Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model

Abstract

Chronic wounds have become an economic, social, and public health burden and need advanced treatment. Platelet-rich plasma (PRP) has been used extensively in treatment of chronic wounds because it contains an abundance of growth factors secreted by platelets. The exosomes derived from PRP (PRP-Exos) have been proven to encapsulate principal growth factors from platelets. This study is the first to show that these exosomes may exert the function of PRP. PRP-Exos can effectively induce proliferation and migration of endothelial cells and fibroblasts to improve angiogenesis and re-epithelialization in chronic wounds. We regulated YAP to verify the PRP-Exos-dependent effect on fibroblast proliferation and migration through YAP activation. In vivo, we observed the cutaneous healing process in chronic wounds treated with PRP-Exos in a diabetic rat model. We provide evidence of the probable molecular mechanisms underlying the PRP effect on healing of chronic ulcers and describe a promising resource of growth factors from exosomes without species restriction.

Keywords: Yes-associated protein; chronic wounds; exosomes; platelet-rich plasma (PRP); re-epithelization..

Figure 1

Characterization of PRP-Exos: (A) Particle size distribution measured by DLS. (B) Morphology observed by TEM. (C) Western blotting and quantitative analysis of the exosome surface markers and cargo. Scale bar: 100 nm. *P < 0.05 compared with PRP-AS.

Figure 2

(A-B) PRP-Exos enhanced the proliferation of HMEC-1 cells and fibroblasts when analyzed by CCK-8 assay with different concentration of PRP-Exos and PRP-AS. (C) Representative photomicrographs showing the effect of different concentrations of PRP-Exos and PRP-AS on the transwell migration (violet-stained cells) of HMEC-1 cells and fibroblasts, and tubule formation of HMEC-1 cells after incubation for 6 h. Scale bar: 100 μm. (D) Quantitative analysis of the transwell assays. *P < 0.05 compared with control. #P < 0.05 comparing PRP-AS and PRP-Exos.

Figure 3

(A) Western blotting showed that exosome-treated fibroblasts induced significantly greater YAP de-phosphorylation and much higher CTGF expression compared with the other three groups, indicating that the Rho/YAP signaling pathway was activated in fibroblasts treated with PRP-Exos and PRP. *P < 0.05 compared with controls. #P < 0.05 comparing PRP-AS and PRP-Exos. (B) YAP expression in fibroblasts after treatment with shYAP. *P < 0.05 compared with empty vector. (C) Proliferation of fibroblasts transfected with shYAP or its empty vector and treated with PRP-Exos, analyzed by cck-8 assay. *P < 0.05 compared with empty vector. (D) Migration of fibroblasts transfected with shYAP or its empty vector and treated by PRP-Exos, analyzed by transwell assay, scale bar: 100 μm. (E) Proliferation of fibroblasts transfected with S127A or its empty vector then treated with PRP-Exos and analyzed by cck-8 assay. *P < 0.05 compared with empty vector. (F) Migration of fibroblasts transfected with S127A or its empty vector and treated with PRP-Exos, analyzed by transwell assay, scale bar: 100 μm. (G) YAP de-phosphorylation following treatment with PRP-Exos was blocked by Y-27632 2HCl. *P < 0.05 compared with control. (H) YAP protein expression in the cytoplasm and nucleus of fibroblasts treated with PRP-Exos was altered by Y-27632 2HCl. *P < 0.05 compared with control. (I) YAP nuclear localization in fibroblasts treated with PRP-Exos was blocked by Y-27632 2HCl. Scale bar: 50 μm. (J) Proliferation of fibroblasts treated with PRP-Exos was blocked by Y-27632 2HCl, as shown by cck-8 assay. *P < 0.05 compared with control. (K) Migration of fibroblasts treated with PRP-Exos was blocked by Y-27632 2HCl, as shown by transwell assay. Scale bar: 100 μm. (L) Erk and Akt phosphorylation level in fibroblasts treated with PRP-Exos or PRP-AS. *P < 0.05 compared with control. #P < 0.05 comparing PRP-AS and PRP-Exos.

Figure 4

(A) Cumulative amount of PRP-Exos released from SAH into serum-free MesenGro hMSC medium as a function of immersion time. (B) Representative images of full-thickness skin defects in a diabetic rat model, left untreated (control) or treated with SAH, PRP or PRP-Exos, at 0, 3, 7 and 14 days after operation. Scale bar: 10 mm. (C) Percentage wound closure of untreated defects and defects treated with SAH, PRP or PRP-Exos at 3, 7 and 14 days after surgery. *P < 0.05 compared with control.

Figure 5

(A) Micro-CT evaluation of blood vessel formation in full-thickness skin defects left untreated (control) or treated with SAH, PRP or PRP-Exos at 14 days after surgery. Three-dimensional reconstructed images showing the new blood vessels. (B) Morphometric analysis of the new blood vessel area and the number of blood vessels. *P < 0.05 compared with control. #P < 0.05 comparing between groups. (C) Transmitted light images of HE-stained sections of the untreated defects (control) and the defects treated with SAH, PRP or PRP-Exos at 14 days after operation (scale bar = 2 mm). The total width of the image represents the initial defect size (1.8 cm) while the black arrows indicate the neo-epithelium. (D) Total neo-epithelium length in the skin defects left untreated (control), treated with SAH, PRP or PRP-Exos at 14 days after operation. *P < 0.05 compared with control. #P < 0.05 between PRP-Exos and PRP.

Figure 6

Transmitted light images of Masson's trichrome-stained sections of the untreated defects (control) and the defects treated with SAH, PRP or PRP-Exos at 7 and 14 days after operation, showing collagen deposition. Scale bar: 100 μm.

Figure 7

(A) IHC staining of CD31 (left) and IF staining of CD31 and a-SMA (right). Newly-formed blood vessels were identified by positive CD31 staining and their typical round or oval structure. ECs (CD31), smooth muscle cells (α-SMA) and cell nuclei are stained green, red and blue, respectively. Red and green co-staining indicates mature blood vessels. (B) Number of newly-formed blood vessels and mature blood vessels in the untreated defects (control) and the defects treated with SAH, PRP or PRP-Exos at 7 and 14 days after operation. Scale bar: 50 μm. *P < 0.05 compared with control.