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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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The induction of  E-selectin on subconfluent  ECs is not mediated through  TNF-α or IL-1. (a) ECs  plated at subconfluent density (0.25 × 105 cells per cm2)  were stimulated with 10 U/ml  TNF-α (TNF) or 1 ng/ml  IL-1β (IL-1) in the presence  of anti–TNF-α (TNF + anti-TNF) (1:1,000), or IL-1ra  (IL-1 + IL-1ra) (100 ng/ml), respectively. Inhibitors and agonists  were added immediately after EC plating and E-selectin expression was assessed 12 h later. (b) ECs were plated at subconfluent  density (stripes, 0.25 × 105 cells per cm2) and confluent density  (solid, 105 cells per cm2). Anti–TNF-α or IL-1ra were added at plating. E-selectin expression was assessed 12 h later. Results shown  are of one representative experiment of four performed.
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Figure 5: The induction of E-selectin on subconfluent ECs is not mediated through TNF-α or IL-1. (a) ECs plated at subconfluent density (0.25 × 105 cells per cm2) were stimulated with 10 U/ml TNF-α (TNF) or 1 ng/ml IL-1β (IL-1) in the presence of anti–TNF-α (TNF + anti-TNF) (1:1,000), or IL-1ra (IL-1 + IL-1ra) (100 ng/ml), respectively. Inhibitors and agonists were added immediately after EC plating and E-selectin expression was assessed 12 h later. (b) ECs were plated at subconfluent density (stripes, 0.25 × 105 cells per cm2) and confluent density (solid, 105 cells per cm2). Anti–TNF-α or IL-1ra were added at plating. E-selectin expression was assessed 12 h later. Results shown are of one representative experiment of four performed.

Mentions: The time course of expression seen in Fig. 2 suggested two phases in the regulation of E-selectin expression: an induction phase, and a maintenance phase. Induction of the expression of adhesion molecules by subconfluent ECs occurred in the absence of exogenous cytokines. As shown in Fig. 5 a, IL-1 receptor antagonist (IL-1ra) or blocking anti–TNF-α antibody potently and specifically inhibited IL-1 or TNF-α–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.


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)

The induction of  E-selectin on subconfluent  ECs is not mediated through  TNF-α or IL-1. (a) ECs  plated at subconfluent density (0.25 × 105 cells per cm2)  were stimulated with 10 U/ml  TNF-α (TNF) or 1 ng/ml  IL-1β (IL-1) in the presence  of anti–TNF-α (TNF + anti-TNF) (1:1,000), or IL-1ra  (IL-1 + IL-1ra) (100 ng/ml), respectively. Inhibitors and agonists  were added immediately after EC plating and E-selectin expression was assessed 12 h later. (b) ECs were plated at subconfluent  density (stripes, 0.25 × 105 cells per cm2) and confluent density  (solid, 105 cells per cm2). Anti–TNF-α or IL-1ra were added at plating. E-selectin expression was assessed 12 h later. Results shown  are of one representative experiment of four performed.
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Related In: Results  -  Collection

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Figure 5: The induction of E-selectin on subconfluent ECs is not mediated through TNF-α or IL-1. (a) ECs plated at subconfluent density (0.25 × 105 cells per cm2) were stimulated with 10 U/ml TNF-α (TNF) or 1 ng/ml IL-1β (IL-1) in the presence of anti–TNF-α (TNF + anti-TNF) (1:1,000), or IL-1ra (IL-1 + IL-1ra) (100 ng/ml), respectively. Inhibitors and agonists were added immediately after EC plating and E-selectin expression was assessed 12 h later. (b) ECs were plated at subconfluent density (stripes, 0.25 × 105 cells per cm2) and confluent density (solid, 105 cells per cm2). Anti–TNF-α or IL-1ra were added at plating. E-selectin expression was assessed 12 h later. Results shown are of one representative experiment of four performed.
Mentions: The time course of expression seen in Fig. 2 suggested two phases in the regulation of E-selectin expression: an induction phase, and a maintenance phase. Induction of the expression of adhesion molecules by subconfluent ECs occurred in the absence of exogenous cytokines. As shown in Fig. 5 a, IL-1 receptor antagonist (IL-1ra) or blocking anti–TNF-α antibody potently and specifically inhibited IL-1 or TNF-α–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.

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