Mesenchymal stem cell-derived extracellular vesicles accelerate diabetic wound healing by inhibiting NET-induced ferroptosis of endothelial cells

Impaired angiogenesis is a major factor contributing to delayed wound healing in diabetes. Dysfunctional mitochondria promote the formation of neutrophil extracellular traps (NETs), obstructing angiogenesis during wound healing. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown promise in promoting tissue repair and regeneration in diabetes; however, the precise pathways involved in this process remain unclear. In this study, NET-induced ferroptosis of endothelial cells (ECs) and angiogenesis were assessed in diabetic wound samples from both patients and animal models. In vitro and in vivo experiments were performed to examine the regulatory mechanisms of NETs in ECs using specific inhibitors and gene-knockout mice. MSC-EVs encapsulating dysfunctional mitochondria were used to trigger mitochondrial fusion and restore mitochondrial function in neutrophils to suppress NET formation. Angiogenesis in wound tissue was evaluated using color laser Doppler imaging and vascular density analysis. Wound healing was evaluated via macroscopic analysis and histological evaluation of the epithelial gap. NET-induced ferroptosis of ECs was validated as a crucial factor contributing to the impairment of angiogenesis in diabetic wounds. Mechanistically, NETs regulated ferroptosis by suppressing the PI3K/AKT pathway. Furthermore, MSC-EVs transferred functional mitochondria to neutrophils in wound tissue, triggered mitochondrial fusion, and restored mitochondrial function, thereby reducing NET formation. These results suggest that inhibiting NET formation and EC ferroptosis or activating the PI3K/AKT pathway can remarkably improve wound healing. In conclusion, this study reveals a novel NET-mediated pathway involved in wound healing in diabetes and suggests an effective therapeutic strategy for accelerating wound healing.

FDR−adjusted p−value < 0.05.Spearman's rank correlation analysis was executed to investigate the correlation among target genes, considering a P−value < 0.05 as statistically significant.

Flow cytometry
Sytox Green, recognized for its high-affinity nucleic acid staining capability, easily permeates cells with compromised plasma membranes while maintaining an inability to penetrate live cell membranes.This characteristic renders it an outstanding DNA counterstain for fixed cells.In a concise procedure, cells were incubated with Sytox, CD73, CD90, CD105, CD34, CD45, and CD44 in the dark for 30 minutes, followed by two washes and subsequent analysis via flow cytometry (Accuri C6, BD Biosciences).For the identification of mitochondrial transfer from hUC-MSC-EVs to neutrophils, MitoTracker Deep Green (Thermo Fisher Scientific, MA, US) was employed to prestain hUC-MSCs.MitoTracker Deep Green-labeled hUC-MSC-EVs were then isolated through two ultracentrifugation steps and administered to the mouse model.Intrahepatic mononuclear cells were collected at 6 hours post-transfusion, stained with APC-Cy5.5-Ly6G for 30 minutes at 4 °C in the dark, and the mean fluorescence intensity (MFI) of MitoTracker Deep Green in neutrophils was analyzed using flow cytometry.Data analysis was conducted using FlowJo (version 10) software, and the experimental process was replicated in triplicate.
Statistical analyses were carried out using GraphPad Prism software.

NET preparation
Venipuncture was employed to collect 5 mL of blood samples from each participant, with EDTA serving as the preservative for subsequent cell isolation.Initial separation of cells utilized Polymorphprep™ (Axis-Shield PoC, Scotland), resulting in two distinct leukocyte fractions: polynuclear cells and monocytes.For enhanced cell purity, the polynuclear fraction was sorted based on positive polymorphonuclear cells and suspended in Roswell Park Memorial Institute Medium 1640 supplemented with 5% fetal bovine serum (FBS) and 1% penicillinstreptomycin.
For NET formation, the cells were seeded at a density of 10 6 /well and stimulated with 100-nM phorbol 12-myristate 13-acetate (PMA, Beyotime, China) for 4 h at 37°C.Confirmation of NET formation was achieved through the visualization of extracellular DNA stained with SYTOX dye.Subsequently, the medium was meticulously removed, and the cell layer was gently washed with 3 mL of PBS without Ca2+ and Mg2+ ions.The PBS solution, collected after vigorous agitation, underwent centrifugation for 10 minutes at 500 g and 4°C to eliminate residual cells and debris.NET concentration was quantified using the Quant-iT PicoGreen dsDNA assay kit (P11496, Thermo Fisher Scientific, Waltham, MA, USA), and NETs were promptly utilized or stored at -80°C.

Cell proliferation assays
During the 5-ethynyl-2′-deoxyuridine (EdU) assay, cells in logarithmic growth phase were seeded into 24-well plates, and the suitable concentration of EDU reagent was introduced for a 4-hour incubation period.To assess cell proliferation, the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, China) was employed, following the manufacturer's protocol.The cells exhibiting proliferation, as indicated by EdU incorporation, displayed a vivid and consistent red fluorescence pattern.Cell nuclei were stained with Hoechst 33258 dye, which emits bright blue fluorescence.

Animal experiments
This study adhered to the ethical guidelines outlined in the Guide for the Care and Use of Laboratory Animals, as published by the National Institutes of Health.
Mice, procured from the Shanghai Laboratory Animal Center, were housed at the Animal Science Center at Shanghai Jiao Tong University School of Medicine.The animal facility maintained a 12-hour light/dark cycle at 22°C, with two mice per cage.
Experimental procedures involving mice were conducted with prior approval from the Ethical Review Board.
Two full-thickness excisional wounds were created in the shaved dorsal skin using a sterile, disposable 5-mm biopsy punch (Kai Industries, Tokyo, Japan), generating one wound on each side of the midline within a midline skin fold.
Photographic documentation of the wounds occurred on days 0, 3, 7, and 14 post-wounding using an Olympus camera.Wound areas were calculated utilizing Vernier calipers to measure the wound diameter and applying the standard formula for the area of an ellipse (semi-major diameter × semi-minor diameter × Pi).Superficial blood flow in the wounds was sequentially assessed by color laser Doppler (Moor, UK), and the ratio of blood flow in the wounds was computed.The analyses of wound size and Doppler assessments were performed in a blinded fashion.Control oligonucleotides (scramble sequence) and SMAD2 siRNA (1 mg/wound) were topically administered into the wound cavity (10 mL in a vehicle of 30% pluronic F-127 gel [liquifies at 4°C but solidifies at body temperature], Sigma-Aldrich) immediately after wounding.

Histology and image analysis
The wounds were harvested, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin.Tissue sections (5 μm) were stained with H&E stain using a commercial kit (DAKO, Denmark) according to the manufacturer's instructions.At day 6 post-wounding, the wounds were harvested and fixed in 10% buffered formalin (16 h at 4℃, Sigma) for embedding in paraffin.Sections were deparaffinized, rehydrated, and stained with anti-CD31 (ab28364, 1:50, Abcam) and α-SMA (ab7817, 1:100, Abcam).Antibodies were used overnight at 4℃. Stained slides were photographed and analyzed in a blinded fashion using a Zeiss LSM 780 confocal microscope (Zeiss, Carl Zeiss, Germany).Vascular density in the wounds was counted after immunostaining for CD31 and α-SMA.Ten fields per section/animal (n = 6; 400 × magnification) were randomly examined and averaged to analyze the number of CD31-positive blood vessels in the wound edges.The images were analyzed using ImageJ software.Vascular density is expressed per square millimeter.
Saturating the samples with 1:1 and 1:2 solutions of acetone and embedding medium followed.Incubation at 37 °C for 12 hours and then at 60 °C for 48 hours ensured complete polymerization.Ultimately, 60-nm ultrathin sections underwent staining with 1% uranyl acetate and were captured using a JEM-1200EX transmission electron microscope (JEOL, Japan).