Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis

Abstract

Background: Enhanced angiogenesis can promote diabetic wound healing. Mesenchymal stem cells (MSCs)-derived exosomes, which are cell-free therapeutics, are promising candidates for the treatment of diabetic wound healing. The present study aimed to investigate the effect of exosomes derived from MSCs pretreated with pioglitazone (PGZ-Exos) on diabetic wound healing.

Results: We isolated PGZ-Exos from the supernatants of pioglitazone-treated BMSCs and found that PGZ-Exos significantly promote the cell viability and proliferation of Human Umbilical Vein Vascular Endothelial Cells (HUVECs) injured by high glucose (HG). PGZ-Exos enhanced the biological functions of HUVECs, including migration, tube formation, wound repair and VEGF expression in vitro. In addition, PGZ-Exos promoted the protein expression of p-AKT, p-PI3K and p-eNOS and suppressed that of PTEN. LY294002 inhibited the biological function of HUVECs through inhibition of the PI3K/AKT/eNOS pathway. In vivo modeling in diabetic rat wounds showed that pioglitazone pretreatment enhanced the therapeutic efficacy of MSCs-derived exosomes and accelerated diabetic wound healing via enhanced angiogenesis. In addition, PGZ-Exos promoted collagen deposition, ECM remodeling and VEGF and CD31 expression, indicating adequate angiogenesis in diabetic wound healing.

Conclusions: PGZ-Exos accelerated diabetic wound healing by promoting the angiogenic function of HUVECs through activation of the PI3K/AKT/eNOS pathway. This offers a promising novel cell-free therapy for treating diabetic wound healing.

Keywords: Angiogenesis; Diabetic wound; Exosomes; Mesenchymal stem cells; Pioglitazone.

Fig. 1

Schematic diagram of PGZ-Exos accelerating diabetic wound repair through enhancing angiogenesis by activation of the PI3K/AKT/eNOS pathway

Fig. 2

Characterization of BMSCs-derived exosomes. A The morphology of the exosomes and PGZ-Exos was visualized with TEM. Scale bar: 50 nm. B The exosome marker proteins CD9 and CD63 in the exosomes and PGZ-Exos were detected with western blotting. C The size distribution of exosomes and PGZ-Exos was examined with NTA. D PKH26-labeled exosomes and PGZ-Exos uptake by HUVECs was investigated with laser scanning confocal microscopy. The cytoskeleton, exosomes and cell nucleus are stained green, red, and blue respectively. Scale bar: 10 m

Fig. 3

PGZ-exos protected against HG-induced inhibition of HUVECs viability and cell proliferation. A CCK8 assay was performed to assess HUVECs viability after treatment with HG and exosomes. B, C EdU incorporation assay was used to evaluate HUVECs proliferation. Scale bar: 50 m. D, E Flow cytometry was used to assess the cell cycle distribution of the HUVECs. F, G Western blotting was used to investigate the protein expression of Cyclin D1, Cyclin D3 and VEGF. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group

Fig. 4

PGZ-exos enhanced the angiogenic ability of HUVECs inhibited by HG. B A transwell assay was used to assess the cell migration of HUVECs. Scale bar: 50 m. B A tube formation assay was performed to visualize the cell capillary network formation of HUVECs. Scale bar: 100 m. C In vitro wound healing assay of the HUVECs. Scale bar: 200 m. D Quantitative analysis of the number of migrating cells in the four groups. E Quantitative analysis of tube formation in the four groups. F Quantitative analysis of the rate of wound closure in the four groups. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group

Fig. 5

PGZ-Exos promoted the angiogenic ability of HUVECs through activation of the PI3K/AKT/eNOS pathway. A Western blotting showing the protein expression of AKT, p-AKT, PI3K and p-PI3K. B Quantitative analysis of the protein level of p-AKT/AKT in the four groups. C Quantitative analysis of the protein level of p-PI3K/PI3K in the four groups. D Western blotting showing the protein expression of eNOS, p-eNOS and PTEN. E Quantitative analysis of the protein level of p-eNOS/eNOS in the four groups. F Quantitative analysis of the protein level of PTEN in the four groups. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group

Fig. 6

LY294002 suppressed PGZ-Exos-enhanced the biological functions of HUVECs. A, B Flow cytometry was used to assess the cell cycle distribution of the HUVECs treated with HG medium supplemented with PGZ-Exos and PGZ-Exos+LY 294002. C, D Western blotting was used to investigate the protein expression of Cyclin D1, Cyclin D3 and VEGF. E, F A transwell assay was used to assess the cell migration of HUVECs. Scale bar: 50 m. G, H A tube formation assay was performed to visualize the cell capillary network formation of HUVECs. Scale bar: 100 m. I, J In vitro wound healing assay of the HUVECs. Scale bar: 200 m. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group

Fig. 7

LY294002 inhibited the PGZ-Exos-induced activation of the PI3K/AKT/eNOS pathway in HUVECs. A Western blotting showing the protein expression of AKT, p-AKT, PI3K and p-PI3K of the HUVECs treated with HG medium supplemented with PGZ-Exos and PGZ-Exos+LY 294002. B Quantitative analysis of the protein level of p-AKT/AKT in the four groups. C Quantitative analysis of the protein level of p-PI3K/PI3K in the four groups. D Western blotting showing the protein expression of eNOS, p-eNOS and PTEN. E Quantitative analysis of the protein level of p-eNOS/eNOS in the four groups. F Quantitative analysis of the protein level of PTEN in the four groups. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group

Fig. 8

PGZ-Exos accelerated diabetic wound healing. A Representative images of full thickness defects in diabetic rats receiving treatment with PBS (Con), exosomes and PGZ-Exo at days 0, 3, 7, 10, and 14 day postoperatively. B Wound healing closure rates was calculated among the different groups using the ImageJ software. C, D Blood perfusion of wounds was assessed with Doppler detection. The results of blood perfusion is presented as the ratio of wound area (ROI-1) to area surrounding the wound (ROI-2). E, F HE staining and quantification of wound length at day 14. Scale bar: 2.5 mm. G Massons trichrome staining at day 14. Scale bar: 50 m

Fig. 9

Immunohistochemical analysis of diabetic wounds post-surgery. A Representative immunohistochemical analysis images of collagen I, collagen III. Scale bar: 50 m. B, C Quantification of the positive areas of the collagen I and collagen III among the different groups using the ImageJ software. D Representative immunohistochemical analysis images of VEGF (Scale bar: 50 m) and CD31 (Scale bar: 100 m). E Quantification of the positive areas of VEGF among the different groups using the ImageJ software. F Quantification of the number of CD31 positive among the different groups using the ImageJ software. Data are presented as meansSD from three independent experiments. *P<0.05 versus the control (con) group