Exosome-shuttled PCBP1 from hypoxic preconditioned urine-derived stem cells accelerates wound healing

Abstract

Background

We previously found that hypoxia-preconditioned human urine-derived stem cells (USCs) accelerated wound healing. Since exosomes are one of the key mediator of USCs to support skin repair, we wonder whether the healing-promoting effects of hypoxia-treated USCs were associated with exosomes.

Methods

A full-thickness cutaneous wound model of female C57BL/6J mice was established. Label-free exosome proteomics was performed to investigate the differentially expression protein from exosomes derived from hypoxia-treated USCs (H-USCs-exo). Transcriptomics was carried out to explore the downstream signaling of exosome recipient cells.

Results

H-USCs-exo facilitated the wound healing via angiogenesis and re-epithelialization. Also, the pro-angiogenesis results were obtained in vitro, as evidenced by the enhancement of proliferation, migration, and angiogenic abilities of human microvascular endothelial cell line HMEC-1. Subsequently, using Label-free exosome proteomics, a protein named poly (RC) binding protein 1 (PCBP1, an RNA-binding protein) was found to be significantly upregulated (Fold change = 3.34, p < 0.05) in H-USCs-exo. Exosomes delivered PCBP1 into endothelial cells, and exosomal PCBP1 mediated the pro-angiogenic effects of H-USCs-exo on HMEC-1 cells. The healing-delaying function was confirmed in mice treated with exosomes secreted by hypoxia-pretreated USCs with PCBP1 knockdown. In addition, as a tissue repair facilitator, hypoxia inducible factor 1 subunit alpha (HIF-1α) was upregulated in USCs under hypoxia condition. HIF-1α potentiated the transcription of PCBP1, leading to the high abundance of PCBP1 in H-USCs and H-USCs-exo. Further high-throughput transcriptomics illustrated that PCBP1 upregulated the expression of WNT5A (Fold change = 2.75, p < 0.05) and its downstream factors including vascular endothelial growth factor A and TEK receptor tyrosine kinase (Fold change = 2.85 and 5.75, respectively; p < 0.05).

Conclusion

Exosome-shuttled PCBP1 from hypoxic preconditioned USCs accelerated wound healing, which might be associated with WNT5A signaling pathway.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05220-0.

Background

Large skin wounds caused by trauma, burns, or chronic diseases (e.g., diabetes, venous ulcers) pose a significant clinical challenge due to their complexity, risk of infection, prolonged healing time, and potential for scarring or functional impairment [1]. Wound healing is a heavy burden for patients, both physically and financially. Conventional wound treatment methods include skin grafting, skin flap transplantation, laser therapy and biological scaffolds, while effective in many cases, these methods have several limitations, such as the risk of atrophic scars, pigmentary abnormalities, and challenges in treating large-scale wounds [2–4]. Nowadays, stem cell therapy has flourished. Multiple types of stem cells have been utilized in preclinical animal models to repair or regenerate tissue, such as, adipose-derived stem cells and bone marrow-derived mesenchymal stromal cells [5–7]. However, the transplantation application of these cells is limited due to low survival rate, tumorigenesis, and immunological rejection [8, 9]. Thus, it is an urgent need to look for an alternative efficient and safe methods to facilitate wound healing.

Paracellular secretion acts as a vital part in the therapeutic function of pluripotent stem cells in many diseases, with exosomes being the key mediator [10]. Exosomes are small extracellular vesicles with a diameter of 40–160 nm that transfer diverse molecules including nucleic acids and proteins to recipient cells [11]. These exogenous exosomal molecules could further regulate the downstream signaling and function of recipient cells [12]. Exosomes are considered a promising treatment in regenerative medicine, as they play a pro-regenerative role similar to stem cells in injured tissues, avoiding the safety concerns associated with stem cell transplantation. For instance, Zhang et al. have reported that exosomes derived from human umbilical cord blood mesenchymal stem cells promote regenerative wound healing by the repression of transforming growth factor-β receptor signaling [13] Similarly, exosomes from adipose-derived stem cells enhance the proliferation and migration of HUVECs, resulting in the angiogenesis and wound repair [14]. Whereas the utilization of these stem cells is impeded due to the limited source, and isolation of stem cells from adipose tissues is invasive [15].

Urine-derived stem cells (USCs) are a subpopulation of cells from urine found in recent years. USCs could be harvested through a safe, simple, low-cost, and noninvasive procedure [16]. Despite the high expansion capacity, they do not form teratomas or tumors, making them safer for regenerative applications compared to some pluripotent stem cells [16, 17]. Both the local transplantation of USCs and the exosomes derived from USCs can accelerate the wound healing by angiogenesis, indicating that exosomes might be one of the mediators for USCs to facilitate wound repair [15, 18, 19].

Hypoxic preconditioning has emerged as a promising strategy to promote the therapeutic potential of mesenchymal stem cells by mimicking the low-oxygen microenvironments they encounter in physiological niches (e.g., bone marrow, adipose tissue) [20]. Mesenchymal stem cells under hypoxic condition possess enhanced angiogenic potential, partly due to the modification in the content and function of their exosomes. 2% oxygen stimulates the exosomes secreted from adipose-derived stem cells, markedly facilitating angiogenesis through stabilizing the hypoxia inducible factor 1 subunit alpha (HIF-1α) [14]. In our previous study, we found that hypoxic preconditioning improved the wound healing potential of USCs by the potentiation of wound angiogenesis and re-epithelialization. The hypoxia responder HIF-1α was upregulated in wound site of mice [18]. We wondered whether the healing promoting effects of hypoxic preconditioning-USCs were associated with exosomes. Herein, a full-thickness cutaneous wound model was established. Exosome proteomic analysis was performed to further explore the changes of exosomes protein secreted from USCs with hypoxia pre-stimulation.

Methods

The work has been reported in line with the ARRIVE guidelines 2.0.

Isolation and identification of USCs

USCs were isolated from urine samples of four healthy adults. Written informed consent was obtained from the donors. This study was approved by the Institutional Review Board of Zhengzhou University (Data of the approval: March 1 (st), 2023) following the Declaration of Helsinki. The isolation of USCs was performed as previously described [18]. Briefly, 600 mL per samples were centrifuged at 400 g for 10 min, and the pellets were collected and rinsed twice with phosphate buffer saline (PBS). 1 mL of medium was added to resuspend the cell pellets and transferred to 12-well plates and cultured at 37 °C in an atmosphere with 5% CO₂. The medium was comprised of 50% keratinocyte serum-free medium (iCell Bioscience Inc, Shanghai, China), 33.75% Dulbecco’s Modified Eagle’s medium (Servicebio, Wuhan, China), 11.25% Ham’s F-12 nutrient mixture (Servicebio), and 5% fetal bovine serum (Sijiqing Biotechnology, Nanjing, China) supplemented with 5 ng/mL epidermal growth factor (Sino Biological Inc., Beijing, China), 50 ng/mL bovine pituitary extract (Absin, Shanghai, China), 0.4 µg/mL hydrocortisone (Solarbio, Beijing, China), 5 µg/mL transferrin (Solarbio), 5 ng/mL bovine insulin (Sigma-Aldrich, St. Louis, MO, USA), 0.18 mM adenine (BioFroxx, Darmstadt, Germany), 2 nM 3,3,5-triiodo-L-thyromine (Aladdin, Shanghai, China), 100 units/mL penicillin (Solarbio), and 100 µg/mL streptomycin (Solarbio). The medium was first refreshed seven days after plating to allow the initial adherence and cell expansion. USCs at passage three were used in the following experiments.

The USCs were identified by the detection of USC markers using flow cytometry. In brief, 1 × 10⁶ cells were incubated with 5 µL FITC Plus Anti-Human CD29 (Proteintech, Wuhan, China), 5 µL FITC Plus Anti-Human CD34 (Thermo Scientific, Pittsburgh, PA, USA), 5 µL APC Plus Anti-Human CD44 (MultiSciences Biotech, Hangzhou, China), 5 µL PE Plus Anti-Human CD45 (MultiSciences Biotech), 5 µL FITC Plus Anti-Human CD73 (Proteintech), or 0.5 µL FITC Plus Anti- Human CD90.2 (Proteintech) at 4 °C for 30 min. Then, cells were conducted to the analysis with NovoCyte flow cytometry (ACEA Biosciences, San Diego, California, USA).

Exosome isolation and characterization

The hypoxia and normoxia treatment of USCs were performed as previously described [18]. For the hypoxia condition, USCs were cultured in the condition of 1% O₂ and 5% CO₂ for 24 h. For the normoxia induction, USCs were cultured in the condition of 21% O₂ and 5% CO₂ for 24 h. Exosomes were isolated and purified from USCs. Briefly, the conditioned medium of USCs was collected and centrifuged at 2,000 g for 30 min at 4 °C to remove the cell debris, followed by the centrifugation at 10,000 g for 45 min at 4 °C to remove the large microvesicles. The supernatant was collected and filtered through a 0.22 μm filter and next centrifuged at 100,000 g for 70 min at 4 °C. The pellets were rinsed with PBS and further centrifuged at 100,000 g for 70 min once again to obtain the exosomes.

The proper size of exosomes derived from USCs (USCs-exo) was detected by the nanoparticle tracking analysis (N30E, NanoFCM, Nottingham, UK), and the morphology was observed using the transmission electron microscopy (HT-7700, Hitachi, Tokyo, Japan). In addition, the exosome isolation was confirmed by the western blotting for exosome markers including CD63, CD81, and TSG101.

Full-thickness cutaneous wound model

Eight-week-old C57BL/6J (female) mice were obtained from Huachuang Sino (Jiangsu, China). Housing female mice minimizes the occurrence of wound reopening, because male mice are more prone to showing aggression when housed with other males [21]. In addition, androgens repress the cutaneous wound healing but estrogens contribute to it [22]. Consistent with the previous studies [15, 23], female mice were used in the present study. The animals were housed under standard barrier conditions: a temperature of 22 °C, 45% humidity, and a 12-hour light/12-hour dark cycle. They were allowed free access to food and water. All efforts were made to minimize animal usage and suffering. Animal experiments were approved by the Institutional Review Board of Zhengzhou University (Data of the approval: March 1 (st), 2023) following The Guideline for the Care and Use of Laboratory Animals. Effect size was calculated to determine the power of the analysis as previously described [24]. The effect size for wound healing analyses was calculated using pilot data, based on the following criteria: power of 80% and a significance level < 0.05. Sample sizes for each experiment are included in the figure legends. The researchers involved in biochemical analysis were blinded to group allocations for the duration of the study. The experimental unit was the individual animal.

Thirty mice were randomly assigned to each group (n = 6/group), and the dosage of exosomes for in vivo experiments was determined based on the previous study [25]. Briefly, (1) Ctrl: 100 µL PBS only, H-USCs-exo: exosomes derived from USCs with hypoxia treatment (100 µg exosomes in 100 µL PBS), N-USCs-exo: exosomes derived from USCs with normoxia treatment (100 µg exosomes in 100 µL PBS); (2) H-Lv-NC^exo: exosomes derived from USCs infected with Lv-NC lentivirus and induced with hypoxia treatment (100 µg exosomes in 100 µL PBS); H-Lv-shPCBP1^exo: exosomes derived from USCs infected with Lv-shPCBP1 lentivirus and induced with hypoxia treatment (100 µg exosomes in 100 µL PBS). All of mice were included in the experiments.

The full-thickness cutaneous wound model was established as previous described [18, 21]. In brief, mice were anesthetized with isoflurane (induction dose: 3%, maintenance dose: 1.5%) and shaved. A full-thickness excisional skin wound (8 mm in diameter) was created on the dorsal bulge of the spine. 100 µg of USC-derived exosomes resuspended in 100 µL PBS was intradermally injected around the wound for four sites. After the operation, the wounds were recovered with Tegaderm (3 M) sterile transparent dressing. A silicon splint ring was used around the wound to prevent skin contraction [26]. The wound area was measured at 0, 7, and 14 days after surgery. Mice were euthanized with 70% vol/min CO₂ at day 14 post-wound, and skin tissues in wound sites were collected for further experiments.

H&E staining

Skin tissues in wound sites were fixed in 4% paraformaldehyde and then embedded in paraffin after dehydration. Section  (5 μm) were deparaffinized in xylene (Aladdin), and rehydrated in an ethanol gradient with distilled water. Sections were incubated with hematoxylin (Solarbio) for 5 min and eosin (Sangon Biotech, Shanghai, China) for 3 min. The wound sites were observed with BX53 microscope (Olympus, Tokyo, Japan).

Immunofluorescence staining

After blocked with 1% bovine serum albumin for 15 min, sections were incubated with anti-KI67 (1:100; Affinity, Changzhou, China), anti-CD31 (1:50; Santa Cruz, CA, USA), anti-keratin 14 (CK14; 1:100; Zenbio, Suzhou, China) at 4 °C overnight. Then, sections were incubated with Cy3-goat anti-rabbit IgG (1:200; Proteintech) or FITC-goat anti-mouse IgG (1:200; Proteintech) at room temperature for 60 min. A DAPI solution was used for counterstaining of nuclear. Images were examined with the BX53 microscope.

Cell culture and treatment of HMEC-1

HMEC-1 cells were obtained from iCell Bioscience Inc. Cells were cultured in MCDB131 medium (Cellverse Bioscience Technology, Shanghai, China) supplemented with 10% fetal bovine serum (Tianhang Biotech, Shanghai, China) and 10 ng/mL epidermal growth factor (Sino Biological Inc) in an incubator of 5% CO₂ at 37 °C.

HMEC-1 cells were treated with 100 µg/mL exosomes from USCs for 0, 6, 12, 48, or 72 h according to the previously study [15]. Then, HMEC-1 cells were subjected to CCK8 assay, scratch wound assay, and tube formation assay.

Lentivirus preparation and infection

For lentivirus infection, the pLKO.1 vector (Fenghuishengwu, Changsha, China) containing the hairpin sequences were utilized together with the packaging vector pSPAX2 (Fenghuishengwu) as well as the envelope vector pMD2.G (Fenghuishengwu). HEK293T cells (iCell Bioscience Inc, Shanghai, China) were employed as the packaging cells that were planted overnight for the transfection. The pLKO.1, pSPAX2, and pMD2.G vectors were mixed in a ratio of 4:3:1 in weight (14 µg/10.5 µg/3.5 µg for a 10 cm culture plate) and transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the user’s protocols. Three poly (RC) binding protein 1 (PCBP1) silencing oligos were utilized to transfect HEK293T cells together to generate a pool of lentivirus containing all three oligos. The supernatant was collected and filtered using a 0.45 μm filter. USCs were infected with the particles at the multiplicity of infection of 10. The sequences of shRNA targeting PCBP1 were as follows.

PCBP1-shRNA1 sense: 5’-GCCTACTCGATTCAAGGACAA-3’; anti-sense: 5’-TTGTCCTTGAATCGAGTAGGC-3’.

PCBP1-shRNA2 sense: 5’-CGGGTGTAAGATCAAAGAGAT-3’; anti-sense: 5’-ATCTCTTTGATCTTACACCCG-3’.

PCBP1-shRNA3 sense: 5’-GCCATCTTTAAGGCTTTCGCT-3’; anti-sense: 5’-AGCGAAAGCCTTAAAGATGGC-3’.

CCK8 assay

CCK8 assay was performed using the CCK8 kit (Biosharp Life Science, Hefei, China) according to the manufacturers’ instructions. Briefly, HMEC-1 cells were planted into 96-well plates (5 × 10³ per well). Cells were incubated with 100 µg/mL exosomes for 0, 12, 24, 48, and 72 h. Then, 10 µL of CCK8 solution was added to cells and cultured for 2 h at 37 °C. The absorbance was detected at 450 nm.

Scratch wound assay

HMEC-1 cells were treated with Mitomycin C (Sigma-Aldrich) for an hour in prior to the scratch wound assay. Cell medium was replaced with serum free medium. A pipette tip was used to create a wound when cell confluence reached to 90%. Cells were further cultured at 37 °C for another 6 h. Images were taken at 0 and 6 h post-scratch using IX53 microscope (Olympus, Tokyo, Japan). The results were quantified with the migration rate. The migration rate (%) = (wound width at 0 h - wound width at 6 h)/ wound width at 0 h *100%. The wound width was measured with Image-Pro Plus v7 software (IPWIN32.exe; Media Cybernetics, Maryland, USA).

Tube formation assay

50 µL of Matrigel were pre-coated onto the 96-well plates at 37 °C for 30 min. 8 × 10³ HMEC-1 cells were then seeded into each well of the 96-well plates, followed by the incubation with 100 µg/mL exosomes for 6 h. The tube formation was observed under a IX53 microscope. The total branching points and total loops were measured with Image-Pro Plus v7 software.

Exosome proteomic analysis

Exosome proteins were reduced with 5 mM dithiothreitol (Amresco, Atlanta, GA, USA) at 37 °C for 1 h and then incubated with 10 mM iodoacetamide (Amresco) as a decontaminant at room temperature for 45 min. The samples were diluted for four times with 25 mM of ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA). The proteins were digested by with trypsin at 50:1 overnight, which were further desalted utilizing a C18 column.

The peptides were dissolved in 10 µL of mobile phase A buffer (0.1% formic acid) and separated employing a RIGOL L-3000 ultra-high-performance liquid phase system (RIGOL Technology, Beijing, China). Mobile phase B was an acetonitrile solution containing 0.1% formic acid. The peptides were subjected to NanoSpray ionization, followed by Orbitrap Eclipse Tribrid Mass Spectrometry (Thermo Scientific) in data-dependent acquisition mode. The electrospray voltage applied was set to 2.0 kV. The data-dependent acquisition regimen included a first-order mass spectrometer survey scan at a resolution of 120,000 full width at half maximum from 350 m/z to 1500 m/z with the automatic gain control set to 4.0E5 (maximum injection time of 50 ms), followed by the top-speed mode at a resolution of 1,5000 full width at half maximum with the automatic gain control set to 5.0E4 (maximum injection time of 22 ms).

The data was search against the database of homo sapiens utilizing the Proteome Discoverer software v2.4. Principal component analysis was employed to assess the quantitative repeatability of proteins. Fold change > 2 or < 0.5, and p < 0.05 were used as screening criteria for the analysis of differentially expressed proteins. The data can be obtained from iProX database with access number IPX0016060000.

Exosome internalization assay

USCs-exo were collected and labeled utilizing a PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) following the user’s protocols. Next, 100 µg/mL of exosomes were incubated with HMEC-1 cells for 0, 3, and 6 h. Nucleus was counterstained with DAPI. Images were taken with laser confocal scanning microscope (Nikon A1, Tokyo, Japan).

Quantitative real-time PCR and RNA-sequencing

Total RNA was extracted using the TRIpure Reagent (BioTeke, Beijing, China) and further reverse transcribed into cDNA utilizing the All-in-One First-Strand SuperMix (Magen Biotechnology, Guangzhou, China) according to the manufacturers’ instructions. The amplification was carried out by using the 2×Fast Taq plus PCR Master Mix (Biosharp Life Science, Hefei, China) and SYBR Green (Solarbio). The relative expression of targeted genes was calculated with the 2^–ΔΔCT method. β-actin was used as an internal control for normalization. The sequences of primers were as follows. PCBP1 forward: 5’-AGTGCGGCTCCCTGATT-3’; PCBP1 reverse: 5’- ACGCCAGCGATGGTGAT-3’. Beta-actin forward: 5’-TCAGGGTGAGGATGCCTCTC-3’; Beta-actin reverse: 5’-CTCGTCGTCGACAACGGCT-3’.

RNA sequencing was performed by Novogene Corporation (Tianjin, China). Briefly, total RNA was extracted from HMEC-1 infected with Lv-PCBP1-OE or Lv-Vector. Selection of mRNA from total RNA was accomplished utilizing the poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was generated using the random primers and then the second strand cDNA synthesis. After terminal repair, tail addition, linker ligation, size selection, amplification and purification, the library was ready. The resulting library was further checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution. Sequencing was performed on the Illumina sequencer. Fold change > 1.5 or < 0.667; and p < 0.05 were considered differentially up-/down-regulated genes. The RNA-seq data generated from this study can be obtained from NCBI GEO database with accession number GSE325302.

Western blot

Total protein was extracted using the radioimmunoprecipitation assay buffer mixed with phenylmethanesulfonyl fluoride buffer (100:1, v/v). Protein concentration was detected with the BCA Protein Assay Kit (Proteintech). The protein was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Thermo Scientific). After blocked with 5% skim milk, membranes were incubated with the primary antibody at 4 °C overnight, followed by the incubation with the horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (1:10000; Proteintech). The bands were visualized employing the Ultrasensitive ECL Detection Kit (Proteintech) and imaged with WD-9413B Gel imaging system (Liuyi biotechnology, Beijing, China). The primary antibodies included anti-PCBP1 (1:1000), anti-HIF-1α (1:5000), anti-CD63 (1:5000), anti-CD81 (1:2000), anti-TSG101 (1:500; an internal control of exosome protein), anti-β-actin (1:1000; an internal control of cellular protein). All the primary antibodies were obtained from Proteintech except for anti-β-actin, which was obtained from CST (Danvers, MA, USA).

Gel-Pro-Analyzer software (Media Cybernetics, Bethesda, MD, USA) was used to analyze the integral optical density (IOD) of bands. Then, the IOD value was normalized with the IOD of each internal control: Relative expression = IOD of targeted protein/IOD of internal control.

Dual luciferase reporter assay

p(HA)HIF-1α (P402A, P564A) was obtained from General biology (Chouzhou, China). The P402A/P564A double mutation prevents the HIF-1α from hydroxylation and degradation. HEK293T cells were transfected with 3 µg of p(HA)HIF-1α (P402A, P564A) and pGL3-basic plasmids containing truncated PCBP1 promoter (-1832 ~ + 15, -1303 ~ + 15, -798 ~ + 15, -358 ~ + 15) using the Lipofectamine 3000. The analysis of firefly versus renilla luciferase activity was performed with the co-transfection with renilla plasmid. The luciferase activity was detected utilizing the Dual-Luciferase Reporter Assay System (Biosharp Life Science, Hefei, China) as per the users’ manuals.

Chromatin immunoprecipitation assay

The chromatin immunoprecipitation (ChIP) assay was carried out by utilizing the ChIP Assay Kit (Beyotime, Beijing, China) following the user’s protocols. Briefly, hypoxia-treated USCs were collected and soniacated to obtain DNA fragments. 20 µL of the samples was used as input. The rest of samples were immunoprecipitated with anti-HIF-1α or IgG for 60 min at 4°C. The immunoprecipitated DNA was further analyzed with PCR. The primers were as follows. Forward 5’-CCCAAGGGTAAGGAAC-3’; Reverse 5’-CCCGCTGAGGAAGTGTC-3’.

Statistical analysis

Data was expressed as mean ± standard deviation. The statistical analysis was performed using the GraphPad software v8.0 (La Jolla, San Diego, CA, America). The data were tested for normality test and homogeneity of variance. Statistical analyses of multiple groups of data were performed using one-way or two-way analysis of variance with Bonferroni correction. Student’s t-test was used to analyze the differences of data between two groups. N = 6 for in vivo experiments, and n = 3 for in vitro experiments. Statistical significance was established at p < 0.05.

Results

Identification of USCs and USCs-exo

In our previous study, we found that hypoxic preconditioning USCs accelerate wound healing [18]. Exosomes are one of the mediators for USCs to facilitate skin repair [15, 18]. Thus, we wonder whether the healing-promoting effects of hypoxia-treated USCs were mediated by exosomes. To begin with, USCs colonies were observed approximately two weeks after initial plating using light microscopy. USCs exhibited a spindle-like morphology (Fig. 1A). Flow cytometry was applied to detect the cell surface markers. The results revealed that USCs stained positive for CD29, CD73, CD90, CD44 antigen and negative for CD45 and CD34 antigen (Fig. 1B). Subsequently, exosomes were derived from USCs in the condition of hypoxia or normoxia, and the protocols were shown in Fig. 1C. USCs-exo expressed classical exosome markers including CD63, CD81, and TSG101 (Fig. 1D). Transmission electron microscopy demonstrated that the exosomes exhibited the presence of round particles with a membrane-like bilayer (Fig. 1E). The size distribution was further assessed with nanoparticle tracking analysis, which was ranged from 50 to 150 nm (Fig. 1F). These characters of USCs-exo were not markedly affected by hypoxia treatment.

Fig. 1.

Identification of USCs and USCs-exo. A USCs showed a spindle-like morphology. Scale bar: 200 μm. B Flow cytometry analysis of the cell surface marker on USCs. C The protocols of exosome extraction. D Protein expression of exosome maker CD63, CD81, and TSG101. E Morphology of USCs-exo under transmission electron Scale bar: 100 nm. F The proper size of USCs-exo. N = 3 biological replicates per group in all panels. Exo exosome, H hypoxia, N normoxia, USCs urine-derived stem cells.

Exosomes from hypoxia-treated USCs accelerate cutaneous wound healing

To investigate the effects of exosomes derived from USCs under hypoxia induction on wound healing, a full-thickness cutaneous wound was created on the back of mice, followed by the intracutaneous injection of exosomes. The wound revealed faster closure rate in mice exposed to exosomes derived from normoxia-treated USCs (N-USCs-exo), and exosomes derived from hypoxia-treated USCs (H-USCs-exo) further accelerated the process, as evidenced by the smaller wound area at 7 th and 14 th post-wound (Fig. 2A). Subsequently, H&E staining was performed to evaluate the results of skin regeneration. A more integrate cutaneous structure with newly formed epithelium and appendages was observed in mice treated with H-USCs-exo at 14 th post-wound (Fig. 2B). Then, the role of exosomes in angiogenesis was explored. The dermal microvessels were visualized with the immunostaining of CD31 (an endothelial marker expressed on the cell membrane). The expression of KI67 (a proliferative marker expressed in the nuclear) in CD31⁺ cells was increased in H-USCs-exo-treated wounds, indicative of revascularization (Fig. 2C and E). In addition, the promoted proliferation of keratinocytes (CK14⁺ positive) was found in wound site (Fig. 2D and F), suggesting the promotion of re-epithelialization.

Fig. 2.

Exosomes from hypoxia-treated USCs accelerate cutaneous wound healing. A Gross view of wounds treated with phosphate buffer saline, H-USCs-exo or N-USCs-exo at day 0, 4, 7, and 14 post-wounding. Wound edge was labeled with blue dotted line. The rate of wound-closure was calculated at the indicated times. **p < 0.01 vs. N-USCs-exo; ##p < 0.01 vs. Ctrl. Two-way ANOVA with Bonferroni correction. B H&E staining of the wound sections at day 14 post-wounding. Scale bar: 500 μm. C, D The expression of KI67 in CD31+ cells and CK14+ cells was detected by immunofluorescence staining. Scale bar: 50 μm. E The quantitative results of KI67 MFI in CD31+ cells by immunofluorescence staining. F The quantitative results of KI67 MFI in CD14+ cells by immunofluorescence staining. **p < 0.01 vs. N-USCs-exo; ##p < 0.01 vs. Ctrl. One-way ANOVA with Bonferroni correction. Data was shown as mean ± SD. N = 6 mice/group in all panels. CK14 keratin 14, Exo exosome H hypoxia, MFI mean fluorescence intensity, N normoxia, USCs urine-derived stem cells

Exosomes from hypoxia-treated USCs facilitate the angiogenic activities of microvascular endothelial cells

We further explored the function of exosomes derived from USCs under hypoxia in angiogenic activities of microvascular endothelial cells HMEC-1. CCK-8 assay demonstrated that H-USCs-exo promoted the proliferated curves of HMEC-1 cells compared to N-USCs-exo (Fig. 3A). Next, a scratch wound assay was performed to assess the migrated ability of HMEC-1 cells. The results revealed that H-USCs-exo treatment facilitated the motility of cells, as determined by the elevated migration rate (Fig. 3B, C). The angiogenesis of HMEC-1 cells was further measured utilizing the tube formation assay on Matrigel. HMEC-1 cells treated with H-USCs-exo exhibited a great number of capillary-like structures, as evidenced by the elevation of total branching points and total loops (Fig. 3D, E). The data suggested that H-USCs-exo facilitated the angiogenic activities of microvascular endothelial cells.

Fig. 3.

H-USCs-exo facilitate the angiogenic activities of endothelial cells. A The proliferation ability of endothelial cell HMEC-1 treated with phosphate buffer saline, H-USCs-exo or N-USCs-exo was measured by CCK-8 assay. *p < 0.05, **p < 0.01 vs. N-USCs-exo; #p < 0.05, ##p < 0.01 vs. Ctrl. Two-way ANOVA with Bonferroni correction. B, C The migration ability of HMEC-1 cells was investigated by wound healing assay. Scale bar: 200 μm. **p < 0.01 vs. N-USCs-exo; ##p < 0.01 vs. Ctrl. One-way ANOVA with Bonferroni correction. D Representative images of the tube formation assay. Scale bar: 100 μm. E Quantitative analyses of the total branching points and total loops. **p < 0.01 vs. N-USCs-exo; #p < 0.05 vs. Ctrl. One-way ANOVA with Bonferroni correction. Data was shown as mean ± SD. N = 3 biological replicates per group in all panels. Exo exosome, H hypoxia, N normoxia, USCs urine-derived stem cells

Proteomic analysis of exosomes from hypoxia-treated USCs

To deeply investigate the functional molecules that mediated the healing promoting effects of H-USCs-exo, proteomic analysis was performed to analyze the differentially protein expression profiles between H-USCs-exo and N-USCs-exo. The procedure of sample preparation was revealed in Fig. 4A. Principal component analysis displayed that H-USCs-exo and N-USCs-exo groups were well clustered in space, implying that each group had a distinctive expression pattern of proteins (Fig. 4B). There were 55 upregulated and 22 downregulated proteins in H-USCs-exo, which were included in the volcano plot (Fig. 4B; Supplemental file 1). Subsequently, the differentially exosome proteins were classified according to their functions. Enzymes are the most abundant, followed by RNA binding protein, cytoskeleton and organelle, transmembrane protein, cells signal transduction, transcription factors, and others (Fig. 4C).

Fig. 4.

Proteomic analysis of H-USCs-exo. A The protocols of sample preparation and proteomic analysis. B Principal component analysis and volcano plot of differentiated proteins (Fold change > 2 or < 0.5, p < 0.05). C Classification of the upregulated and downregulated proteins. D Heat map of RNA-binding proteins. The binding score of HIF-1α on their promoters were obtained from Jaspar database. E Protein expression of PCBP1 in H-USCs-exo and N-USCs-exo. **p < 0.01 vs. N-USCs-exo by Student’s t-test. Data was shown as mean ± SD. N = 3 biological replicates per group in all panels. Exo exosome, HIF-1α hypoxia inducible factor 1 subunit alpha, H hypoxia, N normoxia, PCBP1 poly (RC) binding protein 1, USCs urine-derived stem cells

RNA binding protein is a critical component of post-transcriptional regulation mechanism, involving in many steps of RNA metabolism, including RNA shearing, RNA stability and RNA translation. RNA binding protein is linked with neovascularization, and acts as a vital part in wound healing [27, 28]. Consistent with the current study, RNA bind protein has been detected in exosomes as previously described [12]. The function and regulation mechanism of RNA binding protein in exosomes attracted our great interests. There were 11 RNA binding protein that performed a heatmap according to Z-score (Fig. 4D). In our previously study, HIF-1α was upregulated in wound sites of mice treated with hypoxia-induced USCs [18]. As is known, HIF-1α is a transcription factor that is stabilized under hypoxia condition. And HIF-1α accelerates the wound healing [29]. We wonder whether these differentially expressed factors were associated with HIF-1α. We analyzed the binding sites of HIF-1α on the encoding gene promoter of these exosome proteins by Jaspar dataset. PCBP1 exhibited the highest binding score of HIF-1α (Fig. 4D). This prompted us to investigate the regulation between HIF-1α and PCBP1. Western blot further confirmed the upregulation of PCBP1 in H-USCs-exo (Fig. 4E).

Moreover, the function of other RNA binding proteins is still worth studying. There were five proteins that showed the second binding score of HIF-1α, namely: HNRNPA0, SRSF11, URB1, HNRNPH1, and IMMT (Fig. 4D). HNRNPA0 is a broad-spectrum oncogenic factor, and its tumor-promoting effects have been confirmed in gastrointestinal cancer cells, including pancreatic, esophageal, and gastric, and colorectal cancer [30]. SRSF11, an alternative splicing regulator, acts as a downstream factor of exosomes derived from M1 macrophage. Inhibition of SRSF11 mediates the facilitation of endothelial cell senescence and deep vein thrombosis [31]. URB1 has been found to ensure 3’ ETS rRNA removal to prevent exosome surveillance. Loss of URB1 leads to head malformations in zebrafish and delayed embryonic development in mice [32]. Knockdown of HNRNPH1 has been reported to enhance the endothelial to mesenchymal activation of endothelial cells, together with the promotion of tube formation and migration abilities, indicating the potential on vascular endothelial cell function [33]. IMMT is an inner membrane mitochondrial protein, participating in maintaining the mitochondrial homeostasis and function [34]. The role of these five factors in wound healing remains unclear and merits further investigation, which will be addressed in our future work.

HIF-1α promotes the transcription of PCBP1

We further explore whether the upregulation of PCBP1 in H-USCs-exo was associated with HIF-1α. Both the mRNA and protein expression of PCBP1 was upregulation in USCs in the condition of hypoxia (Fig. 5A). Unsurprisingly, the protein expression of HIF-1α was elevated by hypoxia (Fig. 5B), which was consistent with our previous study. Subsequently, we found that KC7F2 (an inhibitor of HIF-1α) downregulated the expression of PCBP1 (Fig. 5C). Co-transfection of plasmids coding normoxia-stable HIF-1α P402A/P564A together with the luciferase reporter constructs containing the PCBP1 promoter fragments resulted in a strong induction of luciferase activity (Fig. 5D). We concluded from the results that HIF-1α enhanced the PCBP1 transcription, which might be mediated by the binding to promoter fragment from − 1303 to -798. The binding of HIF-1α on the promoter of PCBP1 was further confirmed by ChIP-PCR in USCs with hypoxia induction. The results revealed that the promoter fragment of PCBP1 was co-precipitated with the anti-HIF-1α antibody compared to that with IgG (Fig. 5E). The data suggested that HIF-1α promoted the transcription of PCBP1.

Fig. 5.

HIF-1α promotes the transcription of PCBP1. A mRNA and protein expression of PCBP1 in USCs with the condition of hypoxia and normoxia. *p < 0.05, **p < 0.01 vs. normoxia by Student’s t-test. B protein expression of HIF-1α. **p < 0.01 vs. normoxia by Student’s t-test. C mRNA and protein expression of PCBP1 in USCs treated with HIF-1α inhibitor KC7F2 or DMSO under hypoxia. **p < 0.01 vs. DMSO by Student’s t-test. D Promoter deletion analysis using a dual-luciferase resporter system. Ns, not significance; **p < 0.01 by one-way ANOVA with Bonferroni correction. E The binding of HIF-1α on PCBP1 promoter was determined by ChIP-PCR assay. Data was shown as mean ± SD. N = 3 biological replicates per group in all panels. HIF-1α hypoxia inducible factor 1 subunit alpha, H hypoxia, PCBP1 poly (RC) binding protein 1, USCs urine-derived stem cells

USCs-exo deliver PCBP1 into endothelial cells

We investigated whether USCs-exo could be internalized into endothelial cells. USCs-exo was labeled with PKH67 and incubated with HMEC-1 cells. Fluorescence microscopy analysis demonstrated that the PKH67-labeled USCs-exo transferred to the perinuclear region of HMEC-1 cells at 3 h and 6 h post-incubation in a time-dependent manner (Fig. 6A). At the same time, the protein expression of PCBP1 was upregulated in HMEC-1 cells (Fig. 6B). Then, lentivirus plasmids containing shRNA targeting PCBP1 were constructed, and lentivirus particles were infected with USCs. The protein levels of PCBP1 were downregulated both in hypoxia-treated USCs with PCBP1 knockdown and their exosomes (Fig. 6C, D). After incubated with the exosomes for 3–6 h, the expression of PCBP1 was decreased in HMEC-1 cells (Fig. 6E). The shuttle of exosomes from donor cell USCs to recipient cell HMEC-1 cells was shown in Fig. 6F. These result implied that USCs-exo delivered PCBP1 into microvascular endothelial cells.

Fig. 6.

USCs-exo deliver PCBP1 into endothelial cells. A Fluorescence microscopy analysis of PKH67-labeled USCs-Exo internalization by HMEC-1 cells at 0, 3, 6 h. Scale bar: 50 μm. B The protein expression of PCBP1 in HMEC-1 cells. **p < 0.01 vs. H-exo-0 h by one-way ANOVA with Bonferroni correction. C The protein expression of PCBP1 in USCs transfected with Lv-shPCBP1 or Lv-NC. **p < 0.01 vs. Lv-NC by one-way ANOVA with Bonferroni correction. D The protein expression of PCBP1 in exosomes secreted from USCs transfected with Lv-shPCBP1 or Lv-NC under hypoxia. **p < 0.01 vs. H-Lv-NCexo by Student’s t-test. E The protein expression of PCBP1 in HMEC-1 cells treated with exosomes at the indicated time. **p < 0.01 vs. H-Lv-NCexo by Student’s t-test. F Schematic diagram of the internalization of exosomes. Data was shown as mean ± SD. N = 3 biological replicates per group in all panels. Exo exosome, H hypoxia, MVBs multivesicular bodies, PCBP1 poly (RC) binding protein 1, USCs urine-derived stem cells

PCBP1 mediates the pro-angiogenic effects of hypoxia-treated USCs-exo on microvascular endothelial cells

We further investigated whether exosomal PCBP1 mediated the healing promoting and pro-angiogenic effects of H-USCs-exo. A reduced proliferation ability was observed in HMEC-1 cells incubated with exosomes derived from hypoxia-treated USCs with PCBP1 knockdown (H-Lv-shPCBP1^exo) (Fig. 7A). Treated with H-Lv-shPCBP1^exo repressed the migration and angiogenesis ability of HMEC-1 cells, as evidenced by the reduced migration rate, the decreased number of total loops and branching points (Fig. 7B–F). The data suggested that exosomal PCBP1 at least partly mediated the pro-angiogenic effects of hypoxia-treated USCs-exo on microvascular endothelial cells.

Fig. 7.

PCBP1 mediates the pro-angiogenic effects of H-USCs-exo on endothelial cells. A The proliferation ability of HMEC-1 cells treated with H-Lv-shNCexo or H-Lv-shPCBP1exo was measured by CCK-8 assay. *p < 0.05, **p < 0.01 vs. H-Lv-NCexo by two-way ANOVA with Bonferroni correction. B, C The migration of cells was evaluated by wound healing assay. Scale bar: 200 μm. **p < 0.01 vs. H-Lv-NCexo by Student’s t-test. D-F The angiogenic ability of cells was investigated by tube formation assay, which was further quantified by the total branching points and total loops. Scale bar: 100 μm. **p < 0.01 vs. H-Lv-NCexo by Student’s t-test. Data was shown as mean ± SD. N = 3 biological replicates per group in all panels. Exo exosome, H hypoxia, PCBP1 poly (RC) binding protein 1, USCs urine-derived stem cells

Exosomal PCBP1 potentiates wound healing in vivo

Given the observation that exosomal PCBP1 facilitated the angiogenesis of HMEC-1 cells in vitro, we wonder whether PCBP1 also play a role in wound healing in vivo. Treated with H-Lv-shPCBP1^exo slowed the wound closure rate (Fig. 8A). H&E demonstrated a decrease of skin regeneration in mice injected with H-Lv-shPCBP1^exo (Fig. 8B). KI67-positive cells were reduced in wound sites (Fig. 8C). The data indicated that H-Lv-shPCBP1^exo delayed the wound healing in vivo.

Fig. 8.

Exosomal PCBP1 potentiates wound healing in vivo. A Gross view and wound closure rate of wounds treated with H-Lv-shNCexo or H-Lv-shPCBP1exo at day 0, 7, and 14 post wounding. Wound edge was labeled with blue dotted line. **p < 0.01 vs. H-Lv-NCexo by two-way ANOVA with Bonferroni correction. B H&E staining of the wound sections. Scale bar: 500 μm. C The expression of KI67 was detected by immunofluorescence staining. Scale bar: 50 μm. Data was shown as mean ± SD. N = 6 mice/group in all panels. Exo exosome, H hypoxia, PCBP1 poly (RC) binding protein 1, USCs urine-derived stem cells

PCBP1 facilitates the WNT5A signaling pathways

To investigate the underlying mechanism of the pro-angiogenesis effects of PCBP1, RNA-sequencing was performed. HMEC-1 cells were infected with lentivirus vector carrying a fragment of the PCBP1 CDS to mimic the delivery of exosomal PCBP1 into endothelial cells. The samples were prepared shown in Fig. 9A. Principal component analysis revealed that Lv-vector and Lv-PCPB1-OE groups had a distinctive expression pattern of mRNAs (Fig. 9B). As illustrated in the volcano plot, we screened 2854 upregulated genes and 2325 downregulated genes (Fig. 9C; Supplemental file 1). To analyze the biological function of the differentially expressed genes further, we conducted Gene Ontology enrichment analysis with cellular component, molecular functions, biological processes analysis. We found that mRNAs were mainly enriched in items related to wound healing (such as “wound healing”), angiogenesis (such as “endothelial cell migration” and “vascular endothelial growth factor receptor signaling pathway”), WNT signaling (such as “non-canonical WNT signaling pathway” and “WNT signalosome”), and RNA metabolism (such as “mRNA 5’-UTR binding”) (Fig. 9D). The data indicated the potential of downstream factors of PCBP1 on angiogenesis and provided direction for further investigation. As is known, WNT5A-mediated non-canonical WNT signaling pathway exhibits pro-wound healing and pro-angiogenesis function [35–37], which evoked us much attention. We wonder whether the effects of PCBP1 were associated with WNT5A signaling. The expression of WNT5A and its downstream factors involving in angiogenesis were confirmed. According to the results from RNA-seq, the expression of WNT5A, vascular endothelial growth factor A (VEGFA), and TEK receptor tyrosine kinase (TEK) were upregulated in HMEC-1 cells transfected with Lv-PCBP1-OE (Fig. 9E). The data suggested that exosome-shuttled PCBP1 from hypoxic preconditioned USCs accelerated wound healing, which might be associated with the WNT5A signaling pathways (Fig. 10).

Fig. 9.

Downstream signaling of PCBP1 by RNA sequencing. A Protocols of sample preparing for RNA sequencing. B Principal component analysis to identify the samples from HMEC-1 cells infected with Lv-Vector or Lv-PCBP1-OE. C Volcano plot exhibited the differentially expressed gene (Fold change > 1.5 or < 0.667, p value < 0.05). D Gene ontology enrichment analysis of differentially expressed gene. E The expression of WNT5A and its downstream VEGFA and TEK. N = 3 biological replicates per group in all panels. PCBP1 poly (RC) binding protein 1, TEK TEK receptor tyrosine kinase, VEGFA vascular endothelial growth factor A, WNT5A Wnt family member 5 A

Fig. 10.

Schematic diagram of the potential underlying mechanism of exosomes derived from USCs with hypoxia treatment. HIF-1α hypoxia inducible factor 1 subunit alpha, H hypoxia, PCBP1 poly (RC) binding protein 1, MVBs multivesicular bodies, USCs urine-derived stem cells

Discussion

In our previous study, we found that hypoxic preconditioning USCs accelerated wound healing. Exosomes are one of the mediators of USCs to facilitate wound healing. Thus, we wonder whether the healing-promoting effects of hypoxia-treated USCs were mediated by exosomes. In the current study, exosomes derived from hypoxia-treated USCs accelerated the cutaneous wound healing in vivo and enhanced the angiogenic ability of HMEC-1 cells in vitro. Using exosome Label-free proteomics, a protein named PCBP1 was found to be significantly upregulated in H-USCs-exo. Exosomes delivered PCBP1 into endothelial cells, and exosomal PCBP1 mediated the pro-angiogenic effects of H-USCs-exo on endothelial cells. The similar results were obtained in vivo. In addition, the hypoxia response element HIF-1α was upregulated in USCs under hypoxia condition. HIF-1α potentiated the transcription of PCBP1, leading to the high abundance of PCBP1 in H-USCs and H-USCs-exo. Further high-throughput transcriptomics illustrated that PCBP1 upregulated the expression of WNT5A and its downstream factors including VEGFA and TEK.

PCBP1 is an RNA-binding protein containing KH-RNA binding domain. Consistent with the current study, RNA bind protein and enzymes have been detected in exosomes in previous study [12]. Exosomal PCBP1 was found to be upregulated in H-USCs-exo. PCBP1 has been identified as a multilevel regulator of gene expression and protein function, operating at multiple levels to influence cellular processes. For instance, hepatic PCBP1 deletion triggers chronic liver disease in mice, with steatosis and inflammation [38, 39]. Knockdown of PCBP1 aggravates ox-LDL induced apoptosis and inhibited cell viability of human umbilical vein endothelial cells, which might contribute to the progression of atherosclerosis [40]. Similarly, herein, treated with H-Lv-shPCBP1^exo repressed the proliferation, migration and angiogenesis ability of HMEC-1 cells. H-Lv-shPCBP1^exo also slowed the wound healing in vivo. The data suggested that exosomal PCBP1 at least partly mediates the healing-promoting effects of hypoxia-treated USCs-exo.

The elevated proliferation of keratinocytes induced by H-USCs-exo was found in wound site, suggesting the promotion of re-epithelialization. H-USCs-exo might either direct or indirect affects the keratinocytes. On one hand, exosomes derived from USCs are reported to be internalization by human keratinocytes cell line [15]. Together with our results, the promoted proliferation of keratinocytes induced by N-USCs-exo was further enhanced by H-USCs-exo. It is possible that H-USCs-exo might directly affect the keratinocytes. On the other hand, we found that H-USCs-exo facilitated the revascularization via the internalization by endothelial cells. Endothelial cells and angiogenesis critically support re-epithelialization by supplying oxygen and nutrients to fuel keratinocyte proliferation [41, 42]. They secrete paracrine signals that directly stimulate keratinocyte migration and division. New vessels also create a provisional matrix that guides keratinocyte movement across the wound bed. Thus, we had reason to speculated that H-USCs-exo might also indirectly affects keratinocyte proliferation by the promotion of angiogenesis.

In addition to keratinocytes and endothelial cells, fibroblasts are indispensable for wound healing, as they deposit collagen, drive wound contraction, and cross-talk with other cell types to coordinate tissue repair [43]. In the current study, given the observed pro-revascularization effects of H-USCs-exo, it is plausible that these exosomes may also directly or indirectly modulate fibroblast functions. Directly, exosomal cargo from USCs could be internalized by fibroblasts, potentially regulating their migratory and proliferative capacity [15], as PCBP1 has been reported to participate in post-transcriptional regulation of genes involved in cell motility, extracellular matrix production, and cytoskeletal organization, which are fundamental to fibroblast migration, collagen secretion, and wound contraction [44, 45]. Indirectly, during wound healing, fibroblasts depend critically on angiogenesis. The oxygen and nutrients delivered by newly formed vessels enable them to proliferate and synthesize the extracellular matrix [43, 46]. By promoting angiogenesis, H-USCs-exo may improve the local nutrient and oxygen supply, thereby supporting fibroblast survival and biosynthetic activity. Given that PCBP1 was identified as a key mediator of the pro-angiogenic action of H-USCs-exo, it is tempting to speculate that exosomal PCBP1 may exert dual functions: while promoting endothelial cell tube formation and migration, it might concurrently influence fibroblast behavior, either through shared signaling pathways (e.g., WNT5A signaling) or via distinct intracellular targets. Future studies are warranted to delineate whether exosomal PCBP1 directly acts on fibroblasts and to determine its specific effects on fibroblast proliferation, migration, collagen deposition, and contraction, which would provide a more comprehensive understanding of the therapeutic potential of H-USCs-exo in wound healing.

HIF-1α is a hypoxia-regulated transcriptional activator involving in the hypoxia responses. HIF-1α is subjected to oxygen-dependent ubiquitination and proteasomal degradation by the Von Hippel-Lindau complex [47]. Under hypoxic condition, HIF-1α is stable [47], which is consistent with the results of USCs in the current study. Stabilizing HIF-1α-induced healing-facilitating effects have been reported extensively [29, 48, 49]. We wonder whether the upregulation of PCBP1 was associated with HIF-1α. The results revealed that HIF-1α potentiated the transcription of PCBP1, which might result in the high abundance of PCBP1 in H-USCs and H-USCs-exo.

According to the exosome Label-free proteomics, there were 55 upregulated and 22 downregulated proteins in exosomes derived from USCs pretreated with hypoxia. Among them, there were ten RNA binding proteins except for PCBP1, which were still worth studying. RNA-binding proteins are a crucial class of proteins that interact with RNA molecules to regulate their function, stability, localization, and processing [50]. RNA binding protein involves in neovascularization, and plays an essential role in wound repair [27, 28]. In the current study, the highest up-regulated RNA binding protein in exosomes is HNRNPA0, which is found to enhances the excessive mitosis and growth in colorectal cancer cells [30]. Interestingly, in lung cancer cell line A549 cells, chemical hypoxia did not downregulated the expression of HNRNPA0 [51]. Exosome biogenesis is a tightly regulated process that involves several key steps: formation of early endosomes, formation of multivesicular bodies, and exosome secretion [12]. We speculated that hypoxia might mainly regulate the cargo sorting of HNRNPA0 during exosome formation or the exosome secretion process, but not its transcription process. In addition, the second up-regulated factor in exosomes is CPSF6. Knockdown of CPSF6 triggers antiviral immune responses [52]. Degradation of CPSF6 facilitates alternative polyadenylation and antivirus effects of macrophages [53]. These studies indicate that CPSF6 might serve as a target for developing immune interventions. The expression changes and function of these proteins in exosomes derived from USCs pretreated with hypoxia condition were worth studying.

The WNT family comprises a large group of cysteine-rich secreted glycoproteins that act as a vital part in the regulation cell proliferation, differentiation, developmental patterning, and morphogenetic movement. In general, WNT signaling is divided into two subclasses: β-catenin-dependent signaling (canonical WNT signaling) and β-catenin-independent signaling (non-canonical WNT signaling) [37]. In the current study, differentially expression mRNAs from RNA-sequencing were enriched in non-canonical WNT signaling pathway, implying us that the pro-angiogenesis effects of exosomal PCBP1 might be associated with non-canonical WNT signaling. WNT5A belongs to the noncanonical WNT family and acts as an essential role in the regulation of vascular homeostasis. WNT5A enhances endothelial cell proliferation and reduces the cell apoptosis by the increased expression of TEK [54]. WNT5A also upregulates the VEGFA signaling, which is a well-known angiogenic factor participating in endothelial cell proliferation and migration [37, 55]. Knockdown of WNT5A inhibits the monolayer wound closure [35]. These results indicate the promoting role of WNT5A in angiogenesis and wound healing. Herein, we found that PCBP1 upregulated the expression of WNT5A, TEK, and VEGFA according to the results of RNA-sequencing. We had reason to believe that WNT5A signaling pathway might be at least partly mediated the pro-angiogenesis function of PCBP1.

There were two limitations of the present study. On one hand, the in vivo experiments lack a blank PBS control, which is essential for a complete assessment of exosome effects. On the other hand, the results of RNA-sequencing showed that PCBP1 increased the expression levels of WNT5A and its downstream factors, including TEK and VEGFA, indicating the potential activation of the WNT5A signaling pathway. However, we neither measured the protein expression of WNT5A and its downstream factors nor performed rescue experiments to confirm the function. The direct molecular interaction between PCBP1 and WNT5A remains unclear. These findings attract our attention and warrant further investigation in our future studies.

Conclusion

We presented a non-invasive and cell-free therapeutic method for the wound healing, and the delivery of exosomal PCBP1 mediate the therapeutic efficacy induced by exosomes derived from USCs pretreated with hypoxia.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ChIP

Chromatin immunoprecipitation

HIF-1α

Hypoxia inducible factor 1 subunit alpha

PBS

Phosphate buffer saline

PCBP1

Poly (RC) binding protein 1

TEK

TEK receptor tyrosine kinase

USCs

Urine-derived stem cells

VEGFA

Vascular endothelial growth factor A

Author contributions

XZ: Conceptualization, Investigation, Methodology, Supervision, and Writing - Review & Editing. SZ: Conceptualization, Investigation, Methodology, and Writing - Original Draft. YG: Conceptualization and Methodology. KG and JS: Conceptualization and Investigation. KM and ZG: Conceptualization and Formal analysis.

Funding

This research was supported by Henan Province Science and Technology Project (Grant No. 242102311144).

Data availability

The data of this work is available on request to the corresponding author. The datasets (proteomics and RNA-seq) supporting the conclusions of this article are included within the supplemental files.

Declarations

Ethics approval and consent to participate

USCs were isolated from urine samples of four healthy adults. This study was approved by the Institutional Review Board of Zhengzhou University following the Declaration of Helsinki. The title of the approved project: The effects of exosomes derived from hypoxia-treated human urine-derived stem cells on wound healing. Data of the approval: March 1 (st), 2023. Written informed consent was obtained from the donors. Animal experiments were approved by the Institutional Review Board of Zhengzhou University following The Guideline for the Care and Use of Laboratory Animals. The title of the approved project: The effects of exosomes derived from hypoxia-treated human urine-derived stem cells on wound healing. Data of the approval: March 1 (st), 2023.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.