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Effect and mechanism of thrombospondin-1 on the angiogenesis potential in human endothelial progenitor cells: An in vitro study

PLoS ONE (2014) 9(2)
DOI: 10.1371/journal.pone.0088213
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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). 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. © 2014 Qin et al.

Figures

  • Figure 1. Flow cytometric analysis of CD34+ cells and morphological and immunophenotypical characterization of early and late EPCs. (A) Flow cytometric analysis of CD34 expression after isolation by anti-CD34 microbeads. Shown are representative data from 3 independent experiments using cells isolated from different cord blood with similar results. Isotype controls are used. (B) Early EPCs cultured for 7 days and late EPCs cultured for 14 days (Scale bar = 100 mm, 2006magnification). (C)Early EPCs are shown to uptake DiI-Ac-LDL(red) (Scale bar = 100 mm, 2006magnification). Immunocytochemistry of VEGFR2(red),CD31 (red), and DAPI(blue) was demonstrated in early EPCs (Scale bar = 50 mm,4006 magnification). (D) Immunocytochemistry of VEGFR2(red), vWF(green), CD31(red),and DAPI(blue) was demonstrated in late EPCs(Scale bar = 50 mm,4006magnification). Shown are representative data from 3 independent experiments using early EPCs isolated from different cord blood and 3 independent experiments using late EPCs isolated from different cord blood. doi:10.1371/journal.pone.0088213.g001
  • Figure 2. TSP-1 inhibited early EPCs incorporation into tube-like structure. (A) Early EPCs pretreated with TSP-1 at different concentrations for 1 hr were labeled with a DiI fluorescent marker(red) and coplated with HUVECs(transparent) to form tubule structures on Matrigel(Scale bar = 200 mm,1006magnification). (B–D) Early EPCs pretreated with TSP-1 at different concentrations for 1 hr(B), 6 hrs(C), 12 hrs(D) were coplated with HUVECs on Matrigel. Quantifications of incorporated EPCs per field were presented as mean6S.D of three independent experiments (#p,0.05 versus no TSP-1 intervention). doi:10.1371/journal.pone.0088213.g002
  • Figure 3. TSP-1 inhibited late EPCs tubule formation on Matrigel. (A) Late EPCs were plated on Matrigel with TSP-1 at different concentrations for 8 hrs (Scale bar = 200 mm, 1006magnification). (B) Quantification of total tube length per high power field (HPF, 1006magnification) was presented as mean6S.D. of three independent experiments (#p,0.05 versus no TSP-1 intervention, *p,0.01 versus no TSP-1 intervention). doi:10.1371/journal.pone.0088213.g003
  • Figure 4. Silencing of CD47 attenuated TSP-19s inhibition on angiogenesis of late EPCs. (A) Late EPCs were transfected with negative control siRNA, CD47 siRNA or integrin b1 siRNA, and then plated on Matrigel in the presence or absence of TSP-1(2 mg/ml) as described in methods. EPCs images were captured and analyzed by Leica Qwin system (Scale bar = 400 mm, 506magnification). (B)Total tube length per HPF (1006magnification) was measured. Values are presented as the mean6S.D. of three independent experiments (#p,0.01 versus control siRNA with TSP-1 stimulation,* p,0.01 versus CD47 siRNA without intervention). doi:10.1371/journal.pone.0088213.g004
  • Figure 5. CD47 antibody attenuated TSP-19s inhibition on angiogenesis of late EPCs. (A) Late EPCs were plated on Matrigel and treated with anti-CD47 antibody (2.5 mg/ml), anti-integrin b1 antibody (2 mg/ml) or control IgG for 30 min and then treated with TSP-1(2 mg/ml) as described (Scale bar = 400 mm, 506magnification). (B) Total tube length per HPF (1006magnification) was measured. Values are presented as the mean6S.D. of three independent experiments (#p,0.05 versus control siRNA with TSP-1 stimulation) doi:10.1371/journal.pone.0088213.g005
  • Figure 6. TSP-1 inhibits VEGF induced VEGFR2 phosphorylation through CD47. (A) Late EPCs were treated with VEGF (25 ng/ml) for 0, 5, 10, 15 min respectively. Total protein was extracted and the expression of FLK-1, phospho-VEGFR2 (Tyr1175) was determined by Western blot. (B)Seventy-two hrs after transfection of CD47-specific siRNA or control siRNA, the expression of CD47 in late EPCs was determined by western blotting analysis in three independent experiments (#p,0.05 versus control siRNA). (C)Seventy-two hrs after transfection of CD47-specific siRNA or control siRNA, late EPCs were treated with TSP-1(2 mg/ml) for 30 min and then VEGF(25 ng/ml) for 5 min. Total protein was extracted and the expression of FLK-1, phospho-VEGFR2 (Tyr1175) was determined by Western blot analysis. (D) Quantification of VEGFR2 phosphorylation normalized to VEGFR2 in three independent experiments(#p,0.05 versus control siRNA, *p,0.01 versus control siRNA). doi:10.1371/journal.pone.0088213.g006
  • Table 1. Clinical characteristics and serum TSP-1 level in all patients.

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Qin, Q., Qian, J., Ge, L., Shen, L., Jia, J., Jin, J., & Ge, J. (2014). Effect and mechanism of thrombospondin-1 on the angiogenesis potential in human endothelial progenitor cells: An in vitro study. PLoS ONE, 9(2). https://doi.org/10.1371/journal.pone.0088213

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