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. 2020 Apr 20;10:6646. doi: 10.1038/s41598-020-63716-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Skp2 restores the angiogenic activity of senescent human EPCs in vivo. (A) Laser Doppler perfusion images of hind limb ischemic rats injected with human EPCs to test their angiogenic activity. Young and old EPCs were infected with Gal4 (Gal) or Skp2 and imaged at day 0 and day 21 after EPC injection. PBS was the group without EPCs as mock injection. Right hind limbs were untreated to serve as the control. Right panel: quantification of the ischemia area versus the normal perfusion area. The days on which the images were captured are indicated. *P < 0.05 compared with the PBS group; ^#P < 0.05 compared with the Gal-O-EPC group. (B) Representative images of myocyte and capillary staining. At day 21, tissues were sectioned and stained with laminin (blue) and lectin1 (red) to visualize myocytes and capillaries, respectively. Lower panel: quantification of capillary density, which was derived as the number of myocytes divided by the number of capillaries. Notably, because of tissue atrophy, the peripheries of PBS-treated myocytes appeared to be smaller than those of the EPC-injected myocytes. *P < 0.05 compared with the PBS group; #P < 0.05 compared with the Gal-O-EPC group. Scale bar, 50 μm. (C) Damaged tissue was rescued through the injection of young EPCs and Skp2-infected old EPCs. The degree of tissue damage was classified into three categories: salvageable (green), toe necrotic (yellow), and amputated (red) for each group (n = 10). Notably, the percentage of amputated limbs in the Skp2-treated old EPC group (Skp2-O-EPC) was lower than that in the Gal4-treated old EPC group (Gal-O-EPC).