List of abbreviations

NG: Normal glucose

HG: High glucose

EpiSC: Epidermal stem cell

EpiSC-EXO: Epidermal stem cell derived exosome

Fb-EXO: Fibroblast derived exosome

ROS: Reactive oxygen species

HUVEC: Human umbilical vein endothelial cells

RT–qPCR: Real time quantitative PCR

MAPK: Mitogen-activated protein kinase

SYDE1: Synapse defective Rho GTPase homolog 1

RhoGAP: GTPase-activator protein for Rho-like GTPases

ERK: Extracellular regulated protein kinases

Abstract

     The dysfunction of endothelial cells caused by hyperglycaemia is observed as a decrease in neovascularization in diabetic wound healing. Studies have found that epidermal stem cells (EpiSCs) can promote the angiogenesis of full-thickness wounds. To further explain the therapeutic effect of EpiSCs, epidermal stem cell-derived exosomes (EpiSC-EXOs) are considered the main substance contributing to stem cell effectivity. In our study, EpiSCs and EpiSC-EXOs were supplied to the dorsal wounds of db/db mouse. Results showed that EpiSCs could colonize in the wound area and both EpiSCs and EpiSC-EXOs could accelerate diabetic wound healing by promoting angiogenesis. In vitro, persistent high glucose led to the malfunction and apoptosis of endothelial cells. The apoptosis induced by high glucose is due to excessive autophagy and was alleviated by EpiSCEXOs. RNA sequencing of EpiSC-EXOs showed miR200b-3p was enriched in EpiSC-EXOs and alleviated the apoptosis of endothelial cells. Synapse Defective Rho GTPase Homolog 1 (SYDE1) was identified the target of miR200b-3p and affected the phosphorylation of ERK to regulate intracellular autophagy and apoptosis. Furthermore, animal experiments validated the angiogenic effect of miR200b-3p. Collectively, our results verified the effect of EpiSC-EXOs on apoptosis caused by hyperglycaemia in endothelial cells through the miR200b- 3p/SYDE1/RAS/ERK/autophagy pathway, providing a theoretical basis for EpiSC in treating diabetic wounds.

Introduction

      Chronic wounds constitute a type of age-related disease, and one of its leading causes is diabetic mellitus (DM)(Sun et al., 2022). Currently, the standard treatments for diabetic wounds are debridement, negative pressure aspiration, and skin grafting(Dixon and Edmonds, 2021, Schaper et al., 2020). However, due to the characteristics of diabetic wounds, these treatments have achieved limited benefits(Okonkwo and DiPietro, 2017). As wound healing is a heavy burden for diabetic patients both physically and financially, how to rebalance microenvironments and provide better conditions for wound healing remain major challenges in clinical treatment(Sun et al., 2022).

      In general, the wound healing process can be divided into several stages: immediate haemostasis, acute inflammation, proliferation, and maturation(Patel et al., 2019). In normal wounds, injured tissue proliferates rapidly in granulation tissue, which characterized by intense angiogenesis(Veith et al., 2019). The most important feature of diabetic wounds is the decrease in the number of new blood vessels in the wound, resulting in insufficient blood supply to the wound, which in turn affects cell proliferation and tissue restructuring(Kaushik and Das, 2019). One of the purposes of clinical treatment is to provide a sufficient blood supply to the wound to promote the proliferation of wound cells and the survival of skin grafting(Chang and Nguyen, 2021, Huang X. et al., 2020). Therefore, how to further improve the number of blood vessels on the wound is important to the healing of diabetic wounds.

      Stem cell-based therapy is considered a promising approach for treating diabetic wounds because stem cells are pluripotent, self‐renew and have the ability to regulate their microenvironment(Zarei et al., 2018). Our previous clinical study found that epidermal basal cell suspensions, which contain epidermal stem cells (EpiSCs), can promote wound healing in patients with or without diabetes(Hu et al., 2017, Hu et al., 2015). Additional studies found that EpiSCs can proliferate in the wound area and accelerate wound healing (Huang S. et al., 2020, Wang P. et al., 2019). EpiSCs can promote angiogenesis and regulate inflammation in chronic wounds(Huang S. et al., 2020). However, how EpiSCs affect these biological processes and promote wound healing remains unknown.

      Exosomes, with a diameter of approximately 100 nm, are among the components secreted by almost every cell(Doyle and Wang, 2019). The uptake and release of exosomes is an important means to communicate information between cells over long distances(Kalluri and LeBleu, 2020). Studies have found that exosomes from different kinds of stem cells can regulate wound healing(Li et al., 2018, Lopes et al., 2018, Wang et al., 2022). The role of exosomes in the diagnosis and treatment of clinical diseases has been gradually revealed(Pegtel and Gould, 2019). It is believed that these extracellular vesicles, which are rich in proteins, lipids and nucleic acids, are the main contributors to stem cell efficacy.

      MicroRNAs (miRNAs) are one of the main substances that are abundant in exosomes(Lu and Rothenberg, 2018). MicroRNAs target the 3′ untranslated region (3`UTR) of mRNAs, forming a transient double-stranded miRNA duplex, and then the mature miRNA strand is incorporated into the RNA-induced silencing complex to mediate gene silencing and passenger strand degradation(Bushati and Cohen, 2007). A single miRNA can target hundreds of mRNAs and influence the expression of many genes(Mohr and Mott, 2015). Moreover, miRNAs delivered by exosomes function as posttranscriptional regulators by forming silencing complexes that further affect biological processes within cells(Zhang et al., 2015).

      Apoptosis and autophagy are important intracellular processes that maintain organism homeostasis and regulate survival(Maiuri et al., 2007). Autophagy involves the selective degradation of damaged cellular organelles and protein aggregates, while apoptosis involves the removal of damaged or aged cells(Doherty and Baehrecke, 2018, Thorburn, 2020). Apoptosis and autophagy are in a state of dynamic equilibrium within the cell(Kaminskyy and Zhivotovsky, 2014). Autophagy is induced when there is proper external stimulation, thereby coping with organelle damage, such as endoplasmic reticulum stress(Fernández et al., 2015). Excessive external stimulation such as a high-glucose environment can lead to excessive autophagy, which further leads to autophagy-induced apoptosis(Glick et al., 2010, Wang Y. et al., 2019a, Zhao et al., 2020). We found that excessive autophagy is the reason for endothelial cell apoptosis under high-glucose conditions. Therefore, the inhibition of excessive autophagy within cells under extreme conditions promotes cell survival and further benefits the healing process of diabetic wounds.

      In our study, we found that miR200b-3p delivered by epidermal stem cell-derived exosomes (EpiSC-EXOs) downregulated excessive autophagy-induced endothelial cell apoptosis under high glucose condition, which further explained the effectiveness of EpiSCs in diabetic wound healing.

Results

EpiSCs accelerate diabetic wound healing

      EpiSCs from newborn mouse skin were collected and observed under microscope. The EpiSCs obtained were morphologically uniform, elliptical in shape and had large nuclei (Fig. 1A). The EpiSCs were verified by immunofluorescence microscopy and flow cytometry, which showed high expression of the basement membrane-related marker ITGα6, EpiSC-related markers K15, Ki67 and low expression of the keratinocyte terminal differentiation marker K10 (Fig. 1B-C). The purity reached 96%.

      Next, EpiSCs were labelled with green fluorescence using lentivirus and were sprayed and subcutaneous injected to the dorsal wound model in db/db mouse (Fig. 1D). The wounded tissue was taken 7, 14 and 21 days after surgery for further experiment. We found that EpiSCs could survive in the wound and colonize surround capillary vessel on day 7 (Fig. 1E-F; Fig. S1A). The diabetic wound using EpiSCs healed completely within 3 weeks and had a thicker epidermal layer (Fig. 1G-K; Fig. S1B-C). These results suggested that EpiSCs could accelerate diabetic wound healing and the therapeutic effect was associated with angiogenesis.

Epidermal stem cell derived exosomes accelerate diabetic wound healing by promoting

      Considering that exosomes have been reported to be the main contributor to stem cell efficacy(Wang et al., 2022), EpiSC supernatant was collected and ultracentrifuged to harvest epidermal stem cell derived exosomes (EpiSC-EXO), which were verified by nanoparticle tracking analysis (NTA), scanning electron microscopy, and Western blotting. The extracted exosomes are about 117 nm in diameter and had a high expression of exosome-associated markers (Fig. 2A-D). The collected EpiSC-EXOs were sprayed to the dorsal wound model in db/db mouse, and fibroblast derived exosomes (Fb-EXO) are used as control. Results showed EpiSC-EXOs accelerated the diabetic wound healing rate (Fig.2E-F). The wounded tissue was taken 7 days after surgery and immunohistochemistry staining of microvasculature by CD34 showed that EpiSC-EXOs promotes angiogenesis in diabetic wound (Fig. 2G-H).

High glucose induces endothelial cell malfunction, and EpiSC-EXOs alleviate apoptosis in HUVECs

      To explore the abnormality in the wound healing process of diabetic wound, we used high-glucose medium culturing HUVECs to mimic diabetic conditions in vitro. The results showed that the cell viability and tube formation ability of HUVECs decreased under high-glucose conditions (Fig. 3AC). We then screened for differences in the transcription level between HUVECs under high-glucose (30 mmol/L, HG) and normal glucose (5 mmol/L, NG) conditions by transcriptome sequencing (Fig.3D-E). The differentially expressed genes were analysed for Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment, which showed that these genes were mainly enriched in apoptosis and oxidative phosphorylation (Fig. 3F). Furthermore, elevated ROS and apoptosis rate by high glucose condition was detected by DCFH-DA (Fig. 3G) and ANNEXIN-V/PI staining (Fig.3H-I).

      To explore the function of EpiSC-EXOs on the apoptosis rate of HUVECs under high glucose condition. EpiSC-EXOs were added to high glucose-treated HUVECs at a concentration of 50 µg/ml. We found that HUVECs could uptake EpiSC-EXOs (Fig. 3J, Fig. S1D) and EpiSC-EXOs significantly reduced apoptosis induced by high-glucose conditions in HUVECs (Fig. 3K-L). These results suggested that high glucose induces endothelial cell malfunction and EpiSC-EXOs could accelerate diabetic wound healing and promote angiogenesis by alleviating endothelial cell apoptosis under high glucose condition.

Excessive autophagy is the main cause of apoptosis

      To test whether lowering the ROS level is sufficient to suppress apoptosis of HUVECs in a highglucose environment, we treated HUVECs under high-glucose conditions with N-acetyl-L-cysteine (NAC), a reactive oxygen scavenger. Surprisingly, although NAC indeed decreased the ROS level in HUVECs under high-glucose conditions, it did not affect the apoptosis rate of HUVECs (Fig. 4A-C). Therefore, we assume that the upregulation of ROS may be an outcome of several intracellular processes that lead to apoptosis; simply eliminating ROS is not sufficient to inhibit apoptosis induced by high-glucose conditions.

      It is commonly accepted that the accumulation of ROS indicating a high level of intracellular stress. We also found the increased endoplasmic reticulum (ER) stress (Fig. S1D) and decreased intracellular mitochondrial membrane potentials (φM) (Fig.4D-E) in high glucose treated HUVECs, which indicate the upregulation of intracellular stress. To deal with intracellular stress, autophagy is often upregulated to improve cell survival, however, excessive autophagy can also lead to apoptosis. Increased LC3 expression was also observed in patients with diabetes (Fig. 4F-G). Furthermore, mRFP-eGFP-LC3 plasmid is used to show that autophagy is up regulated in HUVECs under highglucose conditions, and this increase in autophagosomes is not due to the blockade of autophagosome-lysosomal fusion (Fig. 4H-J). We inhibited autophagolysosomal formation using a late-stage autophagy inhibitor, chloroquine (CQ), and detected autophagy-related proteins via Western blotting (Fig. 4K-M). Our results demonstrate that the increase in autophagic flux induced by high glucose treatment is related to increased autophagic flux not to inhibition of autophagolysosomal formation. Given the relationship of autophagy and apoptosis as well as to further explore the main cause of apoptosis in HUVECs under high glucose concentration, we found that rapamycin, an autophagy agonist, significantly reduced the level of ROS but induced apoptosis of HUVECs under high-glucose conditions. In contrast, CQ significantly reduced the apoptosis rate (Fig. 4N-P, Fig. S1F-C). These results indicated that high glucose could induce cell stress levels such as ER stress, ROS production and mitochondria damage. Meanwhile, excessive autophagy in response to cell stress under high glucose is the main reason accounting for the apoptosis of HUVECs.

miR200b-3p, enriched in EpiSC-EXOs, suppresses apoptosis of HUVECs in a high-glucose environment

      To further study the mechanism contributing to the effectiveness of EpiSC-EXOs, we identified 44 upregulated and 32 downregulated miRNAs by comparing the sequencing results between fibroblast-derived exosomes (Fb-EXOs) and EpiSC-EXOs (Fig. 5A-C, Fig. S2A). Among the top 5 upregulated miRNAs, miR200b-3p dramatically decreased the apoptosis rate of HUVECs under high-glucose conditions (Fig. S2B-C). HUVECs stably upregulated or downregulated miR200b-3p cell line also showed the same results (Fig. 5D-E). Consistently, φM was restored, ROS production and ER stress related genes was decreased in HUVECs overexpressing miR200b-3p compared to miR200b-3p downregulation (Fig. 5F-H, Fig. S2D). Therefore, miR200b-3p is a key component in EpiSC-EXOs that inhibits apoptosis of HUVECs in high-glucose environments.

miR200b-3p regulates autophagy through the SYDE1/RAS/ERK pathway.

      To explore how miR200b-3p affects the apoptosis of HUVECs in a high-glucose environment, we constructed a stably upregulated and downregulated miR200b-3p HUVEC cell line (Fig. S2E). After culturing in high glucose for 48 hours, transcriptome sequencing was performed and the differently expressed genes were analysed for KEGG pathway enrichment. As The results revealed that the differentially expressed genes were mainly enriched in the MAPK pathway (Fig. 5I-J), which was consistent with the results of the KEGG enrichment analysis of miR200b-3p potential targets predicted from the online database (Fig. 5K). To identify the specific target genes of miR200b-3p, we selected the common differentially expressed genes among the pools of HG vs. NG, miR200b-3p up vs. miR200b-3p down and miR200b-3p predicted targets (Fig. 5L). 7 genes: JUN, CNKSR3, NYAP1, RUSC2, SYDE1, ERRFI1 and CITED2 were found. RT–qPCR was performed to validate the change in the expression of these 7 candidate genes and SYDE1 had the lowest expression in miR200b-3p-overexpressing cells (Fig. 5M).

      Synapse Defective Rho GTPase Homolog 1 (SYDE1) encodes a RhoGAP that negatively regulates phosphorylation of RAS family proteins such as RHOA(Lo et al., 2017). RAS is an upstream player of the MAPK pathway that regulates the phosphorylation of ERK, which could negatively regulate autophagy(Ba et al., 2019). In line with our previous sequencing results and hypothesis, SYDE1 was confirmed to be a target of miR200b-3p via a dual-luciferase reporter system (Fig. 5N, Fig. S2F). Western blotting of SYDE1, p-ERK, LC3 and P62 was performed to demonstrate the decreased autophagy level caused by miR200b-3p (Fig. 5O-P). Moreover, mRFPeGFP-LC3 plasmid is used to show that autophagy is down regulated by miR200b-3p in HUVECs under high-glucose conditions, and this decrease in autophagosomes does not affect autophagy flux (Fig. 6A-C). SYDE1 was also found upregulated in diabetic skin (Fig. 6D-E). To further confirm the regulatory role of miR200b-3p in the SYDE1/ERK pathway, we transiently transfected SYDE1 plasmids into miR200b-3p overexpressing cells and transiently transfected SYDE1 siRNA into miR200b-3p knockdown cells. We then measured the protein expression levels of P-RHOA, P62, P-ERK, LC3, and LAMP1 to demonstrate that miR200b-3p influences autophagy levels by affecting ERK phosphorylation through SYDE1 (Fig. 6F-I, Fig. S2G-H). In conclusion, miR200b-3p inhibits excessive autophagy of cells under high-glucose conditions through the SYDE1/RAS/ERK pathway, thereby reducing the apoptosis rate of HUVECs.

miR200b-3p promotes neovascularization and wound healing in diabetic mice

     To examine whether miR200b-3p has the same effects in vivo, we used a dorsal wound model of db/db mouse. 8mm circular wounds were created, and then miR200b-3p or NC was administered (Fig. 6J). Results showed miR200b-3p promoted diabetic wound healing rate compared to NC group (Fig. 6K). The wound tissue was taken 3, 7, 14 and 21 days after surgery for HE, IHC and IF analysis. The miR200b-3p group had a thicker granulation tissue on day 7 (Fig. 6L-M). The wound area sections on day 7 were stained with SYDE1 for IHC and CD31 for IF (Fig. 6N-Q). As shown in Fig. 6, miR200b-3p down regulated the SYDE1 level and promoted neovascularization. These results suggested that miR200b-3p could promote neovascularization and improve wound healing in db/db mouse.

Discussion

     Persistent hyperglycaemia is the most important feature of diabetes, which can lead to diabetic vasculopathy. The malfunction of vascular endothelial cells mainly manifested as arteriosclerosis occlusion and vascular lumen narrowing. Delayed cellular proliferation around the wound due to insufficient blood supply leads to delayed wound healing(Okonkwo and DiPietro, 2017). We previously conducted a clinical trial and found that suspensions of epidermal basal cells promoted the healing of acute and chronic wounds(Hu et al., 2015). Further animal experiments demonstrated that epidermal stem cells (EpiSCs) increased the neovascularization in rat wounds, accelerating the healing process(Huang S. et al., 2020). Therefore, exploring the therapeutic effects and mechanisms of ESCs on diabetic wounds may provide a potential new method for treating such wounds in clinical practice.

     At first, we speculated that EpiSCs could proliferate in the wound and differentiate into keratinocytes thus promoting rapid epithelialization of the wound. However, through tracking of EpiSCs, we found that they cannot proliferate fast enough to form epithelium in the wound bed. This phenomenon may be related to the insufficient number of EpiSCs applied to the wound bed. Meanwhile, combined with our observation of neovascularization effect of EpiSCs, we speculate that the promoting effect of EpiSCs on wound healing may be achieved through their paracrine function.

     In vitro, The deceased tube formation ability, increased apoptotic rate and ROS production suggest that the malfunction of endothelial cells under high-glucose environment contribute to delayed diabetic wound healing(Wang et al., 2020, Zhang et al., 2022). Consistent with our animal experiment, the apoptosis induced by high glucose is alleviated by EpiSC-EXOs, which provide evidence of the therapeutic effect of EpiSC-EXOs in vitro. First, we hypothesized that ROS were the main cause of the apoptosis of HUVECs, while the use of the ROS scavenger NAC had no effect on the apoptosis rate of HUVECs in a high-glucose environment. The accumulation of ROS is usually considered a detrimental factor for cell growth(Liu et al., 2021). Literatures also shown that ROS can induce aging of endothelial cells(Khemais-Benkhiat et al., 2020). We believe that this phenomenon may be due to insufficient treatment time in a high glucose environment, which results in the accumulation of ROS has not reach the duration time and amount to affect the biological behaviour of endothelial cells. Therefore, we believe that the main cause of apoptosis in vascular endothelial cells in this experiment should be other biological processes.

     It is widely accepted that a persistent high-glucose environment leads to metabolic stress, endoplasmic reticulum stress, protein unfolding reactions and mitochondrial damage(Fraga et al., 2023, Poznyak et al., 2020, Rizwan et al., 2020, Wang Y. et al., 2019b). All these abnormalities lead to the accumulation of ROS and erroneous metabolites as well as organelle damage, ultimately leading to a decrease in cell viability. These suggestions were further validated by the up regulation of ER stress related genes and decreased mitochondrial membrane potential in high glucose treated HUVECs, demonstrating that these biological processes may be factors leading to decreased viability. In response to various negative aspects of a high-glucose environment, autophagy is induced as an intracellular self-cleaning mechanism to remove impaired organelles. Given that autophagy and apoptosis are in a state of dynamic equilibrium, the upregulation of autophagy by appropriate external stimuli helps to remove impurities that accumulate and further promote cell survival, while excessive autophagy induced by excessive external stimuli leads to autophagy induced apoptosis(Thorburn, 2020). In our study, we found that high glucose condition led to excessive autophagy and the decreases of cell apoptosis by inhibition of autophagy suggesting that excessive autophagy is the main cause of apoptosis in a high-glucose environment.

     Constituting one of the most important components in exosomes, miRNAs function as posttranscriptional regulators to further modulate biological processes within cells. To further explore effective substances in exosomes derived from epidermal stem cells rather than other cell sources in the wound, fibroblasts were chosen as the control group since they are the most common cells in wounds. Our previous experiments confirmed that EpiSC-EXOs promote wound healing more significantly than those derived from fibroblasts(Wang et al., 2022).

      We sequenced differentially expressed miRNAs in EpiSC-EXOs and FB-EXOs and found that miR200b-3p had a significant effect on the apoptosis of HUVECs in a high-glucose environment. To investigate how miR200b-3p affects apoptosis, a sequencing comparison was used to identify target genes. The results revealed 7 genes as potential targets of miR200b-3p, among which SYDE1 was the most downregulated gene. Consistent with the literature and our previous speculation, SYDE1 functions as a negative regulator of RAS and further influences the MAPK pathway and autophagy, which explains the effect of miR200b-3p on the apoptosis of HUVECs in a high-glucose environment(Lo et al., 2017). All these results suggest that EpiSC-EXOs delivered miR200b-3p could alleviate high glucose induced excessive autophagy and reduce apoptosis by targeting SYDE1. Based on the regulatory mode of miRNA, the other 6 genes may also involve in the therapeutic effectiveness of miR200b-3p, particularly the transcription factor JUN. As a component of the AP- 1 complex, JUN participates in various cellular signalling and transcriptional regulation activities. Combined with our findings that miR200b-3p regulate the mitochondrial membrane potential in cells and ER stress, whether this phenomenon is regulated by JUN and further affects the biological behaviour of HUVECs needs further experimental investigation. We also found that CNKSR3, CITED2, ERRFI1 are closely related to diabetes and diabetes related complications(Chen et al., 2014, Dahlström and Sandholm, 2017, Jin et al., 2017, Kunkemoeller et al., 2021). Whether miR200b-3p has an effect on other biological processes through these genes to improve cell biological behaviour remains further study.

      Moreover, there are also deficiencies in our experimental design. The diabetic wound animal model we used is essentially a delayed diabetic wound healing model rather than a chronic wound, so there may be some differences in the mechanisms of wound formation or healing compared to chronic wounds. The healing of wounds involves a multitude of biological processes, and any individual process may only contribute partially to the overall promotion of wound healing. The accelerated wound healing by EpiSCs or Exosome might partly due to increased angiogenesis, however, the involvement of other cells such as macrophage cannot be ruled out. The therapeutic effects of stem cells are diverse, and exosomes are just one way in which stem cells exert their therapeutic effects. Similarly, the contents of exosomes are not limited to miRNA, and their therapeutic effects are not limited to a single type of miRNA. Further research is needed to explore whether these miRNAs or other type of cargos that exosomes carried have effect on cells in the wound healing process. For example, miR183-5p, one of the differentially expressed miRNAs we found through sequencing, also affects apoptosis of HUVECs under high glucose environment. P38, JNK, and ERK are key components of the MAPK pathway which are regulated by different kinases. We have observed that miR200b-3p specifically impacts the phosphorylation of ERK. Considering the functionality of SYDE1 protein and its relationship between RAS/MAPK signalling pathway, we propose that miR200b-3p affects the biological functions of vascular endothelial cells through the SYDE1/RAS/RAF/ERK pathway. The ERK pathway influences various cellular activities, but the specific mechanisms underlying its impact on cellular autophagy remain unclear. Although existing literature reports that phosphorylation of ERK significantly inhibits intracellular autophagy levels, it is still uncertain whether this phenomenon directly stems from the ERK pathway itself or indirectly through the induction of gene expression. Thus, further studies are needed to elucidate the precise mechanisms by which the ERK pathway influences the biological behaviour of autophagy.

      In summary, our study showed that Epidermal stem cells (EpiSCs) could promote angiogenesis and accelerate wound healing in diabetic mouse. High-glucose environment induced the up regulation of excessive autophagy, which is the main cause of apoptosis in HUVECs. miR200b-3p, whose abundance increased in EpiSC-EXOs, could reduce apoptosis in a high-glucose environment through the SYDE1/RAS/ERK/autophagy pathway. Our study provides a theoretical basis for EpiSC effectiveness and suggests the safety of EpiSCs in treating diabetic wounds. The mechanism of the effect of EpiSC-EXOs on wound healing warrants further study to understand the therapeutic effects of EpiSCs and expand their therapeutic scope for diseases.

Materials & Methods Cells and cell culture

      Animal ethics approval was obtained from the First Affiliated Hospital of Sun Yat-sen University of Animal Ethics Committee. Newborn mice were sacrificed by cervical dislocation. The skin was removed and placed in a 15-ml centrifuge tube that contained 1% phosphate-buffered saline (PBS; 10010023; Gibco) and placed on ice. The skin was collected and subcutaneous fascia was removed and then digested with dispease in a cell sorter (MYSEED, Guangzhou Myseed Medical Technonogy Co, Ltd) at 37°C for 30 min so that the epidermis could be scraped off using a sterile scalpel. The epidermis was then collected and digested in 0.25% trypsin-EDTA solution for 5 min. Next, the digested cell suspension was filtered in a 50-ml centrifuge tube using a 200-mesh filter and centrifuged at 1000 r/min for 10 min. The pellet was resuspended in keratinocyte serum-free medium (K-SFM; 17005042; Gibco). T25 culture flasks were pre-coated with 1 ml (0.5 mg/ml) of fibronectin (FN; ~5 µg/cm2 ; Shanghai Fibronectin Biotechnology, Shanghai, China) solution. The basal cell suspension was added to the pre-coated culture flask and incubated at 37°C for 20 min. The supernatant was aspirated and approximately 10% of the cells regarded as EpiSCs adhered to the wall. EpiSCs were then cultured with K-SFM media in a humidified incubator with 5% carbon dioxide added, and the medium was changed every 2 days.

      Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell (#8000, ScienCell). The cells were cultured in endothelial cell medium (ECM, #1001, ScienCell) in a humidified incubator with 5% carbon dioxide added, and the medium was changed every 2 days. For experimental treatment, HUVECs were subjected to normal glucose (NG, 5 mmol/L) or high glucose (HG, 30 mmol/L) for further experiments.

Fluorescence microscopy

      Cells or tissue samples were fixed with 4% paraformaldehyde and 0.5% Triton X-100， then incubated with 5% goat serum (3SL038; Solarbio). Next, the cells were incubated with ITGα6 (1:200; ab181551; Abcam), K15 (1:200; ab52816; Abcam), Ki67 (1:200; ab15580; Abcam), or CD31 (1:200; ab24590; Abcam) antibodies followed by Cy3-conjugated AffiniPure goat anti mouse/rabbit IgG (H+L) (SA00009-1/SA00009-2; Proteintech) or CoraLite 488-conjugated goat anti-mouse/rabbit IgG (H+L) (SA00013-1/SA00013-2; Proteintech). DAPI (C0065; Solarbio) was used for nuclear counterstaining.

Tube formation assays

      Matrigel Matrix (356234, Corning) was plated in 48-well culture plates and then incubated at 37°C for 20 min. HUVECs with the density of 5 × 105 /well treated with different glucose concentrations were cultured on Matrigel. After incubation at 37°C for 4 h, tube formation was assessed under a microscope and analyzed by measuring the tube branches.

Annexin-V/PI staining

     Cells pretreated with high glucose and other treatment components were digested and collected by centrifugation, followed by two rounds of cell pellet washing with PBS. After resuspension with binding buffer, the cells were stained with 10μl of Annexin V-fluorescein isothiocyanate (APC) and 5 μl of propidium iodide (PI) for 10 min in the dark (AP006-100; ESscience). The stained cells were subsequently analyzed by flow cytometry. At least 1 × 104 cells were evaluated using side and forward scattering to identify viable cell populations. Apoptotic cells were identified as AnnexinV+/PI+ or Annexin-V+/PI- group.

Cell viability assays

      HUVECs (4500/well) were subjected to high glucose concentration then incubated with CCK-8 (CK04; Dojindo). The absorbance at 450 nm (OD450) was measured.

RNA sequencing analysis

      Total RNA was extracted from exosomes and HUVRCs using an RNA purification kit (R4013-03, Magen) following the manufacturer’s instructions. The transcripts per million reads were used for the calculation of gene expression, and genes with |log2 (fold change) |≥ 1 and p value < 0.05 were considered statistically significant. Target Scan Human (https://www.targetscan.org/vert_72/) database were performed to screen for miRNA potential binding genes. Kyoto Encyclopaedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) analyses and Gene Ontology (GO; http://geneontology.org/) analyses were performed to elucidate the functions and enriched pathways of statistically significant genes.

Western blots

      50 µg of protein lysates was separated by electrophoresis using 4–12% polyacrylamide gel electrophoresis (PAGE) gels and transferred onto a PVDF membrane (IPVH00010; Millipore). Membranes were blocked with 5% nonfat dried milk and incubated with the following primary antibodies: SYDE1 (1:2000; NBP1-57607; Novus), P-RHOA (1:2000; 9968T; CST), ROHA (1:2000; 9968T; CST), P-ERK (1:2000; #4370; CST), P-P38 (1:2000; #8690; CST), LC3 (1:2000; PD014; MBL), P62(1:3000; ab109012; abcam), LAMP1(1:2000; 21997-1-AP, Proteintech ), and GAPDH (1:5000; 10494-1-AP; Proteintech). Afterward, the membranes were incubated with appropriate secondary IgG antibodies then detected by chemiluminescence, and then analyzed by image j.

Animal experiment

      For animal experiment, 10-week db/db mouse (45g weight at average, male) were anesthetized by inhaling isoflurane (INH). 8mm diameter, full-thickness wound was made on the dorsal skin of each mouse. The mouses were randomly divided into different groups. To explore the function of EpiSCs in diabetic wounds in vivo, 0.5ml of stably expressed green fluorescence EpiSCs (at a density of 2×105 /ml in PBS) were evenly sprayed (0.25ml) in the wound bed and subcutaneous injected (0.25ml) around the wound (0.5mm form the edge, 4 injection site) using a 2-ml syringe instantly after the skin was removed. 0.5ml of PBS was sprayed and subcutaneous injected as control. To explore the function of miR200b-3p in diabetic wounds in vivo, ago miR200b-3p (a chemical modified miRNA, Ribobio), or negative control (NC, Ribobio) were injected to the subcutaneous of the dorsal wound of each mouse at a dose of 60nmol/Kg. The wound healing time was recorded, and the residual wound area rate was calculated as [(day n area) / (day 0 area)] × 100% (n = 0, 3, 7, 10 or 14). Five mouses of each group were sacrificed on days 3, 7, 10 ,14 and 21, respectively, and the wound tissues were harvested and separated into two halves across the center: one half was processed for histological and immunohistochemistry analyses, and the other was rapidly frozen in liquid nitrogen for immunofluorescence.

Statistical analysis

      The results are expressed as the means ± SEMs, unless otherwise indicated. Differences between groups were evaluated using one-way ANOVA. All reported p values were two-sided. A p value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 20.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 8.0.

      See Supplementary Materials and Methods for detailed material and methods.

Data availability statement

      The sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra) under the study accession number PRJNA952792. The data used and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflict of Interest

      The authors declare that they have no conflicts of interest

Acknowledgments

      Corresponding authors include Wuguo Deng, State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China. E-mail: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。,">该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。, and Jiayuan Zhu, The First Affiliated Hospital of Sun Yat-sen University, Burn department, Guangzhou 510080, China. E-mail: 该Email地址已收到反垃圾邮件插件保护。要显示它您需要在浏览器中启用JavaScript。.

      Ethics approval and consent to participate：This study is approved by ICE for Clinical Research and Animal Trials for the First Affiliated Hospital of Sun Yat-sen University. Approval No: [2022]238

      Funding：This work was supported in part by the National Natural Science Foundation of China (81972623, 82172213, 82072180), and the Sun Yat-sen University Clinical Research 5010 Program

(2013001)

      The present work is the result of all joint efforts. We wish to express our sincere appreciation to all those who have offered the invaluable help during this study. The author gratefully acknowledges

the support of Shanhui Ge and Kaixin Yuan, who provide valuable assistance with the laboratory suggestion and accompany during this study.

Author Contributions

Validation: HX, ZW, HY; Writing: HX, HY, QT; Data suggestion: YD, MC, QT, BT, ZX, YR;

Methodology: HX, ZW, CC, DL, XC; Conceptualization&Project administration: ZH, WD, JZ.

References

1. Ba L, Gao J, Chen Y, Qi H, Dong C, Pan H, et al. Allicin attenuates pathological cardiac hypertrophy by inhibiting autophagy via activation of PI3K/Akt/mTOR and MAPK/ERK/mTOR signaling pathways. Phytomedicine : international journal of phytotherapy and phytopharmacology 2019;58:152765.

2. Bushati N, Cohen SM. microRNA functions. Annual review of cell and developmental biology 2007;23:175-205.

3. Chang M, Nguyen TT. Strategy for Treatment of Infected Diabetic Foot Ulcers. Accounts of chemical research 2021;54(5):1080-93.

4. Chen YC, Colvin ES, Griffin KE, Maier BF, Fueger PT. Mig6 haploinsufficiency protects mice against streptozotocin-induced diabetes. Diabetologia 2014;57(10):2066-75.

5. Dahlström E, Sandholm N. Progress in Defining the Genetic Basis of Diabetic Complications. Current diabetes reports 2017;17(9):80.

6. Dixon D, Edmonds M. Managing Diabetic Foot Ulcers: Pharmacotherapy for Wound Healing. Drugs 2021;81(1):29-56.

7. Doherty J, Baehrecke EH. Life, death and autophagy. Nature cell biology 2018;20(10):1110-7.

8. Doyle LM, Wang MZ. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells 2019;8(7).

9. Fernández A, Ordóñez R, Reiter RJ, González-Gallego J, Mauriz JL. Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis. Journal of pineal research 2015;59(3):292-307.

10. Fraga LN, Milenkovic D, Anacleto SL, Salemi M, Lajolo FM, Hassimotto NMA. Citrus flavanone metabolites significantly modulate global proteomic profile in pancreatic β-cells under high-glucose-induced metabolic stress. Biochimica et biophysica acta Proteins and proteomics 2023;1871(3):140898.

11. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. The Journal of pathology 2010;221(1):3-12.

12. Hu Z, Guo D, Liu P, Cao X, Li S, Zhu J, et al. Randomized clinical trial of autologous skin cell suspension for accelerating re-epithelialization of split-thickness donor sites. The British journal of surgery 2017;104(7):836-42.

13. Hu ZC, Chen D, Guo D, Liang YY, Zhang J, Zhu JY, et al. Randomized clinical trial of autologous skin cell suspension combined with skin grafting for chronic wounds. The British journal of surgery 2015;102(2):e117-23.

14. Huang S, Hu Z, Wang P, Zhang Y, Cao X, Dong Y, et al. Rat epidermal stem cells promote the angiogenesis of full-thickness wounds. Stem cell research & therapy 2020;11(1):344.

15. Huang X, Liang P, Jiang B, Zhang P, Yu W, Duan M, et al. Hyperbaric oxygen potentiates diabetic wound healing by promoting fibroblast cell proliferation and endothelial cell angiogenesis. Life sciences 2020;259:118246.

16. Jin L, Wang T, Jiang S, Chen M, Zhang R, Hu C, et al. The Association of a Genetic Variant in SCAF8- CNKSR3 with Diabetic Kidney Disease and Diabetic Retinopathy in a Chinese Population. Journal of diabetes research 2017;2017:6542689.

17. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science (New York, NY) 2020;367(6478).

18. Kaminskyy VO, Zhivotovsky B. Free radicals in cross talk between autophagy and apoptosis. Antioxidants & redox signaling 2014;21(1):86-102.

19. Kaushik K, Das A. Endothelial progenitor cell therapy for chronic wound tissue regeneration. Cytotherapy 2019;21(11):1137-50.

20. Khemais-Benkhiat S, Belcastro E, Idris-Khodja N, Park SH, Amoura L, Abbas M, et al. Angiotensin II-induced redox-sensitive SGLT1 and 2 expression promotes high glucose-induced endothelial cell senescence. Journal of cellular and molecular medicine 2020;24(3):2109-22.

21. Kunkemoeller B, Chen K, Lockhart SM, Wang X, Rask-Madsen C. The transcriptional coregulator CITED2 suppresses expression of IRS-2 and impairs insulin signaling in endothelial cells. American journal of physiology Endocrinology and metabolism 2021;321(2):E252-e9.

22. Li X, Xie X, Lian W, Shi R, Han S, Zhang H, et al. Exosomes from adipose-derived stem cells overexpressing Nrf2 accelerate cutaneous wound healing by promoting vascularization in a diabetic foot ulcer rat model. Experimental & molecular medicine 2018;50(4):1-14.

23. Liu X, Lu B, Fu J, Zhu X, Song E, Song Y. Amorphous silica nanoparticles induce inflammation via activation of NLRP3 inflammasome and HMGB1/TLR4/MYD88/NF-kb signaling pathway in HUVEC cells. Journal of hazardous materials 2021;404(Pt B):124050.

24. Lo HF, Tsai CY, Chen CP, Wang LJ, Lee YS, Chen CY, et al. Association of dysfunctional synapse defective 1 (SYDE1) with restricted fetal growth - SYDE1 regulates placental cell migration and invasion. The Journal of pathology 2017;241(3):324-36.

25. Lopes L, Setia O, Aurshina A, Liu S, Hu H, Isaji T, et al. Stem cell therapy for diabetic foot ulcers: a review of preclinical and clinical research. Stem cell research & therapy 2018;9(1):188.

26. Lu TX, Rothenberg ME. MicroRNA. The Journal of allergy and clinical immunology 2018;141(4):1202-7.

27. Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nature reviews Molecular cell biology 2007;8(9):741-52.

28. Mohr AM, Mott JL. Overview of microRNA biology. Seminars in liver disease 2015;35(1):3-11.

29. Okonkwo UA, DiPietro LA. Diabetes and Wound Angiogenesis. International journal of molecular sciences 2017;18(7).

30. Patel S, Srivastava S, Singh MR, Singh D. Mechanistic insight into diabetic wounds: Pathogenesis, molecular targets and treatment strategies to pace wound healing. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2019;112:108615.

31. Pegtel DM, Gould SJ. Exosomes. Annual review of biochemistry 2019;88:487-514.

32. Poznyak A, Grechko AV, Poggio P, Myasoedova VA, Alfieri V, Orekhov AN. The Diabetes MellitusAtherosclerosis Connection: The Role of Lipid and Glucose Metabolism and Chronic Inflammation. International journal of molecular sciences 2020;21(5).

33. Rizwan H, Pal S, Sabnam S, Pal A. High glucose augments ROS generation regulates mitochondrial dysfunction and apoptosis via stress signalling cascades in keratinocytes. Life sciences 2020;241:117148.

34. Schaper NC, van Netten JJ, Apelqvist J, Bus SA, Hinchliffe RJ, Lipsky BA. Practical Guidelines on the prevention and management of diabetic foot disease (IWGDF 2019 update). Diabetes/metabolism research and reviews 2020;36 Suppl 1:e3266.

35. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al. IDF Diabetes Atlas: Global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes research and clinical practice 2022;183:109119.

36. Thorburn A. Crosstalk between autophagy and apoptosis: Mechanisms and therapeutic implications. Progress in molecular biology and translational science 2020;172:55-65.

37. Veith AP, Henderson K, Spencer A, Sligar AD, Baker AB. Therapeutic strategies for enhancing angiogenesis in wound healing. Advanced drug delivery reviews 2019;146:97-125.

38. Wang J, Shen X, Liu J, Chen W, Wu F, Wu W, et al. High glucose mediates NLRP3 inflammasome activation via upregulation of ELF3 expression. Cell death & disease 2020;11(5):383.

39. Wang P, Hu Z, Cao X, Huang S, Dong Y, Cheng P, et al. Fibronectin precoating wound bed enhances the therapeutic effects of autologous epidermal basal cell suspension for fullthickness wounds by improving epidermal stem cells' utilization. Stem cell research & therapy 2019;10(1):154.

40. Wang P, Theocharidis G, Vlachos IS, Kounas K, Lobao A, Shu B, et al. Exosomes Derived from Epidermal Stem Cells Improve Diabetic Wound Healing. The Journal of investigative dermatology 2022.

41. Wang Y, Xiong H, Liu D, Hill C, Ertay A, Li J, et al. Autophagy inhibition specifically promotes epithelial-mesenchymal transition and invasion in RAS-mutated cancer cells. Autophagy 2019a;15(5):886-99.

42. Wang Y, Xue J, Li Y, Zhou X, Qiao S, Han D. Telmisartan protects against high glucose/high lipidinduced apoptosis and insulin secretion by reducing the oxidative and ER stress. Cell biochemistry and function 2019b;37(3):161-8.

43. Zarei F, Negahdari B, Eatemadi A. Diabetic ulcer regeneration: stem cells, biomaterials, growth factors. Artificial cells, nanomedicine, and biotechnology 2018;46(1):26-32.

44. Zhang J, Li S, Li L, Li M, Guo C, Yao J, et al. Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics, proteomics & bioinformatics 2015;13(1):17-24.

45. Zhang Y, Wang S, Chen X, Wang Z, Wang X, Zhou Q, et al. Liraglutide prevents high glucose induced HUVECs dysfunction via inhibition of PINK1/Parkin-dependent mitophagy. Molecular and cellular endocrinology 2022;545:111560.

46. Zhao X, Su L, He X, Zhao B, Miao J. Long noncoding RNA CA7-4 promotes autophagy and apoptosis via sponging MIR877-3P and MIR5680 in high glucose-induced vascular endothelial cells. Autophagy 2020;16(1):70-85.

Figure legends

Figure 1. Identification of EpiSCs and EpiSCs can proliferate and accelerate wound healing.

a) EpiSC morphology on the 3rd and 7th days (scale bar = 200μm). b) Immunofluorescence identification of third-generation EpiSC via Ki67, ITGα6, and K15 (scale bar = 25μm). c) Flow cytometry to detect the proportion of K10-negative and ITGα6-positive cells. d) Diagram of EpiSCs transfected with lentivirus to stably express green fluorescence and then supplied to diabetic wound (scale bar = 200μm). e) Representative area of wound tissue sections on postinjury day 7 showing EpiSCs (green) colonizing the peri-wound area and wound area in diabetic mouse wounds in the EpiSC group. White arrow indicates EpiSCs (scale bar = 50μm). f) Representative area of wound tissue sections of EpiSC group on postinjury day 7 showing EpiSCs (green) surrounding microvasculature stained with α-SMA (red). White arrow indicates EpiSCs (scale bar = 50μm). g) Images of wounds on the dorsal skin of mice in the EpiSC group and CTR group were taken on postinjury days 0, 3, 7, 10, and 14. h) Analysis of the percentage of residual wound area compared to original area (day 0) (n=3). i) Representative HE picture of wound tissue sections on postinjury day 7, 14 and 21. j) Analysis of epidermal thickness on postinjury day 14 (n=3) showed that EpiSC group had a thicker epithelial layer. k) Analysis of wound healing times (n=3) showed that EpiSC group healed faster than CTR group. CTR: PBS treated group; EpiSC: EpiSC treated group. DAPI: cell nucleus. *, P <0.05; **, P <0.01; ***, P <0.001.

Figure 2. EpiSC-EXOs accelerate wound healing by promoting angiogenesis.

a, b) Exosome size distribution examined through nanoparticle tracking analysis (NTA) showed that the exosomes are about 117 nm in diameter. c) Representative scanning electron microscopy image of EpiSC-EXOs showed that the exosomes are cup-shaped in morphology. d) Exosome-associated proteins (ALIX, TSG101, CD63) were validated with western blotting. Calnexin was used as cell lysis related protein control. e) Images of wounds on the dorsal skin of mice in the EpiSC-EXOs group and control group were taken on postinjury days 0, 3, 7, 10, and 14. f) Analysis of residual wound area compared to original area (day 0) (n=3) showed that EpiSC-EXOs accelerated the diabetic wound healing. g) Representative low-power field and high-power field picture of wound tissue sections stained with microvasculature by CD34 on postinjury day 7. Black arrows indicate the microvasculature. h) Analysis of microvasculature density (amount of microvasculature per high power field) (n=3) showed that EpiSC-EXOs promote angiogenesis in diabetic wound. EXO: exosome; CL: cell lysis; EpiSC-EXO: EpiSC-EXO group; CTR: FB-EXO group; *, P <0.05; **, P <0.01.

Figure 3. High glucose induces the dysfunction and apoptosis of HUVECs and EpiSC-EXOs alleviate the apoptosis of HUVECs under high glucose.

a) CCK-8 assays showed a decrease in the cell viability of HUVECs under high glucose concentrations and durations of glucose treatment (n=5). Control: normal glucose (5mmol/L). b) Representative images of the tube formation ability of HUVECs under high glucose for different treatment times (scale bar = 100μm). c) Analysis of branch points per high power field of tube formation assay (n=3) showed a decrease in tube formation ability of HUVECs under high glucose concentrations. d) Heat map of differential gene cluster analysis of HUVECs under different concentrations of glucose. e) Volcano plot of differentially expressed genes. f) Bubble map of the KEGG pathway enrichment analysis of differentially expressed genes. g) Flow cytometry analysis of intracellular ROS levels in HUVECs subjected to different concentrations of glucose. h) Flow cytometry analysis of the apoptosis rate of HUVECs under different glucose concentrations and durations. i) Analysis of apoptosis rate (n=3) showed an increase of apoptosis rate of HUVECs under high glucose concentration for 48 hours. j) Repetitive picture of the HUVECs (blue: nuclei) uptake PKH67 labelled EpiSC-EXOs (green) after 48 hours coculture. k) Flow cytometry analysis of the apoptosis rate of HUVECs treated with EpiSC-EXOs under high glucose condition. l) Analysis of apoptosis rate (n=3) showed EpiSC-EXOs significantly reduced HUVECs apoptosis under high glucose concentration after 48 hours coculture. NG: normal glucose (5mmol/L); HG: high glucose (30mmol/L); NS: none significant, *, P <0.05; **, P <0.01; ***, P <0.001.

Figure 4. Autophagy decreases ROS and promotes apoptosis under high-glucose conditions.

a) Flow cytometry analysis of the apoptosis rate of HUVECs treated with N-acetyl-L-cysteine (NAC). b) Analysis of apoptosis rate (n=3) showed NAC didn`t changed the apoptosis rate of HUVECs under high glucose concentration. c) Flow cytometry analysis of intracellular ROS levels in HUVECs treated with NAC. d) Representative image of mitochondrial membrane potential. JC- 1 stains mitochondria with a strong membrane potential (red) and mitochondria with a weak membrane potential (green) (scale bar = 200μm). e) Analysis of mitochondrial membrane potential (J-aggregate/J-monomer) (n=3) showed 48 hours high glucose treatment significantly reduced mitochondrial membrane potential of HUVECs. f) Representative picture of diabetic skin dermal sections and normal skin dermal sections stained with LC3 (scale bar = 200μm). g) analysis of the expression levels of LC3 in the dermis (n=3) showed that the expression level of LC3 is higher in diabetic skin, indicating a higher autophagy level. h) Representative mRFP-eGFP-LC3 fluorescence picture of HUVECs under different concentrations of glucose, autophagosome (green), autolysosome (red), (scale bar = 10μm). i) Analysis of autolysosome (red dot) (n=10) showed high glucose promotes intracellular autophagy. j) Analysis of autophagosome/autolysosome ratio (green dot/red dot) (n=10) showed high glucose didn`t affect autophagosome-lysosomal fusion. k) western blot of LC3, P62 and GAPDH expression in HUVECs treated with different glucose concentrations and CQ. l) Analysis of protein level (n=3) showed high glucose concentration promotes LC3-I and LC3-II expression and decrease P62 expression (control: GAPDH; *: compare to NG group; #: compare to NG+CQ group). m) Analysis of LC3-II/LC3-I ratio (n=3) showed high glucose concentration didn`t affect autophagy flux (*: compare to NG group; NS: none significant compare to NG group; #: compare to NG+CQ group). n) Flow cytometry analysis of intracellular ROS levels in HUVECs treated with rapamycin and CQ. o) Flow cytometry analysis of the apoptosis rate of HUVECs treated with rapamycin or CQ under high glucose condition. p) Analysis of apoptosis rate (n=3) showed rapamycin increases apoptosis and CQ decreases apoptosis of HUVECs under high glucose concentration. NG: normal glucose (5mmol/L); HG: high glucose (30mmol/L); NAC: Nacetyl-L-cysteine; CQ: Chloroquine. NS: none significant; *, P <0.05; **/##, P <0.01; ***/###, P <0.001.

Figure 5. EpiSC-EXOs alleviate the apoptosis of HUVECs in a high-glucose environment by miR200b-3p mediated downregulation of the SYDE1/MAPK/autophagy pathway.

a) Differential miRNA cluster analysis of EpiSC-EXOs and Fb-EXOs. b) Differential miRNAs in EpiSC-EXOs and Fb-EXOs. c) Volcano plot of differentially expressed miRNAs in EpiSC-EXOs and Fb-EXOs. d) Flow cytometry of the apoptosis of HUVECs overexpressing miR200b-3p (miR200b-3p up) and knocking down miR200b-3p (miR200b-3p down) and the negative control (NC). e) Analysis of the apoptosis rate (n=3) showed that miR200b-3p overexpression significantly decreased apoptosis rate while miR200b-3p knockdown significantly increased apoptosis rate of HUVECs under high glucose condition. f) Flow cytometry analysis of the intracellular ROS levels of HUVECs differentially expressing miR200b-3p under high glucose condition. g) Representative image and analysis of the mitochondrial membrane potential of HUVECs differentially expressing miR200b-3p under high glucose condition (scale bar = 200μm). h) Analysis of mitochondrial membrane potential (n=3) showed miR200b-3p restored φM of HUVECs under high glucose condition. i) Differential gene cluster analysis of HUVECs differently expressing miR200b-3p (miR200b-3p up, miR200b-3p down) under high glucose condition and HUVECs treated with different concentrations of glucose (HG, NG). j) Bubble map of the pathway enrichment analysis of differential genes in HUVECs differentially expressing miR200b-3p. k) Bubble map of the pathway enrichment analysis of miR200b-3p predicted target. l) Intersection of the differentially expressed genes of HUVECs differentially expressing miR200b-3p and HUVECs treated with different concentrations of glucose and miR200b-3p predicted targets. m) qPCR (n=3) of 7 miR200b-3p potential target genes in HUVECs differentially expressed miR200b-3p and negative control showed SYDE1 had the lowest expression in miR200b-3p-overexpressing cells. n) Dual-luciferase reporter gene assay (n=3) showing that miR200b-3p bind with 3`UTR area of SYDE1. o) Western blot of SYDE1, P-RHOA, RHOA, P-ERK, ERK, P-P38, LC3, P62 and GAPDH expression in HUVECs differentially expressing miR200b-3p and the negative control under high glucose condition. p) Analysis of protein level (n=3) showed the decreased protein level of SYDE1, LC3 and increased protein level of P-RHOA, P-ERK and P62 in miR200b-3p overexpressing HUVECs under high glucose condition (*: compare to NC group; #: compare to miR200b-3p up group). WT: wild type; UTR: untranslated regions; NC: negative control. *, P <0.05; **/##, P <0.01; ***/###, P <0.001.

Figure 6. miR200b-3p-mediated upregulation of the MAPK pathway decreases autophagy through downregulation of SYDE1 and promotes angiogenesis in diabetic wound.

a) Representative mRFP-eGFP-LC3 fluorescence picture of HUVECs differentially expressing miR200b-3p treated with high glucose concentrations (scale bar = 10μm). b) Analysis of autolysosome (red dot) (n=10) showed that miR200b-3p decreased high glucose induced excessive autophagy. c). Analysis of autophagy flux (green dot/red dot) (n=10) showed that miR200b-3p didn`t affect autophagosome-lysosomal fusion. d) Representative area of diabetic wound tissue and normal skin sections stained with SYDE1 (scale bar = 100μm). e) Analysis of the expression levels of SYDE1 in the dermis (n=3) showed that the expression level of SYDE1 is higher in diabetic skin. f) Western blot of SYDE1, P-RHOA, P62, P-ERK, ERK, LC3-I, LC3-II, LAMP1 and GAPDH expression in overexpressing miR200b-3p HUVECs transiently transfected SYDE1 plasmids under high glucose condition. g) Western blot of SYDE1, P-RHOA, P62, P-ERK, ERK, LC3-I, LC3-II, LAMP1 and GAPDH expression in knockdown miR200b-3p HUVECs transiently transfected SYDE1 siRNA under high glucose condition. h) Analysis of protein level (n=3) showed the decreased protein level of P-RHOA, P62, P-ERK and increased protein level of SYDE1, LC3-I, LC3-II, LAMP1 in miR200b-3p overexpressing HUVECs transiently transfected SYDE1 plasmids under high glucose condition (control: VEC). i) Analysis of protein level (n=3) showed the decreased protein level of SYDE1, LC3-I, LC3-II, LAMP1 and increased protein level of P-RHOA, P62, P-ERK in miR200b-3p knockdown HUVECs transiently transfected SYDE1 siRNA under high glucose condition (control: NC). j) Images of wounds on the dorsal skin of mice in the miR200b-3p group and NC group taken on postinjury days 0, 3, 7, 10, and 14. k) Analysis of the percentage of residual wound area compared to original area (day 0) (n=3) showed miR200b-3p promoted diabetic wound healing. l) Representative HE image of wound tissue sections in the miR200b-3p group and NC group on day 3, 7 (scale bar = 800μm). m) Analysis of granulation tissue thickness on day 7 (n=3) showed miR200b-3p group had a thicker granulation tissue indicating a better healing quality. n) Representative picture of peri-wound dermis area stained with SYDE1 (scale bar = 100μm). o) Representative area of wound tissue sections stained with CD31 on postinjury days 7 showing microvascular regeneration in mouse wounds in the miR200b-3p group and NC group (scale bar = 100μm). p) Analysis of microvessel density (n=3) showed miR200b-3p promoted neovascularization in diabetic wound. q) Analysis of SYDE1 positive cells (n=3) showed miR200b-3p down regulated SYDE1 expression in diabetic wound. 200b-3p UP: miR200b-3p upregulated group; 200b-3p DOWN: miR200b-3p downregulated group; NC: negative control. *, P<0.05; **, P <0.01; *** P <0.001. #, P <0.05; ##, P <0.01; ###, P <0.001.

Supplement figure legend:

Figure S1. EpiSCs delivered EpiSC-EXOs promoted diabetic wound healing through inhibition of excessive autophagy in HUVECs.

a) Representative picture of wound area and peri-wound area tissue sections of control group on postinjury day 7. b) Representative Masson staining image of wound tissue sections in the EpiSC group and control group on day 7, 14 and 21 (scale bar = 200μm). c) Analysis of collagen volume fraction of the EpiSC group and control group on day 21 showed that there is no difference in collagen deposition between the two group. d) Representative picture of the HUVECs (blue: nuclei) uptake non labelled EpiSC-EXOs. e) analysis of qPCR result of ER stress related genes in HUVECs treated with different concentrations of glucose. f) Flow cytometry analysis of the apoptosis rate of HUVECs treated with rapamycin and CQ under normal glucose condition. g) Analysis of apoptosis rate (n=3) showed rapamycin and CQ didn`t affect apoptosis of HUVECs under normal glucose concentration. CTR: PBS group. NS: none significant; *, P <0.05; **, P <0.01; *** P <0.001.

Figure S2. EpiSC-EXO delivered miR200b-3p target SYDE1/ERK/autophagy pathway inhibiting excessive autophagy induced apoptosis in HUVECs

a) GO analysis of predicted target genes of all the upregulated miRNA in EpiSC-EXO. b) Flow cytometry analysis of the apoptosis rate of HUVECs transiently transfected with different miRNA mimic/inhibitor under high glucose concentration. c) Analysis of apoptosis rate (n=3) showed miR200b-3p significantly decreased the apoptosis rate of HUVECs under high-glucose conditions. d) qPCR of ER stress related genes in HUVECs differentially expressed miR200b-3p. e) qPCR of miR200b-3p in HUVECs differentially expressed miR200b-3p. f) Predicted SYDE1 3' UTR binding position of miR200b-3p. g) Analysis of LC3-I/LC3-II protein expression ratio (n=3) in miR200b- 3p overexpressing cells transiently transfected SYDE1 plasmids showed SYDE1 didn`t affect autophagy flux. h) Analysis of LC3-I/LC3-II protein expression ratio (n=3) in miR200b-3p knockdown cells transiently transfected SYDE1 siRNA showed SYDE1 didn`t affect autophagy flux. VEC: vector; NC: negative control; NS: none significant; *, P <0.05; **, P <0.01; *** P <0.001.

Methods

Exosomes identification

      The supernatant of EpiSCs at p3 were ultra centrifugated (100000g, 2 hour) and resuspended in DPBS to collect Epidermal stem cell-derived exosomes (EpiSC-EXOs). The EpiSC-EXOs is further identified using nanoparticle tracking analysis (NTA), scanning electron microscope and western blot. For EpiSC-EXO uptake assay, EpiSC-EXO were collected and stained with PKH67 (HR8659; Biorab) following the manufacturer’s instructions and applied to HUVECs for 48 hours and then observed under a confocal laser scanning microscope.

Cell transfection for functional assays

     For animal experiment, EpiSCs at p3 were labeled with green fluorescence using lentivirus (MOI:20, Genechem) and screened using puromycin to stably express green fluorescence following the manufacturer’s instructions.

     For autophagy flux detection, HUVECs were treated with high glucose and rapamycin (final concentration: 10 nM) or chloroquine (final concentration: 10 nM). For functional assay, HUVECs were transiently transfected with miR200b-3p mimic/miR200b-3p inhibitor/negative control (NC) sequence (final concentration: 20nM, Ribobio) or SYDE1 siRNA (final concentration: 50nM, SR313834, SR419690, Origene) or mRFP-eGFP-LC3 plasmid (final concentration: 0.75ug/mL) using Lipofectamine® 3000 (L3000015; Thermo Fisher) according to the manufacturer’s instructions. Two days after transfection, the cells were treated with different glucose concentrations or other treatments for further experiments. For RNA-sequencing, autophagy flux detection and validation of miR200b-3p potential target SYDE1, HUVECs were stably expressed with miR200b-3p using lentivirus (MOI:5, Genechem) and screened by puromycin following the manufacturer’s instructions. The stably expressed cells were treated with different glucose concentrations or other treatments for further experiments.

Cell identification

      EpiSCs were digested and collected by centrifugation followed by two rounds of cell pellet washing with PBS. The cells were then resuspended in 0.1 ml of PBS with K10 (1:50; NBP2-61736, Novus) or ITGα6 (1:50; ab181551; Abcam) for 30 min on ice. Then, the cells were washed and stained with CoraLite594-conjugated goat anti-mouse IgG (H+L) (SA00013-3; Proteintech) on ice and protected from light for 30 min. Afterward, the cells were washed and resuspend in 0.5 ml of PBS and subjected to flow cytometry analysis.

Detection of ROS

      Cells pretreated with high glucose and other treatment components were rinsed and then washed with cold PBS, after which they were incubated with 1 mM 2′,7′-Dichlorofluorescin diacetate (DCFH-DA; D6470; Solarbio) in the dark at 37°C for 20 min. The cells were then digested, washed and resuspended in PBS. The stained cells were analyzed by flow cytometry. At least 1 × 104 cells were evaluated using side and forward scattering to identify viable cell populations. The change of ROS level was identified as the change in the position of the peak of FITC fluorescence.

Mitochondrial membrane potential (φM)

      JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) (HY-15534; MCE), a lipophilic cationic dye that can selectively enter mitochondria, was used for visualization. HUVECs were seeded into 6-well plates and subjected to high glucose concentration and other treatment components. JC-1 (final concentration: 2μM) was added to each well for 15min of incubation and assessed via fluorescence microscopy (the monomeric form: λEm = 527 nm; Jaggregates: λEm = 590 nm). The mitochondrial membrane potential was analyzed by J-aggregate fluorescence density/J-monomer fluorescence density.

RNA extraction and real‑time PCR analysis

     Total RNA was extracted from HUVECs using an RNA purification kit (R4013-03, Magen) following the manufacturer’s instructions. Total RNA was reverse transcribed using a HiScript-TS 5/3’ RACE Kit (RA101-01; Vazyme). Real-time qPCR was performed using ChamQ SYBR Color qPCR Master Mix (Q421-02; Vazyme). The data were obtained as cycle threshold (Ct) values, and the 2−ΔCt method was used for the analysis.

Luciferase activity assays

      We constructed wild-type and mutated luciferase reporter plasmids of SYDE1 3′-UTR binding sites with miR200b-3p and transfected them into HUVECs that stably express miR200b-3p or NC (final concentration: 0.25ug/mL). The firefly luciferase activities were measured by a DualLuciferase Reporter Assay System (E1910; Promega) following the manufacturer’s instructions. Rinella luciferase was used as an internal control. The luciferase activities were calculated by firefly luciferase density/Rinella luciferase density.

IHC, HE, MASSON staining.

      Immunohistochemical staining (IHC), hematoxylin-eosin staining (HE) and MASSON staining are performed according to standard protocols. CD34 (1:100; ab81289; Abcam) and SYDE1 (1:100; PA5-53039; Thermo Fisher) were used as primary antibodies. For IHC analysis, pictures were analyzed using Image Pro Plus software for relative expression density. For MASSON analysis, pictures were analyzed using Image J software for Collagen volume fraction.

Statistical analysis

      The results are expressed as the means ± SEMs, unless otherwise indicated. Differences between groups were evaluated using one-way ANOVA. All reported p values were two-sided. A p value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 20.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 8.0.

Supplementary Figures

Figure S1. EpiSCs delivered EpiSC-EXOs promoted diabetic wound healing through inhibition of excessive autophagy in HUVECs.

a) Representative picture of wound area and peri-wound area tissue sections of control group on postinjury day 7. b) Representative Masson staining image of wound tissue sections in the EpiSC group and control group on day 7, 14 and 21 (scale bar: 200μm). c) Analysis of collagen volume fraction of the EpiSC group and control group on day 21 showed that there is no difference in collagen deposition between the two group. d) Representative picture of the HUVECs (blue: nuclei) uptake non labelled EpiSC-EXOs. e) analysis of qPCR result of ER stress related genes in HUVECs treated with different concentrations of glucose. f) Flow cytometry analysis of the apoptosis rate of HUVECs treated with rapamycin and CQ under normal glucose condition. g) Analysis of apoptosis rate (n=3) showed rapamycin and CQ didn`t affect apoptosis of HUVECs under normal glucose concentration. CTR: PBS group. NS: none significant; *, P <0.05; **, P <0.01; *** P <0.001.

Figure S2. EpiSC-EXO delivered miR200b-3p target SYDE1/ERK/autophagy pathway inhibiting excessive autophagy induced apoptosis in HUVECs

a) GO analysis of predicted target genes of all the upregulated miRNA in EpiSC-EXO. b) Flow cytometry analysis of the apoptosis rate of HUVECs transiently transfected with different miRNA mimic/inhibitor under high glucose concentration. c) Analysis of apoptosis rate (n=3) showed miR200b-3p significantly decreased the apoptosis rate of HUVECs under high-glucose conditions. d) qPCR of ER stress related genes in HUVECs differentially expressed miR200b-3p. e) qPCR of miR200b-3p in HUVECs differentially expressed miR200b-3p. f) Predicted SYDE1 3' UTR binding position of miR200b-3p. g) Analysis of LC3-I/LC3-II protein expression ratio (n=3) in miR200b- 3p overexpressing cells transiently transfected SYDE1 plasmids showed SYDE1 didn`t affect autophagy flux. h) Analysis of LC3-I/LC3-II protein expression ratio (n=3) in miR200b-3p knockdown cells transiently transfected SYDE1 siRNA showed SYDE1 didn`t affect autophagy flux. VEC: vector; NC: negative control; NS: none significant; *, P <0.05; **, P <0.01; *** P <0.001.

This article is excerpted from the 《The Journal of Investigative Dermatology (2023)》by Wound  World.