CD271+ Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promote Angiogenesis via miR-124-3p Mediated PGF Targeting

CD271+ Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promote Angiogenesis via miR-124-3p Mediated PGF Targeting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CD271+ Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promote Angiogenesis via miR-124-3p Mediated PGF Targeting Jing Zhu, Yujie Hang, Nianchun Shan, Chun Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-10006473/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Endometriosis, affecting approximately 10% of reproductive-age women, is critically dependent on angiogenesis for ectopic lesion establishment and growth. While exosomes from mesenchymal stem cells (MSCs) exhibit pro-angiogenic properties, the functional heterogeneity of MSC subpopulations and their distinct angiogenic mechanisms remain poorly understood. CD271 marks a UCMSC subpopulation with enhanced regenerative potential, yet its role in exosome-mediated angiogenesis remains unexplored. Methods Exosomes were isolated from CD271 + UCMSCs via ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. End.3 endothelial cells were treated with UCMSC-Exos or CD271 + UCMSC-Exos, and functional angiogenesis was assessed by scratch wound healing, Transwell migration, and tube formation assays. miRNA cargo was profiled by qRT-PCR, and miR-124-3p inhibition experiments were performed to confirm functional specificity. Putative miR-124-3p targets were identified using miRDB, TargetScan, and the HARRIS_HYPOXIA gene set, and validated by dual-luciferase reporter assay, western blotting, and PGF overexpression rescue experiments. Public transcriptomic datasets (GSE25628) were analyzed for external validation. Results CD271 + UCMSC-Exos were efficiently internalized by End.3 cells and significantly enhanced cell migration and tube formation compared with UCMSC-Exos. miR-124-3p was enriched in CD271 + UCMSC-Exos. Dual-luciferase reporter assays confirmed that miR-124-3p directly targets the 3'-UTR of PGF, leading to post-transcriptional suppression of PGF protein expression. PGF overexpression reversed this effect. miR-124-3p targets were enriched in angiogenesis-related KEGG pathways (HIF-1, VEGF, Rap1; all FDR < 0.05). External validation in endometriosis tissues (GSE25628) confirmed PGF downregulation in ectopic lesions (log2FC = -0.705) with, while angiogenic markers including VEGFA, KDR, and FLT1 were upregulated in the same lesions. Conclusion CD271 + UCMSC-Exos promote angiogenesis through delivery of miR-124-3p, which directly targets PGF in endothelial cells. External transcriptomic validation supports the clinical relevance of this axis in endometriosis, suggesting therapeutic potential. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology Biological sciences/Stem cells Endometriosis CD271 Mesenchymal stem cells Exosomes miR-124-3p Placental growth factor Angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Endometriosis, a common gynecological disorder affecting approximately 10% of reproductive-age women, is characterized by the implantation and growth of endometrial-like tissue outside the uterine cavity [ 1 , 2 ]. The disease imposes substantial physical, psychological, and economic burdens, with diagnosis often delayed by 7–10 years [ 2 ]. Current medical therapies relying on hormonal suppression and surgical excision are limited by high recurrence rates and adverse effects, underscoring the need for mechanism-based therapeutic strategies [ 3 ]. Angiogenesis is a fundamental prerequisite for endometriotic lesion survival and growth [ 4 ]. The peritoneal microenvironment provides a rich milieu of pro-angiogenic factors that facilitate neovascularization of ectopic implants. Among these, placental growth factor (PGF), a member of the VEGF family, has emerged as a key modulator of pathological angiogenesis, acting through VEGFR-1 (FLT1) to amplify VEGF-driven signaling and recruit bone marrow-derived angiogenic cells [ 5 , 20 ]. Mesenchymal stem cell-derived extracellular vesicles, particularly exosomes, have attracted considerable attention as vehicles for intercellular communication in angiogenesis [ 6 – 8 ]. MSC-derived exosomes carry diverse cargo including microRNAs (miRNAs), which can post-transcriptionally regulate angiogenic gene programs in recipient endothelial cells [ 9 , 10 , 16 – 18 ]. Recent studies have demonstrated that exosomes from specific MSC subpopulations exhibit distinct therapeutic profiles [ 11 , 12 ]. CD271 (p75 neurotrophin receptor, NGFR) marks a subset of UCMSCs with enhanced self-renewal, multilineage differentiation, and paracrine activity [ 13 , 14 ]. However, the angiogenic function and molecular mechanisms of CD271 + UCMSC-derived exosomes remain unexplored. In this study, we investigated the pro-angiogenic effects of CD271 + UCMSC-Exos on endothelial cells, identified miR-124-3p as a key effector miRNA cargo, and established PGF as its direct downstream target. Furthermore, we integrated bioinformatics pathway analysis and external endometriosis transcriptomic validation to strengthen the translational relevance of this axis. Materials and Methods Cell culture The mouse microvascular endothelial cell line End.3 was purchased from Procell (China) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified 5% CO₂ atmosphere. CD271 + and unsorted UCMSCs were cultured as previously described [ 15 ]. The End.3 cell line was selected because (i) the miR-124-3p/PGF axis is conserved between mouse and human, (ii) it has been extensively used in published endothelial angiogenesis studies, and (iii) key angiogenic pathways (VEGF, HIF-1, PI3K-Akt) are functionally conserved in this line. Exosome isolation and characterization Exosomes were isolated from the conditioned medium of CD271 + UCMSCs and unsorted UCMSCs by differential ultracentrifugation. Briefly, culture supernatant was subjected to sequential centrifugation at 300 × g for 10 min, 2,000 × g for 20 min, and 10,000 × g for 30 min to remove cells and debris, followed by ultracentrifugation at 100,000 × g for 70 min at 4°C. The exosome pellet was washed with PBS and re-ultracentrifuged. Exosome morphology was examined by TEM after negative staining with uranyl acetate. Particle size distribution and concentration were determined by NTA (NanoSight NS300, Malvern). Exosome markers CD63, CD81, and CD9 were detected by western blotting. Exosome uptake assay Isolated exosomes were labeled with PKH26 fluorescent dye (Sigma-Aldrich) according to the manufacturer's protocol. Labeled exosomes (10 µg/mL) were added to End.3 cells and incubated for 24 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and nuclei were counterstained with DAPI. Fluorescence images were captured using a confocal microscope (Leica). Scratch wound healing assay End.3 cells were seeded in 6-well plates at 2 × 10⁵ cells/well and cultured to confluence. A linear scratch was created using a sterile 200 µL pipette tip. Cells were washed with PBS and cultured in serum-free medium containing UCMSC-Exos or CD271 + UCMSC-Exos (50 µg/mL). Images were captured at 0 h and 24 h, and wound closure was quantified using ImageJ. Transwell migration assay End.3 cells were serum-starved for 12 h, harvested, and resuspended in serum-free medium at 1 × 10⁵ cells/mL. Cell suspension (200 µL) was added to the upper chamber of a 24-well Transwell insert (8 µm pore size, Corning), while 600 µL of medium containing exosomes (50 µg/mL) was added to the lower chamber. After 24 h incubation, cells on the upper surface were removed with a cotton swab, and migrated cells on the lower surface were fixed, stained with 0.1% crystal violet, and counted in five random fields at 200× magnification. Tube formation assay Matrigel (Corning) was thawed at 4°C and added to a pre-chilled 96-well plate (50 µL/well), then polymerized at 37°C for 30 min. End.3 cells (2 × 10⁴ cells/well) were seeded onto the Matrigel in serum-free medium containing exosomes (50 µg/mL). After 6 h, tube formation was imaged and quantified by measuring total tube length and branch points using ImageJ Angiogenesis Analyzer. qRT-PCR Total RNA was extracted from exosomes and cells using TRIzol reagent (Invitrogen). For miRNA detection, cDNA was synthesized using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa), and qRT-PCR was performed with TB Green Premix Ex Taq II (TaKaRa) on a QuantStudio 5 system. U6 was used as an internal control for miRNA. Relative expression was calculated using the 2⁻ΔΔCt method. miR-124-3p inhibition End.3 cells at 70% confluence were transfected with miR-124-3p inhibitor or negative control inhibitor (50 nM, RiboBio) using Lipofectamine 3000 (Invitrogen). After 24 h of transfection, cells were treated with CD271 + UCMSC-Exos (50 µg/mL) for the indicated time points prior to downstream functional assays. Dual-luciferase reporter assay Wild-type (WT) and mutant (MUT) PGF 3'-UTR sequences containing the predicted miR-124-3p binding site were cloned into the pmirGLO dual-luciferase reporter vector (Promega). End.3 cells were co-transfected with the reporter construct and miR-124-3p mimic or negative control (50 nM) using Lipofectamine 3000. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega). PGF overexpression PGF overexpression plasmid (pcDNA3.1-PGF) was constructed by Guangzhou RiboBio. End.3 cells were transfected with pcDNA3.1-PGF or empty vector (2 µg/well) using Lipofectamine 3000. At 24 h post-transfection, cells were treated with CD271 + UCMSC-Exos for downstream functional assays. Bioinformatics pathway enrichment analysis To validate the biological relevance of the miR-124-3p/PGF axis, we performed bioinformatics analysis. Validated miR-124-3p target genes were retrieved from miRTarBase [ 23 ], TargetScan, and miRDB [ 22 ]. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using clusterProfiler in R. Significantly enriched angiogenesis-related pathways were visualized using bubble plots (FDR < 0.05). PGF was confirmed as a validated target of miR-124-3p in the miRTarBase database. The HARRIS_HYPOXIA gene set was included because endometriosis is a hypoxia-driven disease, and genes overlapping between hypoxia signatures and miR-124-3p targets may identify biologically relevant angiogenic effectors in the endometriotic microenvironment. External endometriosis transcriptomic validation To assess the clinical relevance of identified angiogenic markers, we analyzed published transcriptomic datasets of endometriosis (GSE25628 [ 24 , 25 ]). Differential expression of PGF, PECAM1 (CD31), VEGFA, KDR, and FLT1 between ectopic and eutopic endometrium was examined. Correlations between PGF expression and angiogenic markers were calculated using Pearson correlation coefficients in endometriosis tissue samples. Western blotting Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was measured by BCA assay. Protein lysates (30 µg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated with primary antibodies against CD63 (1:1000), CD81 (1:1000), CD9 (1:1000), PGF (1:1000), and GAPDH (1:5000) overnight at 4°C. Following HRP-conjugated secondary antibody incubation, signals were detected using enhanced chemiluminescence (ECL). Statistical analysis Data are expressed as mean ± standard deviation (SD) and analyzed using GraphPad Prism 8. Statistical comparisons between two groups were performed using Student's t-test, and multiple group comparisons used one-way ANOVA with Tukey's post hoc test. P < 0.05 was considered statistically significant. All experiments were performed in at least three independent biological replicates. Results Isolation and characterization of CD271 + UCMSC-derived exosomes Exosomes isolated from both UCMSCs and CD271 + UCMSCs exhibited the characteristic cup-shaped morphology under TEM, with particle sizes ranging from 30–150 nm as determined by NTA (Fig. 1 A). Western blot analysis confirmed the expression of canonical exosomal markers CD63, CD81, and CD9 in both exosome groups (Fig. 1 B). Immunofluorescence imaging demonstrated that PKH26-labeled CD271 + UCMSC-Exos (red) were efficiently internalized by End.3 endothelial cells stained with CD31 (green), with nuclei counterstained with DAPI (Fig. 1 C, D). qRT-PCR analysis revealed that miR-124-3p was significantly enriched in CD271 + UCMSCs compared with unsorted UCMSCs (Fig. 1 E, P < 0.01) and in their secreted exosomes (Fig. 1 F, P < 0.001). CD271 + UCMSC-Exos promote endothelial cell migration and tube formation To evaluate the pro-angiogenic effects of CD271 + UCMSC-Exos, End.3 cells were treated with PBS (Control), UCMSC-Exos, or CD271 + UCMSC-Exos. Scratch wound healing assays demonstrated significantly accelerated wound closure in the CD271 + UCMSC-Exo group compared with the UCMSC-Exo and control groups (Fig. 2 A, B; P < 0.01). Transwell migration assays showed a marked increase in the number of migrated cells in the CD271 + UCMSC-Exo group (Fig. 2 C, D; P < 0.001). In tube formation assays on Matrigel, CD271 + UCMSC-Exo treatment significantly enhanced both total tube length and the number of branch points compared with UCMSC-Exos (Fig. 2 E-G; P < 0.001), indicating superior pro-angiogenic capacity. miR-124-3p mediates the pro-angiogenic effect of CD271 + UCMSC-Exos miR-124-3p was significantly enriched in CD271 + UCMSC-Exos compared with UCMSC-Exos (P < 0.01). To confirm its functional role, End.3 cells were transfected with a miR-124-3p inhibitor prior to CD271 + UCMSC-Exo treatment. Scratch wound healing assays demonstrated that miR-124-3p inhibition significantly attenuated CD271 + UCMSC-Exo-induced wound closure (Fig. 3 A, B; P < 0.01). Transwell migration assays showed a marked reduction in migrated cell numbers upon miR-124-3p inhibition (Fig. 3 C, D; P < 0.01). Tube formation assays further confirmed that miR-124-3p inhibition substantially reduced total tube length and the number of meshes compared with CD271 + UCMSC-Exo treatment alone (Fig. 3 E-G; P < 0.001). These results establish miR-124-3p as a key functional mediator of CD271 + UCMSC-Exo-induced angiogenesis. PGF is a direct target of miR-124-3p Analysis of three miRNA target prediction databases (miRDB, TargetScan, and the HARRIS_HYPOXIA gene set) identified PGF as a conserved target of miR-124-3p (Fig. 4 A). Dual-luciferase reporter assays demonstrated that miR-124-3p mimic significantly repressed the luciferase activity of the wild-type PGF 3'-UTR reporter (P < 0.001), while the mutant reporter was unaffected (Fig. 4 B). Consistently, CD271 + UCMSC-Exo treatment markedly reduced PGF protein expression in End.3 cells, an effect partially reversed by miR-124-3p inhibitor (Fig. 4 C, D). qRT-PCR confirmed reduced PGF mRNA after CD271 + UCMSC-Exo treatment (Fig. 4 E; P < 0.05). Tube formation assays showed that either inhibition of miR-124-3p or overexpression of PGF attenuated the pro-angiogenic effects (Fig. 4 F). PGF overexpression reverses CD271 + UCMSC-Exo-induced angiogenesis To confirm that PGF is the functional downstream effector of the miR-124-3p-mediated angiogenic response, we overexpressed PGF in End.3 cells. CD271 + UCMSC-Exo treatment significantly reduced PGF protein expression compared with the PBS control group (Fig. 5 A, B; P < 0.01). Dual-luciferase reporter assays confirmed direct miR-124-3p binding to the wild-type PGF 3'-UTR but not the mutant construct (Fig. 5 C; P < 0.01). qRT-PCR analysis verified that CD271 + UCMSC-Exo treatment significantly reduced PGF mRNA levels (Fig. 5 D; P < 0.01). In functional tube formation assays, CD271 + UCMSC-Exo promoted endothelial network formation, while PGF overexpression significantly attenuated this effect, as demonstrated by representative images (Fig. 5 E) and quantitative analysis of total tube length (Fig. 5 F) and branch points (Fig. 5 G; P < 0.01). These results establish PGF as a functional downstream target of miR-124-3p in CD271 + UCMSC-Exo-mediated angiogenesis. Bioinformatics validation of the miR-124-3p angiogenic regulatory network To extend the mechanistic findings beyond the single miR-124-3p/PGF axis, we performed systematic pathway enrichment analysis. Integrated analysis of miR-124-3p validated targets from miRTarBase, TargetScan, and miRDB identified 102 target genes, among which PGF was confirmed. KEGG pathway enrichment analysis revealed significant enrichment of miR-124-3p targets in multiple angiogenesis-related pathways, including HIF-1 signaling (FDR = 0.003), VEGF signaling (FDR = 0.008), Rap1 signaling (FDR = 0.012), PI3K-Akt signaling (FDR = 0.018), and focal adhesion (FDR = 0.025) (Fig. 6 A). GO enrichment analysis further revealed overrepresentation in biological processes including endothelial cell migration, blood vessel morphogenesis, and positive regulation of angiogenesis (all FDR < 0.01). These data establish that miR-124-3p broadly regulates angiogenic gene programs beyond PGF, positioning the miR-124-3p/PGF axis within a larger angiogenic regulatory network. To validate the clinical relevance of our findings, we analyzed public endometriosis transcriptomic data. Analysis of GSE25628 revealed PGF downregulation in ectopic versus eutopic endometrium (log2FC = -0.705), consistent with miR-124-3p-mediated suppression. In contrast, VEGFA, KDR, FLT1, MMP9, and ANG2 exhibited upregulation in ectopic lesions (Fig. 6 B), reflecting an active angiogenic state. This pattern—PGF suppression amid broader angiogenic activation—is mechanistically consistent with miR-124-3p targeting PGF while compensatory VEGF family signaling sustains angiogenesis (Fig. 6 C). These results provide independent clinical evidence that the PGF-centered angiogenic program is active in endometriotic lesions. Discussion This study demonstrates that CD271 + UCMSC-derived exosomes promote angiogenesis by delivering miR-124-3p, which directly targets and suppresses PGF expression in endothelial cells. Our findings establish a previously unrecognized miR-124-3p/PGF regulatory axis that underlies the enhanced pro-angiogenic capacity of the CD271 + MSC subpopulation. The CD271 + subpopulation of UCMSCs has been characterized by enhanced self-renewal, multilineage differentiation potential, and superior paracrine activity compared with unsorted MSCs [ 13 , 14 ]. Our finding that CD271 + UCMSC-Exos exhibit significantly stronger pro-angiogenic effects than unsorted UCMSC-Exos extends this functional specialization to the exosome-mediated mode of intercellular communication. This aligns with recent studies demonstrating that exosomes from other MSC subpopulations, such as CD146 + UCMSCs, possess distinct therapeutic activities in tissue repair contexts [ 11 , 15 , 19 , 21 ]. The enrichment of miR-124-3p in CD271 + UCMSC-Exos provides a molecular basis for this subpopulation-specific function and suggests that sorting MSCs by surface markers may be a strategy to obtain exosomes with defined therapeutic properties. The molecular basis for miR-124-3p enrichment in the CD271 + subpopulation remains to be determined. Possible mechanisms include CD271-mediated transcriptional regulation of the pri-miR-124 locus via NF-kB or AP-1 signaling, or selective sorting of miR-124-3p into exosomes through RNA-binding proteins such as hnRNPA2B1. Elucidating this enrichment mechanism may enable engineering of UCMSCs for enhanced therapeutic miRNA loading. The identification of PGF as a direct target of miR-124-3p adds mechanistic depth to the angiogenic function of CD271 + UCMSC-Exos. PGF, as a VEGF family member, primarily binds VEGFR-1 (FLT1) and potentiates VEGF-A-driven angiogenesis by displacing VEGF-A from VEGFR-1, thereby increasing VEGF-A availability for VEGFR-2 activation [ 5 ]. Beyond this competitive mechanism, PGF also directly stimulates endothelial cell proliferation, migration, and survival through VEGFR-1-mediated signaling [ 5 ]. Our dual-luciferase and rescue experiments conclusively establish the functional linkage: miR-124-3p suppresses PGF, which itself acts as a pro-angiogenic factor. While this may appear counterintuitive—exosomes carrying an anti-angiogenic miRNA promoting angiogenesis—it reflects the complex regulatory logic of exosomal miRNA networks. Exosomes deliver a cocktail of miRNAs, and the net biological outcome depends on the integrated action of multiple cargo molecules. miR-124-3p-mediated PGF suppression may function as a homeostatic feedback mechanism or context-dependent regulatory signal within a broader pro-angiogenic program. Our bioinformatics analysis significantly strengthens the findings by expanding the scope from a single miR-124-3p/PGF axis to a broader regulatory network. miR-124-3p targets are enriched in multiple angiogenesis and endothelial function pathways (HIF-1, VEGF, Rap1, PI3K-Akt signaling), suggesting that miR-124-3p functions as a multi-target angiogenic coordinator rather than a single-target regulator. External validation using GSE25628 further supports the translational relevance of the miR-124-3p/PGF axis. The observed PGF downregulation in ectopic versus eutopic endometrium (log2FC = -0.705) aligns with miR-124-3p-mediated suppression, while concurrent upregulation of VEGFA, KDR, and FLT1 indicates that angiogenesis proceeds through compensatory pathways in the endometriotic microenvironment. This underscores the context-dependent role of PGF: miR-124-3p suppresses PGF, yet the broader angiogenic program driven by VEGF family members remains active. These data position the miR-124-3p/PGF axis as a regulatory node rather than a dominant angiogenic switch in endometriosis. Several limitations should be acknowledged. First, while our external validation uses clinical endometriosis tissue transcriptomic data, prospective validation in endometriosis patient samples with matched exosome and PGF measurements would strengthen the translational conclusions. Second, the study focus on miR-124-3p/PGF does not preclude the contribution of other exosomal cargo molecules, and multi-omics profiling of CD271 + UCMSC-Exo cargo is warranted. Third, the complex three-dimensional angiogenic microenvironment of endometriotic lesions cannot be fully recapitulated by in vitro endothelial cell assays. Future studies incorporating in vivo endometriosis models, exosomal miRNA sequencing, and spatial transcriptomic mapping within lesions will be important to validate and extend these findings. Conclusions This study identifies the miR-124-3p/PGF axis as a mechanistic mediator of CD271 + UCMSC-Exo-driven angiogenesis. CD271 + UCMSC-Exos deliver miR-124-3p to endothelial cells, where it directly targets and suppresses PGF, promoting angiogenic activity. External transcriptomic validation supports the clinical relevance of this axis in endometriosis. These findings provide molecular rationale for developing CD271 + subpopulation-derived exosome therapeutics and highlight the miR-124-3p/PGF axis as a potential target for angiogenesis-directed interventions in endometriosis. Declarations Funding None. Clinical trial number N ot applicable. Competing Interests The authors declare no competing interests. Author Contributions C.Z. and N.S. conceived and designed the study. C.Z. performed the experiments and analysed the data. N.S. contributed to bioinformatics analysis and figure preparation. C.Z. drafted the manuscript. N.S. critically revised the manuscript. All authors read and approved the final manuscript. Data Availability All data generated or analyzed during this study are included in this article and its supplementary information files. The public datasets analyzed are available from the Gene Expression Omnibus under accession numbers GSE25628. The full-length western blot images are included in Supplementary Information. The GEO accession number is GSE25628. Ethics Approval and Consent to Participate Not applicable. References Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am. 1997;24:235-58. As-Sanie S, Mackenzie SC, Morrison L et al. Endometriosis: A Review. JAMA. 2025;334:64-78. Taylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397:839-852. Laschke MW, Menger MD. Basic mechanisms of vascularization in endometriosis and their clinical implications. Hum Reprod Update. 2018;24:207-224. Dewerchin M, Carmeliet P. PlGF: a multitasking cytokine with disease-restricted activity. Cold Spring Harb Perspect Med. 2012;2:a011056. Todorova D, Simoncini S, Lacroix R et al. Extracellular vesicles in angiogenesis. Circ Res. 2017;120:1658-1673. Merino-González C, Zuñiga FA, Escudero C et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: potential clinical application. Front Physiol. 2016;7:24. Liang X, Zhang L, Wang S et al. Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis. J Cell Mol Med. 2016;20:937-947. Lee HW, Xu Y, He L et al. Role of venous endothelial cells in developmental and pathological angiogenesis. Circulation. 2021;144:1308-1322. Moirangthem A, Gondaliya P, Yan IK et al. Extracellular vesicle-mediated miR-126-3p transfer contributes to intercellular communication in the liver tumor microenvironment. Int J Oncol. 2023;62:1-13. Sun Y, Zhao J, Liu Q et al. Intranasal delivery of small extracellular vesicles from specific subpopulations of mesenchymal stem cells for the treatment of brain disorders. ACS Nano. 2024;18:3457-3473. Xie Y, Sun Y, Liu Y et al. Targeted delivery of RGD-CD146+CD271+ human umbilical cord mesenchymal stem cell-derived exosomes promotes blood-spinal cord barrier repair after spinal cord injury. ACS Nano. 2023;17:8743-8760. Alvarez-Viejo M, Menendez-Menendez Y, Otero-Hernandez J. CD271 as a marker to identify mesenchymal stem cells from diverse sources. World J Stem Cells. 2015;7:470-476. Barilani M, Banfi F, Sironi S et al. Low-affinity nerve growth factor receptor (CD271) heterogeneous expression in adult and fetal mesenchymal stromal cells. Sci Rep. 2018;8:9321. Zhou Y, Cai X, Zhang X et al. Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the exosomal neuroprotection. J Extracell Vesicles. 2024;13:e12456. Chen S, Polaki V, Bihl JC, Wang J. Compromised endothelial progenitor cell exosomal communication with endothelial cells in hypertensive ischemia conditions. Front Stroke. 2023;1:1015463. Baruah J, Wary KK. Exosomes in the regulation of vascular endothelial cell regeneration. Front Cell Dev Biol. 2019;7:353. Bai S, Yin Q, Dong T et al. Endothelial progenitor cell-derived exosomes ameliorate endothelial dysfunction in a diabetic mouse model. Biomed Pharmacother. 2020;131:110756. Zhang X, Sun S, Ren G et al. Advances in intercellular communication mediated by exosomal ncRNAs in cardiovascular diseases. Int J Mol Sci. 2023;24:16789. Li S, Roberson MS. Dlx3 and GCM-1 functionally coordinate the regulation of placental growth factor in human trophoblast-derived cells. J Cell Physiol. 2017;232:2900-2914. Xie X, Guo LW, Kent CK. miR548ai antagonism attenuates exosome-induced endothelial cell dysfunction. Cell Death Discov. 2021;7:318. Chen Y, Wang X. miRDB: an online database for miRNA target prediction. RNA. 2020;26:205-209. Huang HY, Lin YC, Li J et al. miRTarBase 2020: updates to the experimentally validated microRNA-target interaction database. Nucleic Acids Res. 2020;48:D148-D154. Burney RO, Talbi S, Hamilton AE et al. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology. 2007;148:3814-3826. Crispi S, Piccolo MT, D'Avino A et al. Transcriptional profiling of endometriosis tissues identifies genes related to organogenesis defects. J Cell Physiol. 2013;228:1927-1934. Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51:D587-D592. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 17 Jul, 2026 Editor assigned by journal 24 Jun, 2026 Editor invited by journal 23 Jun, 2026 Submission checks completed at journal 19 Jun, 2026 First submitted to journal 19 Jun, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-10006473","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":680281553,"identity":"fa422b3b-d83c-4f19-9cde-d5d39102d8df","order_by":0,"name":"Jing Zhu","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhu","suffix":""},{"id":680281554,"identity":"20df4b63-70f2-41d8-8c13-a24ff9cc2241","order_by":1,"name":"Yujie Hang","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yujie","middleName":"","lastName":"Hang","suffix":""},{"id":680281555,"identity":"a97e9955-5766-4be8-954a-cb9b4875470a","order_by":2,"name":"Nianchun Shan","email":"","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nianchun","middleName":"","lastName":"Shan","suffix":""},{"id":680281556,"identity":"835c5f89-0ed5-4cf3-9690-38d07db3f2bf","order_by":3,"name":"Chun Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCQY2ECXHwEyqFmPStSQ2EK1Dfnb7swc/ftWmz2/nPfiBocYmmqAWxjkH0g17+47nbjjMlyzBcCwtl6B1zBIJx6QZe47lbmDmMZBgbDhMWAubRGIbSEu6fDOP8Q+itPBIJLNJM/yoSWA4zGNGnC0SEmlskr0NBww3ALVYJBDjF/kZ6c8kfvypk5fvP2N840ONDWEtYMDYdhjCSCBKORj8qSNe7SgYBaNgFIw8AADBBjrGDgPAtgAAAABJRU5ErkJggg==","orcid":"","institution":"Xiangya Hospital Central South University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-06-11 12:54:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-10006473/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-10006473/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":115162206,"identity":"e2e80717-b123-4d3c-8695-ca16e5c98059","added_by":"auto","created_at":"2026-07-20 12:12:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":398066,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of CD271+UCMSC-derived exosomes and miR-124-3p enrichment. (A) TEM images of exosomes. Scale bar = 100 nm. (B) NTA size distribution (30-150 nm). (C) Western blot of CD63, CD81, CD9. (D) Immunofluorescence of PKH26-labeled exosome uptake by CD31+ End.3 cells. Scale bar = 20 um. (E, F) qRT-PCR of miR-124-3p in (E) UCMSCs vs CD271+ UCMSCs, and (F) their exosomes. Mean +/- SD, n = 3. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/f4ec748c758f639290ad9cc2.png"},{"id":115162165,"identity":"5a3a5072-6b3a-42c9-8cb5-08627e00dff4","added_by":"auto","created_at":"2026-07-20 12:12:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1337167,"visible":true,"origin":"","legend":"\u003cp\u003eCD271+UCMSC-Exos promote endothelial cell migration and tube formation. (A) Scratch wound healing at 0/24 h. (B) Wound closure quantification. (C, D) Transwell migration and quantification. (E) Tube formation images. Scale bar = 200 um. (F) Total tube length. (G) Branch points. Mean +/- SD, n = 3. **P \u0026lt; 0.01, ***P \u0026lt; 0.001 vs UCMSC-Exo.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/1c5d25ec5c97af12f7d203d7.png"},{"id":115162168,"identity":"ad26b813-c235-4e53-8786-4b09d8458abb","added_by":"auto","created_at":"2026-07-20 12:12:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1421760,"visible":true,"origin":"","legend":"\u003cp\u003emiR-124-3p mediates CD271+UCMSC-Exo-induced angiogenesis. (A, B) Scratch wound healing with miR-124-3p inhibitor and quantification. (C, D) Transwell migration with inhibitor and quantification. (E) Tube formation images. (F) Total tube length. (G) Number of meshes. Mean +/- SD, n = 3. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/00691d23a1720e4dc4ed43d3.png"},{"id":115162186,"identity":"b9cc3f61-7102-459a-aae4-be10c90934e1","added_by":"auto","created_at":"2026-07-20 12:12:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":298407,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification and validation of PGF as a direct miR-124-3p target. (A) Venn diagram of predicted targets. (B) Dual-luciferase reporter assay (WT vs MUT PGF 3-UTR). (C, D) Western blot of PGF after CD271+UCMSC-Exo treatment and quantification. PGF (22 kDa) and GAPDH (36 kDa) were probed on separate membranes from the same lysates. Full-length blots are provided in Supplementary Information.. (E) qRT-PCR of PGF mRNA. (F) Tube formation rescue by miR-124-3p inhibition or PGF overexpression. Mean +/- SD, n = 3. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/22c55936954d5ed6046ea20a.png"},{"id":115293057,"identity":"84453a78-7e7d-4baa-a0fb-9b669d7f4bc6","added_by":"auto","created_at":"2026-07-21 15:49:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1410616,"visible":true,"origin":"","legend":"\u003cp\u003ePGF overexpression rescues CD271+UCMSC-Exo-suppressed angiogenesis. (A, B) Western blot of PGF after overexpression and quantification. PGF (22 kDa) and GAPDH (36 kDa) were probed on separate membranes from the same lysates. Full-length blots are provided in Supplementary Information.. (C) Dual-luciferase reporter assay. (D) qRT-PCR of PGF mRNA. (E) Tube formation images. (F) Tube length. (G) Branch points. Mean +/- SD.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/12229cc7bc159cc9bfe32d5b.png"},{"id":115292974,"identity":"8745f3d1-47c7-4fdc-8355-1371d60e5d7a","added_by":"auto","created_at":"2026-07-21 15:49:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":367156,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics and external validation. (A) KEGG pathway enrichment of miR-124-3p targets (HIF-1, VEGF, Rap1, PI3K-Akt, focal adhesion; all FDR \u0026lt; 0.05). (B) Differential expression of angiogenic markers in ectopic vs eutopic endometrium (GSE25628). PGF downregulated (log2FC = -0.705); VEGFA, KDR, FLT1 upregulated. (C) Expression patterns of PGF and angiogenic markers in endometriosis tissues.KEGG pathway mapping used with permission from Kanehisa Laboratories [26]\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/aaa00ef1fb2ff8cb7e10321f.png"},{"id":115295475,"identity":"816c5708-7bc2-49e6-bd9f-406bc7833285","added_by":"auto","created_at":"2026-07-21 15:53:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5520192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/ffbae29f-a843-4ee4-83c9-c1ec531abacc.pdf"},{"id":115162208,"identity":"2f7ba4f8-cbfc-4c70-8f6b-1c13289551d3","added_by":"auto","created_at":"2026-07-20 12:12:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":61750,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-10006473/v1/526f3e3fad9a7353ad4b3b70.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CD271+ Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promote Angiogenesis via miR-124-3p Mediated PGF Targeting","fulltext":[{"header":"Background","content":"\u003cp\u003eEndometriosis, a common gynecological disorder affecting approximately 10% of reproductive-age women, is characterized by the implantation and growth of endometrial-like tissue outside the uterine cavity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease imposes substantial physical, psychological, and economic burdens, with diagnosis often delayed by 7\u0026ndash;10 years [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current medical therapies relying on hormonal suppression and surgical excision are limited by high recurrence rates and adverse effects, underscoring the need for mechanism-based therapeutic strategies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAngiogenesis is a fundamental prerequisite for endometriotic lesion survival and growth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The peritoneal microenvironment provides a rich milieu of pro-angiogenic factors that facilitate neovascularization of ectopic implants. Among these, placental growth factor (PGF), a member of the VEGF family, has emerged as a key modulator of pathological angiogenesis, acting through VEGFR-1 (FLT1) to amplify VEGF-driven signaling and recruit bone marrow-derived angiogenic cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMesenchymal stem cell-derived extracellular vesicles, particularly exosomes, have attracted considerable attention as vehicles for intercellular communication in angiogenesis [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. MSC-derived exosomes carry diverse cargo including microRNAs (miRNAs), which can post-transcriptionally regulate angiogenic gene programs in recipient endothelial cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recent studies have demonstrated that exosomes from specific MSC subpopulations exhibit distinct therapeutic profiles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CD271 (p75 neurotrophin receptor, NGFR) marks a subset of UCMSCs with enhanced self-renewal, multilineage differentiation, and paracrine activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the angiogenic function and molecular mechanisms of CD271\u0026thinsp;+\u0026thinsp;UCMSC-derived exosomes remain unexplored.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the pro-angiogenic effects of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos on endothelial cells, identified miR-124-3p as a key effector miRNA cargo, and established PGF as its direct downstream target. Furthermore, we integrated bioinformatics pathway analysis and external endometriosis transcriptomic validation to strengthen the translational relevance of this axis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe mouse microvascular endothelial cell line End.3 was purchased from Procell (China) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. CD271\u0026thinsp;+\u0026thinsp;and unsorted UCMSCs were cultured as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The End.3 cell line was selected because (i) the miR-124-3p/PGF axis is conserved between mouse and human, (ii) it has been extensively used in published endothelial angiogenesis studies, and (iii) key angiogenic pathways (VEGF, HIF-1, PI3K-Akt) are functionally conserved in this line.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExosome isolation and characterization\u003c/h3\u003e\n\u003cp\u003eExosomes were isolated from the conditioned medium of CD271\u0026thinsp;+\u0026thinsp;UCMSCs and unsorted UCMSCs by differential ultracentrifugation. Briefly, culture supernatant was subjected to sequential centrifugation at 300 \u0026times; g for 10 min, 2,000 \u0026times; g for 20 min, and 10,000 \u0026times; g for 30 min to remove cells and debris, followed by ultracentrifugation at 100,000 \u0026times; g for 70 min at 4\u0026deg;C. The exosome pellet was washed with PBS and re-ultracentrifuged. Exosome morphology was examined by TEM after negative staining with uranyl acetate. Particle size distribution and concentration were determined by NTA (NanoSight NS300, Malvern). Exosome markers CD63, CD81, and CD9 were detected by western blotting.\u003c/p\u003e\n\u003ch3\u003eExosome uptake assay\u003c/h3\u003e\n\u003cp\u003eIsolated exosomes were labeled with PKH26 fluorescent dye (Sigma-Aldrich) according to the manufacturer's protocol. Labeled exosomes (10 \u0026micro;g/mL) were added to End.3 cells and incubated for 24 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde, and nuclei were counterstained with DAPI. Fluorescence images were captured using a confocal microscope (Leica).\u003c/p\u003e\n\u003ch3\u003eScratch wound healing assay\u003c/h3\u003e\n\u003cp\u003eEnd.3 cells were seeded in 6-well plates at 2 \u0026times; 10⁵ cells/well and cultured to confluence. A linear scratch was created using a sterile 200 \u0026micro;L pipette tip. Cells were washed with PBS and cultured in serum-free medium containing UCMSC-Exos or CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos (50 \u0026micro;g/mL). Images were captured at 0 h and 24 h, and wound closure was quantified using ImageJ.\u003c/p\u003e\n\u003ch3\u003eTranswell migration assay\u003c/h3\u003e\n\u003cp\u003eEnd.3 cells were serum-starved for 12 h, harvested, and resuspended in serum-free medium at 1 \u0026times; 10⁵ cells/mL. Cell suspension (200 \u0026micro;L) was added to the upper chamber of a 24-well Transwell insert (8 \u0026micro;m pore size, Corning), while 600 \u0026micro;L of medium containing exosomes (50 \u0026micro;g/mL) was added to the lower chamber. After 24 h incubation, cells on the upper surface were removed with a cotton swab, and migrated cells on the lower surface were fixed, stained with 0.1% crystal violet, and counted in five random fields at 200\u0026times; magnification.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTube formation assay\u003c/h2\u003e \u003cp\u003eMatrigel (Corning) was thawed at 4\u0026deg;C and added to a pre-chilled 96-well plate (50 \u0026micro;L/well), then polymerized at 37\u0026deg;C for 30 min. End.3 cells (2 \u0026times; 10⁴ cells/well) were seeded onto the Matrigel in serum-free medium containing exosomes (50 \u0026micro;g/mL). After 6 h, tube formation was imaged and quantified by measuring total tube length and branch points using ImageJ Angiogenesis Analyzer.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eqRT-PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from exosomes and cells using TRIzol reagent (Invitrogen). For miRNA detection, cDNA was synthesized using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa), and qRT-PCR was performed with TB Green Premix Ex Taq II (TaKaRa) on a QuantStudio 5 system. U6 was used as an internal control for miRNA. Relative expression was calculated using the 2⁻ΔΔCt method.\u003c/p\u003e\n\u003ch3\u003emiR-124-3p inhibition\u003c/h3\u003e\n\u003cp\u003eEnd.3 cells at 70% confluence were transfected with miR-124-3p inhibitor or negative control inhibitor (50 nM, RiboBio) using Lipofectamine 3000 (Invitrogen). After 24 h of transfection, cells were treated with CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos (50 \u0026micro;g/mL) for the indicated time points prior to downstream functional assays.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assay\u003c/h2\u003e \u003cp\u003eWild-type (WT) and mutant (MUT) PGF 3'-UTR sequences containing the predicted miR-124-3p binding site were cloned into the pmirGLO dual-luciferase reporter vector (Promega). End.3 cells were co-transfected with the reporter construct and miR-124-3p mimic or negative control (50 nM) using Lipofectamine 3000. Firefly and Renilla luciferase activities were measured 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePGF overexpression\u003c/h2\u003e \u003cp\u003ePGF overexpression plasmid (pcDNA3.1-PGF) was constructed by Guangzhou RiboBio. End.3 cells were transfected with pcDNA3.1-PGF or empty vector (2 \u0026micro;g/well) using Lipofectamine 3000. At 24 h post-transfection, cells were treated with CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos for downstream functional assays.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics pathway enrichment analysis\u003c/h2\u003e \u003cp\u003eTo validate the biological relevance of the miR-124-3p/PGF axis, we performed bioinformatics analysis. Validated miR-124-3p target genes were retrieved from miRTarBase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], TargetScan, and miRDB [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted using clusterProfiler in R. Significantly enriched angiogenesis-related pathways were visualized using bubble plots (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PGF was confirmed as a validated target of miR-124-3p in the miRTarBase database. The HARRIS_HYPOXIA gene set was included because endometriosis is a hypoxia-driven disease, and genes overlapping between hypoxia signatures and miR-124-3p targets may identify biologically relevant angiogenic effectors in the endometriotic microenvironment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExternal endometriosis transcriptomic validation\u003c/h2\u003e \u003cp\u003eTo assess the clinical relevance of identified angiogenic markers, we analyzed published transcriptomic datasets of endometriosis (GSE25628 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]). Differential expression of PGF, PECAM1 (CD31), VEGFA, KDR, and FLT1 between ectopic and eutopic endometrium was examined. Correlations between PGF expression and angiogenic markers were calculated using Pearson correlation coefficients in endometriosis tissue samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was measured by BCA assay. Protein lysates (30 \u0026micro;g) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk and incubated with primary antibodies against CD63 (1:1000), CD81 (1:1000), CD9 (1:1000), PGF (1:1000), and GAPDH (1:5000) overnight at 4\u0026deg;C. Following HRP-conjugated secondary antibody incubation, signals were detected using enhanced chemiluminescence (ECL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed using GraphPad Prism 8. Statistical comparisons between two groups were performed using Student's t-test, and multiple group comparisons used one-way ANOVA with Tukey's post hoc test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All experiments were performed in at least three independent biological replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and characterization of CD271\u0026thinsp;+\u0026thinsp;UCMSC-derived exosomes\u003c/h2\u003e \u003cp\u003eExosomes isolated from both UCMSCs and CD271\u0026thinsp;+\u0026thinsp;UCMSCs exhibited the characteristic cup-shaped morphology under TEM, with particle sizes ranging from 30\u0026ndash;150 nm as determined by NTA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Western blot analysis confirmed the expression of canonical exosomal markers CD63, CD81, and CD9 in both exosome groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Immunofluorescence imaging demonstrated that PKH26-labeled CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos (red) were efficiently internalized by End.3 endothelial cells stained with CD31 (green), with nuclei counterstained with DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). qRT-PCR analysis revealed that miR-124-3p was significantly enriched in CD271\u0026thinsp;+\u0026thinsp;UCMSCs compared with unsorted UCMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and in their secreted exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos promote endothelial cell migration and tube formation\u003c/h2\u003e \u003cp\u003eTo evaluate the pro-angiogenic effects of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos, End.3 cells were treated with PBS (Control), UCMSC-Exos, or CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos. Scratch wound healing assays demonstrated significantly accelerated wound closure in the CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo group compared with the UCMSC-Exo and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Transwell migration assays showed a marked increase in the number of migrated cells in the CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In tube formation assays on Matrigel, CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment significantly enhanced both total tube length and the number of branch points compared with UCMSC-Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating superior pro-angiogenic capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003emiR-124-3p mediates the pro-angiogenic effect of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos\u003c/h2\u003e \u003cp\u003emiR-124-3p was significantly enriched in CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos compared with UCMSC-Exos (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). To confirm its functional role, End.3 cells were transfected with a miR-124-3p inhibitor prior to CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment. Scratch wound healing assays demonstrated that miR-124-3p inhibition significantly attenuated CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo-induced wound closure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Transwell migration assays showed a marked reduction in migrated cell numbers upon miR-124-3p inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Tube formation assays further confirmed that miR-124-3p inhibition substantially reduced total tube length and the number of meshes compared with CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-G; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results establish miR-124-3p as a key functional mediator of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo-induced angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003ePGF is a direct target of miR-124-3p\u003c/h2\u003e \u003cp\u003eAnalysis of three miRNA target prediction databases (miRDB, TargetScan, and the HARRIS_HYPOXIA gene set) identified PGF as a conserved target of miR-124-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Dual-luciferase reporter assays demonstrated that miR-124-3p mimic significantly repressed the luciferase activity of the wild-type PGF 3'-UTR reporter (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the mutant reporter was unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistently, CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment markedly reduced PGF protein expression in End.3 cells, an effect partially reversed by miR-124-3p inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). qRT-PCR confirmed reduced PGF mRNA after CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Tube formation assays showed that either inhibition of miR-124-3p or overexpression of PGF attenuated the pro-angiogenic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePGF overexpression reverses CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo-induced angiogenesis\u003c/h2\u003e \u003cp\u003eTo confirm that PGF is the functional downstream effector of the miR-124-3p-mediated angiogenic response, we overexpressed PGF in End.3 cells. CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment significantly reduced PGF protein expression compared with the PBS control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Dual-luciferase reporter assays confirmed direct miR-124-3p binding to the wild-type PGF 3'-UTR but not the mutant construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). qRT-PCR analysis verified that CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo treatment significantly reduced PGF mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In functional tube formation assays, CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo promoted endothelial network formation, while PGF overexpression significantly attenuated this effect, as demonstrated by representative images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and quantitative analysis of total tube length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and branch points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results establish PGF as a functional downstream target of miR-124-3p in CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo-mediated angiogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eBioinformatics validation of the miR-124-3p angiogenic regulatory network\u003c/h2\u003e \u003cp\u003eTo extend the mechanistic findings beyond the single miR-124-3p/PGF axis, we performed systematic pathway enrichment analysis. Integrated analysis of miR-124-3p validated targets from miRTarBase, TargetScan, and miRDB identified 102 target genes, among which PGF was confirmed. KEGG pathway enrichment analysis revealed significant enrichment of miR-124-3p targets in multiple angiogenesis-related pathways, including HIF-1 signaling (FDR\u0026thinsp;=\u0026thinsp;0.003), VEGF signaling (FDR\u0026thinsp;=\u0026thinsp;0.008), Rap1 signaling (FDR\u0026thinsp;=\u0026thinsp;0.012), PI3K-Akt signaling (FDR\u0026thinsp;=\u0026thinsp;0.018), and focal adhesion (FDR\u0026thinsp;=\u0026thinsp;0.025) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). GO enrichment analysis further revealed overrepresentation in biological processes including endothelial cell migration, blood vessel morphogenesis, and positive regulation of angiogenesis (all FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These data establish that miR-124-3p broadly regulates angiogenic gene programs beyond PGF, positioning the miR-124-3p/PGF axis within a larger angiogenic regulatory network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the clinical relevance of our findings, we analyzed public endometriosis transcriptomic data. Analysis of GSE25628 revealed PGF downregulation in ectopic versus eutopic endometrium (log2FC = -0.705), consistent with miR-124-3p-mediated suppression. In contrast, VEGFA, KDR, FLT1, MMP9, and ANG2 exhibited upregulation in ectopic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), reflecting an active angiogenic state. This pattern\u0026mdash;PGF suppression amid broader angiogenic activation\u0026mdash;is mechanistically consistent with miR-124-3p targeting PGF while compensatory VEGF family signaling sustains angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These results provide independent clinical evidence that the PGF-centered angiogenic program is active in endometriotic lesions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that CD271\u0026thinsp;+\u0026thinsp;UCMSC-derived exosomes promote angiogenesis by delivering miR-124-3p, which directly targets and suppresses PGF expression in endothelial cells. Our findings establish a previously unrecognized miR-124-3p/PGF regulatory axis that underlies the enhanced pro-angiogenic capacity of the CD271\u0026thinsp;+\u0026thinsp;MSC subpopulation.\u003c/p\u003e \u003cp\u003eThe CD271\u0026thinsp;+\u0026thinsp;subpopulation of UCMSCs has been characterized by enhanced self-renewal, multilineage differentiation potential, and superior paracrine activity compared with unsorted MSCs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our finding that CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos exhibit significantly stronger pro-angiogenic effects than unsorted UCMSC-Exos extends this functional specialization to the exosome-mediated mode of intercellular communication. This aligns with recent studies demonstrating that exosomes from other MSC subpopulations, such as CD146\u0026thinsp;+\u0026thinsp;UCMSCs, possess distinct therapeutic activities in tissue repair contexts [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The enrichment of miR-124-3p in CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos provides a molecular basis for this subpopulation-specific function and suggests that sorting MSCs by surface markers may be a strategy to obtain exosomes with defined therapeutic properties. The molecular basis for miR-124-3p enrichment in the CD271\u0026thinsp;+\u0026thinsp;subpopulation remains to be determined. Possible mechanisms include CD271-mediated transcriptional regulation of the pri-miR-124 locus via NF-kB or AP-1 signaling, or selective sorting of miR-124-3p into exosomes through RNA-binding proteins such as hnRNPA2B1. Elucidating this enrichment mechanism may enable engineering of UCMSCs for enhanced therapeutic miRNA loading.\u003c/p\u003e \u003cp\u003eThe identification of PGF as a direct target of miR-124-3p adds mechanistic depth to the angiogenic function of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos. PGF, as a VEGF family member, primarily binds VEGFR-1 (FLT1) and potentiates VEGF-A-driven angiogenesis by displacing VEGF-A from VEGFR-1, thereby increasing VEGF-A availability for VEGFR-2 activation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Beyond this competitive mechanism, PGF also directly stimulates endothelial cell proliferation, migration, and survival through VEGFR-1-mediated signaling [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Our dual-luciferase and rescue experiments conclusively establish the functional linkage: miR-124-3p suppresses PGF, which itself acts as a pro-angiogenic factor. While this may appear counterintuitive\u0026mdash;exosomes carrying an anti-angiogenic miRNA promoting angiogenesis\u0026mdash;it reflects the complex regulatory logic of exosomal miRNA networks. Exosomes deliver a cocktail of miRNAs, and the net biological outcome depends on the integrated action of multiple cargo molecules. miR-124-3p-mediated PGF suppression may function as a homeostatic feedback mechanism or context-dependent regulatory signal within a broader pro-angiogenic program.\u003c/p\u003e \u003cp\u003eOur bioinformatics analysis significantly strengthens the findings by expanding the scope from a single miR-124-3p/PGF axis to a broader regulatory network. miR-124-3p targets are enriched in multiple angiogenesis and endothelial function pathways (HIF-1, VEGF, Rap1, PI3K-Akt signaling), suggesting that miR-124-3p functions as a multi-target angiogenic coordinator rather than a single-target regulator. External validation using GSE25628 further supports the translational relevance of the miR-124-3p/PGF axis. The observed PGF downregulation in ectopic versus eutopic endometrium (log2FC = -0.705) aligns with miR-124-3p-mediated suppression, while concurrent upregulation of VEGFA, KDR, and FLT1 indicates that angiogenesis proceeds through compensatory pathways in the endometriotic microenvironment. This underscores the context-dependent role of PGF: miR-124-3p suppresses PGF, yet the broader angiogenic program driven by VEGF family members remains active. These data position the miR-124-3p/PGF axis as a regulatory node rather than a dominant angiogenic switch in endometriosis.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, while our external validation uses clinical endometriosis tissue transcriptomic data, prospective validation in endometriosis patient samples with matched exosome and PGF measurements would strengthen the translational conclusions. Second, the study focus on miR-124-3p/PGF does not preclude the contribution of other exosomal cargo molecules, and multi-omics profiling of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo cargo is warranted. Third, the complex three-dimensional angiogenic microenvironment of endometriotic lesions cannot be fully recapitulated by in vitro endothelial cell assays. Future studies incorporating in vivo endometriosis models, exosomal miRNA sequencing, and spatial transcriptomic mapping within lesions will be important to validate and extend these findings.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identifies the miR-124-3p/PGF axis as a mechanistic mediator of CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exo-driven angiogenesis. CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos deliver miR-124-3p to endothelial cells, where it directly targets and suppresses PGF, promoting angiogenic activity. External transcriptomic validation supports the clinical relevance of this axis in endometriosis. These findings provide molecular rationale for developing CD271\u0026thinsp;+\u0026thinsp;subpopulation-derived exosome therapeutics and highlight the miR-124-3p/PGF axis as a potential target for angiogenesis-directed interventions in endometriosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;N\u003c/strong\u003eot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003eC.Z. and N.S. conceived and designed the study. C.Z. performed the experiments and analysed the data. N.S. contributed to bioinformatics analysis and figure preparation. C.Z. drafted the manuscript. N.S. critically revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e All data generated or analyzed during this study are included in this article and its supplementary information files. The public datasets analyzed are available from the Gene Expression Omnibus under accession numbers GSE25628. The full-length western blot images are included in Supplementary Information. The GEO accession number is GSE25628.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am. 1997;24:235-58.\u003c/li\u003e\n\u003cli\u003eAs-Sanie S, Mackenzie SC, Morrison L et al. Endometriosis: A Review. JAMA. 2025;334:64-78.\u003c/li\u003e\n\u003cli\u003eTaylor HS, Kotlyar AM, Flores VA. Endometriosis is a chronic systemic disease: clinical challenges and novel innovations. Lancet. 2021;397:839-852.\u003c/li\u003e\n\u003cli\u003eLaschke MW, Menger MD. Basic mechanisms of vascularization in endometriosis and their clinical implications. Hum Reprod Update. 2018;24:207-224.\u003c/li\u003e\n\u003cli\u003eDewerchin M, Carmeliet P. PlGF: a multitasking cytokine with disease-restricted activity. Cold Spring Harb Perspect Med. 2012;2:a011056.\u003c/li\u003e\n\u003cli\u003eTodorova D, Simoncini S, Lacroix R et al. Extracellular vesicles in angiogenesis. Circ Res. 2017;120:1658-1673.\u003c/li\u003e\n\u003cli\u003eMerino-Gonz\u0026aacute;lez C, Zu\u0026ntilde;iga FA, Escudero C et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: potential clinical application. Front Physiol. 2016;7:24.\u003c/li\u003e\n\u003cli\u003eLiang X, Zhang L, Wang S et al. Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis. J Cell Mol Med. 2016;20:937-947.\u003c/li\u003e\n\u003cli\u003eLee HW, Xu Y, He L et al. Role of venous endothelial cells in developmental and pathological angiogenesis. Circulation. 2021;144:1308-1322.\u003c/li\u003e\n\u003cli\u003eMoirangthem A, Gondaliya P, Yan IK et al. Extracellular vesicle-mediated miR-126-3p transfer contributes to intercellular communication in the liver tumor microenvironment. Int J Oncol. 2023;62:1-13.\u003c/li\u003e\n\u003cli\u003eSun Y, Zhao J, Liu Q et al. Intranasal delivery of small extracellular vesicles from specific subpopulations of mesenchymal stem cells for the treatment of brain disorders. ACS Nano. 2024;18:3457-3473.\u003c/li\u003e\n\u003cli\u003eXie Y, Sun Y, Liu Y et al. Targeted delivery of RGD-CD146+CD271+ human umbilical cord mesenchymal stem cell-derived exosomes promotes blood-spinal cord barrier repair after spinal cord injury. ACS Nano. 2023;17:8743-8760.\u003c/li\u003e\n\u003cli\u003eAlvarez-Viejo M, Menendez-Menendez Y, Otero-Hernandez J. CD271 as a marker to identify mesenchymal stem cells from diverse sources. World J Stem Cells. 2015;7:470-476.\u003c/li\u003e\n\u003cli\u003eBarilani M, Banfi F, Sironi S et al. Low-affinity nerve growth factor receptor (CD271) heterogeneous expression in adult and fetal mesenchymal stromal cells. Sci Rep. 2018;8:9321.\u003c/li\u003e\n\u003cli\u003eZhou Y, Cai X, Zhang X et al. Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the exosomal neuroprotection. J Extracell Vesicles. 2024;13:e12456.\u003c/li\u003e\n\u003cli\u003eChen S, Polaki V, Bihl JC, Wang J. Compromised endothelial progenitor cell exosomal communication with endothelial cells in hypertensive ischemia conditions. Front Stroke. 2023;1:1015463.\u003c/li\u003e\n\u003cli\u003eBaruah J, Wary KK. Exosomes in the regulation of vascular endothelial cell regeneration. Front Cell Dev Biol. 2019;7:353.\u003c/li\u003e\n\u003cli\u003eBai S, Yin Q, Dong T et al. Endothelial progenitor cell-derived exosomes ameliorate endothelial dysfunction in a diabetic mouse model. Biomed Pharmacother. 2020;131:110756.\u003c/li\u003e\n\u003cli\u003eZhang X, Sun S, Ren G et al. Advances in intercellular communication mediated by exosomal ncRNAs in cardiovascular diseases. Int J Mol Sci. 2023;24:16789.\u003c/li\u003e\n\u003cli\u003eLi S, Roberson MS. Dlx3 and GCM-1 functionally coordinate the regulation of placental growth factor in human trophoblast-derived cells. J Cell Physiol. 2017;232:2900-2914.\u003c/li\u003e\n\u003cli\u003eXie X, Guo LW, Kent CK. miR548ai antagonism attenuates exosome-induced endothelial cell dysfunction. Cell Death Discov. 2021;7:318.\u003c/li\u003e\n\u003cli\u003eChen Y, Wang X. miRDB: an online database for miRNA target prediction. RNA. 2020;26:205-209.\u003c/li\u003e\n\u003cli\u003eHuang HY, Lin YC, Li J et al. miRTarBase 2020: updates to the experimentally validated microRNA-target interaction database. Nucleic Acids Res. 2020;48:D148-D154.\u003c/li\u003e\n\u003cli\u003eBurney RO, Talbi S, Hamilton AE et al. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology. 2007;148:3814-3826.\u003c/li\u003e\n\u003cli\u003eCrispi S, Piccolo MT, D\u0026apos;Avino A et al. Transcriptional profiling of endometriosis tissues identifies genes related to organogenesis defects. J Cell Physiol. 2013;228:1927-1934.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51:D587-D592.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, CD271, Mesenchymal stem cells, Exosomes, miR-124-3p, Placental growth factor, Angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-10006473/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-10006473/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEndometriosis, affecting approximately 10% of reproductive-age women, is critically dependent on angiogenesis for ectopic lesion establishment and growth. While exosomes from mesenchymal stem cells (MSCs) exhibit pro-angiogenic properties, the functional heterogeneity of MSC subpopulations and their distinct angiogenic mechanisms remain poorly understood. CD271 marks a UCMSC subpopulation with enhanced regenerative potential, yet its role in exosome-mediated angiogenesis remains unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eExosomes were isolated from CD271\u0026thinsp;+\u0026thinsp;UCMSCs via ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. End.3 endothelial cells were treated with UCMSC-Exos or CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos, and functional angiogenesis was assessed by scratch wound healing, Transwell migration, and tube formation assays. miRNA cargo was profiled by qRT-PCR, and miR-124-3p inhibition experiments were performed to confirm functional specificity. Putative miR-124-3p targets were identified using miRDB, TargetScan, and the HARRIS_HYPOXIA gene set, and validated by dual-luciferase reporter assay, western blotting, and PGF overexpression rescue experiments. Public transcriptomic datasets (GSE25628) were analyzed for external validation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos were efficiently internalized by End.3 cells and significantly enhanced cell migration and tube formation compared with UCMSC-Exos. miR-124-3p was enriched in CD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos. Dual-luciferase reporter assays confirmed that miR-124-3p directly targets the 3'-UTR of PGF, leading to post-transcriptional suppression of PGF protein expression. PGF overexpression reversed this effect. miR-124-3p targets were enriched in angiogenesis-related KEGG pathways (HIF-1, VEGF, Rap1; all FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05). External validation in endometriosis tissues (GSE25628) confirmed PGF downregulation in ectopic lesions (log2FC = -0.705) with, while angiogenic markers including VEGFA, KDR, and FLT1 were upregulated in the same lesions.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCD271\u0026thinsp;+\u0026thinsp;UCMSC-Exos promote angiogenesis through delivery of miR-124-3p, which directly targets PGF in endothelial cells. External transcriptomic validation supports the clinical relevance of this axis in endometriosis, suggesting therapeutic potential.\u003c/p\u003e","manuscriptTitle":"CD271+ Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Promote Angiogenesis via miR-124-3p Mediated PGF Targeting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-07-20 12:10:49","doi":"10.21203/rs.3.rs-10006473/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-07-17T10:21:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-06-25T01:15:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-06-23T13:28:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-06-19T04:10:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-06-19T04:00:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc1a0666-d69d-45b7-9441-cd2e7bf7dd10","owner":[],"postedDate":"July 20th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"3","date":"2026-07-17T10:21:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":72317519,"name":"Biological sciences/Cell biology"},{"id":72317520,"name":"Health sciences/Diseases"},{"id":72317521,"name":"Biological sciences/Molecular biology"},{"id":72317522,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-07-20T12:10:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-07-20 12:10:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-10006473","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-10006473","identity":"rs-10006473","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}