Autophagy promotes MSC-mediated vascularization in cutaneous wound healing via regulation of VEGF secretion

Abstract

Vascularization deficiency caused a lot of diseases, such as diabetes ulcer and myocardial infarction. Mesenchymal stem cells (MSCs), with the self-renewal and multipotent differentiation capacities, have been used for many diseases treatment through regulation microenvironment. Numerous studies reported that MSCs transplantation could largely improve cutaneous wound healing via paracrine secretion of growth factors. However, whether MSCs take part in the angiogenesis process directly remains elusive. Previous study proved that autophagy inhibited immunosuppressive function of MSCs and prevented the degradation of MSCs function in inflammatory and senescent microenvironment. Here, we proved that autophagy determines the therapeutic effect of MSCs in cutaneous wound healing through promoting endothelial cells angiogenesis and demonstrated that the paracrine of vascular endothelial growth factor (VEGF) in MSCs was required in wound site. We further revealed that autophagy enhanced the VEGF secretion from MSCs through ERK phosphorylation directly. Collectively, we put forward that autophagy mediated paracrine of VEGF plays a central role in MSCs cured cutaneous wound healing and may provide a new therapeutic method for angiogenesis-related diseases.

Fig. 1. MSCs possess the higher pro-angiogenesis capacity of endothelial cells (ECs) through subcutaneous injection than intravenous injection

a Images of wound size on the back of mouse and quantification of wound healing rate (%) in different groups. Scale bar = 5 mm. b Hematoxylin and eosin (H&E) staining of wound area tissues at 2-week post-operative. The red boxes indicate the healing of wound skin. Scale bar=2 mm. c Vascularization state in wound healing skin and quantification of capillary number at 2-week post-operative. d Immunofluorescence staining on CD31 of wound area tissues and quantification of positive areas of CD31 at 2-week post-operative. Scale bar = 50 μm. e Dio-labeled MSCs (red) and LC3-positive cells (green) at 24 h after injection. The co-stained cells represented the autophagy activation of MSCs, which were then analyzed as the percentage of total detected MSCs (*P < 0.05, **P < 0.01, n = 3). Scale bar = 50 μm. The photo in right corner is the locally enlarged image. Scale bar = 10 μm

Fig. 2. Autophagy enhances the pro-angiogenesis capacity of endothelial cells (ECs) after culturing with MSCs via promoting angiogenesis in injury part

a Dio-labeled MSCs (red) were detected by immunofluorescence assay at 24 h after injection. Scale bar = 50 μm. b The proteins of Atg7, Beclin-1, and LC3I/II of MSCs treated with rapamycin and si-Beclin-1 were tested by western blot. c The genes of Atg7, Beclin-1, and LC3 of MSCs treated with rapamycin and si-Beclin-1 were tested by RT-PCR. d Images of wound size on the back of mouse and quantification of wound healing rate (%) in different groups. Scale bar = 5 mm. e Hematoxylin and eosin (H&E) staining of wound area tissues at 2-week post-operative. The red boxes indicate the healing of wound skin. Scale bar = 2 mm. f Vascularization state in wound healing skin and quantification of capillary number at 2-week post-operative. g Immunofluorescence staining on CD31 of wound area tissues and quantification of positive areas of CD31 at 2-week post-operative (*P < 0.05, **P < 0.01, n = 3). Scale bar = 50 μm

Fig. 3. Autophagy determines the angiogenesis of endothelial cells (ECs) after being co-cultured with MSCs

a Diagram of co-culture system using a Transwell insert. ECs were co-cultured with MSCs (pretreatment with rapamycin or si-Beclin-1) in a Transwell system, and ECs were then collected for further analysis. b In vitro tube formation of ECs at 6 h. Scale bar = 20 μm. c–g Quantification of total branching points, covered areas, total nets, total loops, and total tube length were measured by analytical software Image-Pro Plus 6.0 (IPP) (*P < 0.05, **P < 0.01, n = 3)

Fig. 4. Autophagy had no direct effect on the angiogenesis of endothelial cells (ECs)

a In vitro tube formation of ECs treated with rapamycin or si-Beclin-1 at 6 h. Scale bar = 20 μm. b–f In vitro tube formation of EPCs at 6 h and quantification of total tube length, covered areas, total branching points, total loops, and total nets were measured by analytical software Image-Pro Plus 6.0 (IPP). g The expression of angiogenesis-related proteins VEGF and AngII of ECs were detected by western blot. h, i The expression of angiogenesis-related genes PCNA and VEGF of ECs were detected by RT-PCR. (^NSP > 0.05, n = 3)

Fig. 5. Autophagy promote the secretion of VEGF through phosphorylating ERK1/2

a Screening angiogenesis-related genes (VEGF, AngII, PDGF, FGF, TGF, and PCNA) of MSCs after being treated with rapamycin or si-Beclin-1 by RT-PCR. b The expression of angiogenesis-related proteins VEGF in MSCs as detected by western blot. c The secretion of VEGF in culture medium of MSCs treated with rapamycin or si-Beclin-1 was detected by ELISA assay. d Immunofluorescence staining on VEGF of wound area tissues at 2-week post-operative. Scale bar = 50 μm. e The detection of protein LC3I/II, ERK, and p-ERK in MSCs after being treated with rapamycin or si-Beclin-1 by western blot. f LC3 interacts with ERK in vivo. Immunoblots showing co-immunoprecipitation of LC3 with ERK. g The expression of VEGF was reduced in MSCs treated with ERK inhibitor U0126 by western blot. h The secretion of VEGF was reduced in culture medium of MSCs treated with ERK inhibitor U0126 by ELISA assay (*P < 0.05, **P < 0.01, n = 3)

Fig. 6. The angiogenesis capacity of endothelial cells (ECs) was decreased and the wound healing rate was reduced in MSCs after being treated with si-VEGF

a The secretion of VEGF was reduced in culture medium of MSCs treated with si-VEGF by ELISA assay. b, c In vitro tube formation of EPCs at 6 h and quantification of total tube length, total branching points, covered areas, total nets, and total loops were measured by analytical software Image-Pro Plus 6.0 (IPP). Scale bar = 20 μm. d Images of wound size on the back of mouse and quantification of wound healing rate (%) (*P < 0.05, **P < 0.01, n = 3). Scale bar = 5 mm

Fig. 7. The secreted VEGF from MSCs played a critical importance in the process of angiogenesis of endothelial cells (ECs)

a expression of angiogenesis-related proteins VEGF and AngII in ECs were detected by western blot. b, c In vitro tube formation of ECs at 6 h and quantification of total tube length, total branching points, covered areas, total nets, and total loops were measured by analytical software Image-Pro Plus 6.0 (IPP). Scale bar = 20 μm. d Images of wound size on the back of mouse and quantification of wound healing rate (%) (*P < 0.05, **P < 0.01, n = 3). Scale bar = 5 mm

Fig. 8

Autophagy increase the VEGF secretion from MSCs through regulating ERK phosphorylation, and VEGF further promote vascularization of endothelial cells