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Updated: Aug 16, 2026
In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing
Published on: May 11, 2021
Identification of CD36 molecular features required for its in vitro angiostatic activity
Luca Primo1, Chiara Ferrandi, Cristina Roca
1Institute for Cancer Research and Treatment, Candiolo, and School of Medicine, University of Torino, Italy. luca.primo@ircc.it
Insights
Thrombospondin-1 (TSP-1) inhibits angiogenesis by targeting CD36 on endothelial cells. Specific amino acids in CD36
Area of Science:
- Molecular Biology
- Cell Biology
- Angiogenesis Research
Background:
- Thrombospondin-1 (TSP-1) is a known inhibitor of angiogenesis.
- TSP-1 acts on endothelial cells (ECs) via CD36 to impede migration and morphogenesis.
- Vascular Endothelial Growth Factor-A (VEGF-A) is a key stimulator of angiogenesis.
Purpose of the Study:
- To investigate how CD36, triggered by TSP-1, inhibits VEGF-A-stimulated angiogenesis.
- To identify specific functional components of the CD36 C-terminal cytoplasmic tail involved in TSP-1's angiostatic activity.
- To elucidate the molecular mechanisms underlying CD36-mediated inhibition of VEGF-A signaling.
Main Methods:
- Transduction of CD36 into CD36-deficient endothelial cells.
- Treatment with TSP-1 and an anti-CD36 agonist antibody (SMO).
- Site-directed mutagenesis of the CD36 C-terminal cytoplasmic tail.
- Analysis of VEGF receptor-2 (VEGFR-2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation.
Main Results:
- TSP-1 and SMO inhibited VEGF-A165-induced migration and sprouting of CD36-expressing ECs.
- Amino acids C464, R467, and K469 in CD36 are essential for TSP-1's inhibitory effect.
- TSP-1 activation of CD36 down-modulated VEGFR-2 and p38 MAPK phosphorylation, an effect abolished by C464 mutation.
Conclusions:
- Specific amino acids in the CD36 C-terminal tail are crucial for TSP-1's angiostatic function.
- CD36 plays a key role in mediating TSP-1's inhibition of VEGF-A-driven angiogenesis.
- This study reveals novel insights into the regulation of VEGFR-2 signaling by CD36.
Abstract:
Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.
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