title: Effect and mechanism of thrombospondin-1 on the angiogenesis potential in human endothelial progenitor cells: an in vitro study.
abstract: 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).
  
  
  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).
  
  
  CONCLUSIONS
  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.
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Qing
authors: Q
authors: Qin
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Juying
authors: J
authors: Qian
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Lei
authors: L
authors: Ge
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Li
authors: L
authors: Shen
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Jianguo
authors: J
authors: Jia
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Jianhao
authors: J
authors: Jin
authors: Shanghai Institute of Cardiovascular Disease, Zhongshan Hospital, Fudan University, Shanghai, China.
authors: Junbo
authors: J
authors: Ge
mesh_terms: D000328:Adult; D000368:Aged; D051928:CD47 Antigen / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D018919:Neovascularization, Physiologic; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D019700:Thrombospondin 1 / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D051928:CD47 Antigen; D019700:Thrombospondin 1; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2