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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.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.

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 monoclonal IgG antibodies against CD36. Monoclonal IgG antibodies against CD36, FA6-152, and OKM-5 or an isotype-matched control were tested at 10 μg/ml for ability to block the  inhibition of human microvascular endothelial cell migration towards bFGF by (A) TSP-1 (2 nM) or (B) TSP-1 peptides Mal III (30  μM) or Col overlap (50 μM). Angiostatin (2 μg/ml) served as a control inhibitor. Data from three separate experiments were normalized and reported as in Fig. 1. 100% varied between experiments from 32 to 53 cells migrated/10 high-power fields. *Samples significantly different from parallel condition using control media, P < 0.02.
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Figure 4: Interference with the activity of TSP-1 and its antiangiogenic peptides by monoclonal IgG antibodies against CD36. Monoclonal IgG antibodies against CD36, FA6-152, and OKM-5 or an isotype-matched control were tested at 10 μg/ml for ability to block the inhibition of human microvascular endothelial cell migration towards bFGF by (A) TSP-1 (2 nM) or (B) TSP-1 peptides Mal III (30 μM) or Col overlap (50 μM). Angiostatin (2 μg/ml) served as a control inhibitor. Data from three separate experiments were normalized and reported as in Fig. 1. 100% varied between experiments from 32 to 53 cells migrated/10 high-power fields. *Samples significantly different from parallel condition using control media, P < 0.02.

Mentions: To demonstrate more directly that TSP-1 inhibits angiogenesis via an interaction with CD36, two anti-CD36 monoclonal antibodies, OKM-5 and FA6-152, known to map to an immunodominant epitope on the extracellular region of CD36 (Daviet et al., 1995) as well as to physically displace TSP-1 from CD36 (Asch et al., 1987; Kieffer et al., 1989), were tested. Both antibodies blocked the inhibition of bFGF-induced migration of HMVECs by TSP-1 (Fig. 4 A) and by TSP-1 peptides (Fig. 4 B). This block was specific to TSP-1 as neither antibody affected the inhibition of endothelial cell migration by angiostatin (Fig. 4 A), another well-established inhibitor of endothelial cell migration in vitro (Gately et al., 1996) and of angiogenesis in vivo (O'Reilly et al., 1994). An isotype-matched control monoclonal antibody was without effect in the assay (Fig. 4 A). The two anti-CD36 antibodies were also able to block TSP-1 inhibition of migration induced by angiogenic molecules other than bFGF. For example, antibody FA6-152 blocked TSP-1 inhibition of endothelial cell migration induced by either scatter factor (Lamszus et al., 1996) or vascular endothelial growth factor (Koch et al., 1994) (data not shown).


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 monoclonal IgG antibodies against CD36. Monoclonal IgG antibodies against CD36, FA6-152, and OKM-5 or an isotype-matched control were tested at 10 μg/ml for ability to block the  inhibition of human microvascular endothelial cell migration towards bFGF by (A) TSP-1 (2 nM) or (B) TSP-1 peptides Mal III (30  μM) or Col overlap (50 μM). Angiostatin (2 μg/ml) served as a control inhibitor. Data from three separate experiments were normalized and reported as in Fig. 1. 100% varied between experiments from 32 to 53 cells migrated/10 high-power fields. *Samples significantly different from parallel condition using control media, P < 0.02.
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Related In: Results  -  Collection

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

Figure 4: Interference with the activity of TSP-1 and its antiangiogenic peptides by monoclonal IgG antibodies against CD36. Monoclonal IgG antibodies against CD36, FA6-152, and OKM-5 or an isotype-matched control were tested at 10 μg/ml for ability to block the inhibition of human microvascular endothelial cell migration towards bFGF by (A) TSP-1 (2 nM) or (B) TSP-1 peptides Mal III (30 μM) or Col overlap (50 μM). Angiostatin (2 μg/ml) served as a control inhibitor. Data from three separate experiments were normalized and reported as in Fig. 1. 100% varied between experiments from 32 to 53 cells migrated/10 high-power fields. *Samples significantly different from parallel condition using control media, P < 0.02.
Mentions: To demonstrate more directly that TSP-1 inhibits angiogenesis via an interaction with CD36, two anti-CD36 monoclonal antibodies, OKM-5 and FA6-152, known to map to an immunodominant epitope on the extracellular region of CD36 (Daviet et al., 1995) as well as to physically displace TSP-1 from CD36 (Asch et al., 1987; Kieffer et al., 1989), were tested. Both antibodies blocked the inhibition of bFGF-induced migration of HMVECs by TSP-1 (Fig. 4 A) and by TSP-1 peptides (Fig. 4 B). This block was specific to TSP-1 as neither antibody affected the inhibition of endothelial cell migration by angiostatin (Fig. 4 A), another well-established inhibitor of endothelial cell migration in vitro (Gately et al., 1996) and of angiogenesis in vivo (O'Reilly et al., 1994). An isotype-matched control monoclonal antibody was without effect in the assay (Fig. 4 A). The two anti-CD36 antibodies were also able to block TSP-1 inhibition of migration induced by angiogenic molecules other than bFGF. For example, antibody FA6-152 blocked TSP-1 inhibition of endothelial cell migration induced by either scatter factor (Lamszus et al., 1996) or vascular endothelial growth factor (Koch et al., 1994) (data not shown).

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.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.

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