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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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E-selectin expression varies with EC density.  (a) Flow cytometry profile  of a representative EC line  stained for E-selectin 15 h after plating. Cells were plated  at cobblestone (⋄⋄⋄, 105  cells per cm2) or subconfluent densities (thick line; 0.25 ×  105 cells per cm2). (thin line)  Nonbinding control immunoglobulin, which gave a similar  profile for confluent or subconfluent cells. (b) ECs were  cultured for 15 h after plating  at cell densities ranging from  sparse to confluent (0.125– 2.0 × 105 cells per cm2). The  MFI ± SEM of E-selectin expression is shown for five EC  lines, except for values at densities 0.5 and 2.0 × 105 cells per cm2,  which are triplicates. Asterisks indicate values significantly different (P < 0.003) from confluent density ECs (105 cells per cm2) by  unpaired t test.
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Figure 2: E-selectin expression varies with EC density. (a) Flow cytometry profile of a representative EC line stained for E-selectin 15 h after plating. Cells were plated at cobblestone (⋄⋄⋄, 105 cells per cm2) or subconfluent densities (thick line; 0.25 × 105 cells per cm2). (thin line) Nonbinding control immunoglobulin, which gave a similar profile for confluent or subconfluent cells. (b) ECs were cultured for 15 h after plating at cell densities ranging from sparse to confluent (0.125– 2.0 × 105 cells per cm2). The MFI ± SEM of E-selectin expression is shown for five EC lines, except for values at densities 0.5 and 2.0 × 105 cells per cm2, which are triplicates. Asterisks indicate values significantly different (P < 0.003) from confluent density ECs (105 cells per cm2) by unpaired t test.

Mentions: HUVECs were seeded onto gelatin-coated tissue culture plates at varying numbers (Fig. 1). 15 h after plating, the EC surface expression of E-selectin was measured by flow cytometry. Confluent, cobblestone cultures as seen in Fig. 1 a, showed negligible E-selectin expression (Fig. 2 a). This is in agreement with published work demonstrating a lack of E-selectin on unstimulated ECs (Pober et al., 1986b; Bevilacqua et al., 1989). However, subconfluent ECs (Fig. 1 b) displayed substantial expression of E-selectin (Fig. 2 a). A comparison between subconfluent and confluent density cells using five separate EC lines showed a 17-fold greater induction in subconfluent ECs, (6.4 ± 1.0 vs. 0.37 ± 0.19 mean fluorescence intensity units ± SEM, P = 0.0003, unpaired t test). In these five experiments, 57 ± 4% of ECs at subconfluent density were positive for E-selectin, as opposed to 7 ± 2% of cells in a cobblestone monolayer. The confluency-dependent expression of E-selectin was evident over a range of EC densities (Fig. 2 b) and was seen on HUVECs extracted from their original monolayer culture by treatment with trypsin-EDTA or EDTA alone (data not shown).


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)

E-selectin expression varies with EC density.  (a) Flow cytometry profile  of a representative EC line  stained for E-selectin 15 h after plating. Cells were plated  at cobblestone (⋄⋄⋄, 105  cells per cm2) or subconfluent densities (thick line; 0.25 ×  105 cells per cm2). (thin line)  Nonbinding control immunoglobulin, which gave a similar  profile for confluent or subconfluent cells. (b) ECs were  cultured for 15 h after plating  at cell densities ranging from  sparse to confluent (0.125– 2.0 × 105 cells per cm2). The  MFI ± SEM of E-selectin expression is shown for five EC  lines, except for values at densities 0.5 and 2.0 × 105 cells per cm2,  which are triplicates. Asterisks indicate values significantly different (P < 0.003) from confluent density ECs (105 cells per cm2) by  unpaired t test.
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Related In: Results  -  Collection

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Figure 2: E-selectin expression varies with EC density. (a) Flow cytometry profile of a representative EC line stained for E-selectin 15 h after plating. Cells were plated at cobblestone (⋄⋄⋄, 105 cells per cm2) or subconfluent densities (thick line; 0.25 × 105 cells per cm2). (thin line) Nonbinding control immunoglobulin, which gave a similar profile for confluent or subconfluent cells. (b) ECs were cultured for 15 h after plating at cell densities ranging from sparse to confluent (0.125– 2.0 × 105 cells per cm2). The MFI ± SEM of E-selectin expression is shown for five EC lines, except for values at densities 0.5 and 2.0 × 105 cells per cm2, which are triplicates. Asterisks indicate values significantly different (P < 0.003) from confluent density ECs (105 cells per cm2) by unpaired t test.
Mentions: HUVECs were seeded onto gelatin-coated tissue culture plates at varying numbers (Fig. 1). 15 h after plating, the EC surface expression of E-selectin was measured by flow cytometry. Confluent, cobblestone cultures as seen in Fig. 1 a, showed negligible E-selectin expression (Fig. 2 a). This is in agreement with published work demonstrating a lack of E-selectin on unstimulated ECs (Pober et al., 1986b; Bevilacqua et al., 1989). However, subconfluent ECs (Fig. 1 b) displayed substantial expression of E-selectin (Fig. 2 a). A comparison between subconfluent and confluent density cells using five separate EC lines showed a 17-fold greater induction in subconfluent ECs, (6.4 ± 1.0 vs. 0.37 ± 0.19 mean fluorescence intensity units ± SEM, P = 0.0003, unpaired t test). In these five experiments, 57 ± 4% of ECs at subconfluent density were positive for E-selectin, as opposed to 7 ± 2% of cells in a cobblestone monolayer. The confluency-dependent expression of E-selectin was evident over a range of EC densities (Fig. 2 b) and was seen on HUVECs extracted from their original monolayer culture by treatment with trypsin-EDTA or EDTA alone (data not shown).

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