Limits...
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.

Show MeSH

Related in: MedlinePlus

PECAM-1–transfected  L cells regulate E-selectin expression. (a) EC at subconfluent  density (0.2 × 105 cells per cm2)  were plated in the presence or  absence of various concentrations of PECAM-1–transfected L  cells (○, full-length PECAM-1;  □, domain 2 deletion mutant; ▵,  untransfected L cells). EC were  also plated at high density (♦).  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 (○), or cytoplasmic tail deletion mutant (X). Analysis was as for a and is given as the MFI of a representative experiment of at least four performed.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139821&req=5

Figure 10: PECAM-1–transfected L cells regulate E-selectin expression. (a) EC at subconfluent density (0.2 × 105 cells per cm2) were plated in the presence or absence of various concentrations of PECAM-1–transfected L cells (○, full-length PECAM-1; □, domain 2 deletion mutant; ▵, untransfected L cells). EC were also plated at high density (♦). 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 (○), or cytoplasmic tail deletion mutant (X). Analysis was as for a and is given as the MFI of a representative experiment of at least four performed.

Mentions: The third approach used PECAM-1 transfectants. EC were plated at subconfluent density in the presence of L cells expressing full-length PECAM-1, PECAM-1 lacking domain 2, or lacking the cytoplasmic tail. Both the full length and the truncation of the cytoplasmic tail inhibited E-selectin expression, whereas the mutant lacking domain 2 had no effect (Fig. 10 a). Untransfected L cells (Fig. 10 b) or L cells expressing the L1 adhesion glycoprotein (a member of the Ig superfamily expressed by neural cells and lymphocytes (Hubbe et al., 1993) failed to regulate E-selectin expression.


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)

PECAM-1–transfected  L cells regulate E-selectin expression. (a) EC at subconfluent  density (0.2 × 105 cells per cm2)  were plated in the presence or  absence of various concentrations of PECAM-1–transfected L  cells (○, full-length PECAM-1;  □, domain 2 deletion mutant; ▵,  untransfected L cells). EC were  also plated at high density (♦).  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 (○), or cytoplasmic tail deletion mutant (X). Analysis was as for a and is given as the MFI of a representative experiment of at least four performed.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: PECAM-1–transfected L cells regulate E-selectin expression. (a) EC at subconfluent density (0.2 × 105 cells per cm2) were plated in the presence or absence of various concentrations of PECAM-1–transfected L cells (○, full-length PECAM-1; □, domain 2 deletion mutant; ▵, untransfected L cells). EC were also plated at high density (♦). 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 (○), or cytoplasmic tail deletion mutant (X). Analysis was as for a and is given as the MFI of a representative experiment of at least four performed.
Mentions: The third approach used PECAM-1 transfectants. EC were plated at subconfluent density in the presence of L cells expressing full-length PECAM-1, PECAM-1 lacking domain 2, or lacking the cytoplasmic tail. Both the full length and the truncation of the cytoplasmic tail inhibited E-selectin expression, whereas the mutant lacking domain 2 had no effect (Fig. 10 a). Untransfected L cells (Fig. 10 b) or L cells expressing the L1 adhesion glycoprotein (a member of the Ig superfamily expressed by neural cells and lymphocytes (Hubbe et al., 1993) failed to regulate E-selectin expression.

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