Activation of moderate autophagy promotes survival of fat graft.

Clinically unpredictable retention following fat grafting remains outstanding problems because of the unrevealed mechanism of grafted fat survival. The role of autophagy, a process to maintain cellular homeostasis through recycling cellular debris, has yet been to be reported in fat grafting. This study aims to improve the survival of fat grafting through the autophagy. First, the relationship between cell death and autophagy in the early stage of fat grafting was evaluated through immunostaining, RNA sequencing, and western blot. Next, rapamycin, an autophagic agonist, was used for the culturing of adipose-derived stem cells and adipocytes during ischemia. Cell death, autophagy, and reactive oxygen species (ROS) were assayed. Finally, rapamycin was used to assist fat grafting in nude mice. The results demonstrated that the peak of cell death at the early stage of fat grafting was accompanied by a decrease in autophagy. In vitro, during ischemia, 25 nM was confirmed as the optimal dose of rapamycin that reduces cell death with enhanced autophagy and mitophagy, improved mitochondrial quality as well as decreased ROS accumulation. In vivo, promoted mitophagy, alleviated oxidative stress, and decreased cell apoptosis of rapamycin-treated fat grafts were observed in the early stage. In addition, rapamycin increased the survival of fat grafts with increased neovascularization and reduced fibrosis. We suggested that moderate autophagy induced by rapamycin contribute to enhanced ischemic tolerance and long term survival of fat grafts through mitochondrial quality control.

Adipose-Derived Stem Cell Extracellular Vesicles Improve Wound Closure and Angiogenesis in Diabetic Mice.

BACKGROUND: Currently, there is a lack in therapy that promotes the reepithelialization of diabetic wounds as an alternative to skin grafting. Here, the authors hypothesized that extracellular vesicles from adipose-derived stem cells (ADSC-EVs) could accelerate wound closure through rescuing the function of keratinocytes in diabetic mice. METHODS: The effect of ADSC-EVs on the biological function of human keratinocyte cells was assayed in vitro. In vivo, 81 male severe combined immune deficiency mice aged 8 weeks were divided randomly into the extracellular vesicle-treated diabetes group (n = 27), the phosphate-buffered saline-treated diabetes group (n = 27), and the phosphate-buffered saline-treated normal group (n = 27). A round, 8-mm-diameter, full-skin defect was performed on the back skin of each mouse. The wound closure kinetics, average healing time, reepithelialization rate, and neovascularization were evaluated by histological staining. RESULTS: In vitro, ADSC-EVs improved proliferation, migration, and proangiogenic potential, and inhibited the apoptosis of human keratinocyte cells by suppressing Fasl expression with the optimal dose of 40 µg/mL. In vivo, postoperative dripping of ADSC-EVs at the dose of 40 µg/mL accelerated diabetic wound healing, with a 15.8% increase in closure rate and a 3.3-day decrease in average healing time. ADSC-EVs improved reepithelialization (18.2%) with enhanced epithelial proliferation and filaggrin expression, and suppressed epithelial apoptosis and Fasl expression. A 2.7-fold increase in the number of CD31-positive cells was also observed. CONCLUSION: ADSC-EVs improve diabetic wound closure and angiogenesis by enhancing keratinocyte-mediated reepithelialization and vascularization. CLINICAL RELEVANCE STATEMENT: ADSC-EVs could be developed as a regenerative medicine for diabetic wound care.

Extracellular Vesicles Isolated From Hypoxia-Preconditioned Adipose-Derived Stem Cells Promote Hypoxia-Inducible Factor 1α-Mediated Neovascularization of Random Skin Flap in Rats.

BACKGROUND: Random flaps are widely used for wound repair. However, flap necrosis is a serious complication leading to the failure of operation. Our previous study demonstrated a great proangiogenic potential of hypoxia-treated adipose-derived stem cells-extracellular vesicles (HT-ASC-EVs). Thus, we aim to evaluate the effect of HT-ASC-EVs in the survival and angiogenesis of random skin flap in rats. METHODS: Adipose-derived stem cells-extracellular vesicles were respectively isolated from adipose-derived stem cell culture medium of 3 donors via ultracentrifugation. The expression of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic potential of HT-ASC-EVs and ASC-EVs were compared by co-culturing with human umbilical vein endothelial cells. Forty male Sprague-Dawley rats were randomly divided into 3 group (n = 10/group). A 9 × 3-cm random skin flap was separated from the underlying fascia with both sacral arteries sectioned on each rat. The survival and angiogenesis of flaps treated by ASC-EVs or HT-ASC-EVs were also compared. Laser Doppler flowmetry and immunohistochemistry were used to evaluate skin perfusion and angiogenesis of skin flaps on postoperative day 7. RESULTS: Hypoxia-treated adipose-derived stem cells-extracellular vesicles further improve the proliferation, migration, tube formation with upregulated HIF-1α, and VEGF expression of human umbilical vein endothelial cells in vitro, compared with ASC-EVs. In vivo, postoperatively injecting HT-ASC-EVs suppressed necrosis rate (29.1 ± 2.8% vs 59.2 ± 2.1%) and promoted the angiogenesis of skin flap including improved skin perfusion (803.2 ± 24.3 vs 556.3 ± 26.7 perfusion unit), increased number of CD31-positive cells, and upregulated expression of HIF-1α in vascular endothelium on postoperative day 7, compared with ASC-EVs. CONCLUSIONS: Intradermal injecting HT-ASC-EVs improve the survival of random skin flap by promoting HIF-1α-mediated angiogenesis in rat model.