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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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Inhibition of TSP-1 binding to CD36 and its fusion proteins by antiangiogenic TSP-1 peptides. (A) Binding of 125I–TSP-1 (20 μg/ ml) to a confluent monolayer of Bowes melanoma cells expressing CD36 was determined in the presence of increasing concentrations of  antiangiogenic TSP-1 peptides Mal III (circles, lowest curve), Mal III variant (triangles, second lowest curve), and Col overlap (squares,  third lowest curve) or of control peptide LYPQHKT (diamonds, top curve). Data were normalized as a percentage of TSP-1 bound under control conditions. (B) The effects of TSP-1 peptides Mal III and Col overlap on the binding of 125I–TSP-1 (20 μg/ml) to solid phase  CD36 and CD36 fusion proteins FP67-157 and FP93-120, which contain a TSP-1 binding site, and to FP298-439, which does not, are  shown. Nonspecific binding was determined in the presence of 5 mM EDTA. Bars indicate standard error (n = 3 for A and n = 5 for B).
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Figure 3: Inhibition of TSP-1 binding to CD36 and its fusion proteins by antiangiogenic TSP-1 peptides. (A) Binding of 125I–TSP-1 (20 μg/ ml) to a confluent monolayer of Bowes melanoma cells expressing CD36 was determined in the presence of increasing concentrations of antiangiogenic TSP-1 peptides Mal III (circles, lowest curve), Mal III variant (triangles, second lowest curve), and Col overlap (squares, third lowest curve) or of control peptide LYPQHKT (diamonds, top curve). Data were normalized as a percentage of TSP-1 bound under control conditions. (B) The effects of TSP-1 peptides Mal III and Col overlap on the binding of 125I–TSP-1 (20 μg/ml) to solid phase CD36 and CD36 fusion proteins FP67-157 and FP93-120, which contain a TSP-1 binding site, and to FP298-439, which does not, are shown. Nonspecific binding was determined in the presence of 5 mM EDTA. Bars indicate standard error (n = 3 for A and n = 5 for B).

Mentions: The Mal III and Col overlap peptides physically interacted with CD36 because both were able to competitively displace 125I –TSP-1 from CD36 expressed in its natural context on the surface of CD36 cDNA-transfected Bowes melanoma cells. TSP-1 peptides Mal III and Col overlap, as well as the Mal III variant that lacks the VTCG motif but still inhibits endothelial cell migration (Tolsma, S., and N. Bouck, personal communication), were all able to inhibit binding of TSP-1 to CD36 (Fig. 3 A). Inhibition constants (IC50's) were 8.60 ± 1.12 μM for Mal III, 30.08 ± 8.88 μM for Col overlap, and 15.78 ± 2.08 μM for Mal III variant. A control peptide did not inhibit binding demonstrating specificity. Melanoma cells not transfected with CD36 do not bind TSP-1 (Silverstein et al., 1992). The calculated IC50 of each peptide closely approximated its previously reported ED50 for inhibition of endothelial cell migration (Tolsma et al., 1993).


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)

Inhibition of TSP-1 binding to CD36 and its fusion proteins by antiangiogenic TSP-1 peptides. (A) Binding of 125I–TSP-1 (20 μg/ ml) to a confluent monolayer of Bowes melanoma cells expressing CD36 was determined in the presence of increasing concentrations of  antiangiogenic TSP-1 peptides Mal III (circles, lowest curve), Mal III variant (triangles, second lowest curve), and Col overlap (squares,  third lowest curve) or of control peptide LYPQHKT (diamonds, top curve). Data were normalized as a percentage of TSP-1 bound under control conditions. (B) The effects of TSP-1 peptides Mal III and Col overlap on the binding of 125I–TSP-1 (20 μg/ml) to solid phase  CD36 and CD36 fusion proteins FP67-157 and FP93-120, which contain a TSP-1 binding site, and to FP298-439, which does not, are  shown. Nonspecific binding was determined in the presence of 5 mM EDTA. Bars indicate standard error (n = 3 for A and n = 5 for B).
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Related In: Results  -  Collection

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

Figure 3: Inhibition of TSP-1 binding to CD36 and its fusion proteins by antiangiogenic TSP-1 peptides. (A) Binding of 125I–TSP-1 (20 μg/ ml) to a confluent monolayer of Bowes melanoma cells expressing CD36 was determined in the presence of increasing concentrations of antiangiogenic TSP-1 peptides Mal III (circles, lowest curve), Mal III variant (triangles, second lowest curve), and Col overlap (squares, third lowest curve) or of control peptide LYPQHKT (diamonds, top curve). Data were normalized as a percentage of TSP-1 bound under control conditions. (B) The effects of TSP-1 peptides Mal III and Col overlap on the binding of 125I–TSP-1 (20 μg/ml) to solid phase CD36 and CD36 fusion proteins FP67-157 and FP93-120, which contain a TSP-1 binding site, and to FP298-439, which does not, are shown. Nonspecific binding was determined in the presence of 5 mM EDTA. Bars indicate standard error (n = 3 for A and n = 5 for B).
Mentions: The Mal III and Col overlap peptides physically interacted with CD36 because both were able to competitively displace 125I –TSP-1 from CD36 expressed in its natural context on the surface of CD36 cDNA-transfected Bowes melanoma cells. TSP-1 peptides Mal III and Col overlap, as well as the Mal III variant that lacks the VTCG motif but still inhibits endothelial cell migration (Tolsma, S., and N. Bouck, personal communication), were all able to inhibit binding of TSP-1 to CD36 (Fig. 3 A). Inhibition constants (IC50's) were 8.60 ± 1.12 μM for Mal III, 30.08 ± 8.88 μM for Col overlap, and 15.78 ± 2.08 μM for Mal III variant. A control peptide did not inhibit binding demonstrating specificity. Melanoma cells not transfected with CD36 do not bind TSP-1 (Silverstein et al., 1992). The calculated IC50 of each peptide closely approximated its previously reported ED50 for inhibition of endothelial cell migration (Tolsma et al., 1993).

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