|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1997//219 $5.00
The Journal of Cell Biology, Volume 139, Number 1,
, 1997 219-228
Article |
Novel Cytokine-independent Induction of Endothelial Adhesion Molecules Regulated by Platelet/Endothelial Cell Adhesion Molecule (CD31)
Vascular Biology Laboratory, Baker Medical Research Institute, Prahran Victoria 3181; and Director of Lung Research, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-4283
Tumor necrosis factor–
, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell–cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule–1, and intercellular adhesion molecule–1. In contrast, ECs failed to express E-selectin when plated on poly-L-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 ± 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 ± 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co– culture of subconfluent ECs with PECAM-1– coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin–expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.
Abbreviations used in this paper: CSF, colony stimulating factor; EC, endothelial cell; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule–1; IL-1 and IL-1ra, interleukin-1 and IL-1 receptor agonist; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; PECAM, platelet/endothelial cell adhesion molecule; TNF-, tumor necrosis factor-; VCAM-1, vascular cell adhesion molecule–1.
THE endothelial lining of the vascular system normally displays a nonactivated, nonadhesive phenotype. Stimulation with agents such as tumor necrosis factor- (TNF-)1, interleukin-1 (IL-1), or lipopolysaccharide (LPS) are known to induce the expression of proteins on the endothelial surface that mediate coagulation (Bevilacqua et al., 1986), leukocyte adhesion (Bevilacqua et al., 1985; Gamble et al., 1985; Pober et al., 1986b; Doherty et al., 1989), and leukocyte transendothelial migration (Furie et al., 1989; Moser et al., 1989). The endothelial antigens that are important for the adhesion of leukocytes are members of the selectin family, E- and P-selectin, and the immunoglobulin gene superfamily, vascular cell adhesion molecule–1 (VCAM-1) and intercellular adhesion molecule–1 (ICAM-1) (Carlos and Harlan, 1994; Litwin et al., 1995).
The induction of E-selectin expression on endothelial cells (ECs) in vitro after cytokine stimulation is transient and independent of the continued presence of the stimulant (Pober et al., 1986a). Previous studies have shown that E-selectin mRNA and protein levels peak between 2 and 4 h, respectively, after treatment with an agonist, returning to near basal levels by 24 h (Bevilacqua et al., 1989; Read et al., 1994). VCAM-1 (Osborn et al., 1989) and ICAM-1 (Pober et al., 1986b) are maximal 6 and 12 h, respectively, after stimulation.
In contrast to the transiency of E-selectin and VCAM expression demonstrated by the in vitro data, these antigens have been detected on venular endothelium in chronic inflammatory lesions, such as the synovium in rheumatoid arthritis (Koch et al., 1991), and the skin in psoriasis (Petzelbauer et al., 1994). E-selectin expression is also detected on angiogenic vessels in human hemangiomas, a noninflammatory angiogenic disease (Kraling et al., 1996). Moreover, the architecture and anatomic localization of capillary loops influence the pattern of endothelial expression of E-selectin and VCAM-1, independently of the availability of cytokines (Petzelbauer et al., 1994). Thus it is likely that alternate control mechanisms exist to allow prolonged, locality-based expression of adhesion molecules on the endothelium. At least one of these alternate mechanisms may be flow, since increased shear stress has been shown to selectively modulate adhesion molecule expression, upregulating ICAM-1 but not E-selectin or VCAM-1 (Nagel et al., 1994).
Since sites of inflammation are often associated with morphological changes including cell retraction of the endothelium (Schumacher, 1973), we hypothesized that cell contacts may be important in the regulation of endothelial phenotype. We describe here the central role of the junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1), through the formation of cell–cell interactions, in the maintenance of the functional integrity of the endothelial monolayer. Furthermore, we demonstrate a novel pathway for the induction of adhesion molecules on endothelial cells that is independent of exogenous addition of cytokines, but is related to integrin- and cell shape–associated signaling events.
 |
MATERIALS AND METHODS |
|---|
 TopAbstract MATERIALS AND METHODS RESULTSDISCUSSIONREFERENCES |
|---|
Antibodies
Mouse mAbs directed against E-selectin (49-1B11), VCAM-1 (51-10C9), PECAM-1 (51-9H6, 51-6F6, 55-3D2), VE-cadherin (55-7H1), integrin β1 (61-2C4), colony-stimulating factor (CSF), common βc chain (3D7, supplied by Q. Sun, Hanson Centre for Cancer Research [HCCR], Adelaide, Australia), and Keyhole Limpet Hemacyanin (23-1F11) were raised at the HCCR. The anti–PECAM-1 mAbs were functional in an assay of neutrophil transendothelial migration and anti–VE-cadherin antibody was functional in an assay of EC aggregation. For coating of wells with antibody, plates were coated with rabbit anti–mouse Ig (50 µg/ml) for 18 h, blocked with 1% BSA, and then anti-integrin or anti-CSFβc chain–purified antibodies added for 1 h at 37°C. Wells were then washed and further blocked before addition of cells.
Flow Cytometry
Flow cytometric analysis of in situ endothelial monolayers was performed as previously described (Gamble et al., 1993). EC monolayers were blocked in 5% sheep serum, and then were stained with primary antibody for 30 min at 37°C, washed twice with RPMI 1640 containing 2.5% FCS, and then stained with FITC-conjugated, anti–mouse Ig (Fab2, DAF; Silenus Laboratories, Hawthorn, Australia) for 30 min at room temperature. Cells were washed twice, removed by trypsin-EDTA treatment, and then fixed in 1% formaldehyde, 0.02% azide, and 0.02% glucose. In experiments involving endothelial pretreatment with mAbs, E-selectin was detected using a single layer, FITC-conjugated, anti–E-selectin mAb (49-1B11).
A minimum of 1,000 events per test was analyzed using an EPICS Profile II (Coulter Immunology, Hialeah, FL). Results of individual EC lines are expressed either as a plot of frequency versus log fluorescence, or as the mean fluorescence channel number, subtracting the accompanying value for the negative control Ig. When results from multiple EC lines have been pooled, the mean fluorescence intensity (MFI) represents n cell lines.
In cocultures of ECs and L cells prepared for flow cytometric analysis, the cells were stained with anti–VE-cadherin, detected with phycoerythrin (PE)-conjugated anti–mouse F(ab')2 (DDAPE; Silenus Laboratories, Hawthorn, Australia) and simultaneously stained with goat anti–E-selectin detected with FITC–conjugated, anti–goat antibody (Silenus Laboratories). L cells were negative for VE-cadherin, EC were 100% positive for VE-cadherin and were selected for analysis of E-selectin (FITC staining) using a second fluorescence detector. The flow cytometer was calibrated using single PE- or FITC-stained cells.
Immunofluorescence Confocal Microscopy
Confocal microscopy was performed on ECs cultured on fibronectin-coated glass or Permanox® chamber slides. A staining three layer method was used in wounding assays and involved initial fixation in ice-cold methanol for 5 min and then acetone 1 min and washing in M199-containing 2.5% FCS. Cells were stained with saturating amounts of mAb for 30 min at 25°C. After two washes, cells were incubated with biotin-conjugated, affinity-purified anti–mouse Ig (Vector Labs, Burlingame, CA), washed twice, and incubated with avidin-FITC (Dako Corp., Carpinteria, CA). Slides were mounted using 2% propylgallate in glycerol as an anti-fade agent. Images were captured with a laser scanning confocal microscope (MRC600; Bio Rad Laboratories, Hercules, CA). Comparison images were subjected to equivalent amounts of contrast enhancement.
Neutrophil–Endothelial Adhesion
Peripheral blood from normal volunteers was sedimented on dextran, followed by density-gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway) at 450 g. Contaminating erythrocytes were then lysed by hypotonic 0.2% sodium chloride. Cells were resuspended in RPMI-1640 with 2.5% FCS and yielded a purity of >98%. 5 x 105 neutrophils were added in 125 µl medium to human umbilical vein endothelial cells (HUVECs), which had been plated 16 h earlier onto fibronectin-coated chamber slides at cobblestone and subconfluent densities. After 25 min at 37°C in a humidified 5% CO2 in air atmosphere, the wells were washed three times removing unattached neutrophils. The slides were fixed in 0.5% glutaraldehyde, examined by confocal microscopy, and the number of neutrophils attached per EC counted. At least 160 ECs were assessed.
Preparation of Protein-coupled Beads
Tosyl-activated paramagnetic beads (Dynabeads M-450; DYNAL A.S., Oslo, Norway) were coated with purified platelet PECAM-1 as previously described (Plopper and Ingber, 1993). Essentially 99% of beads were coated with PECAM-1 as assessed by flow cytometry using polyclonal anti–PECAM-1 antibody staining.
Endotoxin Assay
A quantitative, photometric assay (Coatest; Kabi Diagnostica, Stockholm, Sweden) based upon activation of a proenzyme in limulus amoebocyte lysate was used, which detected endotoxin at 0.1–1.2 EU/ml.
Cytokines and Cytokine Antagonists
TNF- (lot S9010AX; sp act 6.27 x 107 U/mg), TGF-β (lot 8987-53), and a monoclonal anti-TNF were gifts from Genentech, Inc. (South San Francisco, CA) IL-1β (108 thymocyte mitogenesis U/mg) was kindly supplied by Immunex (Seattle, WA). IL-1ra was a gift from Synergen (Boulder, CO). All cytokines contained <3 U/ml of LPS.
Reagents and Peptides
Polymyxin B sulfate (Sigma Chemical Co., St. Louis, MO) was used at 10 µg/ml. When added at plating, it effectively abolished induction of E-selectin by LPS on ECs. Soluble PECAM-1 protein was purified from platelet and was used at 0.01–100 µg/ml. Cyclic RGD and RAD peptides (EMD66203, 67679, 69601) were kindly supplied by A. Jonczyk from Merck KGaA (Darmstadt, Germany). These peptides were identical to those used by Brooks et al. (1994) in inhibiting vβ3-dependent angiogenesis.
Enumeration of EC Contacts
Multiple photomicrographs of low power, phase contrast fields (see Fig. 1) were obtained of ECs plated at 0.25 and 105 cells per cm2. The number of cell contacts made with adjoining ECs were counted for 10 ECs per field.
|
Statistics
The statistical significance of results was assessed using the two-tailed Student's t test with either paired or unpaired groups of data as indicated. The frequency histograms of neutrophil adherence to ECs were compared by the Kolmogorov-Smirnov test and the effects of PECAM-1–coated beads on E-selectin expression by the analysis of variance (ANOVA) test.
|
RESULTS |
|---|
|
TopAbstractMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
|
antibody potently and specifically inhibited IL-1 or TNF-–mediated induction of E-selectin, respectively, but these agents were ineffective on the induction of E-selectin by subconfluent ECs (Fig. 5 b). Furthermore, conditioned medium taken from subconfluent ECs or from cells multiply wounded such that the majority of cells were undergoing migration, did not induce E-selectin expression on confluent density ECs (Table I). Thus a role for endogenous endothelial cytokine production, or the release of some other stimulant from the EC, appeared unlikely.
|
|
Low doses of TNF- were able to increase E-selectin expression on both low and high density ECs (Fig. 6 a) suggesting that the cytokine and non-cytokine pathways of induction are at least additive. Although the level of E-selectin expression induced by the cytokine-independent mechanism is less than that induced by TNF or IL-1 (Figs. 5 and 6), it is however, functionally relevant. Assessment of the number of neutrophils adherent per EC showed that there was an increase in the number of neutrophils attached per EC in cells plated at low versus high density EC (Fig. 6 b). In three experiments, the percentage of EC supporting the adhesion of two or more neutrophils was 24.3% ± 14% for low density EC and 1.4% ± 1.0% for high density EC (mean ± SEM; P = 0.02).
|
|
Two molecules known to be concentrated in cell–cell contacts and implicated in establishment of some of the junctional properties of endothelial cell monolayers are PECAM-1 (Albelda et al., 1991; DeLisser et al., 1994) and VE-cadherin (Lampugnani et al., 1992; Ayalon et al., 1994). To determine whether PECAM-1 was involved in the cytokine-independent regulation of E-selectin, three independent methods were used. Firstly, confluent density EC monolayers were exposed to functional antibodies directed to PECAM-1 or VE-cadherin. Monoclonal anti– VE-cadherin antibody (antibody 7H1) had no effect. Polyclonal anti–PECAM-1 resulted in a twofold increase in E-selectin expression (Fig. 8, a and b). Addition of both anti–PECAM-1 and anti–VE-cadherin antibodies produced no further increase than with anti–PECAM-1 antibody alone (data not shown). Although two mAb directed to domain one of PECAM-1 consistently and significantly enhanced E-selectin expression their activity was always less than that seen with the polyclonal anti–PECAM-1 antibody (Fig. 8 b) suggesting the involvement of multiple domains. 55-3D2, an mAb directed to domain two-thirds of PECAM-1 was without function in these assays although it inhibits neutrophil transendothelial cell migration (Yan et al., 1995). The anti–PECAM-1 antibody effect was dose dependent (maximal efficacy at 5 µg/ml) and not due to contaminating endotoxin as polymyxin B did not abrogate the enhancement, the antibodies did not have detectable endotoxin and boiling the antibody abolished its potency (Fig. 8 a). VCAM-1 was also upregulated on cobblestone ECs by polyclonal anti–PECAM-1 antibody by 1.7 ± 0.19-fold (mean ± SEM, n = 3, P = 0.03, paired t test).
|
|
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Type II induction of E-selectin described herein is independent of cell size, cell proliferation, and cell cycle, but is initially integrin mediated and seen in cells plated at both high and low density. Cells plated onto ligands that facilitated integrin-mediated attachment induce E-selectin expression (Fig. 7). However if the initial attachment is blocked, as with the addition of cyclic RGD peptides to cells on fibrinogen, E-selectin induction is inhibited. Binding of RGD to cells per se is insufficient to inhibit E-selectin induction since RGD peptides have no effect on cells plated onto collagen.
The intensity and duration of induction of type II responses was related to the density of cell plating. Cells plated at confluent density expressed less E-selectin when measured 8–48 h after plating, compared to cells plated at subconfluent density (Fig. 3). Morphologically, cells plated at high density form rapid cell–cell interactions in contrast to low density cells. Thus, junctional control of EC adhesion molecule expression was considered. Two molecules have been described that are known to be important in endothelial junctional integrity (Albelda et al., 1991; Lampugnani et al., 1992; Ayalon et al., 1994; DeLisser et al., 1994): PECAM-1, a transmembrane glycoprotein belonging to the Ig gene superfamily containing six extracellular, Ig-like domains; and VE-cadherin, an endothelium-specific member of the cadherin family of cell junctional molecules. The involvement of PECAM-1 in the control of E-selectin expression was shown by three independent methods. Firstly, anti–PECAM-1 but not anti–VE-cadherin antibodies induced E-selectin expression on confluent ECs (Fig. 8). Although the increases achieved with the antibodies were not to the level seen on low density cells, they were consistent. This may suggest that molecules other than PECAM-1 could exert secondary events that influence the level of expression of E-selectin. Secondly, PECAM-coated beads, when added at plating, inhibited the level of E-selectin expression on low density cells compared to BSA-coated beads (Fig. 9). Thirdly, transfectants expressing full-length PECAM-1, or those expressing the extracellular domains but lacking the cytoplasmic domain, inhibited E-selectin expression on subconfluent ECs (Fig. 10). In contrast, L cells expressing the domain 2 deletion mutant failed to effect E-selectin expression, suggesting a critical involvement of domain 2 in this regulation. These results are in agreement with other studies showing the importance of domain 2 in cell aggregation (Sun et al., 1996). Together with our antibody studies showing that antibodies to domain 1 increased the level of E-selectin expression on high density cells, our results suggest that both domains 1 and 2 of PECAM-1 are critical in regulation of E-selectin expression.
Cell–cell interactions are known to regulate a number of events, including cell proliferation (Gradl et al., 1995), release of bFGF from astrocytes (Murphy et al., 1988), responsiveness of fibroblasts to TGF-β (Paulsson et al., 1988), and the regulation of intracellular pH (Galkina et al., 1995). The mechanisms that underlie such regulation however have not been elucidated, although autocrine release of growth inhibitory factors (Antonelli-Orlidge et al., 1989; Gradl et al., 1995) and gap junction changes (Chen et al., 1995) have been proposed. As described here, a cell surface junctional protein, PECAM-1, plays a central role in the density-dependent regulation of endothelial E-selectin expression. The possibility of a signaling role for PECAM-1 has been raised previously in a number of different systems. These include activation of integrins on T cells (Tanaka et al., 1992), natural killer cells (Berman et al., 1996), monocytes and neutrophils (Berman and Muller, 1995), and on CD34+ hematopoietic progenitor cells (Leavesley et al., 1994), inhibition of EC proliferation (Fawcett et al., 1995), and platelet aggregation (Newman et al., 1992). The association of the tyrosine phosphatase-SHP2 with aggregated platelets (demonstrated recently by Jackson et al. [1997]) has put credence to a signaling pathway associated with PECAM-1 itself. Of interest to our study here is the recent observation by Lu et al. (1996) that integrin engagement and cell spreading results in PECAM-1 dephosphorylation in EC. Thus, the likely cross-talk between integrin and PECAM-1 is further strengthened.
Our in vitro model of cell wounding displays similar features to that described in the density-dependent regulation of adhesion molecules. Firstly, the induction of E-selectin expression is restricted to the migrating front and is associated with a change in cell shape and PECAM-1 redistribution away from the cell junction. Secondly, the E-selectin expression on these migrating cells is maintained at least up to 30 h after the wound signal. Thus we would suggest that this model may reflect identical signaling pathways as those operating in our plating experiments, and may therefore be appropriate as a model for in vivo endothelial regulation at wound sites.
Pathological tissue inflammation is characterized by increased and chronic expression of endothelial adhesion molecules (Koch et al., 1991; Petzelbauer et al., 1994; Kraling et al., 1996), changes in endothelial morphology and angiogenesis (Fitzgerald et al., 1991). These morphological changes include the formation of high endothelial venules (Freemont et al., 1983) and cell retraction with associated intercellular gaps (Nagel et al., 1994). In balloon angioplasty, expression of adhesion molecules at the wound front associated with morphological changes has been observed in contrast to the lack of expression in the area behind the wound edge (Tanaka et al., 1993). More recently, a role for E-selectin in angiogenesis has been postulated (Nguyen et al., 1993; Koch et al., 1995; Kraling et al., 1996). The results reported here, demonstrate: (a) a cytokine-independent mechanism of adhesion molecule expression, and (b) that cell–cell interactions influence the duration and magnitude of this expression, may explain some of these in vivo observations. Furthermore, the observation that restoration of PECAM-1 interactions can downregulate adhesion molecule expression on ECs offers the promise that manipulation of EC junctional molecules may permit the development of novel therapeutics.
|
Acknowledgments |
|---|
This work was supported by the National Health and Medical Research Council (Australia), the Anti-Cancer Foundation of the Universities of South Australia, and the National Heart Foundation of Australia.
Submitted: 23 July 1996
Revised: 6 December 1996
M. Vadas and J. Gamble contributed equally to this paper.
|
REFERENCES |
|---|
|
TopAbstractMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Albelda SM, Muller WA, Buck CA & Newman PJ. Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell– cell adhesion molecule, J Cell Biol, 1991, 114, 1059–1068.
Antonelli-Orlidge A, Saunders KB, Smith SR & D'Amore PA. An activated form of transforming growth factor β is produced by cocultures of endothelial cells and pericytes, Proc Natl Acad Sci USA, 1989, 86, 4544–4548.
Ayalon O, Sabanai H, Lampugnani M-G & Dejana E. Spatial and temporal relationships between cadherins and PECAM-1 in cell–cell junctions of human endothelial cells, J Cell Biol, 1994, 126, 247–258.
Berman ME & Muller WA. Ligation of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) on monocytes and neutrophils increases binding capacity of leukocyte CR3 (CD11b/CD18), J Immunol, 1995, 154, 299–307.[Abstract]
Berman ME, Xie Y & Muller WA. Roles of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) in natural killer cell transendothelial migration and β2 integrin activation, J Immunol, 1996, 156, 1515–1524.[Abstract]
Bevilacqua MP, Pober JS, Wheeler ME, Cotran RS & Gimbrone MA Jr. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes and related leukocyte lines, J Clin Invest, 1985, 76, 2003–2009.[Medline]
Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Cotran RS & Gimbrone MA Jr. Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterisation and comparison with the actions of interleukin 1, Proc Natl Acad Sci USA, 1986, 83, 4533–4537.
Bevilacqua MP, Stengelin S, Gimbrone MA Jr & Seed B. Endothelial leukocyte adhesion molecule-1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins, Science (Wash DC), 1989, 243, 1160–1165.
Brooks PC, Montgomery AMP, Rosenfeld M, Reisfeld T, Hu G, Klier G & Cheresh DA. Integrin vβ3antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels, Cell, 1994, 79, 1157–1164.[Medline]
Carlos TM & Harlan JM. Leukocyte-endothelial adhesion molecules, Blood, 1994, 84, 2068–2101.
Chen SC, Pelletier DB, Ao P & Boynton AL. Connexin 43 reverses the phenotype of transformed cells and alters their expression of cyclin/cyclin-dependent kinases, Cell Growth Diff, 1995, 6, 681–690.[Abstract]
DeLisser HM, Yan HC, Newman PJ, Muller WA, Buck CA & Albelda SM. Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans, J Biol Chem, 1993, 168, 16037–16046.
DeLisser HM, Newman PJ & Albelda SM. Molecular and functional aspects of PECAM-1/CD31, Immunol Today, 1994, 15, 490–495.[Medline]
Doherty DE, Zagarella L, Henson PM & Worthen GS. Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell, J Immunol, 1989, 143, 3673–3679.[Abstract]
Fawcett J, Buckley C, Holness CL, Bird IN, Spragg JH, Saunders J, Harris A & Simmons DL. Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts, J Cell Biol, 1995, 128, 1229–1241.
Fitzgerald O, Soden M, Yanni G, Robinson R & Bresnihan B. Morphometric analysis of blood vessels in synovial membranes obtained from clinically affected and unaffected knee joints of patients with rheumatoid arthritis, Ann Rheum Dis, 1991, 50, 792–796.
Freemont AJ, Jones CJP, Bromley M & Andrews P. Changes to vascular endothelium related to lymphocyte collections in diseased synovia, Arthritis Rheum, 1983, 26, 1427–1433.[Medline]
Furie MB & McHugh DD. Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor-, J Immunol, 1989, 143, 3309–3317.[Abstract]
Galkina SI, Sud'ina GF, Dergacheva GB & Margolis LB. Regulation of intracellular pH by cell–cell adhesive interactions, FEBS (Fed Eur Biol Soc) Lett, 1995, 374, 17–20.
Gamble JR, Harlan JM, Klebanoff SJ & Vadas MA. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor, Proc Natl Acad Sci USA, 1985, 82, 8667–8671.
Gamble JR, Khew-Goodall Y & Vadas MA. Transforming growth factor-β inhibits E-selectin expression on human endothelial cells, J Immunol, 1993, 150, 4494–4503.[Abstract]
Gradl G, Faust D, Oesch F & Wieser RJ. Density-dependent regulation of cell growth by contactinhibin and the contactinhibin receptor, Curr Biol, 1995, 5, 526–535.[Medline]
Heimark RL, Twardzik DR & Schwartz SM. Inhibition of endothelial regeneration by type-β transforming growth factor from platelets, Science (Wash DC), 1986, 233, 1078–1080.
Hubbe M, Kowitz A, Schirrmacher V, Schachner M & Altevogt P. L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding, Eur J Immunol, 1993, 23, 2927–2931.[Medline]
Jackson DE, Ward CM, Wang R & Newman PJ. The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation, J Biol Chem, 1997, 272, 6986–6993.
Kato J, Matsuoka M, Polyak K, Massague J & Sherr CJ. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27KIP1) of cyclin-dependent kinase 4 activation, Cell, 1994, 79, 487–496.[Medline]
Koch AE, Burrows JC, Haines GK, Carlos TM, Harlan JM & Leibovich SJ. Immunolocalisation of endothelial and leukocyte adhesion molecules in human rheumatoid and osteoarthritic synovial tissues, Lab Invest, 1991, 64, 313–320.[Medline]
Koch AE, Halloren MM, Haskell CJ, Shah MR & Polverini PJ. Angiogenesis is mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1, Nature (Lond), 1995, 376, 517–519.[Medline]
Korpelainen EI, Gamble JR, Smith WB, Goodall GJ, Qiyu S, Woodcock JM, Dottore M, Vadas MA & Lopez AF. The receptor for interleukin 3 is selectively induced in human endothelial cells by tumor necrosis factor and potentiates interleukin 8 secretion and neutrophil transmigration, Proc Natl Acad Sci USA, 1993, 90, 11137–11141.
Kraling BM, Razon MJ, Boon LM, Zurakowski D, Seachord C, Darveau RP, Mulliken JB, Corless CL & Bischoff J. E-selectin is present in proliferating endothelial cells in human hemangiomas, Am J Pathol, 1996, 148, 1181–1191.[Abstract]
Lampugnani MG, Resnati M, Raiteri M, Pigott R, Piscane A, Houen G, Ruco LP & Dejana E. A novel endothelial-specific membrane protein is a marker of cell–cell contacts, J Cell Biol, 1992, 118, 1511–1522.
Leavesley DI, Oliver JM, Swart BW, Berndt MC, Haylock DN & Simmons PJ. Signals from platelet/endothelial cell adhesion molecule enhance the adhesive activity of the very late antigen-4 integrin of human CD34+ hemopoietic progenitor cells, J Immunol, 1994, 153, 4673–4683.[Abstract]
Litwin, M.S., J.R. Gamble, and M.A. Vadas. 1995. Role of cytokines in endothelial cell functions. In Human Cytokines: Their Role in Disease and Therapy. B.B. Aggarawal, and R.K. Puri, editors. Blackwell Science, Cambridge, Massachusetts. 101–130.
Lu TT, Yan LG & Madri JA. Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1, Proc Natl Acad Sci USA, 1996, 93, 11808–11813.
Moser R, Schleiffenbaum B, Groscurth P & Fehr J. Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage, J Clin Invest, 1989, 83, 444–455.[Medline]
Murphy PR, Sato R, Sato Y & Friesen HG. Fibroblast growth factor messenger ribonucleic acid expression in a human astrocytoma cell line: regulation by serum and cell density, Mol Endocrinol, 1988, 2, 591–598.
Nagel T, Resnick N, Atkinson WJ, Dewey CF & Gimbrone MA. Shear stress selectively upregulates intercellular adhesion molecule expression in cultured human vascular endothelial cells, J Clin Invest, 1994, 94, 885–891.[Medline]
Newman PJ, Hillery CA, Albrecht R, Parise LV, Berndt MC, Mazurov AV, Dunlop LC, Zhang J & Rittenhouse SE. Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution, J Cell Biol, 1992, 119, 239–246.
Nguyen M, Strubel NA & Bischoff J. A role for sialyl lewis-x/a glycoconjugates in capillary morphogenesis, Nature (Lond), 1993, 365, 267–269.[Medline]
Osborn L, Hession C, Tizard R, Vassallo C, Luhowskyj S, Chi-Rosso G & Lobb R. Direct expression cloning of Vascular Cell Adhesion Molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes, Cell, 1989, 59, 1203–1211.[Medline]
Paulsson Y, Beckman M, Westermark B & Heldrin C-H. Density-dependent inhibition of cell growth by transforming growth factor β1 in normal human fibroblasts, Growth Factors, 1988, 1, 19–27.[Medline]
Petzelbauer P, Pober JS, Keh A & Braverman IM. Inducibility and expression of microvascular endothelial adhesion molecules in lesional, perilesional and uninvolved skin of psoriatic patients, J Invest Dermatol, 1994, 103, 300–305.[Medline]
Plopper G & Ingber DE. Rapid induction of focal adhesion complexes, Biochem Biophys Res Commun, 1993, 193, 571–578.[Medline]
Pober JS, Bevilacqua MP, Mendrick DL, Lapierre LA, Fiers W & Gimbrone MA Jr. Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells, J Immunol, 1986a, 136, 1680–1687.[Abstract]
Pober JS, Gimbrone MA Jr, Lapierre LA, Mendrick DL, Fiers W, Rothlein R & Springer TA. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor and immune interferon, J Immunol, 1986b, 137, 1893–1896.[Abstract]
Read MA, Whitley MZ, Williams AJ & Collins T. NF-kB and IkBa: an inducible regulatory system in endothelial activation, J Exp Med, 1994, 179, 503–512.
Schimmenti LA, Yan H-C, Madri JA & Albelda SM. Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration, J Cell Physiol, 1992, 153, 417–428.[Medline]
Schumacher HR. Joint involvement in progressive systemic sclerosis (scleroderma), Am J Clin Pathol, 1973, 60, 593–600.[Medline]
Sun J, Williams J, Yan H-C, Amin KM, Albelda SM & DeLisser HM. Platelet endothelial cell adhesion molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression, J Biol Chem, 1996, 271, 18561–18570.
Tanaka Y, Albelda SM, Horgan KJ, van Seventer GA, Shimizu Y, Newman W, Hallam J, Newman PJ, Buck CA & Shaw S. CD31 expressed on distinctive T cell subsets is a preferential amplifier of β1integrin-mediated adhesion, J Exp Med, 1992, 176, 245–253.
Tanaka H, Sukhova GK, Swanson SJ, Clinton SK, Ganz P, Cybulsky MI & Libby P. Sustained activation of vascular cells and leukocytes in the rabbit aorta after balloon injury, Circulation, 1993, 88, 1788–1803.
Taylor WR & Alexander RW. Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells, Am J Physiol, 1993, 265, 801–805.
Wall RT, Harker LA, Quadricci LJ & Striker GE. Factors influencing endothelial cell proliferation in vitro, J Cell Physiol, 1978, 96, 203–213.[Medline]
Yan H-C, Pilewski JM, Zhang Q, DeLisser HM, Romer L & Albelda SM. Localization of multiple functional domains on human PECAM-1 (CD31) by monoclonal antibody epitope mapping, Cell Adhes Commun, 1995, 3, 45–66.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
, 105 cells per cm2) and subconfluent (
, 0.25 x 105 cells per cm2) densities. The expression of E-selectin was assayed by flow cytometry at specified times after plating. The MFI (± SEM) of three to five cell lines is shown but values at 2, 32, and 72 h after plating are singlicates. Asterisks denote values significantly different between the two cell densities (P < 0.03) by paired t test.
, full-length PECAM-1; 
, domain 2 deletion mutant; 
). A ratio of 1 EC to 8 L cells would be equivalent in cell number to a high density culture of EC. 18 h later, cells were harvested and stained with goat anti–E-selectin polyclonal antibody and detected using an anti–goat labeled polyclonal antibody together with a mouse anti–VE-cadherin antibody and detected with an anti–mouse, FITC-conjugated antibody. Cells were analyzed by two color analysis, and only the fluorescein positive (i.e., EC) were analyzed for E-selectin expression. The results are given as the MFI of one representative experiment of at least four performed where a similar trend was seen in each. (b) Subconfluent EC were plated as in a but either the full-length, PECAM-1 L cell transfectants (