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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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Time course of  E-selectin expression after  EC plating. ECs were plated  at confluent (▵, 105 cells per  cm2) and subconfluent (▪,  0.25 × 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.
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Figure 3: Time course of E-selectin expression after EC plating. ECs were plated at confluent (▵, 105 cells per cm2) and subconfluent (▪, 0.25 × 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.

Mentions: The expression of E-selectin was measured on ECs at varying times after plating. Fig. 3 shows that cells plated at high and low densities both displayed significant levels of E-selectin early after plating that is within 4 to 12 h. However, expression of E-selectin on high density ECs was fourfold less at its maximum 8 h after plating, was transient and returned to basal levels by 24 h. In contrast, E-selectin expression on ECs plated at low density peaked at 12 h after plating and was still evident at 32 h. ECs at very sparse densities, such that single cells were maintained over the course of the experiment, displayed a persistent and significant expression of E-selectin even 72 h after plating (data not shown). There was considerable variation in the absolute levels of E-selectin between different EC preparations, the reason for which is not known. However, the fold induction calculated between high and low density cells was relatively consistent (21.1 ± 3.6%, n = 30) when measured 16–20 h after plating.


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)

Time course of  E-selectin expression after  EC plating. ECs were plated  at confluent (▵, 105 cells per  cm2) and subconfluent (▪,  0.25 × 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139821&req=5

Figure 3: Time course of E-selectin expression after EC plating. ECs were plated at confluent (▵, 105 cells per cm2) and subconfluent (▪, 0.25 × 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.
Mentions: The expression of E-selectin was measured on ECs at varying times after plating. Fig. 3 shows that cells plated at high and low densities both displayed significant levels of E-selectin early after plating that is within 4 to 12 h. However, expression of E-selectin on high density ECs was fourfold less at its maximum 8 h after plating, was transient and returned to basal levels by 24 h. In contrast, E-selectin expression on ECs plated at low density peaked at 12 h after plating and was still evident at 32 h. ECs at very sparse densities, such that single cells were maintained over the course of the experiment, displayed a persistent and significant expression of E-selectin even 72 h after plating (data not shown). There was considerable variation in the absolute levels of E-selectin between different EC preparations, the reason for which is not known. However, the fold induction calculated between high and low density cells was relatively consistent (21.1 ± 3.6%, n = 30) when measured 16–20 h after plating.

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