Effect and Mechanism of Thrombospondin-1 on the Angiogenesis Potential in Human Endothelial Progenitor Cells: An In Vitro Study

OBJECTIVE: Coronary collateral circulation plays a protective role in patients with coronary artery disease (CAD). We investigated whether thrombospondin-1(TSP-1) has an inhibitory effect on angiogenesis potential in endothelial progenitor cells(EPCs) and tested whether TSP-1 are altered in plasma of patients who had chronic total occlusion (CTO) in at least one coronary artery and with different collateral stages(according to Rentrop grading system). METHODS AND RESULTS: We isolated early and late EPCs from human cord blood and investigated a dose-dependent effect of TSP-1 on their angiogenesis potential by Matrigel angiogenesis assay. We found that TSP-1 (5 µg/ml) inhibited early EPCs incorporation into tubules after pretreatment for 1, 6 and 12 hours, respectively (83.3±11.9 versus 50.0±10.1 per field for 1 hour,161.7±12.6 versus 124.0±14.4 for 6 hours, 118.3±12.6 versus 68.0±20.1 for 12 hours, p<0.05). TSP-1 also inhibited late EPCs tubule formation at 1 µg/ml (6653.4±422.0 µm/HPFversus 5552.8±136.0 µm/HPF, p<0.05), and the inhibition was further enhanced at 5 µg/ml (6653.4±422.0 µm/HPF versus 2118.6±915.0 µm/HPF p<0.01). To explore the mechanism involved, a small interfering RNA was used. In vitro, CD47 siRNA significantly attenuated TSP-1's inhibition of angiogenesis on late EPCs and similar results were obtained after functional blocking by anti-CD47 antibody. Then we investigated pathways downstream of CD47 and found TSP-1 regulated VEGF-induced VEGFR2 phosphorylation via CD47. Furthermore, we examined plasma TSP-1 levels in patients with CTO who developed different stages of collaterals and found a paradoxical higher level of TSP-1 in patients with good collaterals compared with bad ones (612.9±554.0 ng/ml versus 224.4±132.4 ng/ml, p<0.05). CONCLUSION: TSP-1 inhibited angiogenesis potential of early and late EPCs in vitro. This inhibition may be regulated by TSP-1's interaction with CD47, resulting in down regulation of VEGFR2 phosphorylation. In patients with CTO, there may be a self-adjustment mechanism in bad collaterals which is shown as low level of angiogenesis inhibitor TSP-1, and thus favoring collateral formation.