The Inhibitory Effect of Lysophosphatidylcholine on Proangiogenesis of Human CD34+ Cells Derived Endothelial Progenitor Cells

Figures

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  FIGURE 1. Identification of CD34+ hematopoietic stem cells and differentiation of CD34+ hematopoietic stem cells to early EPC. (A) The purity of CD34+ hematopoietic stem cells was accessed by a flow cytometry analysis. (B) Flow cytometry analysis was used to detect the expression of surface markers (CD34, KDR and CD31) of EPCs derived from CD34+ cells. (C) The immunofluorescence staining was used to examine the specific biomarkers of the endothelial cells on the cell surface (VE-Cadherin, vWF and eNOS). (D) The morphological features of EPCs derived from CD34+ hematopoietic stem cells were observed using a microscope.

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  FIGURE 2. The expression of inflammatory cytokines after LPC or PC treatment in EPCs. RT-qPCR and western blotting analyses were used to detect the expression of inflammatory factors including CCL2 (A), IL-8 (B), CCL4 (C) in EPCs after 10 μg/ml of LPC or PC treatment for 1, 5 or 24 h **p < 0.01, ***p < 0.001 compared with the control, # p < 0.05, ### p < 0.001 compared with the LPC group.

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  FIGURE 3. The expression of factors associated with angiogenesis after LPC or PC treatment in EPCs. RT-qPCR and western blotting was used to access the levels of angiogenesis-associated genes including VEGF (A), Ang-1 (B), Ang-2 (C) in EPCs after 10 μg/ml of LPC or PC treatment for 1, 5 and 24 h *p < 0.05, **p < 0.01 compared with the control, ## p < 0.01 compared with the LPC group.

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  FIGURE 4. The suppressive effect of LPC on the proangiogenesis of EPCs. (A) A tube formation assay was used to examine the angiogenesis potential of EPCs in vitro. (B) Tubes developed by HUVECs in vitro. (C) Tubes developed by HUVECs cultured with the supernatant of EPCs in vitro. (D) After the PC treatment or (E) LPC treatment, the tubes formed by HUVECs that were cultured with supernatant of EPCs. (F,G) Tube count and capillary length of tubes in each group. ***p < 0.001 compared with the HUVECs group, # p < 0.05, ## p < 0.01 compared with the EPCs + HUVECs group.

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  FIGURE 5. The promotive effect of LPC on the activation of the PKC signaling in EPCs. (A,B) The mRNA expression of PKC-β1 and TGF- β1 after LPC or PC treatment in EPCs were measured by RT-qPCR. (C,D) The protein levels of p-PKC and TGF-β1 were in EPCs treated with LPC or PC were accessed using western blot. (E) Immunofluorescence staining was used to determine the location of PKC-β1 after the LPC or PC treatment in EPCs (Figure 5D). **p < 0.01, ***p < 0.001 compared with the control, # p < 0.05, ## p < 0.01 compared with the LPC group.