVECs using an anti-TF antibody of high affinity and specificity. Tissue factor was not detected in intact endothelial cells but was localized to abundant small granules within the cytoplasm in Triton X-100-permeabilized cultured HUVECs. The J82 bladder carcinoma cell line, which constitutively expresses TF on the plasma membrane, was used as a positive control for surface expression of TF and an isotype-matched non-immune antibody used as a negative control. These results indicate the presence of a preformed intracellular pool of TF within HUVECs that under resting conditions is not detectable on the cell surface. Given the rapid time course of TF expression and platelet accumulation in vivo following laser-induced injury of the endothelium, any TF expression on the vessel wall must be preformed and not derived via endothelial protein synthesis. Although to date, we have not been able to detect TF antigen on the plasma membrane of laser-activated HUVECs it is possible that the amount of TF antigen is below the limits of sensitivity of immunodetection. Alternatively, TF, like P-selectin, may be rapidly recycled from the plasma membrane to the cell interior. Nonetheless, we suspect that tissue factor is translocated to the endothelial cell surface following cell activation and that preformed endothelial cell tissue factor, following laser-induced injury of the endothelium, plays a critical role in thrombin generation and fibrin formation in this thrombosis model.

Non-DNA-Binding Antibodies in Patients with Lupus Nephritis Could Recognize Membrane Proteins of Glomerular Mesangial Cells

Lupus nephritis (LN) is a prototypic autoimmune disease, however, the precise immuno-pathogenesis of LN remains to be elucidated. In our previous studies, autoantibodies against mesangial cells had been identified in sera from patients with lupus nephritis and could bind the membrane proteins of human mesangial cells (HMC) directly through antigen-antibody interaction without DNA bridge. The current study is to investigate whether the autoantibodies were associated with anti-DNA antibodies and their target antigens distribution in different cell types. Sera from nine patients with renal biopsy proven lupus nephritis with positive anti-dsDNA antibodies and four healthy subjects were collected. IgG was isolated by Protein G affinity chromatography and then non-DNA-binding IgG fractions were obtained after deletion of anti-DNA antibodies using a DNA-cellulose affinity column. Membrane proteins, obtained from HMC, human umbilical vein endothelial cells (HUVEC), peripheral mononuclear cells by sonication and sequential centrifugation, were solubilized and applied in Western-blot analysis to characterize the target antigens. In results, the non-DNA-binding IgG fractions from sera of patients with lupus nephritis could blot the protein(s) of HMC membrane at 74, 63, and 42 kD. However, only a similar 74-kD protein could be blotted on membrane of HUVEC, and the target antigens on membranes of mononuclear cells were heterogeneous. In conclusion, our preliminary study had demonstrated that non-DNA binding autoantibodies against mesangial cells could be found in sera from patients with lupus nephritis. Although the target antigens might not be cell specific, the roles of these autoantibodies in the pathogenesis of lupus nephritis need further investigation.

Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves αv integrins and the participation of MMP2

The growth of ovarian carcinoma is dependent upon their vascularistion, but the interaction of ovarian cancer cells with the endothelium and their invasion through an endothelial environment remain poorly understood at the molecular level. To investigate adhesive events underlying this process with focusing on the role of alphav integrins and MT1MMP-MMP2 proteinases, we used in vitro models of cocultures of human ovarian adenocarcinoma cell lines (IGROV1 and SKOV3) with human umbilical vein endothelial cells (HUVECs). Immunostaining of HUVECs revealed the network organisation of fibrillar fibronectin (Fn) and pericellular vitronectin (Vn). During coculture, IGROV1 and SKOV3 cells gain access to subendothelial basement membrane of HUVECs and dislocated endothelial Fn without affecting endothelial Vn. Transmigration assays revealed that tumour cells invade Vn and, with an higher efficiency, Fn. Our data also highlighted that ovarian carcinoma cells migrated through the Fn-rich HUVEC-ECM. The expression of MMP2 and MT1-MMP was revealed in tumour cells within an endothelial environment. Furthermore, we found that cell migration through the endothelial ECM was almost totally dependent on alphav integrin function, whereas beta1 integrins were not solicited. In addition, inhibitors of MMP2 activity (alone or combined with anti-alphav integrin MAb) or TSRI265 (which blocks MMP2-alphavbeta3 association) were found to impede this process. Finally, alphav integrins, MT1-MMP and MMP2 were found in ovarian carcinoma cells within the 3-dimensional architecture of intraperitoneal tumour nodes collected from nude mice xenografted with IGROV1 or SKOV3 cell lines or within human tumour tissues. alphav integrins therefore appear as essential to the migration properties of human ovarian carcinoma cells, especially in an endothelial environment, with MMP2 participating to this process.

Recombinant prolylcarboxypeptidase activates plasma prekallikrein

AbstractThe serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. (Blood. 2004;103:4554-4561)

Antibodies to membranes of endothelial cells and fibroblasts in scleroderma.

Anti-endothelial and other cell membrane-reactive antibodies in scleroderma were characterized by immunoblotting sera with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), dermal fibroblasts and a T cell lymphoma HUT78. Antibodies reactive with HUVEC membranes were found in 17 of 20 patients with scleroderma (33 bands) in contrast to only two of 20 controls (two bands; P < 0.01) and three of 11 patients with myocardial infarction (four bands). Eleven of the 20 patients possessed antibodies that were specific for HUVEC membrane and did not cross-react with other cell lines. Analysis of patient subgroups showed that HUVEC membrane antibodies were present in nine of 11 patients with systemic sclerosis and in all nine with the CREST syndrome, and were HUVEC-specific in five and six of these cases, respectively. Although considerable heterogeneity was seen, antibodies to an 18-19-kD membrane epitope were found in 11 of the 20 patients but in none of the controls (P < 0.01). This antibody which reacted particularly with HUVEC (n = 9) and HUT78 membranes (n = 9) was associated with CREST syndrome rather than systemic sclerosis (9/9 versus 1/11; P < 0.01), and after elution was shown to possess anticentromere activity. In addition, antibodies reactive with both fibroblast (n = 11; 18 bands) and HUT78 membranes (n = 18; 42 bands) were detected and were specific for either fibroblast or HUT78 membranes in nine and 14 patients, respectively. There was no significant difference in the incidence of these fibroblasts and HUT78 membrane antibodies in the two patient subgroups. These findings support the concept that membrane-reactive antibodies, including anticentromeric antibodies, may play a central role in the pathogenesis of scleroderma, through their ability to react with endothelial cells.

Proteinase 3 interacts with a 111-kD membrane molecule of human umbilical vein endothelial cells.

Proteinase 3 (PR3) is the major autoantigen of antineutrophil cytoplasmic antibodies in Wegener's granulomatosis. Previously, it was demonstrated that PR3 induces apoptosis of human endothelial cells and that PR3 contributes to endothelial cell activation by enhancing interleukin-8 production. The present study demonstrates that PR3 binds specifically to human umbilical vein endothelial cells (HUVEC). Digoxigenin (DIG)-labeled PR3 bound readily to HUVEC cultured on coverslips. By fluorescence-activated cell sorter analysis, a homogeneous binding of PR3 to HUVEC, using either DIG-labeled or unlabeled PR3, was observed. No detectable membrane expression of PR3 was observed after either tumor necrosis factor-alpha stimulation or in nonstimulated HUVEC. The binding of PR3-DIG to HUVEC was dose-dependent and was inhibited by unlabeled PR3. Scatchard analysis revealed 2000 binding sites per cell, with a Kd of 0.1 microM. Affinity precipitation of biotin-labeled HUVEC membrane proteins with protein G-Sepharose bearing PR3 resulted in specific precipitation of a membrane molecule with a molecular weight of 111 kD under nonreducing conditions and 52 and 63 kD under reducing conditions. It is hypothesized that PR3, either released systemically or locally at inflammatory sites following activation of primed polymorphonuclear neutrophils, may lead to endothelial cell injury and activation of endothelial cells.

Interactions of PC/Chol and PS/Chol Liposomes with Human Cells in Vitro

Interactions between phosphatidylcholine (PC) or phosphatidylserine (PS) liposomes and human umbilical vein endothelial cells (HUVEC) or human promyelocyte leukemia cells (HL60) were investigated. Pyramine encapsulating or rhodamine incorporating small unilamellar liposomes with mean diameters around 80 nm (demonstrated to retain encapsulated material and to be nontoxic under experimental conditions) were used. Liposome uptake by both types of cells increased when increasing amounts of vesicles were co-incubated. For both lipid compositions, the interaction with HUVEC was very fast (association reached a plateau within 5 min) and so was the release of internalized vesicles (90% within 10min at 37°C). The reduced association values at 4°C and the punctuate fluorescence observed in the cell cytoplasm after interaction, were indicative of whole liposome internalization. This internalization was clathrin-independent, since it was not inhibited by sodium azide and deoxyglucose. Pretreatment of HUVEC with filipin or NEM resulted in modification of the interaction, something that could be due to alterations in the biochemical characteristics of HUVEC membranes that inhibit vesicular processes. In HL-60 cells, a slower association and faster release of PC/Chol liposomes was demonstrated, while association of both liposomes with these cells was energy-and temperature-independent. Nevertheless, morphological studies revealed differences in the interactions: A bright fluorescent rim observed after interaction with PC/Chol liposomes, suggests that these liposomes were adsorbed on the surface of HL60 cells, while the uniform cytoplasmic fluorescence observed after incubation with PS/Chol liposomes was indicative of fusion as the interaction mechanism.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Signal transduction pathways of membrane expression of proteinase 3 (PR-3) in human endothelial cells.

At present, the exact mechanism of the pathogenic effect of anti-PR-3 antibodies remains unknown. Interaction of anti-neutrophil cytoplasmic antibodies (ANCAs) with human umbilical vein endothelial cells (HUVECs) may play a key role. Recently we were able to show that ANCAs recognize their target antigen, PR-3, translocated into the membrane of HUVECs. The objective of this study was to investigate regulation, i.e. signal transduction pathways, of PR-3 expression in endothelial cells. HUVECs were isolated according to the method of Jaffe et al. and cultured under standard conditions. A cyto-enzyme-linked immunosorbent assay (ELISA) with unfixed cells was performed. Membrane-expressed PR-3 was detected by affinity-purified and monoclonal anti-PR-3 Ab. Tumour necrosis factor alpha (TNF-alpha)-induced membrane expression of PR-3 could be blocked with the RNA synthesis inhibitor actinomycin D, the protein kinase C (PKC) and proteinase A (PKA) inhibitor staurosporine, the specific PKA inhibitor calphostin C, the c-AMP-dependent PKA inhibitor KT5720 and the tyrosine kinase inhibitor genistein in a dose-dependent manner. The effect of calphostin C was the most significant. In addition, the effect of phorbol 12-myristate 13-acetate (PMA), a mediator of intracellular second messengers, was investigated. In our study, pretreatment of cells with PMA for 48 h led to a down-regulation of PR-3 expression. This effect, however, could be overridden by TNF-alpha stimulation, i.e. TNF-alpha-induced membrane expression of PR-3 was resistant to down-regulation of PKC. In conclusion, our data suggest that translocation of PR-3 in HUVECs is an active process depending on protein synthesis. PR-3 expression by HUVECs may involve a PKC reactive to cytokines such as TNF-alpha which induces PR-3 expression at a transcriptional level.

Antibodies to proteinase-3 mediate expression of intercellular adhesion molecule-1 (ICAM-1, CD 54).

We investigated the role of intercellular adhesion molecule-1 (ICAM-1) in the adhesion of polymorphonuclear neutrophils (PMN) to classic antineutrophil cytoplasmic antibody (C-ANCA)-treated endothelial cells, independently of cytokines. Human umbilical vein endothelial cells (HUVEC) grown to confluence in cytokine-free conditions were stimulated with C-ANCA sera and affinity-purified anti-proteinase 3 antibodies (PR3) from Wegener's granulomatosis (WG) patients. Non-activated PMN were added to treated HUVEC and adhesion was measured. In parallel experiments, treated HUVEC were fixed and ICAM-1 and E-selectin were assayed by cyto-ELISA; in other experiments anti-ELAM-1 and anti-ICAM-1 antibodies were assessed. In this in vitro model, adhesion of non-activated PMN to anti-PR3-stimulated HUVEC was enhanced. Adhesion was greater with anti-PR3 antibodies than with control and normal immunoglobulins, and correlated with the level of anti-PR3 antibodies. Neutralization of anti-PR3 antibodies by neutrophil azurophilic granule proteins abolished adhesion. This adhesion increased at the fourth hour after simulation, peaked at the twelfth hour and then decreased. This phenomenon occurred mainly through endothelial expression of ICAM-1 (the main counter-receptor for integrins, involved in firm PMN adhesion and migration) and E-selectin on HUVEC membranes. Anti-adhesion molecule antibodies inhibited this adhesion. This work supports the hypothesis of a direct effect of C-ANCA in endothelial stimulation, namely, on endothelium-PMN adhesion, and strengthens the major role of ICAM-1, directly involved in firm sticking of PMN to HUVEC, besides E-selectin. C-ANCA upregulate endothelial adhesiveness and thus participate in inflammatory reactions by providing endothelial adhesive structures for neutrophils. This might be one of the first steps leading to clinical expression of the disease. These results provide new insights into the pathogenesis of C-ANCA-related diseases.

Regulated exocytosis in vascular endothelial cells can be triggered by intracellular guanine nucleotides and requires a hydrophobic, thiol-sensitive component. Studies of regulated von Willebrand factor secretion from digitonin permeabilized endothelial cells.

To study the intracellular events leading to regulated exocytosis in human umbilical vein endothelial cells (HUVEC) the plasma membrane of HUVEC was selectively permeabilized with digitonin while retaining secretory function. Fusion of Weibel-Palade bodies, the secretory organelle of HUVEC, with the plasma membrane was detected by assaying the media for von Willebrand factor (vWF). The secretion from permeabilized cells faithfully reflects that in intact cells by a number of criteria. First, in the presence of calcium, permeabilized HUVEC secreted vWF with the same kinetics and to the same extent as intact cells stimulated with secretagogue. In addition, the vWF secreted by permeabilized cells after stimulus was exclusively the processed mature form found in Weibel-Palade bodies. Release required micromolar levels of calcium. In addition, GTPgammaS could also stimulate release by a parallel pathway. Both calcium- and GTPgammaS-stimulated secretion required a thiol-sensitive component. The hydrophobic thiol alkylating agent U73122 inhibited calcium-dependent and GTPgammaS-stimulated secretion. Surprisingly, N-ethylmaleimide, a hydrophilic alkylating agent, did not inhibit secretion. The N-ethylmaleimide-sensitive fusion protein (NSF), a protein implicated in a variety of vesicle fusion events, did not appear to be the target of U73122. These data strongly suggests the participation of a non-NSF, membrane-associated protein in regulated secretion in endothelial cells. Further, there appear to be two parallel pathways leading to secretion in HUVEC, one stimulated by elevated levels of calcium and the other mediated by a GTP-binding protein.

Functional Role of Cation-Independent Mannose 6-Phosphate/Insulin-Like Growth Factor II Receptor in Cell Adhesion and Proliferation of a Human Myeloma Cell Line OPM-2

The molecular mechanism underlying the interaction between myeloma cells and stromal cells was investigated by using a human myeloma cell line (OPM-2) and human umbilical vein endothelial cells (HUVECs). Adhesion of OPM-2 cells to HUVECs was found to be significantly augmented with treatment of OPM-2 cells with an alpha-glycosidase inhibitor, castanospermine (CSP). The treatment of OPM-2 cells with CSP resulted in alteration of oligosaccharide structures of cell surface glycoproteins particularly at molecular weight of 220 kD (GP220). To determine if GP220 was involved in the adhesion of OPM-2 cells to HUVECs, cell surface glycoproteins of HUVECs were labeled by biotin and were incubated with the PVDF membrane to which cell surface glycoproteins of OPM-2 cells were blotted. The biotinylated glycoproteins at the plasma membrane of HUVECs specifically bound to GP220 of OPM-2 cells. Purification and partial amino acid sequencing of GP220 revealed that GP220 had a structure homologous to cation-independent mannose 6-phosphate/insulin-like growth factor-II (CIM6P/IGF-II) receptor. Furthermore, an antibody against CIM6P/IGF-II receptor was reactive with GP220, indicating that GP220 was a CIM6P/IGF-II receptor. The adhesion of OPM-2 cells to HUVECs was inhibited by mannose 6-phosphate. Moreover, M6P was found to suppress the adhesion of human myeloma cell lines, OPM-2 and RPMI 8226, to bone marrow stromal cells that was established from the patients with multiple myeloma. In addition, proliferation of OPM-2 was stimulated in response to IGF-II. These results suggest that CIM6P/IGF-II receptor may be functional in terms of supporting cell adhesion and proliferation of myeloma cells.