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CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells.

Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP - J. Cell Biol. (1997)

Bottom Line: Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells.Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells.Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology and Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.

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Interference with the activity of TSP-1 and its antiangiogenic peptides by soluble GST–CD36 fusion proteins. Increasing concentrations of CD36 fusion proteins that contain a TSP-1 binding site, FP93-120 (circles) or FP93-298 (triangles), or a CD36 fusion protein that lacks a TSP-1 binding site, FP298-439 (squares), were preincubated for 2 h at 4°C with (A) 2 nM TSP-1, (B) 10 μM Mal III peptide, (C) 30 μM Col overlap peptide, or (D) control media. Each mixture was then tested for the ability to block bovine capillary  endothelial cell migration towards bFGF (solid symbols) or influence background migration in the absence of bFGF (D, open symbols).  Data, accumulated from nine experiments, are reported as a percentage of maximum migration, where 100% represents the number of  cells migrating towards the inducer bFGF alone, and 0% corresponds to the number of cells migrating randomly in the absence of inducer (Bkgd). 100% varied between experiments from 32 to 81 cells migrated/10 high-power fields. Bars indicate standard errors.
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Figure 2: Interference with the activity of TSP-1 and its antiangiogenic peptides by soluble GST–CD36 fusion proteins. Increasing concentrations of CD36 fusion proteins that contain a TSP-1 binding site, FP93-120 (circles) or FP93-298 (triangles), or a CD36 fusion protein that lacks a TSP-1 binding site, FP298-439 (squares), were preincubated for 2 h at 4°C with (A) 2 nM TSP-1, (B) 10 μM Mal III peptide, (C) 30 μM Col overlap peptide, or (D) control media. Each mixture was then tested for the ability to block bovine capillary endothelial cell migration towards bFGF (solid symbols) or influence background migration in the absence of bFGF (D, open symbols). Data, accumulated from nine experiments, are reported as a percentage of maximum migration, where 100% represents the number of cells migrating towards the inducer bFGF alone, and 0% corresponds to the number of cells migrating randomly in the absence of inducer (Bkgd). 100% varied between experiments from 32 to 81 cells migrated/10 high-power fields. Bars indicate standard errors.

Mentions: To determine if an interaction between CD36 and TSP-1 could block the antiangiogenic activity of TSP-1, soluble GST–CD36 fusion proteins were tested for their ability to block the inhibition of bovine capillary endothelial cell migration by TSP-1. This assay measures the migration of cultured endothelial cells toward a known angiogenic factor (bFGF) and consistently parallels angiogenic activity seen in vivo (Folkman and Klagsbrun, 1987; Klagsbrun and D'Amore, 1991; Bouck et al., 1996). TSP-1 was previously shown to interact in a specific, saturable, and reversible manner with a minimal region of CD36 encompassing amino acids 93–120 (Pearce et al., 1995). As shown in Fig. 2 A, GST–CD36 fusion proteins FP93-120 and FP93-298, both of which contained the TSP-1 binding domain, were able to block TSP-1 inhibition of bFGF-induced migration in a dose-dependent fashion, whereas GST alone (data not shown) and fusion protein FP298-439, which lacked the TSP-1 binding domain (Fig. 2 A), were ineffective.


CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells.

Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP - J. Cell Biol. (1997)

Interference with the activity of TSP-1 and its antiangiogenic peptides by soluble GST–CD36 fusion proteins. Increasing concentrations of CD36 fusion proteins that contain a TSP-1 binding site, FP93-120 (circles) or FP93-298 (triangles), or a CD36 fusion protein that lacks a TSP-1 binding site, FP298-439 (squares), were preincubated for 2 h at 4°C with (A) 2 nM TSP-1, (B) 10 μM Mal III peptide, (C) 30 μM Col overlap peptide, or (D) control media. Each mixture was then tested for the ability to block bovine capillary  endothelial cell migration towards bFGF (solid symbols) or influence background migration in the absence of bFGF (D, open symbols).  Data, accumulated from nine experiments, are reported as a percentage of maximum migration, where 100% represents the number of  cells migrating towards the inducer bFGF alone, and 0% corresponds to the number of cells migrating randomly in the absence of inducer (Bkgd). 100% varied between experiments from 32 to 81 cells migrated/10 high-power fields. Bars indicate standard errors.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141641&req=5

Figure 2: Interference with the activity of TSP-1 and its antiangiogenic peptides by soluble GST–CD36 fusion proteins. Increasing concentrations of CD36 fusion proteins that contain a TSP-1 binding site, FP93-120 (circles) or FP93-298 (triangles), or a CD36 fusion protein that lacks a TSP-1 binding site, FP298-439 (squares), were preincubated for 2 h at 4°C with (A) 2 nM TSP-1, (B) 10 μM Mal III peptide, (C) 30 μM Col overlap peptide, or (D) control media. Each mixture was then tested for the ability to block bovine capillary endothelial cell migration towards bFGF (solid symbols) or influence background migration in the absence of bFGF (D, open symbols). Data, accumulated from nine experiments, are reported as a percentage of maximum migration, where 100% represents the number of cells migrating towards the inducer bFGF alone, and 0% corresponds to the number of cells migrating randomly in the absence of inducer (Bkgd). 100% varied between experiments from 32 to 81 cells migrated/10 high-power fields. Bars indicate standard errors.
Mentions: To determine if an interaction between CD36 and TSP-1 could block the antiangiogenic activity of TSP-1, soluble GST–CD36 fusion proteins were tested for their ability to block the inhibition of bovine capillary endothelial cell migration by TSP-1. This assay measures the migration of cultured endothelial cells toward a known angiogenic factor (bFGF) and consistently parallels angiogenic activity seen in vivo (Folkman and Klagsbrun, 1987; Klagsbrun and D'Amore, 1991; Bouck et al., 1996). TSP-1 was previously shown to interact in a specific, saturable, and reversible manner with a minimal region of CD36 encompassing amino acids 93–120 (Pearce et al., 1995). As shown in Fig. 2 A, GST–CD36 fusion proteins FP93-120 and FP93-298, both of which contained the TSP-1 binding domain, were able to block TSP-1 inhibition of bFGF-induced migration in a dose-dependent fashion, whereas GST alone (data not shown) and fusion protein FP298-439, which lacked the TSP-1 binding domain (Fig. 2 A), were ineffective.

Bottom Line: Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells.Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells.Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology-Immunology and Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA.

ABSTRACT
Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase-CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.

Show MeSH
Related in: MedlinePlus