Neurovascular effects of CD47 signaling: promotion of cell death, inflammation, and suppression of angiogenesis in brain endothelial cells in vitro

Abstract

The concept of the neurovascular unit emphasizes that common signals and substrates underlie the physiology and pathophysiology of neuronal and endothelial compartments in brain. Recent data suggest that activation of the integrin-associated protein CD47 promotes neuronal cell death. Is it possible that CD47 may also negatively affect cerebral endothelial cells? Exposure of wild-type primary mouse cerebral endothelial cells to the CD47 ligand thrombospondin 1 (TSP-1) induced an increasing amount of cell death, whereas cytotoxicity was significantly decreased in cerebral endothelial cells derived from CD47 knockout mice. The specific CD47-activating peptide, 4N1K, similarly induced cell death in human brain microvascular endothelial cells. Promotion of inflammation was also involved because lower TSP-1 was able to up-regulate the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. Finally, CD47 signaling may suppress angiogenesis because 4N1K significantly inhibited endothelial cell migration and tube formation in vitro. We conclude that CD47 signaling can negatively affect the viability and function of cerebral endothelial cells, further supporting the notion that CD47 may be a potential neurovascular target for stroke and brain injury.

Fig. 1

CD47-mediated TSP-1-induced cytotoxicity in endothelial cells. A: Exposure to CD47 ligand TSP-1 for 24 hr induced an increasing amount of cell death in wild-type cerebral endothelial cells. TSP-1-induced cytotoxicity was significantly decreased in brain endothelial cells from CD47 knockout mice. B: CD47-specific activating peptide 4N1K similarly induced a cytotoxic response in human brain endothelial cells. *P < 0.05, **P < 0.01 compared with the controls; ●P<0.01 between wild-type and CD47 knockout groups.

Fig. 2

TSP-1 up-regulates the expression of ICAM-1 and VCAM-1 in brain endothelial cells. Exposure of human brain endothelial cells to a low dose of the CD47 ligand TSP-1 (500 ng/ml) triggers an up-regulation in the inflammatory markers ICAM-1 and VCAM-1 within 2 to 8 hr. No changes are detected for E-selectin; n = 3 independent experiments in triplicate. *P < 0.05 compared with controls.

Fig. 3

The CD47 activating peptide 4N1K inhibits cell migration in brain endothelial cells. A: Representative photographs showed decreased migration of human brain endothelial cells across a wound scratch line after treatment with 4N1K (100 μg/ml). B: Quantified cell counts showed a significant difference in cell migration between controls and 4N1K-treated cells. **P < 0.01.

Fig. 4

The CD47 activating peptide 4N1K inhibits Matrigel tube formation in vitro. A: Representative photographs of human brain endothelial cells seeded on Matrigel-coated wells after 18 hr. Untreated cells formed the connected tubular networks. 4N1K (100 μg/ml) attenuated network formation. B: Quantified cell counts showed a significant difference in tube formation between controls and 4N1K-treated cells. Data are expressed as a percentage of the number of tubes in untreated wells. *P < 0.05 compared with the controls.