P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/ CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts

Abstract Background Persistent hyperglycaemia in diabetes causes functional abnormalities of human dermal fibroblasts (HDFs), partially leading to delayed skin wound healing. Extracellular vesicles (EVs) containing multiple pro-healing microRNAs (miRNAs) have been shown to exert therapeutic effects on diabetic wound healing. The present study aimed to observe the effects of EVs derived from placental mesenchymal stem cells (P-MSC-EVs) on diabetic wound healing and high glucose (HG)-induced senescent fibroblasts and to explore the underlying mechanisms. Methods P-MSC-EVs were isolated by differential ultracentrifugation and locally injected into the full-thickness skin wounds of diabetic mice, to observe the beneficial effects on wound healing in vivo by measuring wound closure rates and histological analysis. Next, a series of assays were conducted to evaluate the effects of low (2.28 x 1010 particles/ml) and high (4.56 x 1010 particles/ml) concentrations of P-MSC-EVs on the senescence, proliferation, migration, and apoptosis of HG-induced senescent HDFs in vitro. Then, miRNA microarrays and real-time quantitative PCR (RT–qPCR) were carried out to detect the differentially expressed miRNAs in HDFs after EVs treatment. Specific RNA inhibitors, miRNA mimics, and small interfering RNA (siRNA) were used to evaluate the role of a candidate miRNA and its target genes in P-MSC-EV-induced improvements in the function of HG-induced senescent HDFs. Results Local injection of P-MSC-EVs into diabetic wounds accelerated wound closure and reduced scar widths, with better-organized collagen deposition and decreased p16INK4a expression. In vitro, P-MSC-EVs enhanced the antisenescence, proliferation, migration, and antiapoptotic abilities of HG-induced senescent fibroblasts in a dose-dependent manner. MiR-145-5p was found to be highly enriched in P-MSC-EVs. MiR-145-5p inhibitors effectively attenuated the P-MSC-EV-induced functional improvements of senescent fibroblasts. MiR-145-5p mimics simulated the effects of P-MSC-EVs on functional improvements of fibroblasts by suppressing the expression of cyclin-dependent kinase inhibitor 1A and activating the extracellular signal regulated kinase (Erk)/protein kinase B (Akt) signaling pathway. Furthermore, local application of miR-145-5p agomir mimicked the effects of P-MSC-EVs on wound healing. Conclusions These results suggest that P-MSC-EVs accelerate diabetic wound healing by improving the function of senescent fibroblasts through the transfer of miR-145-5p, which targets cyclin-dependent kinase inhibitor 1A to activate the Erk/Akt signaling pathway. P-MSC-EVs are promising therapeutic candidates for diabetic wound treatment.


Background
Diabetes mellitus is a chronic and multifaceted metabolic disease characterized by continuously and rapidly increasing morbidity, especially in the elderly population.Nearly 20% of diabetic patients worldwide develop delayed or nonhealing chronic wounds.In China, diabetes has also become the major cause of chronic wounds in hospitalized patients [1][2][3][4].Leg and foot ulcers are the most common chronic wounds in diabetic patients.If not properly diagnosed and treated, diabetic foot ulceration results in amputation in 15-27% of patients [5].Conventional treatments [6], including skin substitutes, debridement and pressure offloading, are sometimes ineffective and fail to provide optimum clinical outcomes for diabetic foot ulcers, which increases the risk of limb amputation.
Human dermal fibroblasts (HDFs) are involved in and responsible for normal wound healing.They can migrate to the wound bed to proliferate and differentiate into myofibroblasts, providing a scaffold for repopulating cells through synthesizing and secreting extracellular matrix, as well as expressing cytokines and growth factors, thereby promoting wound closure.However, substantial evidence suggests that a high-glucose (HG) microenvironment impairs the proliferation, migration, and antiapoptotic abilities of fibroblasts and inhibits fibroblast differentiation into myofibroblasts, as well as extracellular matrix production, thus blocking wound repair [7].Therefore, improving the functional state of fibroblasts in a HG environment is crucial to promoting diabetic wound healing.
Increasing lines of evidence indicate that mesenchymal stem cells (MSCs) are beneficial for regenerative medicine due to their self-renewal and multilineage differentiation abilities.However, many studies have proven that the benefits of MSCbased cell transplantation are mainly attributed to paracrine products rather than direct differentiation.Extracellular vesicles (EVs), which are paracrine factors released from MSCs, have been demonstrated to possess almost equivalent biological effects to their parent cells [8], representing a prospective stem cell-free therapeutic option [9].EVs derived from MSCs with different tissue origins have been reported to accelerate diabetic wound healing.For example, EVs derived from bone marrow MSCs promote the proliferation and migration of chronic wound fibroblasts and endothelial cells [10].EVs derived from induced pluripotent stem cells tend to enhance collagen maturity and re-epithelization [11].EVs derived from placental MSCs (P-MSC-EVs) have great potential in promoting the proliferation and migration of endothelial cells and reducing scar formation [12,13].However, the effects of P-MSC-EVs on chronic diabetic healing and the function of senescent HDFs need to be confirmed, and the potential mechanisms also need to be elucidated.
EVs contain a variety of endogenous biological cargos, such as proteins, lipids and microRNAs (miRNAs), involved in mediating intercellular crosstalk.miRNAs are highly conserved noncoding small RNAs (18-24 nucleotides) that can bind to the 3 -untranslated regions (3 -UTRs) of mRNAs to cause mRNA degradation or translation inhibition.Further study demonstrated that miRNAs in EVs have a crucial regulatory roles in wound healing [14].For example, miR-19b in EVs derived from human adipose-derived stem cells promoted fibroblast proliferation and migration by targeting CC chemokine ligand 1 and regulating the transforming growth factor-β (TGF-β) pathway, which in turn accelerated skin wound healing [15].EVs derived from human adiposederived stem cells transferred miRNA-125a to endothelial cells and promoted angiogenesis in wounds byinhibiting Delta-like 4 expression.
In the present study, we observed the effects of P-MSC-EVs on diabetic wound healing and collagen deposition in wounds.P-MSC-EVs were used to treat HG-induced senescent fibroblasts and the antisenescence, proliferation, migration and antiapoptotic abilities of fibroblasts were examined.Next, the candidate miRNAs responsible for the function of P-MSC-EVs were confirmed in vitro and in vivo.Furthermore, the target genes of candidate miRNAs were identified by RNAi experiments.Finally, we found that P-MSC-EVs accelerated diabetic wound healing by improving the function of senescent fibroblasts through the transfer of miR-145-5p, which targets cyclin-dependent kinase inhibitor 1A (CDKN1A) to activate the extracellular signal regulated kinase (Erk)/protein kinase B (Akt) signaling pathway.

Methods
Cell culture P-MSCs isolated from placenta were obtained from our laboratory and cultured in Dulbecco's modified Eagle's medium/F12 (Gibco, USA) containing 10% EVs-free fetal bovine serum (FBS; Gibco, USA) and 1% penicillin and streptomycin (Gibco, USA) [32].Cells were cultured at 37 • C with 5% CO 2 in a humidified environment, and cells at passages 3-6 were employed for isolation of EVs.HDFs were isolated using previously described protocols [33] and cultured at 37 • C with 5% CO 2 in a humidified environment.For HGinduced experiments, HDFs at passage 6 were cultured in Dulbecco's modified Eagle's medium with glucose at final concentrations of 5.5 and 35 mM [32] for 10 days during which the medium was changed every 72 h.After the desired time, cell senescence was assessed by senescence-associated beta-galactosidase (SA-β-gal) staining, cell proliferation was evaluated by EdU (5-ethynyl-2'-deoxyuridine) and cell counting kit-8 (CCK-8) assay, cell cycle and cell apoptosis were analyzed by flow cytometry, and cell migration was observed by scratch and transwell assays.

Isolation and identification of P-MSC-EVs
EVs were harvested according to previously described methods [34].Briefly, the cell culture medium was collected and centrifuged at 2000 x g for 10 min to remove cellular debris.The supernatants were then collected and centrifuged at 10,000 x g for 30 min, and the new supernatants were collected and ultracentrifuged at 100,000 x g for 75 min.Next the deposit was obtained and resuspended with 1 ml phosphate-buffered saline (PBS) and the resuspension was ultracentrifuged again at 100,000 x g for 75 min after filtering with a 0.22 μm filter (Steritop™ Millipore, MA, USA).The new deposit (P-MSC-EVs) was resuspended with PBS and stored at −80 • C. All the centrifugation steps were conducted at 4 • C.
In addition, for the identification of P-MSC-EVs, a transmission electron microscope (HITACHI HT7700, Hitachi High-Technologies, Japan) was used to capture the morphology, a Zetaview instrument (Particle Metrix, Meerbusch, Germany) was used to measure the density and size, and western blotting was used to examine the expression of CD9, tumor susceptibility gene101 (TSG101), and calnexin in the cell lysis, EVs-depleted medium and P-MSC-EVs.

In vivo administration of P-MSC-EVs
Eight-week-old male diabetic mice (BKS-Dock Leprem2Cd479, db/db, weighing 26-30 g) were purchased from The Center for Experimental Animals, Jicuikang Company.All procedures followed the guidelines of the Animal Research Committee of the Chinese PLA General Hospital.After the mice were shaved and anesthetized, full-thickness excisional skin wounds on the dorsum (10 mm in diameter) were created.All animals were randomized into control (PBS) and P-MSC-EVs groups (n = 5).P-MSC-EVs were labeled with PKH26 (Sigma-Aldrich, Germany) according to the manufacturer's protocol.Observation of the fluorescent image was carried out using a Bruker in vivo imaging system Fx Pro two days after injection.P-MSC-EVs (100 μl; 4.56 x 10 10 particles/ml) were injected around the wounds at four injection sites (25 μl per site) every two days for 14 consecutive days.The wounds of each group were photographed on Days 0, 4, 8, 12, and 16 after surgery and the wound closure rate was measured using Image-Pro Plus 6 software (Media Cybernetics, Bethesda, USA) and calculated using the equation: wound closure rate (%) = [(initial wound area − actual wound area at day x)/initial wound area] × 100%.

Histological analysis
After different treatments, the mice were sacrificed at the projected time points (Days 4, 8, 12 and 16) and skin specimens were obtained and fixed in 4% paraformaldehyde postoperatively.Then, tissues were dehydrated using graded ethanol, embedded in paraffin and subsequently cut into 4-μmthick sections, followed by hematoxylin and eosin staining (Solarbio, Beijing, China) and Masson's trichrome staining (Solarbio, Beijing, China) according to the manufacturer's instructions.Scar width and the degree of collagen maturity or collagen volume fraction were evaluated according to the literature as previously described [35,36].Images of stained sections were captured using a digital imaging scanning system (Precipoint M8; Precipoint, Freising, Germany) and the results were analyzed using Image-Pro Plus 6 software.

Immunofluorescence staining
Immunofluorescent staining was performed as described elsewhere [37].Briefly, paraffin-embedded slides of skin samples obtained on Day 16 post-wounding were processed with 1 h heating at 60 • C, deparaffinization in xylene, rehydration in graded ethanol, 15 min antigen retrieval in citrate buffer, 15 min cell permeation using 0.3% Triton x-100 and 2 h of unspecific antigen blocking using 10% goat serum, and then were incubated with primary antibodies against p16INK4a (1 : 50; sc-1661, Santa Cruz Biotechnology) at 4 • C overnight.The next day, the slides were washed three times with PBS and incubated with Alexa Fluor 647 fluorescence secondary antibody (1 : 200, Invitrogen) for 1 h in dark at room temperature.Next, the slides were stained with DAPI (4',6-diamidino-2-phenylindole) and mounted.Finally, immunofluorescence images were recorded using confocal microscope (Leica, Germany).
SA-β-gal staining SA-β-gal staining was used to evaluate SA-β-gal expression in HG-induced HDFs and was carried out according to the manufacturer's instructions of the SA-β-gal staining kit (Sigma-Aldrich, Germany).Briefly, HDFs were washed and fixed with 4% paraformaldehyde for 20 min and then incubated with the SA-β-gal staining solution overnight at 37 • C under CO 2 -free conditions.Subsequently, HDFs were observed under a phase-contrast microscope (Leica DMI 3000B, Solms, Germany).The proportion of SA-β-galpositive cells was measured by counting the blue cells vs total cells.

Uptake assay of P-MSC-EVs
For the EV uptake assay, 4 μg PKH67 (Sigma-Aldrich, Germany) was used to label 50 μg of P-MSC-EVs at room temperature for 5 min.Then, the EVs were washed with PBS and recollected via ultracentrifugation to remove nonsolubilized material, followed by sterilization through a 0.22 μm membrane filter.HDFs were co-incubated in 24well plates with PKH67-labeled EVs.The internalization of P-MSC-EVs by HDFs was counterstained with phalloidinrhodamine B (cytoskeleton) and DAPI (cell nucleus) and observed under confocal microscope (Leica, Germany).The HG-induced HDFs were treated with P-MSC-EVs at different concentrations (2.28 × 10 10 particles/ml, 4.56 × 10 10 particles/ml) for the cell proliferation assay, cell cycle, scratch assay, transwell assay, and apoptosis assay.At the same time, the expression of cyclinD1, Bcl-2 and Bax was also analyzed by real-time quantitative PCR (RT-qPCR) and western blotting.

Cell proliferation assay
CCK-8 and EdU assays were conducted to evaluate the proliferative capacity of HDFs.For the CCK-8 assay, HDFs were seeded into 96-well plates at a density of 2 × 10 3 cells/well (four replicates per group) and cultured in medium supplemented with or without P-MSC-EVs, miRNA and siRNA.At a planned time point, CCK-8 reagent was added to the cells in serum-free medium and incubated for 3 h, followed by measurement of absorbance at 450 nm.For the EdU assay, 1 × 10 4 HDFs/well were added to 24-well plates and EdU (Beyotime, Shanghai, China) staining was carried out 48 h after P-MSC-EVs, miRNA or siRNA treatment according to the provided protocols.Then the results were visualized by a fluorescence microscope.

Cell migration assay
The migration of HDFs was evaluated by means of scratch and transwell assays.For the scratch assay, 2 × 10 5 HDFs/well were plated into a 12-well plate (three replicates per group).When 90% confluence was reached, cells were scratched with a 1-ml pipette tip and then gently washed with PBS to remove floating cells.After different treatment as described above, the cells were photographed at 0, 24 or 32 h post-scratch and measured by Image-Pro Plus 6.0 software.The migration area was calculated as (%) = [(original gap area − gap area at x h)/original gap area] × 100%.For the transwell assay, 24-well transwell inserts (Corning, NY, USA) were used with 8-μmpore-sized filters.HDFs (1 × 10 4 cells/well) were suspended in 100 μl of low-serum medium (containing 5% FBS), and then plated into the upper chamber filled with 600 μl of HG medium (containing 10% FBS) supplemented with or without P-MSC-EVs, miRNA, and siRNA.After 24 h incubation, cotton swabs were used to scrape off cells attached to the upper surface of the filter membrane.The cells attaching on the lower surface were then stained using 1% crystal violet (Solarbio, Beijing, China) for several minutes at room temperature.Finally, the migrated cells were photographed and counted under an optical microscope.

Flow cytometry
Cell cycle and cell apoptosis assays were performed by flow cytometry.For the cell cycle assay, HDFs (8 × 10 5 cells/well) were seeded in six-well plates and cultured by different treatments for 48 h.Then, the cells were collected and fixed in 70% cold ethanol at 4 • C overnight, followed by resuspension with DNase-free RNase at 37 • C for 30 min and subsequent addition of propidium iodide (PI) for DNA staining (30 min, 4 • C).PI fluorescence was examined with a flow cytometer (BD FACS Calibur™, BectonDickinson, NJ, USA).
For the cell apoptosis assay, HDFs that had received different treatments for 48 h, as described above, were treated with carbonyl cyanide m-chlorophenyl hydrazine (10 μM; Solarbio, Beijing, China) in an incubator for 20 min to induce apoptosis.The cells were then collected and washed with icecold PBS and resuspended with the buffer provided in the cell apoptosis kit (Solarbio, Beijing, China).Subsequently, 100 μl of cell suspension, 200 μl of PBS, 5 μl of Annexin V-FITC (Fluorescein Isothiocyanate) reagent and 5 μl of PI regent were sequentially mixed and incubated for 15 min at room temperature in the dark.Cell apoptosis was then immediately detected via flow cytometry.

RT-qPCR
Total RNA was extracted from cells and P-MSC-EVs using an miRNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.For mRNA detection, cDNA was synthesized using a FastKing gDNA Dispelling RT Super Mix kit (Tiangen, Beijing, China), followed by qPCR reactions using a SYBR Green Super-Real PreMix Plus kit (Tiangen, Beijing, China), which was performed on an ABI Real-Time PCR Detection System (ABI7500 FAST, Thermo Fisher Scientific, MA, USA).For determination of miRNA expression in P-MSC-EVs, a synthetic analog of non-human cel-miR-39 (QIAGEN, Hilden, Germany) was spiked in 10 μl of a 5-fmol/μl stock to normalize RNA extraction efficiency.Then, cDNA synthesis and subsequent qPCR reactions were conducted using a Hairpin-itTM microRNA and U6 snRNA Normalization RT-PCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer's protocol.For determination of miRNA expression in cells, cDNA synthesis of miRNAs and qPCR reactions were performed as described above.The mRNA-specific forward and reverse primers as well as miRNA-specific forward primers and the universal reverse primer were designed and synthesized by Sangon Biotech (Shanghai, China).The primer sequences are listed in Tables S2 and S3 (see online supplementary material), respectively.The relative mRNA and miRNA expression levels were normalized to β-actin and U6 levels" respectively, and were quantified according to the 2 − CT method.

Application of miR-145-5p in vivo
Animal experiment with mice was also performed to evaluate the potential role of miR-145-5p in P-MSC-EVs.

Statistical analysis
All quantitative data are presented as the mean ± SD.Student's t-test was used to analyze significant differences between two groups.One-way ANOVA (Analysis of Variance) with the Dunnett post hoc test was adopted to compare differences of more than two groups at the same point and two-way ANOVA with the Dunnett post hoc test was performed to analyze differences between multiple groups at different time points.All statistical analyses were conducted using GraphPad Prism 7.0 software.Differences were considered statistically significant at a level of * p < 0.05, * * p < 0.01, * * * p < 0.001 and * * * * p < 0.0001.

Characterization of P-MSC-EVs
The conditioned medium of P-MSCs was collected for EV isolation.The characteristics of P-MSC-EVs were evaluated by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting.The ultrastructure of P-MSC-EVs was revealed by TEM and is presented in Figure 1a and is cup shaped with a diameter of ∼100 nm.NTA showed that the size of P-MSC-EVs basically ranged from 30 to 200 nm, and the average particle size was 115.5 nm (Figure 1b), which is consistent with the previously reported size distribution of EVs [41].Representative markers of EVs including CD9 and TSG101, were expressed by P-MSC-EVs.Calnexin, which is a negative protein marker of EVs, was not detectable in our study (Figure 1c).These results demonstrate that the characteristics of the isolated nanoparticles from P-MSCs matched the criteria of EVs.

P-MSC-EVs promoted cutaneous wound healing in diabetic mice
We next explored the ability of P-MSC-EVs to promote diabetic wound healing by creating full-thickness cutaneous wounds on the dorsal skin of diabetic mice.In vivo image analysis confirmed the continuous retention of P-MSC-EVs two days after injection (Figure S1, see online supplementary material).In addition, as shown in Figure 1d, gross observations indicated significantly accelerated wound closure rates  1e, f).Moreover, P-MSC-EV-treated wounds were characterized by larger amounts of wavy collagen fibers and better-organized collagen deposition than the control wounds (Figure 1g).In addition, immunofluorescence staining showed a large number of p16INK4a-positive HDFs in the wound beds in the PBS group, while p16INK4a-positive HDFs were barely found in the P-MSC-EV group (Figure 1h).These data indicate that P-MSC-EV treatment improved collagen deposition and HDF senescence and accelerated diabetic wound healing.

HG induced senescence and impaired the cell function of HDFs
To simulate a HG microenvironment in vitro, HDFs were cultured with glucose at a final concentration of 35 mM as previously reported [34,42].After 10 days of HG treatment, the expression of SA-β-gal was increased in the HG group compared with the normal glucose (NG) group (Figure 2a, b), which is consistent with the previously published reports [42].Next, we observed the effects of HG on the proliferation and migration of HDFs.The results of the CCK-8 assay and EdU incorporation assay showed lower proliferation in the HG group (Figure 2c, d).The result was further confirmed using a cell cycle assay.The results revealed that the HG group exhibited lower proportions of S and G 2 /M subpopulations, as well as higher proportions of G 0 /G 1 subpopulation than the NG group (Figure 2e, p < 0.05).The migration of HDFs was evaluated with wound scratch and transwell assays.The scratch assays demonstrated that the migration rates in the HG group were decreased significantly compared with those in the NG group (Figure 2f, g).Consistent with thisfinding, the number of migrated HG-treated HDFs was less than the number of migrated NG-treated HDFs in transwell assays (Figure 2h, i).Additionally, a correspondingly increased cell apoptosis rate was observed in the HG group (Figure 2j, p < 0.001).Together these results suggest that glucose at a concentration of 35 mM induced senescence and impaired the function of HDFs.

P-MSC-EVs improved the function of HG-induced senescent HDFs
To illustrate the multiple effects of P-MSC-EVs on HDFs, we first determined whether P-MSC-EVs could be internalized by HDFs.P-MSC-EVs were labeled with PKH67 and then incubated with HDFs for 12 h.After fixation, recipient cells were stained with phalloidin.As shown in (Figure 3a), all HDFs were stained green after incubation with labeled P-MSC-EVs, demonstrating that PKH67-tagged P-MSC-EVs had been transferred to the cells and were mainly localized around the perinuclear region in HDFs.Next, P-MSC-EVs at different concentrations were added to the culture medium of HG-induced HDFs. Figure 3b shows that the SA-β-gal expression in HDFs was decreased in the P-MSC-EV group.The results of EdU incorporation and CCK-8 assay indicated that treatment with 2.28-4.56× 10 10 particles/ml P-MSC-EVs enhanced the proliferation of HGinduced HDFs in a dose-dependent manner (Figure 3c and Figure S2, see online supplementary material), which was confirmed by a cell cycle assay showing that more cells were in the S and G 2 /M phases than HDFs treated with HG alone (Figure 3d).The results of the scratch assay indicated that the migration of HG-induced HDFs was improved after P-MSC-EV treatment (Figure 3e, p < 0.01).
In accordance with this finding, many more migrated HDFs were observed in the P-MSC-EV-treated group (Figure 3f, p < 0.0001).Additionally, P-MSC-EVs were capable of lowering the apoptotic rate of HG-induced HDFs (Figure 3g).Consistent with these results, we observed elevated expression of proliferation-related protein cyclin D1, upregulated antiapoptotic Bcl-2 expression, and decreased pro-apoptotic Bax expression at the mRNA and protein levels after P-MSC-EV treatment (Figure 3h-j).Together, these findings indicate that P-MSC-EVs could improve the antisenescence, proliferation, migration, and antiapoptotic abilities of HG-induced senescent HDFs.

Detection of miRNAs in P-MSC-EVs and the prediction of their targets
MiRNAs, which are important cargo in EVs, are able to exert a regulatory effects on a wide array of biological processes by binding with target mRNAs to regulate their expression.

P-MSC-EVs improved the function of HG-induced HDFs by transferring miR-145-5p
To verify the role of miR-145-5p in the P-MSC-EV-mediated functional improvements of HG-induced HDFs, we initially confirmed the increased expression of miR-145-5p in HGinduced HDFs treated with P-MSC-EVs (Figure 5a, b).MiR-145-5p inhibitors were used to knock down miR-145-5p expression (Figure 5c) in HG-induced HDFs after treatment with P-MSC-EVs.Subsequently, a series of functional assays were conducted to test the regenerative effects of  P-MSC-EV-derived miR-145-5p.Figure 5d shows that miR-145-5p inhibitors could block the effect of P-MSC-EVs on SA-β-gal expression in HDFs.EdU incorporation and cell cycle assays were carried out to evaluate the proliferation of HDFs, and the results showed that EdU-positive HDFs and cells in S and G 2 /M phase were increased after P-MSC-EV stimulation but were reduced by the transfection of miR-145-5p inhibitors (Figure 5e, f).These results were also confirmed by the CCK8 cell viability assay (Figure S3, see online supplementary material).Next the migratory ability of HGinduced HDFs was attenuated after miR-145-5p inhibition, as shown in in Figure 5g, h.We further quantified the apoptosis rate and found that the effect of P-MSC-EV-induced protection against apoptosis was diminished (Figure 5i).In addition, transfection of the miR-145-5p inhibitors resulted in decreased cyclin D1 and Bcl-2 expression and increased Bax expression, verifying that the enhanced proliferation and antiapoptotic effects on HDFs induced by P-MSC-EVs were partially reversed by the miR-145-5p inhibitors (Figure 5j-l).
Collectively, these data demonstrate that miR-145-5p in P-MSC-EVs exert a protective effects on HG-induced senescent HDFs.
MiR-145-5p mimicked the effects of P-MSC-EVs to improve the function of HG-induced HDFs Subsequently, we further investigated whether miR-145-5p could mimic the effects of P-MSC-EVs on HG-induced HDFs.MiR-145-5p mimics or miR-NC were transfected into HGinduced HDFs, and the transfection rate was shown to be 93.45%(Figure 6a, b).After transfection, SA-β-gal staining results verified the beneficial effects of miR-145-5p on improving HDF senescence (Figure 6c).The EdU and CCK-8 results confirmed the ability of miR-145-5p to enhance the proliferation of HG-induced senescent HDFs (Figure 6d and Figure S4, see online supplementary material).Consistently, in miR-145-5p mimic-treated HDFs, more cells were in the S and G 2 /M phases than in the control and miR-NC groups (Figure 6e).The results of the cell migration assay showed that HG-induced HDFs transfected with miR-145-5p mimics migrated faster than cells in control and miR-NC groups (Figure 6f, g).As expected, fewer apoptotic cells were found in the miR-145-5p mimics group (Figure 6h).Additionally, we observed that miR-145-5p mimics upregulated cyclin D1 and Bcl-2 expression and downregulated Bax expression at the mRNA and protein levels (Figure 6i-k).Numerous   studies have reported that MSC-EV-derived miRNAs could enhance wound healing at least in part by activating the Erk/Akt signaling pathway [59,60].Therefore, the expression levels of Erk1/2, p-Erk1/2, Akt and p-Akt were detected to determine whether miR-145-5p could activate the Erk/Akt signaling pathway.As expected, miR-145-5p mimics notably increased the phosphorylation of Erk1/2 and Akt, although there was no significant change in the expression of Erk1/2 or Akt (Figure 6l, m).In summary, miR-145-5p mimics could activate the Erk/Akt signaling pathway and mimic the effects of P-MSC-EVs on improving the functions of HG-induced senescent HDFs in terms of inhibiting senescence and apoptosis and inducing proliferation and migration in vitro.
MiR-145-5p improved the function of HG-induced HDFs by targeting CDKN1A to activate the Erk/Akt pathway CDKN1A was further confirmed to be the target gene of miR-145-5p (Figure 7a).To test whether knocking down the expression of CDKN1A could achieve comparable effects as miR-145-5p on cell function, si-CDKN1A #1 was selected to inhibit the expression of CDKN1A in HG-induced HDFs (Figure 7b).The transfection rate was shown to be 90.13%(Figure 7c, d). Figure 7e shows reduced SA-β-gal expression in HDFs transfected with si-CDKN1A #1.The EdU and CCK-8 results indicated that si-CDKN1A #1-transfected HDFs exhibited higher cell proliferation (Figure 7f and Figure S5, see online supplementary material).Correspondingly, we found the majority of si-CDKN1A #1-transfected HDFs in the S and G 2 /M phases (Figure 7g).Furthermore, the migration ability of HDFs detected by scratch and transwell assays was also increased in the si-CDKN1A #1 group (Figure 7h, i).Unexpectedly, we did not find a decrease in apoptosis after ransfection with si-CDKN1A (Figure S6, see online supplementary material), indicating that other target genes of miR-145-5p may be involved in regulating the antiapoptotic effects on HG-induced HDFs.CDKN1A is involved in arresting cell cycle progression and has been reported to be a negative regulator of the Erk/Akt signaling pathway in some tumor cells [61].Consistent with their reports, CDKN1A inhibition induced significant increases in the phosphorylation of Erk1/2 and Akt in our study, indicating that activation of the Erk/Akt pathway may be the underlying mechanism by which CDKN1A inhibition enhances fibroblast function (Figure 7j, k).CAMK1D was confirmed as another target gene of miR-145-5p (Figure 8a).Similarly, we chose the most effective siRNA, si-CAMK1D #2, to inhibit CAMK1D expression in functional assays of HG-induced HDFs (Figure 8b).The transfection rate was shown to be 89.59%(Figure 8c, d).Disappointingly, we found that the transfection of si-CAMK1D #2 had no effects on cell senescence, cell cycle or cell apoptosis of HG-induced HDFs (Figure 8e-g), indicating that CAMK1D was not involved in the functional improvements of HG-induced HDFs induced by miR-145-5p derived from P-MSC-EVs.
These results indicate that miR-145-5p is responsible for the P-MSC-EV-induced functional improvements of HG-induced HDFs by targeting CDKN1A to activate the Erk/Akt pathway.

MiR-145-5p mimicked the effects of P-MSC-EVs to promote wound healing in vivo
A previous experiment revealed that miR-145-5p could mimic the effects of P-MSC-EVs to improve the function of HGinduced HDFs.Next, we assessed the effects of miR-145-5p on diabetic wound healing.Full-thickness cutaneous wounds were made on the backs of diabetic mice and were injected with PBS, agomiR-145-5p, and antagomiR-145-5p every two days after wounding.In vivo image analysis confirmed the continuous retention of agomiR-145-5p two days after injection (Figure S7, see online supplementary material).Additionally, as shown in Figure 9a, the area of the wounds in the agomiR-145-5p group was significantly decreased on Days 8, 12, and 16 after the operation relative to the other groups.Furthermore, the narrowest scar widths, as well as much longer, thicker, and better-organized fibers, were observed in wounds treated with agomiR-145-5p than in the control and antagomiR-145-5p groups on Day 16 after wounding (Figure 9b, c).Moreover, we evaluated p16INK4a expression at the wound site, and the results confirmed that agomiR-145-5p was able to improve the fibroblast senescence state in vivo (Figure 9d).Collectively, these data demonstrate that miR-145-5p could mimic the effects of P-MSC-EVs to promote diabetic wound healing and collagen deposition and improve fibroblast senescence, highlighting its great potential in diabetic wound treatment.

Discussion
In this study, we first proved that local injection of P-MSC-EVs into cutaneous wound sites in diabetic mice leads to rapid wound closure and better-organized collagen deposition.In vitro, P-MSC-EVs could be internalized by HGinduced senescent HDFs and subsequently enhanced the cell antisenescence, proliferation, migration, and antiapoptotic abilities of HDFs.Next, the potential underlying mechanisms were explored, and miR-145-5p was shown to be abundant in P-MSC-EVs, which could be delivered into HDFs to activate the Erk/Akt signaling pathway by targeting CDKN1A.With agomirs and antagomirs, we further confirmed that miR-145-5p plays a key role in the positive effects of P-MSC-EVs on diabetic wounds.
Diabetic wounds are associated with great healing difficulties, high costs, severe disability due to amputation and intense care.Over the past decades, accumulating evidence has indicated that cellular senescence is one of the causes of diabetic wound healing [62].Cellular senescence is traditionally defined as permanent cell growth arrest and is divided into two main types: telomere-dependent replicative senescence and stress-induced premature senescence [63].Many studies have proven that persistent hyperglycaemia is involved in accelerating the shortening of telomere length and inducing stress-induced premature senescence, eventually giving rise to large-scale cellular senescence.These senescent cells were characterized by impaired cell proliferation, migration, and antiapoptotic abilities, thereby inhibiting wound healing [32,34].HDFs serve as one of the main repair cells, and functional defects in any biological behavior can lead to healing failure.In the present study, we confirmed that HG at a concentration of 35 mM could induce HDF senescence.Moreover, HG-induced HDFs exhibited lower proliferation, migration, and antiapoptotic abilities, which was consistent with previous reports [32,42].Therefore, it is of pivotal importance to protect HDFs from HG insult to achieve rapid wound healing.
Stem cells derived from different tissues have been reported to have positive effects on diabetic wound repair.However, the actual effects of stem cell transplantation were confined to donor condition and functional debilitation after long-term culture and successive passages [64].The application of these cells poses uncertain risks of immune rejection, tumorigenicity, and ethical issues [65].Recently, many studies have proved that the beneficial effects of stem cells are mainly attributable to their exocrine function rather than direct differentiation into target cells [10,59].EVs are key paracrine factors secreted from stem cells that can regulate the functions of target cells during wound repair [66][67][68].MSC-EVs originating from bone marrow, umbilical cord, placenta, and adipose tissue, as well as EVs derived from human amniotic epithelial cells [12,59,69,70], have been reported to accelerate wound healing and have regenerative and protective effects on wound repair cells, especially on fibroblasts [71,72].In the current study, we demonstrated the beneficial effects of P-MSC-EVs on diabetic wound healing and functional improvements of HG-induced senescent HDFs.Likewise, P-MSC-EVs were reported to stimulate angiogenesis in ischaemic diseases [12] and exert therapeutic effects on Duchenne muscular dystrophy patients [73].
We investigated the mechanism responsible for the positive effects of P-MSC-EVs on HG-induced senescent HDFs.EVs contain a variety of components in addition to miR-NAs that can protect miRNAs from extracellular degradation.MiRNAs are involved in regulating multiple biological processes in type II diabetes including insulin secretion, immune inflammatory response, angiogenesis and diabetic wound healing [14,74].In this study, candidate miR-NAs within P-MSC-EVs were identified using RT-qPCR.We first reported that miRNA-145-5p was highly enriched in P-MSC-EVs.Currently, during tumor treatment, miR-145-5p has been reported to have controversial effects.For example, miR-145-5p serves as a tumor suppressor in melanoma, tongue and laryngeal squamous cell carcinoma, hepatocellular carcinoma, colorectal cancer, pancreatic ductal adenocarcinoma, and lung cancer by inhibiting the proliferation, migration, and antiapoptotic status of tumor cells [17,[75][76][77][78][79][80], while Zhou et al. found that miR-145-5p could enhance the proliferation, migration, and invasion of Wilms' tumor cells [81].Apart from mediating tumorigenic functions, miR-145-5p was found to be down-regulated in hypertrophic scar tissues, psoriatic lesional skin, hepatopulmonary syndrome tissues, lung tissues of smokers and atherosclerotic plaques of atherosclerosis mice, and miR-145-5p overexpression was able to attenuate the proliferation, migration, and antiapoptotic abilities of hypertrophic scar fibroblasts, keratinocytes, pulmonary microvascular endothelial cells, astrocytes, and cardiac cells [82][83][84][85][86][87][88][89][90].Nevertheless, miR-145-5p overexpression has also been demonstrated to be capable of enhancing the proliferation and migration of trophoblasts cells, and airway smooth muscle cells in vitro [91][92][93].Moreover, Yang et al. and Condorelli et al. found that miR-145-5p overexpression improved the viability and migration of lung fibroblasts and recessive dystrophic epidermolysis bullosa skin fibroblasts and led to the establishment and maintenance of fibrotic traits of contraction, which was accompanied by upregulated expression of α-smooth muscle actin [53,55].Additionally, miR-145-5p could be induced by TGF-β and dihydroartemisinin.However, dihydroartemisinin could significantly reverse the proliferation and fibrosis of tenon fibroblasts caused by TGF-β [94,95].These results indicate that miR-145-5p may be a potential target in the development of novel gene therapies to treat pathological fibrotic diseases.

Figure 1 .
Figure 1.P-MSC-EVs promoted cutaneous wound healing in diabetic mice.(a) Morphology of P-MSC-EVs observed with transmission electron microscopy.Scale bar: 100 nm.(b) Particle size distribution of P-MSC-EVs measured by NTA.(c) Expression levels of CD9, TSG101 and Calnexin.(d) General view and the rate of wound closure with different treatments on Days 0, 4, 8, 12, and 16 after wounding, n = 5 per group.(e, f) H&E staining of wound sections treated with PBS and P-MSC-EVs on Day 16 after the operation.Scale bar: 1 mm.(g) Masson's trichrome staining on Day 16 post-wounding.Collagen fiber is stained in blue.Scale bars: 2 mm (upper), 500 μm (lower).(h) Expression of p16INK4a in fibroblasts in vivo after treatment with P-MSC-EVs.Scale bar: 50 μm.Compared with the control group, * * * * p < 0.0001; ns no significance.P-MSC-EVs extracellular vesicles derived from human placental mesenchymal stem cells, NTA nanoparticle tracking analysis, H&E hematoxylin and eosin, PBS phosphate-buffered salin, TSG101 tumor susceptibility gene 101

Figure 4 .
Figure 4. Detection of miRNAs in P-MSC-EVs and prediction of their targets.(a) Expression detection of the selected miRNAs with RT-qPCR.n = 3 per group.(b) Venn diagram for the intersection of putative miR-145-5p targets predicted using miRwalk, starBase, miRTarbase and DIANA Tarbase.A total of 21 co-predicted genes (red circle) were found including CDKN1A.(c) Putative miR-145-5p binding sites in the 3 -UTRs of CDKN1A.(d) Venn diagram for the intersection of putative miR-145-5p targets predicted using miRDB, Targetscan, miRwalk and DIANA Tarbase.A total of 33 co-predicted genes (red circle) were identified including CAMK1D.(e) Putative miR-145-5p binding sites in the 3 -UTRs of CAMK1D.CAMK1D Calcium/calmodulin dependent protein kinase 1D, CDKN1A Cyclin dependent kinase inhibitor 1A, P-MSC-EVs extracellular vesicles derived from human placental mesenchymal stem cells