Resveratrol Improves Endothelial Progenitor Cell Function through miR-138 by Targeting Focal Adhesion Kinase (FAK) and Promotes Thrombus Resolution In Vivo

Figures

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  Figure 1. Characterization of endothelial progenitor cells (EPCs). (A) EPCs colony exhibited a central cluster on day 4 (a) and formed a spindle shaped, endothelial cell-like morphology on day 14 (b) (100×). (B) Double staining of FITC-UEA-1 and DiI-ac-LDL and merge image (400×). (C) Expression of CD31, CD34 and CD309 of EPCs at day 14.

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  Figure 2. Resveratrol regulates migration, tube formation of endothelial progenitor cells. (A, B) Migrated cells counting (200×). ** p<0.01 versus control (n=3). (C, D) Cell migration distance (200×). * p<0.05 versus control (n=3). (E, F) Relative tube number (100×). ** p<0.01 versus control (n=3).

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  Figure 3. Resveratrol promotes function of endothelial progenitor cells (EPCs) by regulating expression level of miR-138. (A) Time course of miRNA-138 expression in EPCs treated with RSV. * p<0.05, ** p<0.01. (B–D), EPCs were transfected with miR-138 mimics or inhibitor or negative control. (B) Migrated cells counting (200×). # p<0.05 versus NC mimics (n=3), ** p<0.01 versus NC inhibitor (n=3). (C) Cell migration distance (200×). # p<0.05 versus. NC mimics (n=3), ** p<0.01 versus. NC inhibitor (n=3). (D) Relative tube number (100×). # p<0.05 versus. NC mimics (n=3), * p<0.05 versus. NC inhibitor (n=3).

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  Figure 4. Upregulation of focal adhesion kinase (FAK) by resveratrol (RSV) enhances migration and tube formation of endothelial progenitor cells (EPCs). (A) The protein level of FAK in EPCs treated by RSV or vehicle. (B–D) RSV-induced EPCs were transfected with FAK siRNA or vehicle. (B) Decreased migrated cells in EPCs transfected with FAK siRNA. (n=3, * p<0.05 versus vehicle group). (C) Decreased migration distance in EPCs transfected with FAK siRNA at 24 hours and 48 hours. (n=3, * p<0.05 versus vehicle group). (D) Decreased tube number in EPCs transfected with FAK siRNA (n=3, * p<0.05 versus vehicle group). (E) A schematic representation showing the putative target site of FAK and mutated target site for miR-138 with the seed region and base substitutions (upper). Luciferase report assays were performed on HEK 293 T cells with co-transfection of FAK-UTR-WT or FAK-UTR-MUT. (F) Luciferase assay of the FAK 3′UTR showed increased activity when EPCs were treated with RSV, as compared to control (vehicle).

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  Figure 5. Resveratrol (RSV)-induced endothelial progenitor cells (EPCs) promotes recanalization of venous thrombosis in vivo. (A) Weight of the venous thrombi at day 7 post the transplantation. Data are expressed as mean ±SEM (n=10, * p<0.05 versus blank control; ** p<0.005 versus blank control). (B) Hematoxylin and eosin (H & E) staining of thrombus sects acquired on 7 day after transplantation (200×). (C) DSA image revealed significantly increase of thrombus recanalization and resolution in rats transplanted with RSV-induced EPCs compared to blank control on day 7.