Topical application of ex vivo expanded endothelial progenitor cells promotes vascularisation and wound healing in diabetic mice

Abstract

Impaired wound healing leading to skin ulceration is a serious complication of diabetes and may be caused by defective angiogenesis. Endothelial progenitor cells (EPCs) can augment neovascularisation in the ischaemic tissue. Experiments were performed to test the hypothesis that locally administered EPCs can promote wound healing in diabetes. Full-thickness skin wounds were created on the dorsum of diabetic mice. EPCs were obtained from bone marrow mononuclear cells (BMMNCs) and applied topically to the wound immediately after surgery. Vehicle and non-selective BMMNCs were used as controls. Wound size was measured on days 5, 10 and 14 after treatment, followed by resection, histological analysis and quantification of vascularity. Topical application of EPCs significantly promoted wound healing, as assessed by closure rate and wound vascularity. Immunostaining revealed that transplanted EPCs induced increased expression of vascular endothelial growth factor and basic fibroblast growth factor. Few EPCs were observed in the neovasculature based on in vivo staining of the functional vasculature. Ex vivo expanded EPCs promote wound healing in diabetic mice via mechanisms involving increased local cytokine expression and enhanced neovascularisation of the wound. This strategy exploiting the therapeutic capacity of autologously derived EPCs may be a novel approach to skin repair in diabetes.

Keywords: Basic fibroblast growth factor; Endothelial progenitor cells; Neovascularisation; Vascular endothelial growth factor; Wound healing.

Figure 1

Effects of endothelial progenitor cell (EPC) transplantation on full‐thickness skin wound closure in genetically diabetic mice. EPCs, bone marrow mononuclear cells (BMMNCs) or phosphate‐buffered saline (PBS) were applied to the wound. (A) Macroscopic appearance of wounds after each treatment. (B) % wound closure. Topical application of ex vivo expanded EPCs significantly accelerated wound closure compared to controls (n = 5 per group, *P < 0·001 versus PBS, ^† P < 0·001 versus BMMNC).

Figure 2

Topical application of endothelial progenitor cells (EPCs) accelerated formation of granulation tissue. (A) Sections from wounds of diabetic mice treated with EPCs, bone marrow mononuclear cells (BMMNCs) and phosphate‐buffered saline (PBS) were stained with H&E. Wound margins on days 5, 10 and 14 showed an improved wound appearance and increased granulation tissue with time for EPC‐treated wounds. In the PBS group, the wound bed shows a thin layer of granulation tissue over adipose tissue. In the EPC group, the wound bed shows a thick layer of granulation tissue covered with epithelium. (B) Histological score. The EPC group had a significantly higher histological score compared with the other two groups (n = 5 per group, *P < 0·001 versus PBS, ^† P < 0·001 versus BMMNC).

Figure 3

Effects of topical application of endothelial progenitor cells (EPCsI on wound vascularity. (A) The sections were stained with an antibody against CD31. On day 14, tiny and narrow new vessels at the wound margin were present in the bone marrow mononuclear cell (BMMNC) and phosphate‐buffered saline (PBS) groups, while larger and thicker vessels were growing widely in the centre of the wound in the EPC group. Original magnification ×100. (B) Topical application of EPCs significantly promoted wound vascularity (n = 5 per group, *P < 0·001 versus PBS, ^† P < 0·001 versus BMMNC).

Figure 4

Endothelial progenitor cell (EPC) tracking. For EPC tracking, cells were marked with red fluorescent carbocyanine dioctadecyl tetramethylindocarbocyanine (DiI) dye‐labelled acetylated LDL. (A) Many DiI‐labelled EPCs were observed around the neovasculature by in vivo staining of the functional vasculature with FITC‐labelled Bandeiraea simplicifolia lectin I on day 10. In contrast, only a few DiI‐labelled EPCs were observed in the neovasculature on day 14 (red arrow). (B) Immunostaining revealed that DiI‐labelled EPCs expressed vascular endothelial growth factor‐A (VEGF‐A) and basic fibroblast growth factor (bFGF).