ox-LDL-Induced Endothelial Progenitor Cell Oxidative Stress via p38/Keap1/Nrf2 Pathway

Figure 4

ox-LDL impaired EPC migration and tube formation and induced apoptosis via Nrf2 or p38 pathway. EPCs were pretreated with Keap1 siRNA or 1 μM SB203580 and then exposed to ox-LDL (20 μg/mL) for 6 hours. (a) Apoptosis was determined through Annexin V-FITC and PI double-staining using flow cytometry after ox-LDL or 1 μM SB203580 treatment for 6 hours. ox-LDL increased the apoptotic rate of EPCs. Pretreatment of SB203580 reduced the apoptotic rate of EPCs. (b, c) EPC apoptosis was quantified by manually counting pyknotic nuclei after DAPI (Sigma-Aldrich) staining. ox-LDL increased the apoptotic rate of EPCs. Pretreatment of SB203580 reduced the apoptotic rate of EPCs. Scale: 100 μm. (d) Represented Western blot images of the protein expressions of Bax and Bcl-2 in EPCs after treatment of ox-LDL or Keap1 siRNA. (e) Quantification of relative quantity of Bax and Bcl-2 showed that ox-LDL significantly increased Bax/Bcl-2 ratio, which was blocked by the pretreatment of Keap1 siRNA transfection. Migration of EPCs was examined by transwell chemotaxis assay. (f) The representative images of migrated EPCs in the control group, ox-LDL group, and Keap1 siRNA pretreatment plus the ox-LDL group (magnification: ×100). Scale: 50 μm. (g) The presentative photomicrographs of tube formation of EPCs under the treatment of ox-LDL and Keap1 siRNA pretreatment. Scale: 200 μm. (h) Quantitative data show that ox-LDL significantly reduced the numbers of migrated EPCs, but Keap1 siRNA significantly increased the numbers of migrated cells. Values are the from 3 independent experiments. The angiogenic function of EPCs under exposure to ox-LDL or Keap1 siRNA was determined by tube formation assay. (i) Quantification analysis of the number of tube branches showed that Keap1 siRNA pretreatment ameliorated EPC angiogenic function which were inhibited by ox-LDL. vs. control. ^# vs. ox-LDL group.

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