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Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Litwin M, Clark K, Noack L, Furze J, Berndt M, Albelda S, Vadas M, Gamble J - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Immunology, Hanson Centre for Cancer Research, Adelaide, South Australia.

ABSTRACT
Tumor necrosis factor-alpha, 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.

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Anti–PECAM-1 antibody upregulates E-selectin on  ECs plated at cobblestone density. (a) ECs at cobblestone density (105 cells per cm2) were treated with Ig-purified rabbit polyclonal antibody to PECAM-1 (5 μg/ml) or a control nonimmune  rabbit Ig at the time of plating. E-selectin expression was assessed  by flow cytometry 12 h later. The MFI (± SEM ) of two to six experiments is given where untreated confluent density ECs have  been normalized to 1.0. Subconfluent ECs showed a 5.1 ± 1.4-fold increase in E-selectin expression relative to confluent EC.  Asterisks denote a significant difference (P < 0.05) compared  with control rabbit Ig by unpaired t test. (b) ECs at confluent  density were treated with 5 μg/ml Ig-purified mAbs to PECAM-1  or VE-cadherin at the time of plating or polyclonal anti–PECAM-1  antibody. E-selectin expression was measured 12 h later. The  MFI (± SEM) of three experiments is given where untreated  confluent density ECs have been normalized to 1.0. Subconfluent  ECs showed a 4.0 ± 0.7-fold relative increase in E-selectin expression relative to confluent EC. Asterisks denote a significant  difference (P < 0.05) compared with cobblestone EC group.
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Figure 8: Anti–PECAM-1 antibody upregulates E-selectin on ECs plated at cobblestone density. (a) ECs at cobblestone density (105 cells per cm2) were treated with Ig-purified rabbit polyclonal antibody to PECAM-1 (5 μg/ml) or a control nonimmune rabbit Ig at the time of plating. E-selectin expression was assessed by flow cytometry 12 h later. The MFI (± SEM ) of two to six experiments is given where untreated confluent density ECs have been normalized to 1.0. Subconfluent ECs showed a 5.1 ± 1.4-fold increase in E-selectin expression relative to confluent EC. Asterisks denote a significant difference (P < 0.05) compared with control rabbit Ig by unpaired t test. (b) ECs at confluent density were treated with 5 μg/ml Ig-purified mAbs to PECAM-1 or VE-cadherin at the time of plating or polyclonal anti–PECAM-1 antibody. E-selectin expression was measured 12 h later. The MFI (± SEM) of three experiments is given where untreated confluent density ECs have been normalized to 1.0. Subconfluent ECs showed a 4.0 ± 0.7-fold relative increase in E-selectin expression relative to confluent EC. Asterisks denote a significant difference (P < 0.05) compared with cobblestone EC group.

Mentions: 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).


Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).

Litwin M, Clark K, Noack L, Furze J, Berndt M, Albelda S, Vadas M, Gamble J - J. Cell Biol. (1997)

Anti–PECAM-1 antibody upregulates E-selectin on  ECs plated at cobblestone density. (a) ECs at cobblestone density (105 cells per cm2) were treated with Ig-purified rabbit polyclonal antibody to PECAM-1 (5 μg/ml) or a control nonimmune  rabbit Ig at the time of plating. E-selectin expression was assessed  by flow cytometry 12 h later. The MFI (± SEM ) of two to six experiments is given where untreated confluent density ECs have  been normalized to 1.0. Subconfluent ECs showed a 5.1 ± 1.4-fold increase in E-selectin expression relative to confluent EC.  Asterisks denote a significant difference (P < 0.05) compared  with control rabbit Ig by unpaired t test. (b) ECs at confluent  density were treated with 5 μg/ml Ig-purified mAbs to PECAM-1  or VE-cadherin at the time of plating or polyclonal anti–PECAM-1  antibody. E-selectin expression was measured 12 h later. The  MFI (± SEM) of three experiments is given where untreated  confluent density ECs have been normalized to 1.0. Subconfluent  ECs showed a 4.0 ± 0.7-fold relative increase in E-selectin expression relative to confluent EC. Asterisks denote a significant  difference (P < 0.05) compared with cobblestone EC group.
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Figure 8: Anti–PECAM-1 antibody upregulates E-selectin on ECs plated at cobblestone density. (a) ECs at cobblestone density (105 cells per cm2) were treated with Ig-purified rabbit polyclonal antibody to PECAM-1 (5 μg/ml) or a control nonimmune rabbit Ig at the time of plating. E-selectin expression was assessed by flow cytometry 12 h later. The MFI (± SEM ) of two to six experiments is given where untreated confluent density ECs have been normalized to 1.0. Subconfluent ECs showed a 5.1 ± 1.4-fold increase in E-selectin expression relative to confluent EC. Asterisks denote a significant difference (P < 0.05) compared with control rabbit Ig by unpaired t test. (b) ECs at confluent density were treated with 5 μg/ml Ig-purified mAbs to PECAM-1 or VE-cadherin at the time of plating or polyclonal anti–PECAM-1 antibody. E-selectin expression was measured 12 h later. The MFI (± SEM) of three experiments is given where untreated confluent density ECs have been normalized to 1.0. Subconfluent ECs showed a 4.0 ± 0.7-fold relative increase in E-selectin expression relative to confluent EC. Asterisks denote a significant difference (P < 0.05) compared with cobblestone EC group.
Mentions: 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Human Immunology, Hanson Centre for Cancer Research, Adelaide, South Australia.

ABSTRACT
Tumor necrosis factor-alpha, 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.

Show MeSH
Related in: MedlinePlus