Nanotherapeutic Strategy via ADSC-mitoEVs Rescues Ischemic Angiogenesis through Mitophagy and Mitochondrial Metabolic Reprogramming

Nanotherapeutic Strategy via ADSC-mitoEVs Rescues Ischemic Angiogenesis through Mitophagy and Mitochondrial Metabolic Reprogramming | 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 Research Article Nanotherapeutic Strategy via ADSC-mitoEVs Rescues Ischemic Angiogenesis through Mitophagy and Mitochondrial Metabolic Reprogramming Yuan-zheng Zhu, Min-chen Zhang, Xue-er Li, Xing-hong Zeng, Xue-ting Gong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7849775/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 12 You are reading this latest preprint version Abstract Ischemic vascular diseases are critically associated with mitochondrial dysfunction in endothelial cells, leading to impaired angiogenesis and compromised tissue repair. Although mitochondrial transfer has emerged as a promising therapeutic strategy for revascularization, its clinical translation has been hindered by inefficient delivery systems. In this study, we show that mitochondrial-enriched extracellular vesicles derived from adipose-derived stem cells (ADSC-mitoEVs) function as an efficient nanotherapeutic, restoring angiogenic function both in vitro and in a murine model of diabetic hindlimb ischemia. Mechanistically, the uptake of ADSC-mitoEVs triggered PINK1/Parkin-mediated mitophagy in recipient endothelial cells, a process essential for initiating angiogenesis. Moreover, ADSC-mitoEVs directly delivered functional mitochondrial proteins, including superoxide dismutase 2 (SOD2), into the endogenous mitochondrial network, which enhanced antioxidant defense and improved bioenergetic capacity independently of mitophagy, as shown by reduced reactive oxygen species and elevated ATP production even in PINK1-silenced cells. Our results demonstrate ADSC-mitoEVs as a versatile, cell-free nanotherapeutic that promotes mitochondrial quality control and metabolic reprogramming, offering a potent therapeutic avenue for ischemic vascular diseases. mitochondrial transfer extracellular vesicles adipose-derived stem cells mitophagy ischemic vascular diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Ischemic diseases, encompassing conditions such as peripheral artery disease and coronary artery disease, represent a leading cause of global morbidity and mortality[ 1 ]. These pathologies are defined by a critical imbalance between oxygen supply and metabolic demand within affected tissues[ 2 , 3 ]. The therapeutic induction of robust and functional angiogenesis remains a paramount, yet persistently elusive, goal in vascular medicine. The ischemic microenvironment is notoriously pathological, characterized by profound hypoxia, elevated levels of reactive oxygen species (ROS), nutrient deprivation and sustained inflammation[ 4 ]. These factors collectively impose severe bioenergetic stress on resident vascular endothelial cells, compromising their capacity for repair and regeneration. Central to cellular survival and function, mitochondria are indispensable for energy production, redox homeostasis and critical signaling processes that govern angiogenesis, including proliferation, migration and tube formation [ 5 – 7 ]. Compelling evidence now indicates that mitochondrial dysfunction is a fundamental hallmark of ischemic tissues, directly contributing to cellular apoptosis and the failure of re-vascularization[ 5 – 7 ]. Consequently, innovative strategies designed to directly ameliorate mitochondrial bioenergetics within ischemic cells present a compelling and targeted therapy. Adipose-derived stem cells (ADSCs) have emerged as a potent source for regenerative therapy, prized for their abundance, accessibility and pro-angiogenic secretome[ 8 – 10 ]. However, a groundbreaking paradigm shift has occurred with the recognition of horizontal mitochondrial transfer as a fundamental mechanism of ADSC-mediated cytoprotection and repair[ 11 ]. Intercellular mitochondrial trafficking can occur via several routes, including the formation of tunneling nanotubes (TNTs), gap junctions and cell fusion. While physiologically relevant, these direct cell-contact mechanisms are inherently limited in a therapeutic context by their low efficiency and spatial constraints[ 11 , 12 ]. In contrast, the transfer of mitochondria via extracellular vesicles (EVs) represents a sophisticated, distance-independent delivery system[ 13 ]. These mitochondrial-enriched extracellular vesicles (mitoEVs) are naturally packaged by donor cells and can be efficiently internalized by recipient cells, conferring a full complement of functional proteins, lipids and mitochondrial DNA (mtDNA) to resuscitate bioenergetics[ 13 ]. This vesicle-mediated mechanism offers distinct advantages for therapy, including enhanced biological stability in circulation, a reduced risk of immunogenicity compared to whole cells, and the potential for targeted delivery to sites of injury, thereby bypassing the limitations of direct cell-cell contact. In this study, we isolated and characterized mitoEVs from ADSC-conditioned media through Nano Flow Cytometry and analyzed their protein components. We then investigated their efficacy in promoting angiogenesis in a murine model of critical hindlimb ischemia. Utilizing integrated proteomic profiling and real-time analysis of cellular metabolism via Seahorse extracellular flux technology, we further elucidate the core mechanism through which ADSC-mitoEVs reprogram cellular energy metabolism and activate potent angiogenic pathways in ischemic VECs. Results Characterization and Function Analysis of Mitochondrial Components in ADSC-EVs Proteomic profiling identified 390 mitochondrial proteins (8.2%) in ADSC-EVs (Supplementary Fig. 1A). Bioinformatic analysis via GO and KEGG pathways highlighted significant involvement in energy metabolism remodeling including oxidative phosphorylation, fatty acid beta-oxidation, glycolysis and regulation of reactive oxygen species (Supplementary Fig. 1B). Further functional annotations underscored roles in mitochondrion targeting, antioxidant, proton motive force and electron transport, supporting the potential of ADSC-mitoEVs to modulate cellular bioenerg Characterization of ADSC-mitoEVs (Supplementary Fig. 1C-F). Characterization of ADSC-mitoEVs Both mitoEVs and mito-free EVs from ADSCs exhibited characteristic cup-shaped morphology under transmission electron microscopy (Fig. 1 B). MitoEVs shown increased mean particle size of 218.6 ± 32.5 nm compared with that of mito-free EVs (105.3 ± 24.1 nm) as determined by nanoparticle tracking analysis (Fig. 1 C). Western blot analysis, as shown in Fig. 1 D, confirmed positive expression of EV markers (CD63 and TSG101) in both mitoEVs and mito-free EVs, while positive expression Mitochondrial outer membrane protein (TOM20) only observed in mitoEVs. Ischemia Accelerates the Uptake and Mitochondrial Binding of ADSC-mitoEVs by HUVECs We simulated in vivo ischemia by constructing a cell culture microenvironment with hypoxia and serum deprivation. Colocalization analysis indicated that internalized mitoEVs preferentially bind the endogenous mitochondrial network within 12 hours of co-culture, as visualized via confocal microscopy using miRFP670-labeled mitoEVs and DsRed-labeled mitochondria in HUVECs. Increased uptake rate and mitochondrial binding efficiency of mitoEVs was observed in HUVECs cultured under ischemic condition, compared to Normal condition (Fig. 1 E-F). ADSC-mitoEVs Facilitates the Survival and Angiogenetic Potential of Ischemic HUVECs Apoptosis analysis by flow cytometry indicated that ADSC-mitoEVs significantly increased the viability of ischemic HUVECs (under hypoxia and serum deprivation) in a dose-dependent manner up to 40 µg/ml (Fig. 2 A-C). Similarly, scratch wound healing and tube formation assays demonstrated markedly enhanced migration and angiogenic capacity in mitoEV-treated groups compared to controls under ischemic conditions, with efficacy plateauing beyond 40 µg/ml (Fig. 2 D-G). Doses exceeding 40 µg/ml provided minimal additional benefit to either cell survival or angiogenesis. Therefore, 40 µg/ml was selected as the optimal concentration of ADSC-mitoEVs for all subsequent experiments. ADSC-mitoEVs Enhances the Revascularization in vivo ADSC-mitoEVs significantly improved the angiogenic potential of HUVECs in vivo. Subcutaneous grafts co-implanted with HUVECs and ADSC-mitoEVs exhibited extensive networks of perfused microvessels by day 7, in contrast to control grafts, which showed poor vascularization (Fig. 3 A-H). Furthermore, ADSC-mitoEVs administration promoted functional recovery in ischemic hindlimbs, as evidenced by markedly improved perfusion over a three-week period and prevention of foot necrosis (Fig. 3 I-N). Histological analysis of muscle tissues retrieved from the ADSC-mitoEV-treated group revealed increased capillary density (CD31⁺ structures) and higher skeletal muscle density (α-SMA⁺) (Fig. 3 O-Q). These findings demonstrate that ADSC-mitoEVs not only stimulate robust vascular network formation but also enhance tissue perfusion and protect against ischemic injury, highlighting their therapeutic potential in promoting vascular regeneration and functional recovery. Proteomics Indicates Improved Mitophagy and Remodeled Energy Metabolism by ADSC-mitoEVs As shown in Fig. 4 , proteomic profiling of hypoxic HUVECs treated with ADSC-mitoEVs identified 845 significantly upregulated proteins (fold change > 1.25, p < 0.05) associated with autophagy, ubiquitin-dependent protein catabolism, cellular redox homeostasis, mitochondrial ATP synthesis, oxidant detoxification, and inhibition of apoptosis. Key pathways such as oxidative phosphorylation were notably enriched. Additionally, 419 proteins were downregulated (fold change < 0.8, p < 0.05), including those involved in fatty acid β-oxidation. Importantly, GO Cellular Component analysis revealed that upregulated proteins were enriched in both extracellular exosome and mitochondrial compartments, supporting successful transfer and uptake of mitoEVs. These findings indicate that ADSC-mitoEVs induce extensive reprogramming of cellular metabolism and enhance cytoprotective responses in endothelial cells under hypoxia, highlighting their role in promoting metabolic adaptation and survival under ischemic conditions. ADSC-mitoEVs rescues the mitochondrial dysfunction of ischemic HUVECs Using JC-1 staining, we observed that ADSC-mitoEVs markedly enhanced the mitochondrial membrane potential (MMP) and increased total ATP production in ischemic HUVECs (Fig. 5 A-C). Furthermore, ADSC-mitoEV treatment significantly reduced mitochondrial ROS levels (Fig. 5 D-E). Mitochondrial stress assays demonstrated that ADSC-mitoEVs improved basal oxygen consumption rate (OCR), mitochondrial ATP production, spare respiratory capacity, and non-mitochondrial ATP generation (Fig. 5 F-G). These results indicate that ADSC-mitoEVs restore mitochondrial bioenergetics, enhance redox balance, and boost metabolic flexibility in hypoxic endothelial cells, underscoring their therapeutic potential in ameliorating ischemia-induced mitochondrial dysfunction. ADSC-mitoEVs Promotes the Mitophagy via PINK1/Parkin Signaling Pathway To investigate the impact of ADSC-mitoEVs on mitochondrial quality control under ischemic conditions, we first employed mt-Keima lentiviral transduction to monitor mitophagy flux. The results indicated that ADSC-mitoEVs significantly enhanced mitophagy, as evidenced by an increased ratio of mitochondria within lysosomes to those in the neutral cytosol (Fig. 6 A-B). Moreover, ADSC-mitoEV treatment improved mitochondrial morphology under ischemic stress, promoting increased mitochondrial network length and size (Fig. 6 C-D). We next evaluated the persistence and integration of transferred mitochondria. Using droplet digital PCR (ddPCR) with primers specific to mtDNA and ADSCs and HUVECs (Supplementary Data 1), we detected approximately 32,000 copies of ADSC-derived mtDNA in HUVECs after 12 hours of co-culture with ADSC-mitoEVs. This copy number declined to 22,400 at 24 hours and 12,800 at 48 hours, becoming undetectable by 72 hours (Supplementary Fig. 2 and Fig. 6 E). To elucidate the mechanisms underlying enhanced mitophagy, we analyzed markers of autophagic flux and mitophagy pathways (Fig. 6 F). ADSC-mitoEVs treatment increased the LC3-II/I ratio and decreased p62 expression, confirming enhanced autophagic activity. Western blot analysis further revealed elevated levels of PINK1, phospho-ubiquitin, and phospho-Parkin, indicating activation of the PINK1/Parkin-mediated mitophagy pathway. In contrast, no significant activation was observed in the BNIP3/NIX pathway. ADSC-mitoEVs Enhances the Angiogenesis in PINK1 Depend Manner To elucidate the mechanistic dependence of ADSC-mitoEV-mediated angiogenesis and mitochondrial function repair, we genetically knocked down PINK1 in HUVECs (Supplementary Fig. 3A). Loss of PINK1 abolished the pro-angiogenic effects of ADSC-mitoEVs, as demonstrated by significantly impaired tube formation and reduced endothelial cell migration (Fig. 7 C-F). However, PINK1 knockdown did not fully abrogate the beneficial effects of ADSC-mitoEVs. Anti-apoptotic activity, reduction in oxidative stress, and improvements in cellular energy metabolism were partially retained, suggesting these effects are mediated through both PINK1-dependent and -independent mechanisms (Fig. 7 A-B, G-O). ADSC-mitoEVs Facilitates the Survival of Ischemic HUVECs through SOD2 Transfer To further elucidate the mechanistic basis of ADSC-mitoEV-mediated mitochondrial functional recovery, we performed Venn analysis to identify mitochondrial proteins enriched in ADSC-mitoEVs and concurrently upregulated in recipient HUVECs (Supplementary Data 2). This approach revealed 43 candidate mitochondrial proteins potentially transferred via mitoEVs (Fig. 8 A-B). Subsequent protein-interaction and functional enrichment analyses indicated that these proteins are primarily involved in cellular respiration, mitochondrial organization, antioxidant responses, and anti-apoptotic pathways. Among these, SOD2 emerged as a central node capable of modulating all aforementioned processes (Fig. 8 C). To experimentally validate the functional significance of SOD2 transfer, we generated SOD2-overexpressing ADSCs and isolated SOD2-enriched mitoEVs (Supplementary Fig. 3B-D). Flag-tagged SOD2 was detected within HUVECs following co-culture, confirming vesicle-mediated delivery (Supplementary Fig. 3E). Western blot analysis further corroborated the increase in SOD2 expression in recipient cells (Supplementary Fig. 3F-G). Treatment with SOD2-enriched mitoEVs significantly reduced apoptosis and mitochondrial ROS levels (Fig. 9 A-D), while enhancing mitochondrial membrane potential in ischemic HUVECs with PINK1 silence (Fig. 9 E-F). Seahorse assays demonstrated improved basal OCR, ATP-linked respiration, spare respiratory capacity, and non-mitochondrial ATP production (Fig. 9 G-K). These results identify SOD2 as a key functional mediator within ADSC-mitoEVs that orchestrates enhanced mitochondrial function and redox balance. Importantly, SOD2-enriched mitoEVs directly improved energy metabolism and mitochondrial performance in recipient cells independent of the PINK1/Parkin-mediated mitophagy. Discussion In this study, we demonstrate that mitochondrial-enriched extracellular vesicles derived from adipose-derived stem cells serve as an efficient bioenergetic cargo system capable of rescuing ischemic endothelial cells through enhanced mitophagy and mitochondrial protein transfer. These vesicles promote angiogenesis, improve mitochondrial function, and facilitate functional recovery in models of hindlimb ischemia, thereby presenting a novel cell-free strategy for ischemic vascular diseases. Our experimental strategy was designed to systematically elucidate the functional and mechanistic contributions of ADSC-mitoEVs. Beginning with proteomic characterization, we identified a significant enrichment of mitochondrial proteins within ADSC-mitoEVs, which contribute to oxidative phosphorylation, redox homeostasis, and anti-apoptotic processes. This initial profiling suggested that mitoEVs might directly supplement dysfunctional mitochondria in recipient cells. However, internalized ADSC-mitoEVs did not integrate into the mitochondrial network of recipient endothelial cells. Instead, they rapidly activated the PINK1/Parkin-mediated mitophagy pathway within 12 hours and were completely degraded within 72 hours. This activation promotes mitochondrial turnover and quality control, served as a prerequisite for initiating angiogenesis in ischemic endothelial cells. Concurrently, mitophagy induction enhanced reactive oxygen species clearance, alleviating mitochondrial stress[ 14 , 15 ]. These effects align with mechanisms reported for both free mitochondrial transfer from ADSCs and TNT-mediated mitochondrial delivery[ 11 ]. Although the internalization routes differ (endocytosis for EVs versus direct intercellular transfer for TNTs) both pathways converge on a shared functional outcome: activation of mitophagy and subsequent cytoprotection. We propose that this convergence is mediated by the delivery of mitochondrial components, such as some kinds of mitochondrial proteins and mtDNA, which are recognized as “non-self” motifs by the recipient cell’s surveillance system. These motifs trigger PINK1 stabilization on the outer mitochondrial membrane, Parkin recruitment, ubiquitination of mitochondrial substrates, and ultimately autophagosome formation around the exogenous material[ 16 – 18 ]. The PINK1/Parkin pathway, central to mitochondrial quality control via mitophagy, plays a critical and predominantly restrictive role in angiogenesis by clearing dysfunctional mitochondria in endothelial cells. This pathway maintains cellular fitness by limiting reactive oxygen species, preventing apoptosis, and supporting the necessary metabolic reprogramming for cell migration and proliferation[ 19 – 22 ]. Moderate activation of mitophagy is beneficial, as it facilitates the removal of dysfunctional organelles and stimulates mitochondrial biogenesis, leading to increased ATP production[ 19 – 22 ]. Indeed, following mitoEV treatment, we observed increased total mitochondrial content, well-developed mitochondrial network and enhanced ATP generation in endothelial cells, indicating improved bioenergetic capacity as well as enhanced cellular fitness under stress conditions. Notably, unlike free mitochondrial transfer as reported previously[ 11 ], ADSC-mitoEVs retained partial functionality, such as antioxidant capacity and improvements in energy metabolism, even in PINK1-silenced endothelial cells. We attribute this PINK1-independent effect to the efficient vesicle-mediated delivery of functional mitochondrial enzymes, particularly superoxide dismutase 2 (SOD2). Owing to the protective bilayer phospholipid membrane of extracellular vesicles[ 23 , 24 ], the structural and functional integrity of SOD2 and other oxidative phosphorylation-related proteins is preserved during intercellular transit, enabling them to remain catalytically active upon delivery. Mechanistically, once internalized, SOD2-enriched ADSC-mitoEVs preferentially localize to the mitochondrial compartments of recipient endothelial cells, where the encapsulated SOD2 is released and integrates into the endogenous mitochondrial matrix. SOD2 anions dismutates superoxide anions into hydrogen peroxide and oxygen, thereby markedly reducing mitochondrial reactive oxygen species levels and protecting electron transport chain complexes from oxidative damage, stabilizing mitochondrial membrane potential and enhancing ATP synthesis independently of PINK1/Parkin signaling[ 25 – 28 ]. Proteomic analyses further suggest that ADSC-mitoEVs carry additional mitochondrial enzymes, such as ubiquinone oxidoreductase subunits and ATP synthase, which may act synergistically with SOD2 to ameliorate bioenergetic deficits and enhance mitochondrial resilience under ischemic stress. This multi-protein delivery mechanism distinguishes ADSC-mitoEVs from conventional mitochondrial transplantation and underscores their potential as a comprehensive mitochondrial therapy for vascular diseases. Mitochondrial transplantation has emerged as a promising treatment for conditions such as cancer[ 29 ], Parkinson’s disease[ 30 ], and myocardial infarction[ 31 ]. ADSC-mitoEVs offer a cell-free strategy that combines the advantages of mitochondrial transplantation and conventional EV therapies while overcoming following limitations. Unlike direct mitochondrial transplantation, which suffers from poor stability and short retention times, ADSC-mitoEVs provide a membrane-protected biological vector that enhances protein stability and delivery efficiency of multiple mitochondrial-derived enzymes. Moreover, compared with heterogeneous conventional MSC-EV preparations, ADSC-mitoEVs represent a defined subpopulation with elevated mitochondrial content, enabling more targeted bioenergetic modulation. Despite these promising findings, our study has several limitations. First, advanced purification strategies, such as immunoaffinity-based sorting for mitochondrial markers, could yield more homogeneous mitoEV populations if the membrane protein profile of mitoEVs can be characterized. Additionally, while diabetic murine models of hindlimb ischemia offer useful insights, they hardly recapitulate the complex pathophysiology of human vascular diseases, particularly in aging or comorbid settings. Further validation in large animal models is essential before clinical translation. Finally, the long-term safety, immunogenicity, and fate of administered mitoEVs warrant systematic investigation. Conclusion In this study, we demonstrate that ADSC-mitoEVs, a novel cell-free therapeutic platform, ameliorates ischemic endothelial damage through dual mechanisms: activation of PINK1/Parkin-mediated mitophagy and direct transfer of functional mitochondrial proteins. This strategy enhances mitochondrial quality control, redox homeostasis and energy metabolism reprogramming, offering a promising approach for ischemic vascular diseases treatment. Materials and methods Isolation and Characteristics of ADSC-mitoEVs Human lipoaspirates were harvested from the thigh region of healthy female donors (aged 25 years) using water jet-assisted liposuction (Body-Jet System; Human Med AG, Schwerin, Germany). All procedures complied with the principles outlined in the Declaration of Helsinki (1975) and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (IIT-0-2025-100). Adipose-derived stem cells (ADSCs) were isolated following a previously established protocol. Primary ADSCs were transfected with pLV-mito-miRFP670 lentiviral particles (Han Heng Biotechnology, Shanghai, China) to enable mitochondrial labeling via miRFP670 fluorescence. Transfected mito-miRFP670-ADSCs (passages 3–5) were cultured in mesenchymal stem cell medium supplemented with 5% extracellular vesicle (EV)-depleted serum at 37°C under 5% CO₂ and 95% humidified air. The conditioned medium was collected and subjected to sequential centrifugation steps at 800×g and 3,000×g at 4°C to remove cellular debris. The resulting supernatant was concentrated using an ultrafiltration centrifuge tube equipped with a 100 kDa molecular weight cut-off filter (Millipore, MA, USA) by centrifugation at 3,000×g for 30 minutes at 4°C. The EV suspension was then processed using a CytoFLEX SRT cell sorter (Beckman Coulter, CA, USA) to isolate miRFP670-positive vesicles. Finally, the sorted suspension was filtered through a 0.22 µm membrane and subjected to ultracentrifugation at 110,000×g for 90 minutes at 4°C (Beckman Coulter, CA, USA) to pellet the ADSC-derived mitochondrial extracellular vesicles (ADSC-mitoEVs). (Fig. 1 A) The characterization of ADSC-mitoEVs was carried out using transmission electron microscopy (Hitachi, Tokyo, Japan) and Nanoparticle Tracking Analysis (NTA, ZetaVIEW, Meerbusch, Garmany) for morphology and particle size distribution analysis. Antibodies against CD63, TOM20 and tumor susceptibility gene 101 protein (TSG101) (Abcam, Cambridge, UK) were used in the Western blot to detect the expression of mito-EV-specific markers. The protein concentration was evaluated using a BCA Protein Assay Kit (Sigma, MA, USA). Proteomic Analysis Reveals Mitochondrial Components in ADSC-EVs Proteomic profiling was employed to characterize the mitochondrial components within ADSC-EVs. Total protein was extracted from isolated ADSC-EVs and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, MA, USA). The acquired data were processed with MaxQuant software and matched against the UniProt human database. Mitochondrial proteins were identified based on subcellular localization predictions. Subsequent bioinformatic analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, were performed to delineate significantly enriched pathways associated with the identified mitochondrial proteins. Cell Culture Human umbilical vein endothelial cells (HUVECs) were acquired from the Chinese Academy of Sciences cell bank and maintained in endothelial cell medium (ECM, ScienCell, CA, USA) supplemented with 5% fetal bovine serum (FBS) at 37°C under normoxic conditions (20% O₂). To mimic ischemia, HUVECs were subjected to hypoxia (1% O₂) in serum-free ECM at 37°C. MitoEVs Uptake HUVECs were transfected with pLV-mito-DsRed lentiviral particles (Han Heng Biotechnology, Shanghai, China) to achieve mitochondrial labeling with DsRed fluorescence. The transfected mito-DsRed-HUVECs were then co-cultured with ADSC-mitoEVs (10 µg/mL) for 12 hours under both normal and ischemic conditions. Cellular uptake of ADSC-mitoEVs was visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany). The area of both miRFP670 and DsRed signal were counted in ten random cells per group. Three independent experiments were performed. Cell Apoptosis Assay Apoptosis was evaluated using an Annexin V/7-AAD Apoptosis Detection Kit (Beyotime Biotechnology, Shanghai, China). After treatment with ADSC-mitoEVs (10, 20, 40 and 80 µg/mL) for 48 hours under ischemic conditions. HUVECs cultured at normal condition were served as positive control. HUVECs were harvested, washed with PBS, and then were stained with Annexin V and 7-AAD for 20 minutes at 37°C in the dark. Apoptotic rates were analyzed using a flow cytometer (BD biosciences, NJ, USA). Three independent experiments were performed. Scratch Test A scratch test was performed to evaluate cell migration. HUVECs were seeded in 24-well plates and grown to confluence. A sterile 100 µL pipette tip was used to create a linear scratch. After washing with PBS to remove detached cells, the cells were incubated with ADSC-mitoEVs (10, 20, 40 and 80 µg/mL) in serum-free medium under hypoxia. HUVECs cultured at normal condition were served as positive control. Wound closure was monitored at 0 and 24 hours using an inverted microscope (Carl Zeiss, Oberkochen, Germany). Migration rate was quantified with ImageJ software. Three independent experiments were performed. Tube Formation The angiogenic capability of HUVECs was assessed using a tube formation assay on Matrigel (Corning, NY, USA). Briefly, 96-well plates were coated with Matrigel and polymerized for 30 minutes at 37°C. HUVECs were incubated with ADSC-mitoEVs (10, 20, 40 and 80 µg/mL) under hypoxia for 12 hours. Tubular structures were imaged under an inverted microscope (Carl Zeiss, Oberkochen, Germany). Total tube length was analyzed with Angiogenesis Analyzer tool in Image J. Three independent experiments were performed. Animal Models All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang University (NCULAE-20250912001) and complied with the NIH Guide for the Care and Use of Laboratory Animals. For tube formation in vivo, five male BALB/c nude mice (8 weeks old) anesthetized by inhalation of isoflurane (induced at 4% and maintained at 2% at a flow rate of 1.5 L/min). 5×10 5 HUVECs with or without ADSC-mitoEVs (40 µg/ml) were coated with 100 µL Matrigel (Corning, NY, USA) with investigators blinded to group allocation during procedures and assessments, and then injected subcutaneously into both armpits of nude mice. After one week, neoformations were harvested and weighted. Surrounding neovascularization were analyzed with Angiogenesis Analyzer tool in Image J. Diabetes was induced in 8-week-old male BALB/c nude mice by intraperitoneal injection of streptozotocin (STZ; Sigma, MA, USA) at the dose of 100 mg/kg. Blood glucose levels were monitored from the tail vein using a glucometer (Omron, Kyoto, Japan) on days 14 post-initial injection. Mice with two consecutive fasting (6-hour) blood glucose measurements of ≥ 16.7 mmol/L were considered diabetic and included in the subsequent experimental cohort. For mouse hindlimb ischemia model, ten diabetic BALB/c nude mice were randomly assigned to mitoEVs group (n = 5) or PBS group (n = 5), with investigators blinded to group allocation during procedures and assessments. Mice were anesthetized by inhalation of isoflurane (induced at 4% and maintained at 2% at a flow rate of 1.5 L/min), and unilateral hindlimb ischemia was induced by ligating the femoral artery. ADSC-mitoEVs (40 µg/ml) in 100 µL PBS or an equal volume of PBS as control were administered via intramuscular injection at three sites in the ischemic muscle immediately after surgery. Blood perfusion was monitored on days 0, 7, 14 and 21 using a Laser Speckle Contrast Imaging system (Perimed, Stockholm, Sweden). The thigh muscle tissue was harvested on the 21st day after surgery and used for subsequent experiments. Whole mount staining Neoformation and thigh muscle tissues from nude mice were fixed in 4% paraformaldehyde for 24 h, followed by dehydration in 30% sucrose for 72 h. The samples were then embedded and cryosectioned into 150 µm-thick slices. For immunofluorescence staining, neoformation sections were incubated with an anti-CD31 primary antibody (R&D Systems, MN, USA). Thigh muscle sections were co-stained with anti-CD31 and anti-α-SMA primary antibodies (R&D Systems and Sigma, respectively). Neovascularization density and tube length were visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany) and then quantified using the Angiogenesis Analyzer tool in ImageJ software, while muscle density was assessed using Image J. Proteomic Analysis of ADSC-mitoEVs Regulating Ischemic Vascular Endothelial Cells HUVECs were treated with ADSC-mitoEVs (40 µg/mL) under ischemic conditions for 24 hours. Total proteins were extracted. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, MA, USA). Data were analyzed using MaxQuant software and searched against the UniProt human database. Bioinformatic analysis including GO and KEGG enrichment was conducted to identify significantly altered pathways. Three independent experiments were performed. Mitochondrial Membrane Potential (MMP) Assay MMP was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, Shanghai, China). After treatment, HUVECs were incubated with JC-1 staining solution for 20 minutes at 37°C, and then was visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany). The ratio of red (aggregates) to green (monomers) fluorescence was used to indicate MMP. Three independent experiments were performed. Mitochondrial Oxidative Stress Assay Mitochondrial respiration was assessed using a Seahorse XFe24 Analyzer (Agilent, CA, USA). HUVECs were seeded in XF24 plates and treated with ADSC-mitoEVs (40 µg/mL) under ischemic conditions. Oxygen consumption rate (OCR) was measured under basal conditions and in response to oligomycin, FCCP and rotenone/antimycin A. For mitochondrial superoxide detection, cells were stained with mitoSOX Red (Invitrogen, CA, USA) and analyzed by flow cytometry (BD biosciences, NJ, USA). Three independent experiments were performed. ATP measurement Intracellular ATP levels were quantified using an ATP Assay Kit (Abcam, UK) based on luciferase-luciferin reaction. Treated HUVECs were lysed, and luminescence was measured with a microplate reader (ShanpuBiotech, Shanghai, China). Three independent experiments were performed. Mitophagy Flow Assay HUVECs were transfected with mt-Keima lentivirus (Han Heng Biotechnology, China) following the manufacturer’s protocol. After treatment with ADSC-mitoEVs under ischemic conditions, mitophagy was assessed by laser scanning confocal microscopy (LSCM; Carl Zeiss, Germany). Mitophagy activity was quantified based on the red-to-green fluorescence intensity ratio (550/440 nm) in ten random cells per group per time point, corresponding to acidified mitochondria within lysosomes versus mitochondria in the neutral cytoplasmic environment. Furthermore, key proteins associated with mitophagy pathways including PINK1, phospho-ubiquitin, phospho-Parkin (PINK1/Parkin pathway), BNIP3/NIX, as well as LC3 and P62 were analyzed by western blot. Three independent experiments were performed. Exogenous mtDNA Assay To evaluate the persistence of transferred mitochondria in recipient HUVECs over time, we employed a mitochondrial DNA (mtDNA) genotyping strategy based on the detection of heteroplasmy. mtDNA was isolated from ADSCs and HUVECs using a Mitochondrial DNA Isolation Kit (MCE, NJ, USA). Regions with sequence divergence between the two cell types were identified by Sanger sequencing. Specific primers were designed to target these differential sites, and their amplification efficiency was validated by Droplet Digital PCR (ddPCR) on a QuantStudio Absolute Q system (Thermo Fisher Scientific, MA, USA). Using these primers, we quantified the copy number of ADSC-derived mtDNA in HUVECs co-cultured with ADSC-mitoEVs at 12, 24, 48, and 72 h post-treatment via ddPCR. Three independent experiments were performed. Silencing of PINK1 by shRNA ShRNA lentiviral particles targeting human PINK1, along with negative control shRNA constructs, were obtained from Han Heng Biotechnology (Shanghai, China). HUVECs were transduced with lentiviral particles in the presence of polybrene. Following a 72-hour incubation, transduced cells were selected using puromycin. Silencing efficiency was confirmed by qRT–PCR analysis of mRNA extracted from selected cells. Three independent experiments were performed. Generation of SOD2-overexpressed ADSC-mitoEVs To determine whether ADSC-mitoEVs confer mitochondrial protection in recipient cells via delivery of mitochondrial proteins, we identified overlapping mitochondrial proteins between the mitoEVs and upregulated mitochondrial proteins in HUVECs treated with mitoEVs using Venn analysis. This set was defined as candidate mitochondrial factors. Protein-protein interaction and functional enrichment analyses were performed using STRING ( https://cn.string-db.org/ ) to predict the biological roles of these candidates, leading to the identification of SOD2 as a central node protein. Lentiviral particles encoding human SOD2-Flag and the corresponding empty vector controls were obtained from Han Heng Biotechnology (Shanghai, China). ADSCs were transduced with the lentiviral particles in the presence of polybrene. After 72 hours, stable cell lines were selected using puromycin. Overexpression efficiency was confirmed at the mRNA level by qRT‑PCR and at the protein level by western blot and immunofluorescence staining, using antibodies against SOD2 (Santa Cruz Biotechnology, Dallas, USA) and Flag (Proteintech, Wuhan, China). SOD2-overexpressing ADSCs were cultured, and the conditioned medium was collected for the isolation of SOD2‑overexpressing ADSC‑mitoEVs (SOD2‑mitoEVs). The uptake of SOD2‑mitoEVs by HUVECs was analyzed via Flag immunofluorescence staining. Additionally, SOD2 expression in HUVECs that had taken up SOD2‑mitoEVs was further evaluated by western blot. Three independent experiments were performed. Statistical Analysis Data are presented as mean ± SD. Comparisons were analyzed via unpaired t-tests or one-way ANOVA with Fisher’s LSD post hoc test (GraphPad Prism 9). Significance was defined as P < 0.05. Declarations Acknowledgements Not applicable. Author contributions Yuan-zheng Zhu contributed to the study design, isolation and characteristics ADSC-mitoEVs, cell culture and functional assay in vitro, proteomics assay and manuscript writing. Min-chen Zhang contributed to Seahorse assay and ATP detection. Xue-er Li contributed to the heterogeneity analysis of mtDNA and generation of PINK1 silenced and SOD2 overexpressed cells. Xing-hong Zeng contributed to the animal study. Xue-ting Gong and Yu-zi Wu contributed to the western blotting. Ze-jun Dong, Shu Wu and Xue-fei Liu contributed to the data statistics. Yang-yan Yi contributed to the conception and design, financial support and final approval of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China [Grant number: 82460448]; Natural Science Foundation of Jiangxi Province [Grant number: 20242BAB26140 and 20252BAC240585]; Jiangxi Province Key laboratory of Precision Cell Therapy [Grant number: 2024SSY06241]. Data availability All data generated and analyzed during this research are included in this published article. Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang University (NCULAE-20250912001) and complied with the NIH Guide for the Care and Use of Laboratory Animals. Human lipoaspirates complied with the principles outlined in the Declaration of Helsinki (1975) and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (IIT-0-2025-100). Consent for publication All authors agree for publication. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Contributor Information Yang-yan Yi; Email: [email protected] References Sayed A, Michos ED, Navar AM, Virani SS, Brewer LC, Manson JE. Global Sociodemographic Disparities in Ischemic Heart Disease Mortality According to Sex, 1980 to 2021. Circ Cardiovasc Qual Outcomes. 2025;18:e011648. Slater J, Maron DJ, Jones PG, Bangalore S, Reynolds HR, Fu Z, Stone GW, Kirby R, Hochman JS, Spertus JA, Group IR. 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Supplementary Files SupplementaryData1.docx Supplementary Data 1 SFigure1.jpg Supplementary Figure 1.Proteomic analysis of ADSC-EVs (A) Subcellular localization of proteins carried by ADSC-EVs; (B) Top 20 enrichment terms of GO and KEGG of 390 mitochondrial proteins carried by ADSC-EVs; (C-F) Chord diagrams shown the key mitochondrial proteins which contribute to mitochondrion targeting, antioxidant, proton motive force and mito-electron transport. SFigure2.jpg Supplementary Figure 2.Quantify exogenous mtDNA through ddPCR (A) Study design for exogenous mtDNA quantization; (B) Verification of the specificity of primer probes through ddPCR; (C) The results of ddPCR indicated copy number of mtDNA from ADSCs internalized by HUVECs (ADSC-derived mtDNA were highlighted by blue triangle). SFigure3.jpg Supplementary Figure 3. (A) The results of qRT-PCR confirmed that PINK1 was successfully knocked down in HUVECs, *p-value < 0.05; (B) The results of qRT-PCR confirmed that SOD2 was successfully overexpressed in HUVECs, p-value < 0.05; (C) Increased SOD2 expression in SOD2-Lentiviral-transfected ADSCs via flag immunofluorescence staining, scale bar = 50 μm; (D) The results of western blotting confirmed the overexpression of SOD2 in ADSC-mitoEVs; (E-F) HUVECs co-culturing with SOD2-overexpressed mitoEVs shown increased SOD2 expression as validated by flag immunofluorescence staining and western blotting, scale bar = 50 μm, * p-value < 0.05. GraphicalAbstract.pdf Cite Share Download PDF Status: Published Journal Publication published 25 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 02 Nov, 2025 Reviews received at journal 02 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviews received at journal 29 Oct, 2025 Reviewers agreed at journal 26 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviewers agreed at journal 21 Oct, 2025 Reviewers invited by journal 21 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Submission checks completed at journal 14 Oct, 2025 First submitted to journal 13 Oct, 2025 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-7849775","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":537501400,"identity":"d155d60e-3a5b-4bfd-93ec-33108dc2c11d","order_by":0,"name":"Yuan-zheng Zhu","email":"","orcid":"","institution":"Department of Plastic Surgery, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yuan-zheng","middleName":"","lastName":"Zhu","suffix":""},{"id":537501401,"identity":"da830442-71a9-449f-bc67-d6b6e83fe6ae","order_by":1,"name":"Min-chen 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07:01:23","extension":"xml","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120359,"visible":true,"origin":"","legend":"","description":"","filename":"282cb04590514d0abfc086eaba0a3d371structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/746ef73bf2870ce69c256712.xml"},{"id":94884161,"identity":"b0b91067-222a-4eb3-b140-60a01bdebd76","added_by":"auto","created_at":"2025-10-31 17:40:29","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132378,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/fa3cd4977140606c2908f4e6.html"},{"id":94884113,"identity":"3e656cce-6e30-4f72-b18d-f3352894820b","added_by":"auto","created_at":"2025-10-31 17:40:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2295874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and Characterization of ADSC-mitoEVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The isolation and sorting process of ADSC-mitoEVs; (B) Microscopic morphology of mitoEVs and mito-free EVs under transmission electron microscopy, scale bar = 200 nm; (C) Nano particle size distribution of mitoEVs and mito-free EVs; (D) Western blotting for markers of EVs (CD63 and TSG101) and mitochondria outer membrane (TOM20); (E) Uptake of ADSC-mitoEVs (red) in mito-DsRed-labeled HUVECs (yellow) under ischemia (1%O\u003csub\u003e2\u003c/sub\u003e, FBS-free) and normal conditions (20% O\u003csub\u003e2\u003c/sub\u003e, 10%FBS). MitoEVs that binding on mitochondrial network of HUVECs were highlighted by white triangle symbol, scale bar = 20 μm; (F) The line chart shown the proportion of exogenous mitoEVs bound to the HUVECs mitochondrial network over a period of 4 to 12 hours under both ischemia and normal conditions, *p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/0565ffe6441c06ae77efc0d3.jpg"},{"id":94986934,"identity":"a80e3459-0461-47da-87e8-698eb5ea2945","added_by":"auto","created_at":"2025-11-03 07:00:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2671111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell Protective and Proangiogenic Effects of ADSC-mitoEVsin Vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) The effect of ADSC-mitoEVs on apoptosisof ischemic endothelial cells was evaluated by flow cytometry. The results indicated reduced apoptosis rate led by ADSC-mitoEVs treatment, *p-value \u0026lt;0.05; (D-E) The migration rate of ischemic endothelial cells treated with ADSC-mitoEVs was assayed by scratch test. The results shown that ADSC-mitoEVs enhanced migration of ischemic endothelial cells, scale bar = 200 μm, *p-value \u0026lt;0.05; (F-G) ADSC-mitoEVs promoted the tube formation potential of ischemic endothelial cells on Matrige, scale bar = 500 μm, *p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/5070808622c0171866567eb8.jpg"},{"id":94884124,"identity":"57cc6837-6a29-4545-bd5a-b33b9cbb03b0","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3759117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSC-mitoEVs Promoted Revascularization in Vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Study design of Matrigel angiogenesis test in nude mice; (B) Image of new organisms treated with ADSC-mitoEVs or not, scale bar = 0.5 mm; (C) Surrounding vascular density of new organisms obtained through Image J software statistics, *p-value \u0026lt;0.05; (D) The mass of new organisms, *p-value \u0026lt;0.05; (E) Whole mount immunofluorescence staining for CD31 of new organisms, scale bar = 200 μm; (F-G) Vascular density (CD31 positive) and total tube length obtained through Image J software statistics, *p-value \u0026lt;0.05; (H) Study design for evaluate the effect of ADSC-mitoEVs on ischemic hindlimbs in nude mice; (I-J) The results of laser speckle imaging indicated improved blood flow in mitoEV-treated hindlimbs, *p-value \u0026lt;0.05; (K) Statistics of individuals with hind limb disabilities; (L-M) The results of laser speckle imaging indicated improved blood flow in mitoEV-treatedfeet, *p-value \u0026lt;0.05; (N-P) Whole mount immunofluorescence staining for CD31 and α-SMA of ischemic hind limb muscle tissue indicated enhanced revascularization in mitoEV-treated tissue, scale bar = 200 μm, *p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/9298bc901c0002f13acbc843.jpg"},{"id":94884141,"identity":"1ae0d93b-c5d2-4bd0-8e9b-4186caa9cb93","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5546475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomics Indicated Improved Mitophagy and Remodeled Energy Metabolism by ADSC-mitoEVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Volcano plot shown 845 upregulated proteins (fold change \u0026gt; 1.25, p \u0026lt; 0.05) and 419 downregulated proteins (fold change \u0026lt; 0.8, p \u0026lt; 0.05) of hypoxic HUVECs treated with ADSC-mitoEVs or not; (B-D) The results of the analysis of GO-Biological Process/ Cellular Component and KEGG-pathways shown the function of 845 upregulated proteins as well as 419 downregulated proteins.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/200f9ebe22f2b63cd3b7d0e3.jpg"},{"id":94986834,"identity":"11b4d802-5748-4b33-8e59-b23870dd5c60","added_by":"auto","created_at":"2025-11-03 07:00:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2669404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSC-mitoEVs Rescued the MitochondrialDysfunction of Ischemic HUVECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) JC-1 staining indicated the mitochondrial membrane potential of ischemic HUVECs treated with ADSC-mitoEVs or not, scale bar = 50 μm; (B) Bar chart shown the rate of JC-1 aggregates to monomers, *p-value \u0026lt;0.05; (C) The results of Intracellular ATP quantification based on luciferase-luciferin reaction indicated increased ATP level in mitoEV-treated HUVECs, *p-value \u0026lt;0.05; (D-E) The results of mitoSOX Red stained HUVECs analyzed by flow cytometry shown reduced mitoROS level in mitoEV-treated HUVECs, *p-value \u0026lt;0.05; (F-J) Mitochondrial respiration assay on ischemic HUVECs shown enhanced basal OCR, mito-ATP production, spare capacity and non-mito OCR, *p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/5fb9e2f2217a7a7eea85913c.jpg"},{"id":94884136,"identity":"6ae604f4-6ceb-49bf-b82c-15f281859007","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1781531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSC-mitoEVs Promoted Mitophagy of Ischemic HUVECs through PINK1/Parkin Pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) The images of mt-Keima lentiviral transfected HUVECs shown that ADSC-mitoEVs accelerate the mitophagy flux with increased rate of mitochondria within lysosomes to those in the neutral cytosol, and also improved the mito-network size and length, scale bar = 10 μm, *p-value \u0026lt;0.05; (E) The results of ddPCR indicated decreased ADSC-mtDNA retain in HUCECs from co-culturing 12 to 72 h; (F) Western blotting for markers of autophagic flux and mitophagy pathways demonstrated that \u0026nbsp;ADSC-mitoEVs treatment enhance autophagy with increased the LC3-II/I ratio and decreased p62 expression, and enhance mitophagy through PINK1/Parkin pathway with upregulated PINK1, phospho-ubiquitin, and phospho-Parkin expression. However, no significant activation was observed in the BNIP3/NIX pathway, *p-value \u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/a65b1cda7651e813daed0b3d.jpg"},{"id":94884127,"identity":"66e16e26-126c-4bba-93e3-3192b46b06be","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2743286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADSC-mitoEVs Promote Angiogenesis and Rescue Mitochondrial Dysfunction in a PINK1-dependent Manner\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional and metabolic improvements in ischemic HUVECs treated with ADSC-mitoEVs, with and without PINK1 knockdown. All data are vs. respective NC (*p-value \u0026lt; 0.05). MitoEVs enhanced cell migration (C-D, scratch assay) and angiogenic potential (E-F, tube formation) only in PINK1-competent cells. MitoEVs reduced apoptosis (A-B) and mito-ROS (G-H), and improved mitochondrial membrane potential (I-J) and mitochondrial energy metabolism (K-O) irrespective of PINK1 status.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/10106598401afe10a8ec2ae0.jpg"},{"id":94986967,"identity":"8b2a255d-2ab9-41cb-b7a8-d2fb660284cc","added_by":"auto","created_at":"2025-11-03 07:01:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1184549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOD2 as a critical candidate protein for ADSC-mitoEVs to rescue mitochondrial damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Venn analysis of mitochondrial proteins in ADSC-EVs and upregulated proeins in mitoEV-treated HUVECs identified 43 candidate proteins; (B) Heatmap of candidate proteins in HUVECs and mitoEV-treated HUVECs; (C) Protein interaction and functional analysis of 43 candidate proteins, SOD2 is served as a key node proteins that involved in multiple biological processes including cellular respiration, mitochondrion organization, antioxidant process, cellular biosynthetic process and apoptotic process.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/c9ab5e0d12f4df5c876cc09e.jpg"},{"id":94986829,"identity":"1822bf6e-b43c-4aab-aecc-a92146c83d6a","added_by":"auto","created_at":"2025-11-03 07:00:50","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1881578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOD2-overexpressed-ADSC-mitoEVs Further promoted the Survival and Mitochondrial Homeostasis of Ischemic HUVECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurvival and metabolic improvements in ischemic HUVECs treated with ADSC-mitoEVs or SOD2-overexpressed-ADSC-mitoEVs. All data are vs. respective control (*p-value \u0026lt; 0.05). Compared with mitoEVs, SOD2-overexpressed-mitoEVs further reduced apoptosis (A-B) and mito-ROS (C-D), as well as improved mitochondrial membrane potential (E-F) and mitochondrial energy metabolism(G-K).\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/deba84aa88b2ba5af4876d94.jpg"},{"id":103765675,"identity":"fcb2bef9-c56c-46b4-b790-09d5b7b206a3","added_by":"auto","created_at":"2026-03-02 16:07:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25916972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/29db243c-9143-4c7f-b0ef-a8eff1ada380.pdf"},{"id":94884128,"identity":"21ef6fc1-21f4-437a-820b-cbecc8be9c98","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16744,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 1\u003c/p\u003e","description":"","filename":"SupplementaryData1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/d3719342aef489fda5707c47.docx"},{"id":94884115,"identity":"2c3ef10e-e455-407f-a702-7be7234256b4","added_by":"auto","created_at":"2025-10-31 17:40:27","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4920617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1.Proteomic analysis of ADSC-EVs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Subcellular localization of proteins carried by ADSC-EVs; (B) Top 20 enrichment terms of GO and KEGG of 390 mitochondrial proteins carried by ADSC-EVs; (C-F) Chord diagrams shown the key mitochondrial proteins which contribute to mitochondrion targeting, antioxidant, proton motive force and mito-electron transport.\u003c/p\u003e","description":"","filename":"SFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/cfcce98427dcaee2b1d40f40.jpg"},{"id":94884120,"identity":"62964052-86de-4758-af3c-056c4b016f09","added_by":"auto","created_at":"2025-10-31 17:40:28","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1638505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2.Quantify exogenous mtDNA through ddPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Study design for exogenous mtDNA quantization; (B) Verification of the specificity of primer probes through ddPCR; (C) The results of ddPCR indicated copy number of mtDNA from ADSCs internalized by HUVECs (ADSC-derived mtDNA were highlighted by blue triangle).\u003c/p\u003e","description":"","filename":"SFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/215fb3e3381c5501d2cee766.jpg"},{"id":94884152,"identity":"1a80e684-20d0-4cce-b3d5-f1e81a127343","added_by":"auto","created_at":"2025-10-31 17:40:29","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1359753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The results of qRT-PCR confirmed that PINK1 was successfully knocked down in HUVECs, *p-value \u0026lt; 0.05; (B) The results of qRT-PCR confirmed that SOD2 was successfully overexpressed in HUVECs, p-value \u0026lt; 0.05; (C) Increased SOD2 expression in SOD2-Lentiviral-transfected ADSCs via flag immunofluorescence staining, scale bar = 50 μm; (D) The results of western blotting confirmed the overexpression of SOD2 in ADSC-mitoEVs; (E-F) HUVECs co-culturing with SOD2-overexpressed mitoEVs shown increased SOD2 expression as validated by flag immunofluorescence staining and western blotting, scale bar = 50 μm, * p-value \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"SFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/4008f688181daa63fdc127c3.jpg"},{"id":94884155,"identity":"ea5f828f-e144-4db9-bd11-5a41c28951e7","added_by":"auto","created_at":"2025-10-31 17:40:29","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1141051,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7849775/v1/63bb146ecda692d59544d7c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nanotherapeutic Strategy via ADSC-mitoEVs Rescues Ischemic Angiogenesis through Mitophagy and Mitochondrial Metabolic Reprogramming","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemic diseases, encompassing conditions such as peripheral artery disease and coronary artery disease, represent a leading cause of global morbidity and mortality[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These pathologies are defined by a critical imbalance between oxygen supply and metabolic demand within affected tissues[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The therapeutic induction of robust and functional angiogenesis remains a paramount, yet persistently elusive, goal in vascular medicine. The ischemic microenvironment is notoriously pathological, characterized by profound hypoxia, elevated levels of reactive oxygen species (ROS), nutrient deprivation and sustained inflammation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These factors collectively impose severe bioenergetic stress on resident vascular endothelial cells, compromising their capacity for repair and regeneration.\u003c/p\u003e\u003cp\u003eCentral to cellular survival and function, mitochondria are indispensable for energy production, redox homeostasis and critical signaling processes that govern angiogenesis, including proliferation, migration and tube formation [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Compelling evidence now indicates that mitochondrial dysfunction is a fundamental hallmark of ischemic tissues, directly contributing to cellular apoptosis and the failure of re-vascularization[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, innovative strategies designed to directly ameliorate mitochondrial bioenergetics within ischemic cells present a compelling and targeted therapy.\u003c/p\u003e\u003cp\u003eAdipose-derived stem cells (ADSCs) have emerged as a potent source for regenerative therapy, prized for their abundance, accessibility and pro-angiogenic secretome[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, a groundbreaking paradigm shift has occurred with the recognition of horizontal mitochondrial transfer as a fundamental mechanism of ADSC-mediated cytoprotection and repair[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Intercellular mitochondrial trafficking can occur via several routes, including the formation of tunneling nanotubes (TNTs), gap junctions and cell fusion. While physiologically relevant, these direct cell-contact mechanisms are inherently limited in a therapeutic context by their low efficiency and spatial constraints[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In contrast, the transfer of mitochondria via extracellular vesicles (EVs) represents a sophisticated, distance-independent delivery system[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These mitochondrial-enriched extracellular vesicles (mitoEVs) are naturally packaged by donor cells and can be efficiently internalized by recipient cells, conferring a full complement of functional proteins, lipids and mitochondrial DNA (mtDNA) to resuscitate bioenergetics[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This vesicle-mediated mechanism offers distinct advantages for therapy, including enhanced biological stability in circulation, a reduced risk of immunogenicity compared to whole cells, and the potential for targeted delivery to sites of injury, thereby bypassing the limitations of direct cell-cell contact.\u003c/p\u003e\u003cp\u003eIn this study, we isolated and characterized mitoEVs from ADSC-conditioned media through Nano Flow Cytometry and analyzed their protein components. We then investigated their efficacy in promoting angiogenesis in a murine model of critical hindlimb ischemia. Utilizing integrated proteomic profiling and real-time analysis of cellular metabolism via Seahorse extracellular flux technology, we further elucidate the core mechanism through which ADSC-mitoEVs reprogram cellular energy metabolism and activate potent angiogenic pathways in ischemic VECs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization and Function Analysis of Mitochondrial Components in ADSC-EVs\u003c/h2\u003e\u003cp\u003eProteomic profiling identified 390 mitochondrial proteins (8.2%) in ADSC-EVs (Supplementary Fig.\u0026nbsp;1A). Bioinformatic analysis via GO and KEGG pathways highlighted significant involvement in energy metabolism remodeling including oxidative phosphorylation, fatty acid beta-oxidation, glycolysis and regulation of reactive oxygen species (Supplementary Fig.\u0026nbsp;1B). Further functional annotations underscored roles in mitochondrion targeting, antioxidant, proton motive force and electron transport, supporting the potential of ADSC-mitoEVs to modulate cellular bioenerg Characterization of ADSC-mitoEVs (Supplementary Fig.\u0026nbsp;1C-F).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCharacterization of ADSC-mitoEVs\u003c/h3\u003e\n\u003cp\u003eBoth mitoEVs and mito-free EVs from ADSCs exhibited characteristic cup-shaped morphology under transmission electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). MitoEVs shown increased mean particle size of 218.6\u0026thinsp;\u0026plusmn;\u0026thinsp;32.5 nm compared with that of mito-free EVs (105.3\u0026thinsp;\u0026plusmn;\u0026thinsp;24.1 nm) as determined by nanoparticle tracking analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Western blot analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, confirmed positive expression of EV markers (CD63 and TSG101) in both mitoEVs and mito-free EVs, while positive expression Mitochondrial outer membrane protein (TOM20) only observed in mitoEVs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eIschemia Accelerates the Uptake and Mitochondrial Binding of ADSC-mitoEVs by HUVECs\u003c/h3\u003e\n\u003cp\u003eWe simulated in vivo ischemia by constructing a cell culture microenvironment with hypoxia and serum deprivation. Colocalization analysis indicated that internalized mitoEVs preferentially bind the endogenous mitochondrial network within 12 hours of co-culture, as visualized via confocal microscopy using miRFP670-labeled mitoEVs and DsRed-labeled mitochondria in HUVECs. Increased uptake rate and mitochondrial binding efficiency of mitoEVs was observed in HUVECs cultured under ischemic condition, compared to Normal condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F).\u003c/p\u003e\n\u003ch3\u003eADSC-mitoEVs Facilitates the Survival and Angiogenetic Potential of Ischemic HUVECs\u003c/h3\u003e\n\u003cp\u003eApoptosis analysis by flow cytometry indicated that ADSC-mitoEVs significantly increased the viability of ischemic HUVECs (under hypoxia and serum deprivation) in a dose-dependent manner up to 40 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). Similarly, scratch wound healing and tube formation assays demonstrated markedly enhanced migration and angiogenic capacity in mitoEV-treated groups compared to controls under ischemic conditions, with efficacy plateauing beyond 40 \u0026micro;g/ml (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-G). Doses exceeding 40 \u0026micro;g/ml provided minimal additional benefit to either cell survival or angiogenesis. Therefore, 40 \u0026micro;g/ml was selected as the optimal concentration of ADSC-mitoEVs for all subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eADSC-mitoEVs Enhances the Revascularization in vivo\u003c/h3\u003e\n\u003cp\u003eADSC-mitoEVs significantly improved the angiogenic potential of HUVECs in vivo. Subcutaneous grafts co-implanted with HUVECs and ADSC-mitoEVs exhibited extensive networks of perfused microvessels by day 7, in contrast to control grafts, which showed poor vascularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-H). Furthermore, ADSC-mitoEVs administration promoted functional recovery in ischemic hindlimbs, as evidenced by markedly improved perfusion over a three-week period and prevention of foot necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-N). Histological analysis of muscle tissues retrieved from the ADSC-mitoEV-treated group revealed increased capillary density (CD31⁺ structures) and higher skeletal muscle density (α-SMA⁺) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO-Q). These findings demonstrate that ADSC-mitoEVs not only stimulate robust vascular network formation but also enhance tissue perfusion and protect against ischemic injury, highlighting their therapeutic potential in promoting vascular regeneration and functional recovery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProteomics Indicates Improved Mitophagy and Remodeled Energy Metabolism by ADSC-mitoEVs\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, proteomic profiling of hypoxic HUVECs treated with ADSC-mitoEVs identified 845 significantly upregulated proteins (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) associated with autophagy, ubiquitin-dependent protein catabolism, cellular redox homeostasis, mitochondrial ATP synthesis, oxidant detoxification, and inhibition of apoptosis. Key pathways such as oxidative phosphorylation were notably enriched. Additionally, 419 proteins were downregulated (fold change\u0026thinsp;\u0026lt;\u0026thinsp;0.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), including those involved in fatty acid β-oxidation. Importantly, GO Cellular Component analysis revealed that upregulated proteins were enriched in both extracellular exosome and mitochondrial compartments, supporting successful transfer and uptake of mitoEVs. These findings indicate that ADSC-mitoEVs induce extensive reprogramming of cellular metabolism and enhance cytoprotective responses in endothelial cells under hypoxia, highlighting their role in promoting metabolic adaptation and survival under ischemic conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eADSC-mitoEVs rescues the mitochondrial dysfunction of ischemic HUVECs\u003c/h3\u003e\n\u003cp\u003eUsing JC-1 staining, we observed that ADSC-mitoEVs markedly enhanced the mitochondrial membrane potential (MMP) and increased total ATP production in ischemic HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Furthermore, ADSC-mitoEV treatment significantly reduced mitochondrial ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Mitochondrial stress assays demonstrated that ADSC-mitoEVs improved basal oxygen consumption rate (OCR), mitochondrial ATP production, spare respiratory capacity, and non-mitochondrial ATP generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). These results indicate that ADSC-mitoEVs restore mitochondrial bioenergetics, enhance redox balance, and boost metabolic flexibility in hypoxic endothelial cells, underscoring their therapeutic potential in ameliorating ischemia-induced mitochondrial dysfunction.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eADSC-mitoEVs Promotes the Mitophagy via PINK1/Parkin Signaling Pathway\u003c/h3\u003e\n\u003cp\u003eTo investigate the impact of ADSC-mitoEVs on mitochondrial quality control under ischemic conditions, we first employed mt-Keima lentiviral transduction to monitor mitophagy flux. The results indicated that ADSC-mitoEVs significantly enhanced mitophagy, as evidenced by an increased ratio of mitochondria within lysosomes to those in the neutral cytosol (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Moreover, ADSC-mitoEV treatment improved mitochondrial morphology under ischemic stress, promoting increased mitochondrial network length and size (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next evaluated the persistence and integration of transferred mitochondria. Using droplet digital PCR (ddPCR) with primers specific to mtDNA and ADSCs and HUVECs (Supplementary Data 1), we detected approximately 32,000 copies of ADSC-derived mtDNA in HUVECs after 12 hours of co-culture with ADSC-mitoEVs. This copy number declined to 22,400 at 24 hours and 12,800 at 48 hours, becoming undetectable by 72 hours (Supplementary Fig.\u0026nbsp;2 and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eTo elucidate the mechanisms underlying enhanced mitophagy, we analyzed markers of autophagic flux and mitophagy pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). ADSC-mitoEVs treatment increased the LC3-II/I ratio and decreased p62 expression, confirming enhanced autophagic activity. Western blot analysis further revealed elevated levels of PINK1, phospho-ubiquitin, and phospho-Parkin, indicating activation of the PINK1/Parkin-mediated mitophagy pathway. In contrast, no significant activation was observed in the BNIP3/NIX pathway.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eADSC-mitoEVs Enhances the Angiogenesis in PINK1 Depend Manner\u003c/h2\u003e\u003cp\u003eTo elucidate the mechanistic dependence of ADSC-mitoEV-mediated angiogenesis and mitochondrial function repair, we genetically knocked down PINK1 in HUVECs (Supplementary Fig.\u0026nbsp;3A). Loss of PINK1 abolished the pro-angiogenic effects of ADSC-mitoEVs, as demonstrated by significantly impaired tube formation and reduced endothelial cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-F). However, PINK1 knockdown did not fully abrogate the beneficial effects of ADSC-mitoEVs. Anti-apoptotic activity, reduction in oxidative stress, and improvements in cellular energy metabolism were partially retained, suggesting these effects are mediated through both PINK1-dependent and -independent mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B, G-O).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eADSC-mitoEVs Facilitates the Survival of Ischemic HUVECs through SOD2 Transfer\u003c/h2\u003e\u003cp\u003eTo further elucidate the mechanistic basis of ADSC-mitoEV-mediated mitochondrial functional recovery, we performed Venn analysis to identify mitochondrial proteins enriched in ADSC-mitoEVs and concurrently upregulated in recipient HUVECs (Supplementary Data 2). This approach revealed 43 candidate mitochondrial proteins potentially transferred via mitoEVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequent protein-interaction and functional enrichment analyses indicated that these proteins are primarily involved in cellular respiration, mitochondrial organization, antioxidant responses, and anti-apoptotic pathways. Among these, SOD2 emerged as a central node capable of modulating all aforementioned processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo experimentally validate the functional significance of SOD2 transfer, we generated SOD2-overexpressing ADSCs and isolated SOD2-enriched mitoEVs (Supplementary Fig.\u0026nbsp;3B-D). Flag-tagged SOD2 was detected within HUVECs following co-culture, confirming vesicle-mediated delivery (Supplementary Fig.\u0026nbsp;3E). Western blot analysis further corroborated the increase in SOD2 expression in recipient cells (Supplementary Fig.\u0026nbsp;3F-G). Treatment with SOD2-enriched mitoEVs significantly reduced apoptosis and mitochondrial ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D), while enhancing mitochondrial membrane potential in ischemic HUVECs with PINK1 silence (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE-F). Seahorse assays demonstrated improved basal OCR, ATP-linked respiration, spare respiratory capacity, and non-mitochondrial ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG-K). These results identify SOD2 as a key functional mediator within ADSC-mitoEVs that orchestrates enhanced mitochondrial function and redox balance. Importantly, SOD2-enriched mitoEVs directly improved energy metabolism and mitochondrial performance in recipient cells independent of the PINK1/Parkin-mediated mitophagy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that mitochondrial-enriched extracellular vesicles derived from adipose-derived stem cells serve as an efficient bioenergetic cargo system capable of rescuing ischemic endothelial cells through enhanced mitophagy and mitochondrial protein transfer. These vesicles promote angiogenesis, improve mitochondrial function, and facilitate functional recovery in models of hindlimb ischemia, thereby presenting a novel cell-free strategy for ischemic vascular diseases.\u003c/p\u003e\u003cp\u003eOur experimental strategy was designed to systematically elucidate the functional and mechanistic contributions of ADSC-mitoEVs. Beginning with proteomic characterization, we identified a significant enrichment of mitochondrial proteins within ADSC-mitoEVs, which contribute to oxidative phosphorylation, redox homeostasis, and anti-apoptotic processes. This initial profiling suggested that mitoEVs might directly supplement dysfunctional mitochondria in recipient cells. However, internalized ADSC-mitoEVs did not integrate into the mitochondrial network of recipient endothelial cells. Instead, they rapidly activated the PINK1/Parkin-mediated mitophagy pathway within 12 hours and were completely degraded within 72 hours. This activation promotes mitochondrial turnover and quality control, served as a prerequisite for initiating angiogenesis in ischemic endothelial cells.\u003c/p\u003e\u003cp\u003eConcurrently, mitophagy induction enhanced reactive oxygen species clearance, alleviating mitochondrial stress[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These effects align with mechanisms reported for both free mitochondrial transfer from ADSCs and TNT-mediated mitochondrial delivery[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although the internalization routes differ (endocytosis for EVs versus direct intercellular transfer for TNTs) both pathways converge on a shared functional outcome: activation of mitophagy and subsequent cytoprotection. We propose that this convergence is mediated by the delivery of mitochondrial components, such as some kinds of mitochondrial proteins and mtDNA, which are recognized as \u0026ldquo;non-self\u0026rdquo; motifs by the recipient cell\u0026rsquo;s surveillance system. These motifs trigger PINK1 stabilization on the outer mitochondrial membrane, Parkin recruitment, ubiquitination of mitochondrial substrates, and ultimately autophagosome formation around the exogenous material[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The PINK1/Parkin pathway, central to mitochondrial quality control via mitophagy, plays a critical and predominantly restrictive role in angiogenesis by clearing dysfunctional mitochondria in endothelial cells. This pathway maintains cellular fitness by limiting reactive oxygen species, preventing apoptosis, and supporting the necessary metabolic reprogramming for cell migration and proliferation[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moderate activation of mitophagy is beneficial, as it facilitates the removal of dysfunctional organelles and stimulates mitochondrial biogenesis, leading to increased ATP production[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Indeed, following mitoEV treatment, we observed increased total mitochondrial content, well-developed mitochondrial network and enhanced ATP generation in endothelial cells, indicating improved bioenergetic capacity as well as enhanced cellular fitness under stress conditions.\u003c/p\u003e\u003cp\u003eNotably, unlike free mitochondrial transfer as reported previously[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], ADSC-mitoEVs retained partial functionality, such as antioxidant capacity and improvements in energy metabolism, even in PINK1-silenced endothelial cells. We attribute this PINK1-independent effect to the efficient vesicle-mediated delivery of functional mitochondrial enzymes, particularly superoxide dismutase 2 (SOD2). Owing to the protective bilayer phospholipid membrane of extracellular vesicles[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the structural and functional integrity of SOD2 and other oxidative phosphorylation-related proteins is preserved during intercellular transit, enabling them to remain catalytically active upon delivery. Mechanistically, once internalized, SOD2-enriched ADSC-mitoEVs preferentially localize to the mitochondrial compartments of recipient endothelial cells, where the encapsulated SOD2 is released and integrates into the endogenous mitochondrial matrix. SOD2 anions dismutates superoxide anions into hydrogen peroxide and oxygen, thereby markedly reducing mitochondrial reactive oxygen species levels and protecting electron transport chain complexes from oxidative damage, stabilizing mitochondrial membrane potential and enhancing ATP synthesis independently of PINK1/Parkin signaling[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Proteomic analyses further suggest that ADSC-mitoEVs carry additional mitochondrial enzymes, such as ubiquinone oxidoreductase subunits and ATP synthase, which may act synergistically with SOD2 to ameliorate bioenergetic deficits and enhance mitochondrial resilience under ischemic stress. This multi-protein delivery mechanism distinguishes ADSC-mitoEVs from conventional mitochondrial transplantation and underscores their potential as a comprehensive mitochondrial therapy for vascular diseases.\u003c/p\u003e\u003cp\u003eMitochondrial transplantation has emerged as a promising treatment for conditions such as cancer[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], Parkinson\u0026rsquo;s disease[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and myocardial infarction[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. ADSC-mitoEVs offer a cell-free strategy that combines the advantages of mitochondrial transplantation and conventional EV therapies while overcoming following limitations. Unlike direct mitochondrial transplantation, which suffers from poor stability and short retention times, ADSC-mitoEVs provide a membrane-protected biological vector that enhances protein stability and delivery efficiency of multiple mitochondrial-derived enzymes. Moreover, compared with heterogeneous conventional MSC-EV preparations, ADSC-mitoEVs represent a defined subpopulation with elevated mitochondrial content, enabling more targeted bioenergetic modulation.\u003c/p\u003e\u003cp\u003eDespite these promising findings, our study has several limitations. First, advanced purification strategies, such as immunoaffinity-based sorting for mitochondrial markers, could yield more homogeneous mitoEV populations if the membrane protein profile of mitoEVs can be characterized. Additionally, while diabetic murine models of hindlimb ischemia offer useful insights, they hardly recapitulate the complex pathophysiology of human vascular diseases, particularly in aging or comorbid settings. Further validation in large animal models is essential before clinical translation. Finally, the long-term safety, immunogenicity, and fate of administered mitoEVs warrant systematic investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we demonstrate that ADSC-mitoEVs, a novel cell-free therapeutic platform, ameliorates ischemic endothelial damage through dual mechanisms: activation of PINK1/Parkin-mediated mitophagy and direct transfer of functional mitochondrial proteins. This strategy enhances mitochondrial quality control, redox homeostasis and energy metabolism reprogramming, offering a promising approach for ischemic vascular diseases treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIsolation and Characteristics of ADSC-mitoEVs\u003c/h2\u003e\u003cp\u003eHuman lipoaspirates were harvested from the thigh region of healthy female donors (aged 25 years) using water jet-assisted liposuction (Body-Jet System; Human Med AG, Schwerin, Germany). All procedures complied with the principles outlined in the Declaration of Helsinki (1975) and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (IIT-0-2025-100). Adipose-derived stem cells (ADSCs) were isolated following a previously established protocol. Primary ADSCs were transfected with pLV-mito-miRFP670 lentiviral particles (Han Heng Biotechnology, Shanghai, China) to enable mitochondrial labeling via miRFP670 fluorescence.\u003c/p\u003e\u003cp\u003eTransfected mito-miRFP670-ADSCs (passages 3\u0026ndash;5) were cultured in mesenchymal stem cell medium supplemented with 5% extracellular vesicle (EV)-depleted serum at 37\u0026deg;C under 5% CO₂ and 95% humidified air. The conditioned medium was collected and subjected to sequential centrifugation steps at 800\u0026times;g and 3,000\u0026times;g at 4\u0026deg;C to remove cellular debris. The resulting supernatant was concentrated using an ultrafiltration centrifuge tube equipped with a 100 kDa molecular weight cut-off filter (Millipore, MA, USA) by centrifugation at 3,000\u0026times;g for 30 minutes at 4\u0026deg;C. The EV suspension was then processed using a CytoFLEX SRT cell sorter (Beckman Coulter, CA, USA) to isolate miRFP670-positive vesicles. Finally, the sorted suspension was filtered through a 0.22 \u0026micro;m membrane and subjected to ultracentrifugation at 110,000\u0026times;g for 90 minutes at 4\u0026deg;C (Beckman Coulter, CA, USA) to pellet the ADSC-derived mitochondrial extracellular vesicles (ADSC-mitoEVs). (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003c/p\u003e\u003cp\u003eThe characterization of ADSC-mitoEVs was carried out using transmission electron microscopy (Hitachi, Tokyo, Japan) and Nanoparticle Tracking Analysis (NTA, ZetaVIEW, Meerbusch, Garmany) for morphology and particle size distribution analysis. Antibodies against CD63, TOM20 and tumor susceptibility gene 101 protein (TSG101) (Abcam, Cambridge, UK) were used in the Western blot to detect the expression of mito-EV-specific markers. The protein concentration was evaluated using a BCA Protein Assay Kit (Sigma, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eProteomic Analysis Reveals Mitochondrial Components in ADSC-EVs\u003c/h2\u003e\u003cp\u003eProteomic profiling was employed to characterize the mitochondrial components within ADSC-EVs. Total protein was extracted from isolated ADSC-EVs and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, MA, USA). The acquired data were processed with MaxQuant software and matched against the UniProt human database. Mitochondrial proteins were identified based on subcellular localization predictions. Subsequent bioinformatic analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, were performed to delineate significantly enriched pathways associated with the identified mitochondrial proteins.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were acquired from the Chinese Academy of Sciences cell bank and maintained in endothelial cell medium (ECM, ScienCell, CA, USA) supplemented with 5% fetal bovine serum (FBS) at 37\u0026deg;C under normoxic conditions (20% O₂). To mimic ischemia, HUVECs were subjected to hypoxia (1% O₂) in serum-free ECM at 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMitoEVs Uptake\u003c/h2\u003e\u003cp\u003eHUVECs were transfected with pLV-mito-DsRed lentiviral particles (Han Heng Biotechnology, Shanghai, China) to achieve mitochondrial labeling with DsRed fluorescence. The transfected mito-DsRed-HUVECs were then co-cultured with ADSC-mitoEVs (10 \u0026micro;g/mL) for 12 hours under both normal and ischemic conditions. Cellular uptake of ADSC-mitoEVs was visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany). The area of both miRFP670 and DsRed signal were counted in ten random cells per group. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCell Apoptosis Assay\u003c/h2\u003e\u003cp\u003eApoptosis was evaluated using an Annexin V/7-AAD Apoptosis Detection Kit (Beyotime Biotechnology, Shanghai, China). After treatment with ADSC-mitoEVs (10, 20, 40 and 80 \u0026micro;g/mL) for 48 hours under ischemic conditions. HUVECs cultured at normal condition were served as positive control. HUVECs were harvested, washed with PBS, and then were stained with Annexin V and 7-AAD for 20 minutes at 37\u0026deg;C in the dark. Apoptotic rates were analyzed using a flow cytometer (BD biosciences, NJ, USA). Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eScratch Test\u003c/h2\u003e\u003cp\u003eA scratch test was performed to evaluate cell migration. HUVECs were seeded in 24-well plates and grown to confluence. A sterile 100 \u0026micro;L pipette tip was used to create a linear scratch. After washing with PBS to remove detached cells, the cells were incubated with ADSC-mitoEVs (10, 20, 40 and 80 \u0026micro;g/mL) in serum-free medium under hypoxia. HUVECs cultured at normal condition were served as positive control. Wound closure was monitored at 0 and 24 hours using an inverted microscope (Carl Zeiss, Oberkochen, Germany). Migration rate was quantified with ImageJ software. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eTube Formation\u003c/h2\u003e\u003cp\u003eThe angiogenic capability of HUVECs was assessed using a tube formation assay on Matrigel (Corning, NY, USA). Briefly, 96-well plates were coated with Matrigel and polymerized for 30 minutes at 37\u0026deg;C. HUVECs were incubated with ADSC-mitoEVs (10, 20, 40 and 80 \u0026micro;g/mL) under hypoxia for 12 hours. Tubular structures were imaged under an inverted microscope (Carl Zeiss, Oberkochen, Germany). Total tube length was analyzed with Angiogenesis Analyzer tool in Image J. Three independent experiments were performed.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eAnimal Models\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang University (NCULAE-20250912001) and complied with the NIH Guide for the Care and Use of Laboratory Animals. For tube formation in vivo, five male BALB/c nude mice (8 weeks old) anesthetized by inhalation of isoflurane (induced at 4% and maintained at 2% at a flow rate of 1.5 L/min). 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e HUVECs with or without ADSC-mitoEVs (40 \u0026micro;g/ml) were coated with 100 \u0026micro;L Matrigel (Corning, NY, USA) with investigators blinded to group allocation during procedures and assessments, and then injected subcutaneously into both armpits of nude mice. After one week, neoformations were harvested and weighted. Surrounding neovascularization were analyzed with Angiogenesis Analyzer tool in Image J.\u003c/p\u003e\u003cp\u003eDiabetes was induced in 8-week-old male BALB/c nude mice by intraperitoneal injection of streptozotocin (STZ; Sigma, MA, USA) at the dose of 100 mg/kg. Blood glucose levels were monitored from the tail vein using a glucometer (Omron, Kyoto, Japan) on days 14 post-initial injection. Mice with two consecutive fasting (6-hour) blood glucose measurements of \u0026ge;\u0026thinsp;16.7 mmol/L were considered diabetic and included in the subsequent experimental cohort. For mouse hindlimb ischemia model, ten diabetic BALB/c nude mice were randomly assigned to mitoEVs group (n\u0026thinsp;=\u0026thinsp;5) or PBS group (n\u0026thinsp;=\u0026thinsp;5), with investigators blinded to group allocation during procedures and assessments. Mice were anesthetized by inhalation of isoflurane (induced at 4% and maintained at 2% at a flow rate of 1.5 L/min), and unilateral hindlimb ischemia was induced by ligating the femoral artery. ADSC-mitoEVs (40 \u0026micro;g/ml) in 100 \u0026micro;L PBS or an equal volume of PBS as control were administered via intramuscular injection at three sites in the ischemic muscle immediately after surgery. Blood perfusion was monitored on days 0, 7, 14 and 21 using a Laser Speckle Contrast Imaging system (Perimed, Stockholm, Sweden). The thigh muscle tissue was harvested on the 21st day after surgery and used for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eWhole mount staining\u003c/h2\u003e\u003cp\u003eNeoformation and thigh muscle tissues from nude mice were fixed in 4% paraformaldehyde for 24 h, followed by dehydration in 30% sucrose for 72 h. The samples were then embedded and cryosectioned into 150 \u0026micro;m-thick slices. For immunofluorescence staining, neoformation sections were incubated with an anti-CD31 primary antibody (R\u0026amp;D Systems, MN, USA). Thigh muscle sections were co-stained with anti-CD31 and anti-α-SMA primary antibodies (R\u0026amp;D Systems and Sigma, respectively). Neovascularization density and tube length were visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany) and then quantified using the Angiogenesis Analyzer tool in ImageJ software, while muscle density was assessed using Image J.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eProteomic Analysis of ADSC-mitoEVs Regulating Ischemic Vascular Endothelial Cells\u003c/h2\u003e\u003cp\u003eHUVECs were treated with ADSC-mitoEVs (40 \u0026micro;g/mL) under ischemic conditions for 24 hours. Total proteins were extracted. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, MA, USA). Data were analyzed using MaxQuant software and searched against the UniProt human database. Bioinformatic analysis including GO and KEGG enrichment was conducted to identify significantly altered pathways. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eMitochondrial Membrane Potential (MMP) Assay\u003c/h2\u003e\u003cp\u003eMMP was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, Shanghai, China). After treatment, HUVECs were incubated with JC-1 staining solution for 20 minutes at 37\u0026deg;C, and then was visualized using laser scanning confocal microscopy (LSCM; Carl Zeiss, Oberkochen, Germany). The ratio of red (aggregates) to green (monomers) fluorescence was used to indicate MMP. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eMitochondrial Oxidative Stress Assay\u003c/h2\u003e\u003cp\u003eMitochondrial respiration was assessed using a Seahorse XFe24 Analyzer (Agilent, CA, USA). HUVECs were seeded in XF24 plates and treated with ADSC-mitoEVs (40 \u0026micro;g/mL) under ischemic conditions. Oxygen consumption rate (OCR) was measured under basal conditions and in response to oligomycin, FCCP and rotenone/antimycin A. For mitochondrial superoxide detection, cells were stained with mitoSOX Red (Invitrogen, CA, USA) and analyzed by flow cytometry (BD biosciences, NJ, USA). Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eATP measurement\u003c/h2\u003e\u003cp\u003eIntracellular ATP levels were quantified using an ATP Assay Kit (Abcam, UK) based on luciferase-luciferin reaction. Treated HUVECs were lysed, and luminescence was measured with a microplate reader (ShanpuBiotech, Shanghai, China). Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eMitophagy Flow Assay\u003c/h2\u003e\u003cp\u003eHUVECs were transfected with mt-Keima lentivirus (Han Heng Biotechnology, China) following the manufacturer\u0026rsquo;s protocol. After treatment with ADSC-mitoEVs under ischemic conditions, mitophagy was assessed by laser scanning confocal microscopy (LSCM; Carl Zeiss, Germany). Mitophagy activity was quantified based on the red-to-green fluorescence intensity ratio (550/440 nm) in ten random cells per group per time point, corresponding to acidified mitochondria within lysosomes versus mitochondria in the neutral cytoplasmic environment. Furthermore, key proteins associated with mitophagy pathways including PINK1, phospho-ubiquitin, phospho-Parkin (PINK1/Parkin pathway), BNIP3/NIX, as well as LC3 and P62 were analyzed by western blot. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExogenous mtDNA Assay\u003c/h3\u003e\n\u003cp\u003eTo evaluate the persistence of transferred mitochondria in recipient HUVECs over time, we employed a mitochondrial DNA (mtDNA) genotyping strategy based on the detection of heteroplasmy. mtDNA was isolated from ADSCs and HUVECs using a Mitochondrial DNA Isolation Kit (MCE, NJ, USA). Regions with sequence divergence between the two cell types were identified by Sanger sequencing. Specific primers were designed to target these differential sites, and their amplification efficiency was validated by Droplet Digital PCR (ddPCR) on a QuantStudio Absolute Q system (Thermo Fisher Scientific, MA, USA). Using these primers, we quantified the copy number of ADSC-derived mtDNA in HUVECs co-cultured with ADSC-mitoEVs at 12, 24, 48, and 72 h post-treatment via ddPCR. Three independent experiments were performed.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eSilencing of PINK1 by shRNA\u003c/h2\u003e\u003cp\u003eShRNA lentiviral particles targeting human PINK1, along with negative control shRNA constructs, were obtained from Han Heng Biotechnology (Shanghai, China). HUVECs were transduced with lentiviral particles in the presence of polybrene. Following a 72-hour incubation, transduced cells were selected using puromycin. Silencing efficiency was confirmed by qRT\u0026ndash;PCR analysis of mRNA extracted from selected cells. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eGeneration of SOD2-overexpressed ADSC-mitoEVs\u003c/h2\u003e\u003cp\u003eTo determine whether ADSC-mitoEVs confer mitochondrial protection in recipient cells via delivery of mitochondrial proteins, we identified overlapping mitochondrial proteins between the mitoEVs and upregulated mitochondrial proteins in HUVECs treated with mitoEVs using Venn analysis. This set was defined as candidate mitochondrial factors. Protein-protein interaction and functional enrichment analyses were performed using STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict the biological roles of these candidates, leading to the identification of SOD2 as a central node protein.\u003c/p\u003e\u003cp\u003eLentiviral particles encoding human SOD2-Flag and the corresponding empty vector controls were obtained from Han Heng Biotechnology (Shanghai, China). ADSCs were transduced with the lentiviral particles in the presence of polybrene. After 72 hours, stable cell lines were selected using puromycin. Overexpression efficiency was confirmed at the mRNA level by qRT‑PCR and at the protein level by western blot and immunofluorescence staining, using antibodies against SOD2 (Santa Cruz Biotechnology, Dallas, USA) and Flag (Proteintech, Wuhan, China).\u003c/p\u003e\u003cp\u003eSOD2-overexpressing ADSCs were cultured, and the conditioned medium was collected for the isolation of SOD2‑overexpressing ADSC‑mitoEVs (SOD2‑mitoEVs). The uptake of SOD2‑mitoEVs by HUVECs was analyzed via Flag immunofluorescence staining. Additionally, SOD2 expression in HUVECs that had taken up SOD2‑mitoEVs was further evaluated by western blot. Three independent experiments were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Comparisons were analyzed via unpaired t-tests or one-way ANOVA with Fisher\u0026rsquo;s LSD post hoc test (GraphPad Prism 9). Significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuan-zheng Zhu contributed to the study design, isolation and characteristics ADSC-mitoEVs, cell culture and functional assay in vitro, proteomics assay and manuscript writing. Min-chen Zhang contributed to Seahorse assay and ATP detection. Xue-er Li contributed to the heterogeneity analysis of mtDNA and generation of PINK1 silenced and SOD2 overexpressed cells. Xing-hong Zeng contributed to the animal study. Xue-ting Gong and Yu-zi Wu contributed to the western blotting. Ze-jun Dong, Shu Wu and Xue-fei Liu contributed to the data statistics. Yang-yan Yi contributed to the conception and design, financial support and final approval of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [Grant number: 82460448]; Natural Science Foundation of Jiangxi Province [Grant number: 20242BAB26140 and 20252BAC240585]; Jiangxi Province Key laboratory of Precision Cell Therapy [Grant number: 2024SSY06241].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated and analyzed during this research are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Nanchang University (NCULAE-20250912001) and complied with the NIH Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003eHuman lipoaspirates complied with the principles outlined in the Declaration of Helsinki (1975) and were approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (IIT-0-2025-100).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang-yan Yi; Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSayed A, Michos ED, Navar AM, Virani SS, Brewer LC, Manson JE. 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Neonatal-Tissue-Derived Extracellular Vesicle Therapy (NEXT): A Potent Strategy for Precision Regenerative Medicine. Adv Mater. 2023;35:e2300602.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaza AK, Wamala I, Friehs I, Kuebler JD, Rathod RH, Berra I, Ericsson M, Yao R, Thedsanamoorthy JK, Zurakowski D, Levitsky S, Del Nido PJ, Cowan DB, McCully JD. Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia/reperfusion. J Thorac Cardiovasc Surg. 2017;153:934\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"mitochondrial transfer, extracellular vesicles, adipose-derived stem cells, mitophagy, ischemic vascular diseases","lastPublishedDoi":"10.21203/rs.3.rs-7849775/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7849775/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic vascular diseases are critically associated with mitochondrial dysfunction in endothelial cells, leading to impaired angiogenesis and compromised tissue repair. Although mitochondrial transfer has emerged as a promising therapeutic strategy for revascularization, its clinical translation has been hindered by inefficient delivery systems. In this study, we show that mitochondrial-enriched extracellular vesicles derived from adipose-derived stem cells (ADSC-mitoEVs) function as an efficient nanotherapeutic, restoring angiogenic function both in vitro and in a murine model of diabetic hindlimb ischemia. Mechanistically, the uptake of ADSC-mitoEVs triggered PINK1/Parkin-mediated mitophagy in recipient endothelial cells, a process essential for initiating angiogenesis. Moreover, ADSC-mitoEVs directly delivered functional mitochondrial proteins, including superoxide dismutase 2 (SOD2), into the endogenous mitochondrial network, which enhanced antioxidant defense and improved bioenergetic capacity independently of mitophagy, as shown by reduced reactive oxygen species and elevated ATP production even in PINK1-silenced cells. Our results demonstrate ADSC-mitoEVs as a versatile, cell-free nanotherapeutic that promotes mitochondrial quality control and metabolic reprogramming, offering a potent therapeutic avenue for ischemic vascular diseases.\u003c/p\u003e","manuscriptTitle":"Nanotherapeutic Strategy via ADSC-mitoEVs Rescues Ischemic Angiogenesis through Mitophagy and Mitochondrial Metabolic Reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 17:40:23","doi":"10.21203/rs.3.rs-7849775/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-02T14:43:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-02T08:53:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T12:48:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T02:48:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217596327749396183094610745674058865000","date":"2025-10-26T14:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142216789785593285464474487920914682753","date":"2025-10-24T03:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34795516490109348074126668963586200213","date":"2025-10-22T14:03:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175786774789988037666786982674202082377","date":"2025-10-21T15:59:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T13:56:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T12:33:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T12:32:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-10-13T13:47:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"241da198-2f71-4536-bff0-d824a61561db","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:09+00:00","versionOfRecord":{"articleIdentity":"rs-7849775","link":"https://doi.org/10.1186/s12951-026-04183-x","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-25 15:57:47","publishedOnDateReadable":"February 25th, 2026"},"versionCreatedAt":"2025-10-31 17:40:23","video":"","vorDoi":"10.1186/s12951-026-04183-x","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04183-x","workflowStages":[]},"version":"v1","identity":"rs-7849775","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7849775","identity":"rs-7849775","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}