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WHAT IS ALREADY KNOWN ABOUT THIS TOPIC Persistent exposure to elevated glucose levels disrupts endothelial integrity, resulting in aberrant proliferation and apoptosis of endothelial cells.Orai1 overexpression under high glucose (HG) conditions synergizes with insulin-like growth factor-binding protein 3 (IGFBP3) to form pathogenic Orai1-IGFBP3 complexes, exacerbating diabetic vasculopathy.Exosomes derived from endothelial progenitor cells (EPCs-EXOs) enhance endothelial function through various mechanisms.WHAT THIS STUDY ADDS EPCs-EXOs can attenuate HG-induced endothelial dysfunction in human coronary artery endothelial cells and reduce atherosclerotic plaque formation in mouse models of type 2 diabetes.EPCs-EXOs suppress the HG-induced upregulation of Orai1 and IGFBP3 proteins, key mediators of store-operated calcium entry.miR-7116-3p within EPCs-EXOs acts as a critical microRNA that directly targets Orai1 and IGFBP3 messenger RNAs.HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY miR-7116-3p in EPCs-EXOs specifically acts on the Orai1-IGFBP3 complex, thereby inhibiting abnormal proliferation of coronary endothelial cells induced by a high-sugar environment and delaying the onset and development of atherosclerosis in patients with diabetes, offering a promising strategy for the treatment and prevention of diabetic vascular complications.Introduction Patients with type 2 diabetes mellitus may experience multiple complications and face premature mortality due to atherosclerotic cardiovascular disease, hospitalization for heart failure, and/or chronic kidney disease. 1 Diabetes, a chronic metabolic disorder characterized by sustained hyperglycemia, initiates a cascade of vascular complications, with endothelial dysfunction serving as a central pathological feature. The endothelium—a monolayer of cells lining the vasculature—is critical for preserving vascular homeostasis.2 However, persistent exposure to elevated glucose levels disrupts endothelial integrity, resulting in aberrant proliferation and apoptosis of endothelial cells. These cellular perturbations impair endothelial repair capacity and promote vascular injury, thereby accelerating the progression of atherosclerosis (AS) and other diabetes-related vascular complications. Despite considerable advances, the precise molecular mechanisms driving hyperglycemia-induced endothelial dysfunction remain incompletely elucidated.Store-operated calcium entry (SOCE) is primarily mediated by the Ca2+ release-activated calcium (Orai) channels and stromal interaction molecules (STIM), which couple intracellular Ca2+ depletion to extracellular Ca2+ influx.3 Accumulating evidence suggests that high glucose (HG) activates SOCE pathways and endoplasmic reticulum stress (ERS), both of which are major contributors to endothelial dysfunction.4 Inhibition of sarcoplasmic/ER Ca2+-ATPase prevents ER Ca2+ reuptake, depleting internal stores and enhancing SOCE, thereby elevating intracellular Ca2+ levels and influencing endothelial function. Studies have shown that prolonged HG exposure increases SOCE activity and upregulates Orai channel isoforms (Orai1, Orai2, Orai3) as well as STIM1/2 proteins in endothelial cells.5 6 Our previous findings demonstrated that dysregulated Ca2+ signaling via Orai1-3 contributes to hyperglycemia-induced endothelial dysfunction. Notably, Orai1 overexpression under HG conditions synergizes with insulin-like growth factor-binding protein 3 (IGFBP3) to form pathogenic Orai-IGFBP3 complexes, exacerbating diabetic vasculopathy.7 Targeted disruption of this interaction may therefore represent a promising therapeutic strategy.Endothelial progenitor cells (EPCs) possess distinct vascular reparative capabilities through their ability to differentiate into mature endothelial cells and restore damaged vasculature.8 Exosomes—nanosized vesicles (30–120 nm) enriched with bioactive cargos such as proteins, lipids, and non-coding RNAs—have emerged as critical mediators of intercellular communication.9 10 Recently, circulating microRNAs (miRNAs) have emerged as novel molecular players in cardiometabolic diseases.11 Multiple studies have demonstrated that exosomes derived from EPCs (EPCs-EXOs) enhance endothelial function through various mechanisms. For example, EPCs-EXOs promote endothelial cell proliferation, migration, and angiogenesis in an miR-126-dependent manner.12 Additionally, EPCs-EXOs suppress carotid intimal hyperplasia following balloon injury, facilitate endothelial repair at the site of intimal damage, and improve endothelial function.13Our previous work revealed that EPCs-EXOs reduce lipid droplet accumulation in the thoracic aorta of mice with diabetes and hyperlipidemia, indicating their potential utility in managing diabetic vascular complications (DVCs).14 miRNA profiling of EPCs-EXOs further identified a broad spectrum of miRNAs with regulatory effects on vascular endothelial cells. We also found that elevated Orai expression under hyperglycemic conditions cooperates with IGFBP3 to form pathogenic Orai-IGFBP3 complexes that aggravate diabetic vasculopathy.7 Further analysis of EPCs-EXOs revealed a subset of miRNAs capable of targeting and regulating both Orai and IGFBP3 transcripts. Based on these findings, we hypothesized that miRNAs enriched in EPCs-EXOs may attenuate HG-induced endothelial dysfunction by disrupting Orai-IGFBP3 complex formation. To test this hypothesis, we employed both in vitro endothelial cell models and a type 2 diabetic mouse (DM) model. Our results confirmed that EPCs-EXO-derived miRNAs mitigate diabetic vascular injury. Furthermore, we identified specific miRNAs involved in this process and elucidated their mechanisms of action. This study provides new mechanistic insight into diabetes-associated AS and proposes novel therapeutic targets for the clinical treatment of DVCs.Materials and methods Animals Male C57BL/6J mice aged 6 weeks were obtained from the Guangdong Provincial Laboratory Animal Center and housed under specific pathogen-free conditions.Mouse model of type 2 diabetes To establish the diabetic model, mice were fasted for 12 hours and then intraperitoneally injected with streptozotocin at 40 mg/kg for five consecutive days. Control mice received equivalent volumes of citrate buffer under the same fasting conditions. Starting 1 week after the final injection, fasting blood glucose levels were measured daily. Mice in the model group with fasting glucose levels >11.1 mM for three consecutive days were selected for subsequent experiments.Oil Red O staining After sacrifice, hearts were harvested, processed into frozen sections, and stained with Oil Red O followed by hematoxylin counterstaining. Aortic images were captured using a ZEISS microscope camera and quantified using ImageJ software. Lesion areas were expressed as a percentage of the total vascular surface area.Cell culture and characterization of EPCs Human coronary artery endothelial cells (HCAECs) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing normal glucose (NG, 5.6 mM D-glucose), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. To control for osmolarity, 19.5 mM mannitol was added to NG medium (NG+M); for simplicity, this condition is referred to as ‘NG’. The HG group was cultured in DMEM containing 25 mM D-glucose, with 10% FBS and 1% antibiotics. Cells were maintained in a humidified incubator at 37°C with 5% CO 2.Male C57BL/6J mice were humanely euthanized. Under sterile conditions, the femur or tibia was dissected from the mouse skeleton. The ends of the bone were cut with scissors to expose the medullary cavity. Using a syringe, aspirate 1 mL of homogenization buffer to wash out the bone marrow from the cavity. Collect the bone marrow or bone marrow suspension into a suitable centrifuge tube and repeatedly pipette to generate a single-cell suspension. Isolate monocytes and culture them in EBM-2 endothelial basal medium supplemented with endothelial cell growth medium-2. EPCs were identified based on their morphological features during differentiation: (1) initial-stage EPCs appeared polygonal with linear arrangements; (2) early stage EPCs formed typical colonies; (3) mid-stage EPCs exhibited a cobblestone-like morphology; and (4) late-stage EPCs developed vascular ring structures.Isolation and characterization of exosomes When EPCs reached approximately 90% confluence, the culture medium was replaced with serum-free medium. After 24 hours of incubation, the cell culture supernatant was collected and centrifuged at 2000×g for 30 min to remove cellular debris, followed by filtration through a 0.22 μm membrane. The filtered supernatant was mixed with an exosome isolation reagent and incubated at 4°C overnight. Exosomes were then quantified using a BCA protein assay kit and stored at −80°C for subsequent use. Exosomal identity was confirmed by detecting the exosomal surface markers CD63 and CD81. For in vivo experiments, exosomes were administered intravenously at a dosage of 20 μg/100 g body weight (based on exosomal protein content) every 3 days for a total duration of 5 weeks.Transmission electron microscopy For transmission electron microscopy (TEM), freshly isolated exosomes were placed on copper grids precoated with 0.125% Formvar dissolved in chloroform. Grids were stained with 1% (v/v) uranyl acetate in double-distilled water and immediately visualized to assess exosome morphology and size.Reverse transcription real-time fluorescence quantitative PCR analysis Total RNA was extracted using the Total RNA Isolation Kit (FOREGENE, Cat# RE-03011). Complementary DNA (cDNA) was synthesized from messenger RNA (mRNA) using the miRNA cDNA First-Strand Synthesis Mix (Stem-Loop Method) (Accurate Biology, Cat# AG11748). Gene expression was analyzed using SYBR Green Premix Pro Taq HS quantitative PCR (qPCR) Kit (Accurate Biology, Cat# AG11719), with U6 serving as the internal reference gene. Primer sequences are listed in online supplemental table 1 and were purchased from Sangon Biotech (Shanghai, China). qPCR was performed using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA), and gene expression levels were calculated using the comparative Ct (ΔΔCt) method.SP110.1136/bmjdrc-2025-005556.supp1Supplementary dataWestern blot analyses For protein extraction from cells, samples were washed three times with prechilled phosphate-buffered saline (PBS) and lysed in radio-immunoprecipitation assay buffer on ice for 10 min. For tissue samples, thawed tissues were placed on ice and homogenized with magnetic beads using a tissue grinder. The lysates were centrifuged at 12 000 rpm for 15 min at 4°C, and the resulting supernatants were transferred to fresh tubes. Protein concentrations were determined using a BCA (Bicinchoninic acid) Protein Assay Kit (Beyotime, Cat# P0010). Samples were denatured by boiling at 100°C for 15 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for protein separation. Proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk for 1–2 hours at room temperature. After washing three times with Tris-buffered saline with Tween 20 (TBST) (7–10 min per wash), membranes were incubated overnight at 4°C with anti-β-actin primary antibody on a shaker. The following day, membranes were incubated with horseradish peroxidase-conjugated goat antimouse IgG or goat antirabbit IgG for 1–2 hours at room temperature. After three additional washes with TBST (10 min each), immunoreactive bands were visualized using a chemiluminescence imaging system (Tanon 5200Multi, Shanghai) and an enhanced chemiluminescence Western blot detection kit.Immunohistochemistry staining Tissue sections were baked in a 60°C oven for 3 hours, then deparaffinized in xylene and rehydrated through a graded ethanol series to water. Antigen retrieval was performed according to the manufacturer’s instructions for the primary antibody. A hydrophobic barrier was drawn around each tissue section using an immunohistochemical pen, and sections were placed in a humid chamber. Immunohistochemistry was performed using a commercial kit (ORIGENE, Cat# PV-9000). Endogenous peroxidase activity was blocked by incubation with peroxidase blocker at 25°C for 10 min, followed by three washes with PBS (3 min each). Primary antibodies were applied and incubated overnight at 4°C. The next day, sections were washed with PBS and incubated with secondary antibody at 37°C for 20 min. After washing, DAB substrate solution (diluted 1:20) was added, and color development was monitored under a microscope. The reaction was terminated by rinsing with distilled water for 5 min. Sections were counterstained with hematoxylin for 1 min until nuclear staining appeared blue. They were then rinsed with tap water for 5 min, differentiated with 1% hydrochloric acid ethanol for 30 s, returned to blue in a bluing solution for 30 s and finally washed in tap water for 3–4 min. After drying, the sections were mounted and visualized under a light microscope.Measurement of intracellular Ca2+ concentrationFor intracellular calcium measurements, HCAECs were seeded onto 30 mm Petri dishes and cultured in HG medium. Cytosolic calcium concentration ([Ca2+]i) was measured using Fluo-4 acetoxymethyl ester (AM). Cells were incubated with 2 μM Fluo-4 AM and 0.02% Pluronic F-127 (Invitrogen) for 30 min at 37°C. To deplete intracellular Ca2+ stores, cells were treated with either 2 μM thapsigargin or 100 μM ATP for 10 min in Ca2+-free saline solution (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 glucose; pH 7.4). Ca2+ influx was then induced by adding 2 mM extracellular Ca2+. Fluorescence was detected and recorded using a Nikon T200 fluorescence microscope (Tokyo, Japan) equipped with a xenon lamp. The excitation and emission wavelengths were set at 488 nm and 515 nm, respectively. Changes in intracellular calcium concentration ([Ca2+]i) were quantified as the ratio of fluorescence intensity following extracellular Ca2+ addition to the baseline intensity prior to Ca2+ application (F1/F0).Cell viability and proliferation HCAECs were seeded into 96-well plates at a density of 1500 cells per well and cultured for 24 hours under standard conditions. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Vazyme, Nanjing, China). 100 μL of culture medium containing 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 1 hour. The resulting formazan product, which is directly proportional to the number of viable cells, was quantified by measuring absorbance at 450 nm using an automated microplate reader (Rayto, Shandong, China).Migration assays HCAECs in logarithmic growth phase were seeded into 6-well plates and grown to 90%–100% confluence. A straight scratch was made across the cell monolayer using a sterile pipette tip. After rinsing with PBS to remove detached cells, serum-free medium was added. Images were captured at 0 and 24 hours. The migration rate was calculated based on the change in scratch width between time points.Apoptosis analysis For apoptosis analysis, treated cells were harvested by enzymatic digestion, centrifuged, and washed twice with PBS. The cell suspension was adjusted to a concentration of 1×10⁶ cells per tube. Annexin V-fluorescein isothiocyanate and propidium iodide (PI) were added to the cell suspension and incubated in the dark at room temperature for 15–20 min. After staining, PBS was added, and the samples were analyzed by flow cytometry within 1 hour to ensure data integrity.Statistical analysis All data are presented as mean±SEM for the indicated number of biological replicates. Statistical analysis was performed using unpaired two-tailed Student’s t-tests in GraphPad Prism V.9.0 (GraphPad Software, San Diego, California, USA). A p value <0.05 was considered statistically significant.Results Identification of EPCs and characterization of their derived exosomes EPCs were isolated by inducing the differentiation of mouse bone marrow mononuclear cells. Morphological evaluation revealed four distinct stages of EPCs development: (1) initial-stage EPCs exhibited polygonal morphology with spindle-like projections; (2) early stage EPCs formed typical cell colonies; (3) metaphase-stage EPCs displayed a cobblestone-like appearance; and (4) late-stage EPCs differentiated into vascular ring-like structures ( figure 1A). Immunofluorescence staining confirmed that the majority of cells expressed the surface markers CD133 and CD34 (figure 1B). Western blot analysis of the isolated exosomes verified the presence of exosomal markers CD9, CD63, and CD81 (figure 1C). Nanoparticle tracking analysis (NTA) showed that exosome particle sizes were primarily distributed within the 60–100 nm range (figure 1D). TEM revealed that the exosomes displayed a characteristic ‘teacup-like’ morphology (figure 1E). These findings collectively confirm the successful isolation and characterization of EPCs-EXOs.Figure 1Identification of endothelial progenitor cells (EPCs) and characterization of their derived exosomes. (A) Morphological stages of EPCs during differentiation: initial stage (spindle-like projections), early stage (colony formation), metaphase stage (cobblestone-like morphology), and late stage (vascular ring structures). Scale bar=100 µm. (B) Immunofluorescence staining of EPCs showing expression of CD34 (red) and CD133 (green), with DAPI (4’,6-Diamidino-2-phenylindole, blue) for nuclear staining; blank control images are included. Scale bar=100 µm. (C) Western blot analysis of EPC-derived exosomes (EPCs-EXOs) confirming the presence of exosomal markers CD9, CD63, and CD81. (D) Nanoparticle tracking analysis of EPCs-EXOs showing particle size distribution primarily between 60 and 100 nm. (E) Transmission electron microscopy image of EPCs-EXOs displaying typical cup-shaped morphology. Scale bar=200 nm.EPCs-EXOs reduce HG-induced endothelial cell dysfunction and plaque in diabetes combined with atherosclerosis To investigate the effect of EPCs-EXOs on HG-induced endothelial dysfunction, HCAECs were cultured in HG medium for 7 days and subsequently treated with EPCs-EXOs. Functional assays indicated that HG exposure significantly increased HCAEC proliferation, apoptosis, and migration, whereas these effects were markedly attenuated following EPCs-EXOs treatment. Specifically, EPCs-EXOs reduced HG-induced proliferation ( figure 2E), decreased apoptosis rates (figure 2C,D), and suppressed the enhanced migratory capacity of HCAECs under HG conditions (figure 2A,B). Additionally, we overexpressed or silenced miR-7116-3p in HCAEC cells cultured under NG conditions to assess its impact on cell viability. The results indicate that miR-7116-3p exhibits no potential toxicity toward normal cells (online supplemental figure 2A). To further assess the in vivo effects of EPCs-EXOs on HG-induced AS, a DM model was established. Longitudinal measurements confirmed persistent hyperglycemia in mice with diabetes compared with controls (figure 2F), along with progressive body weight loss (figure 2G). Oil Red O staining of cardiac coronary arteries revealed substantial reductions in lipid droplet accumulation within the vessel wall following EPCs-EXOs treatment (figure 2H,I). Through histopathological examination, we observed no abnormal lesions in major organs such as the heart, liver, kidneys, spleen, and lungs (online supplemental figure 2B). Together, these findings indicate that EPCs-EXOs effectively mitigate atherosclerotic lesion formation by alleviating HG-induced endothelial dysfunction in coronary arteries.Figure 2EPCs-EXOs reduce plaque and HG-induced endothelial cell dysfunction in diabetes combined with atherosclerosis. (A) Wound-healing assay images of HCAECs cultured under NG, NG with EPCs-EXOs, HG, and HG with EPCs-EXOs at 0 hour and 24 hours. Scale bar=100 µm. (B) Quantification of wound-healing rates showing increased migration in HG-treated cells, with EPCs-EXOs attenuating this effect. (C) Flow cytometry analysis of apoptosis in HCAECs under NG, HG, and HG with EPCs-EXOs, stained with Annexin V and PI. (D) Quantification of apoptosis rates indicating increased apoptosis under HG conditions, mitigated by EPCs-EXOs. (E) Cell viability assessment at 0 hour, 24 hours, 48 hours, and 72 hours in NG, HG, and HG with EPCs-EXOs, showing increased proliferation in HG, reversed by EPCs-EXOs. (F) Blood glucose levels from day 0 to day 11 in CT and mice with diabetes (DM), with DM showing sustained hyperglycemia. (G) Body weight changes over time in CT and DM mice, with DM exhibiting progressive weight loss. (H) Oil Red O staining of cardiac coronary arteries in CT, CT with EPCs-EXOs, DM, and DM with EPCs-EXOs, showing lipid droplet accumulation. Scale bar=10 µm. (I) Quantification of lipid droplet area, demonstrating reduced accumulation in DM with EPCs-EXOs treatment. Values are mean±SEM (n=3–5 samples). *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared with NG-cultured cells or control group. CT, control; DM, diabetes mellitus; EPCs-EXOs, endothelial progenitor cell-derived exosomes; HCAECs, human coronary artery endothelial cells; HG, high glucose; NG, normal glucose; PI, propidium iodide.EPCs-EXOs can alleviate high glucose-induced endothelial cell dysfunction by reducing Orai1 and IGFBP3 expression Our previous studies demonstrated that HG exposure enhances SOCE in HCAECs by upregulating the expression of Orai1, Orai2, Orai3, and IGFBP3. To investigate whether EPCs-EXOs mitigate HG-induced endothelial dysfunction by suppressing Orai1 and IGFBP3 expression, HCAECs were cultured in NG or HG medium for 7 days. SOCE levels and the expression of Orai channel proteins were subsequently assessed. HCAECs were incubated with 2 μM Fluo-4 AM for 30 min, followed by treatment with 100 μM ATP for 10 min in a Ca 2+-free saline solution to deplete intracellular Ca2+ stores. This depletion led to a transient rise in cytosolic Ca2+, indicating ER Ca2+ release. The subsequent addition of 2 mM extracellular Ca2+ induced a secondary increase in intracellular Ca2+, consistent with SOCE-mediated Ca2+ influx. SOCE activity was significantly enhanced and sustained in HCAECs exposed to HG compared with NG controls. However, treatment with EPCs-EXOs substantially attenuated this HG-induced SOCE increase (figure 3A,B). To further elucidate the therapeutic potential of EPCs-EXOs, we examined their effect on the HG-induced upregulation of Orai1, Orai2, Orai3, and the key SOCE-associated protein IGFBP3. Western blot analysis revealed significantly elevated protein levels of all four markers in HCAECs cultured under HG conditions relative to NG. Notably, EPCs-EXOs treatment effectively suppressed this upregulation, with reductions of 43.82% in Orai1, 34.46% in Orai2, and 42.38% in Orai3, with the strongest inhibitory effect observed on Orai1 (figure 3C–G). These findings were further corroborated by immunohistochemical analysis, which displayed consistent trends in protein expression (figure 3H–L).Figure 3EPCs-derived exosomes can alleviate HG-induced endothelial cell dysfunction by reducing Orai1 and IGFBP3 expression. (A) Fluorescence imaging of intracellular Ca2+ in HCAECs under CT, NG, HG, and HG with EPCs-EXOs, using 2 mM Ca2+. Scale bar=100 µm. (B) Bar graph comparing RFI of Ca2+ under NG, HG, and HG with EPCs-EXOs conditions. (C) Western blot analysis showing protein expression of Orai1, Orai2, Orai3, IGFBP3, and β-actin under NG, HG, and HG with EPCs-EXOs. (D–G) Bar graphs quantifying Orai1, Orai2, Orai3, and IGFBP3 protein levels normalized to β-actin across NG, HG, and HG with EPCs-EXOs conditions. (H) Immunohistochemical staining of Orai1, Orai2, Orai3, and IGFBP3 in cardiac tissue from CT, mice with diabetes (DM), and DM with EPCs-EXOs mice; arrows indicate positive staining. Scale bar=20 µm. (I–L) Bar graphs showing staining intensity of Orai1, Orai2, Orai3, and IGFBP3 across CT, DM, and DM with EPCs-EXOs conditions. (M) Western blot analysis showing IGFBP3 and Orai1 protein bands under HG, HG with Orai1 OE, and HG with IGFBP3 OE conditions. (N–O) Bar graphs quantifying IGFBP3 and Orai1 levels normalized to β-actin across HG, HG with Orai1 OE, and HG with IGFBP3 OE conditions. (P) Wound-healing assay images of HCAECs under HG, HG with Orai1 OE, and HG with IGFBP3 OE, with or without EPCs-EXOs at 0 hour and 24 hours. Scale bar=100 µm. (Q) Bar graph comparing wound-healing rates across HG, HG with Orai1 OE, and HG with IGFBP3 OE conditions, with or without EPCs-EXOs. (R) Oil Red O staining images of cardiac tissue from CT, DM, DM with Orai1 OE, and DM with IGFBP3 OE, with or without EPCs-EXOs. Scale bar=100 µm. (S) Bar graph quantifying Oil Red O-positive area across CT, DM, DM with Orai1 OE, and DM with IGFBP3 OE conditions, with or without EPCs-EXOs. (T) Immunohistochemical staining of Orai1 and IGFBP3 in cardiac tissue under CT, DM, DM with Orai1 OE, and DM with IGFBP3 OE conditions, with or without EPCs-EXOs; arrows indicate positive staining. Scale bar=20 µm. (U) Bar graphs comparing staining intensity of Orai1 and IGFBP3 under CT, DM, DM with Orai1 OE, and DM with IGFBP3 OE conditions, with or without EPCs-EXOs. (V) Wound-healing assay images of HCAECs under NG, NG with Orai1 OE, and NG with IGFBP3 OE at 0 hour and 24 hours. Scale bar=100 µm. (W) Bar graph comparing wound-healing rates under NG, HG, NG with Orai1 OE, and NG with IGFBP3 OE conditions. (A’) Flow cytometry analysis of apoptosis in HCAECs under NG, NG with Orai1 OE, and NG with IGFBP3 OE, stained with Annexin V and PI. (B’) Bar graph comparing apoptosis rates across NG, NG with Orai1 OE, and NG with IGFBP3 OE conditions. (C’) Line graph showing cell viability at 0 hour, 24 hours, 48 hours, and 72 hours under NG, NG with Orai1 OE, and NG with IGFBP3 OE. (D’) Oil Red O staining images of heart tissue from CT, Orai1 OE, and IGFBP3 OE groups. Scale bar=100 µm. (E’) Bar graph comparing Oil Red O-positive area in CT, Orai1 OE, and IGFBP3 OE groups. (F’) Immunohistochemical staining of Orai1 and IGFBP3 in heart tissue from CT, Orai1 OE, and IGFBP3 OE mice; arrows indicate positive staining. Scale bar=20 µm. (G’) Bar graph comparing staining intensity of Orai1 and IGFBP3 across CT, Orai1 OE, and IGFBP3 OE conditions. Values are presented as mean±SEM (n=3–10 samples). *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared with NG-cultured cells or control groups. CT, control; DM, diabetes mellitus; EPCs-EXO, endothelial progenitor cell-derived exosomes; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor binding protein 3; IOD, integrated optical density; NG, normal glucose; OE, overexpression; PI, propidium iodide; RFI, relative fluorescence intensity; SOCE, store-operated Ca2+ entry; STIM1, stromal interaction molecule 1.To further investigate the roles of IGFBP3 and Orai1 in HG-induced endothelial dysfunction, both proteins were overexpressed in HCAECs prior to treatment with EPCs-EXOs. Western blot analysis (figure 3M–O) confirmed that HG elevated IGFBP3 and Orai1 protein levels, which were suppressed by EPCs-EXOs in vector-transfected control cells. However, in cells with IGFBP3 or Orai1 overexpression, EPCs-EXOs did not significantly reduce protein expression levels. Consistent with these findings, wound-healing assays showed that EPCs-EXOs inhibited cell migration under HG conditions in control cells, but exerted no significant effect in cells overexpressing IGFBP3 or Orai1—suggesting that downregulation of these proteins is essential for the antimigratory effect of EPCs-EXOs (figure 3P,Q). To further validate the contribution of IGFBP3 and Orai1 to endothelial dysfunction, HCAECs were cultured in NG medium and transfected to overexpress either IGFBP3 or Orai1. Functional assays demonstrated that overexpression of these proteins significantly altered endothelial behavior: proliferation was increased (figure 3C’), apoptotic rates were elevated (figure 3A’,B’), and migration capacity was enhanced (figure 3V,W). Mouse models with cardiac-specific overexpression of Orai1 or IGFBP3 were then generated to examine in vivo relevance. Oil Red O staining revealed that hearts from overexpression groups showed markedly higher lipid droplet accumulation compared with controls (figure 3D’,E’), and immunohistochemistry confirmed target protein overexpression (figure 3F’,G’). To evaluate whether Orai1 and IGFBP3 overexpression interferes with EPCs-EXO-mediated protection, DM models with Orai1 or IGFBP3 overexpression were established (figure 3R,S). In standard DM mice, EPCs-EXOs reduced lipid accumulation in coronary arteries; however, this effect was not observed in mice overexpressing Orai1 or IGFBP3. Immunohistochemical analysis confirmed sustained protein overexpression in these groups (figure 3T–U). These results collectively indicate that Orai1 and IGFBP3 are essential mediators of HG-induced endothelial dysfunction. The therapeutic benefit of EPCs-EXOs appears to rely, at least in part, on the suppression of Orai1 and IGFBP3 expression.EPCs-EXOs miR-7116-3p can target both Orai1 and IGFBP3 mRNAs To identify the specific miRNA within EPCs-EXOs responsible for alleviating endothelial cell dysfunction in the coronary arteries of mice with diabetes, candidate miRNAs predicted to simultaneously bind at least two of the following targets—Orai1, Orai2, Orai3, and IGFBP3—were screened using the miRDB (miRNA Target Prediction Database) online database. Three candidates, miR-669c-3p, miR-6951-3p, and miR-7116-3p, were predicted to target both Orai1 and IGFBP3 mRNAs ( online supplemental figure 1A). A comparison with the miRNA expression profile of EPCs-EXOs confirmed the presence of all three candidate miRNAs within the exosomal cargo (online supplemental figure 1B). Binding site predictions for Orai1 and IGFBP3 mRNAs were further verified using miRDB (online supplemental figure 1C), suggesting direct targeting by miR-669c-3p, miR-6951-3p, and miR-7116-3p. To assess the regulatory impact of these miRNAs on Orai1 and IGFBP3 expression, coronary artery endothelial cells were transfected with either mimics or inhibitors of each candidate miRNA. Transfection with mimics resulted in reduced expression of both proteins, while inhibitor transfection led to increased levels of Orai1 and IGFBP3 (online supplemental figure 1D–H). Among the three, miR-7116-3p demonstrated the most significant suppressive effect on target protein expression. The qPCR results were consistent with the western blot analysis (online supplemental figure 1I–L) and, therefore, miR-7116-3p was selected for subsequent investigation. To determine whether miR-7116-3p within EPCs-EXOs contributes to the attenuation of HG-induced endothelial dysfunction through regulation of Orai1 and IGFBP3, EPCs were transfected with either miR-7116-3p mimics or inhibitors. Exosomes were then isolated and characterized. Transfection efficiency was confirmed (figure 4A). These modified exosomes were applied to HG-cultured coronary artery endothelial cells, and their effects on proliferation, migration, and apoptosis were assessed. Under HG conditions, coronary endothelial cells exhibited significantly increased proliferation, migration, and apoptosis. Treatment with EPCs-EXOs suppressed these pathological responses. Notably, miR-7116-3p-enriched exosomes further enhanced the inhibitory effect, whereas depletion of miR-7116-3p reversed the suppression, restoring proliferative, migratory, and apoptotic activity (figure 4B–F). Consistently, expression levels of Orai1 and IGFBP3 were reduced in response to miR-7116-3p-enriched exosomes but were elevated when miR-7116-3p was depleted (figure 4G–I). These findings collectively indicate that miR-7116-3p within EPCs-EXOs plays a pivotal role in mitigating HG-induced endothelial dysfunction by inhibiting Orai1 and IGFBP3 expression in endothelial cells.Figure 4miR-7116-3p in EPC-derived exosomes attenuates HG-induced endothelial dysfunction by inhibiting Orai1 and IGFBP3 expression in endothelial cells. (A) Bar graph showing miR-7116-3p expression levels in EPCs transfected with miR-7116-3p mimics or inhibitors. (B) Line graph depicting cell viability at 0 hour, 24 hours, 48 hours, and 72 hours under HG, HG with EPCs-EXOs, HG with miR-7116-3p-overexpressing EPCs-EXOs, and HG with miR-7116-3p-knockdown EPCs-EXOs. (C) Wound-healing assay images of HCAECs under HG, HG with EPCs-EXOs, HG with miR-7116-3p-overexpressing EPCs-EXOs, and HG with miR-7116-3p-knockdown EPCs-EXOs at 0 hour and 24 hours. Scale bar=100 µm. (D) Bar graph comparing wound-healing rates across the indicated conditions. (E) Flow cytometry analysis of apoptosis in HCAECs under the same conditions, stained with Annexin V and PI. (F) Bar graph comparing apoptosis rates across groups. (G) Western blot analysis showing protein bands of Orai1, IGFBP3, and β-actin under HG, HG with EPCs-EXOs, HG with miR-7116-3p-overexpressing EPCs-EXOs, and HG with miR-7116-3p-knockdown EPCs-EXOs. (H–I) Bar graphs quantifying Orai1 and IGFBP3 protein expression normalized to β-actin across conditions. Values are presented as mean±SEM (n=3 samples). *P<0.05; **p<0.01; ***p<0.001; ****p<0.0001 compared with NG-cultured cells or control groups. EPCs-EXOs, endothelial progenitor cell-derived exosomes; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor-binding protein 3; NG, normal glucose; PI, propidium iodide.Discussion Diabetes mellitus, a growing global health burden, markedly increases the risk of diverse complications, particularly through its detrimental impact on cardiovascular health via both microvascular and macrovascular pathologies. 15 Exosomes and other small extracellular vesicles constitute a unique mechanism of intercellular communication, whereby miRNAs released from donor cells can be internalized by distant recipient cells to modulate gene expression.16 The present study investigated the therapeutic potential of EPCs-EXOs in ameliorating HG-induced endothelial dysfunction, with a focus on identifying the bioactive cargo responsible for the observed protective effects. The central finding of this investigation is that miR-7116-3p, encapsulated within EPCs-EXOs, directly targets the Orai1-IGFBP3 signaling axis, thereby alleviating HG-induced endothelial dysfunction. This finding uncovers a novel molecular mechanism underlying diabetic endothelial injury and highlights EPCs-EXOs as a promising therapeutic modality. The key supporting evidence includes: (1) EPCs-EXOs significantly ameliorated HG-induced endothelial dysfunction and reduced atherosclerotic plaque formation in a combined diabetes–AS mouse model; (2) EPCs-EXO treatment suppressed the HG-induced upregulation of Orai1 and IGFBP3 protein expression in coronary endothelial cells, implicating a role for these proteins in the pathogenesis of diabetic vascular damage; and (3) EPCs-EXOs attenuated the HG-driven enhancement of endothelial proliferation, migration, and apoptosis. Notably, exosomes enriched with miR-7116-3p exerted stronger inhibitory effects on these cellular processes, whereas miR-7116-3p knockdown reversed these effects, restoring pathological proliferation, migration, and apoptosis under HG conditions.DVCs, encompassing both macrovascular lesions and microvascular injury, remain a primary cause of mortality among patients with diabetes mellitus. Endothelial dysfunction is widely recognized as an early and critical event in the initiation and progression of DVCs.17 Bone marrow-derived EPCs play a central role in endogenous vascular repair and neovascularization.18 Previous studies, including that by He et al, demonstrated that EPCs overexpressing miR-126-3p significantly enhanced vascular repair in diabetic rat models.19 However, HG environments compromise both the function and number of EPCs.20 Exosomes, released into the extracellular space by most cell types, retain molecular signatures reflective of their cellular origin and serve as key mediators of paracrine signaling.21 Recent studies have demonstrated that EPCs-EXOs can promote angiogenesis and alleviate endothelial oxidative stress and dysfunction through diverse mechanisms.22 23 Despite these advances, the contribution of EPCs-EXOs to the prevention of diabetic coronary endothelial injury remains insufficiently characterized. The current findings suggest that EPC-derived exosomes can functionally restore endothelial integrity under diabetic conditions. Nevertheless, further mechanistic investigations are warranted to fully elucidate the molecular pathways governing this reparative process.Hyperglycemia, a hallmark of diabetes mellitus, induces vascular dysfunction through a variety of complex molecular pathways.24 25 Extensive evidence supports the reparative potential of EPCs-EXOs in restoring endothelial function. For example, Pan et al showed that miR-17-5p mediates the protective effects of ACE2-enriched EPCs-EXOs against cerebral ischemic injury in aged mice.26 Lin et al further revealed that Y-box binding protein 1 promotes the selective loading of miR-133 into EPCs-EXOs under hypoxia/reoxygenation conditions, enhancing fibroblast angiogenesis and mesenchymal-endothelial transition.27 Previous findings have established that HG-induced endothelial dysfunction is primarily driven by upregulated expression and interaction of Orai1 and IGFBP3. In this study, we further discovered that miR-7116-3p packaged within EPCs-EXOs directly targets the Orai1-IGFBP3 signaling axis, thereby alleviating high-glucose-induced endothelial dysfunction.Exosomes play a crucial role in mediating intercellular communication by transferring various biomolecules between cells, including proteins, lipids, RNA (such as miRNA, long non-coding RNA, circular RNA, and mRNA), and other molecular components.28 Among these, miRNAs may be crucial in initiating vascular calcification by regulating a network of specific cellular targets.29 EPCs-EXOs promote diabetic wound healing by delivering miR-182-5p.30 Circulating exosomal miR-16-2-3p is associated with coronary microvascular dysfunction in diabetes through regulating the fatty acid degradation of endothelial cells. EPCs-EXOs contain a complex miRNA population. Beyond miR-7116-3p, which is the focus of our study, other miRNAs likely promote endothelial repair through synergistic or independent mechanisms. For instance, Zhang et al found that exosomes derived from endothelial progenitor cells improve Lipopolysaccharide-induced damage in cerebral microvascular endothelial cells by delivering miR-126a-5p.31 Wu et al found that astragaloside IV promotes endothelial progenitor cell secretion of exosomes regulating the PI3KR2/SPRED1 pathway to inhibit pyroptosis in diabetic endothelial cells.32 This study primarily focuses on miR-7116-3p. Our preliminary screening and bioinformatics analysis indicate it exhibits the most significant changes in mediating the Orai1-IGFBP3 complex to alleviate endothelial cell dysfunction. However, limiting the scope to a single key molecule may not fully reveal the complete functional landscape of EPCs-derived exosomes. Further exploration in future studies is warranted. While the role of exosomal miRNAs in improving endothelial function under diabetic conditions has been widely recognized, the present study specifically identifies miR-7116-3p, encapsulated within EPCs-EXOs, as a key modulator that attenuates HG-induced endothelial dysfunction by suppressing Orai1 and IGFBP3 expression in endothelial cells.Our system still has several limitations that need to be addressed in future iterations. The Orai protein family includes multiple subtypes such as Orai1, Orai2, and Orai3. However, existing research consistently indicates that Orai1 is the most thoroughly studied and functionally characterized representative member of this family, serving as the primary calcium ion entry point in the SOCE signaling pathway. Recent studies have also extensively documented the physiological and pathological roles of Orai1 as the primary protein in the cardiovascular system.33–36 In this study, we found that Orai1 exhibited the most significant expression changes, leading us to select Orai1 as the primary target for subsequent investigations. Although our in vitro results indicate that Orai1 is the primary factor responsible for high-glucose-induced endothelial dysfunction, Orai2 and Orai3 also showed downregulation. We cannot rule out the potential involvement of Orai2 and Orai3 in this process. Additionally, despite demonstrating significant therapeutic potential, the clinical translation of EPCs-EXOs faces numerous challenges. These include standardizing administration protocols (dosage, frequency, and route), optimizing in vivo biodistribution and targeting efficiency, and evaluating potential off-target effects and long-term immune responses. Future research must advance their application through exosome engineering, delivery system optimization, and systematic preclinical evaluation. Furthermore, without directly comparing EPCs-EXOs with standard therapies for diabetic vascular complications—such as sodium-glucose cotransporter 2 inhibitors and glucagon-like peptide-1 receptor agonists—their clinical value cannot be quantified. This aspect also warrants in-depth exploration in future studies.In summary, As shown in figure 5, HG significantly enhances Orai-mediated SOCE activity and upregulates IGFBP3 expression, thereby promoting aberrant proliferation and migration of HCAECs. Encapsulated within EPCs-EXOs, miR-7116-3p effectively mitigates these pathological responses. These findings uncover a novel regulatory mechanism contributing to endothelial dysfunction in diabetes and highlight miR-7116-3p as a potential therapeutic target.Figure 5Schematic diagram of the proposed miR-7116-3p-mediated regulation of the Orai-Ca2+-IGFBP3 signaling pathway in EPC-derived exosomes. miR-7116-3p in EPC-derived exosomes inhibits HG-induced overexpression and interaction of IGFBP3 and Orai proteins in HCAECs, thereby suppressing SOCE-mediated intracellular Ca2+ elevation. This, in turn, attenuates endothelial cell proliferation, apoptosis, and migration driven by elevated intracellular Ca2+ levels. EPC, endothelial progenitor cell; EXO, exosomes; ER, endoplasmic reticulum; HCAECs, human coronary artery endothelial cells; HG, high glucose; IGFBP3, insulin-like growth factor-binding protein 3; SOCE, sarcoplasmic reticulum-operated Ca2+ influx; STIM1, stromal interaction molecule 1.Conclusion In conclusion, the data presented support the therapeutic potential of EPCs-derived exosomes and their cargo miR-7116-3p in attenuating HG-induced endothelial dysfunction. This effect is mediated through inhibition of the Orai1-IGFBP3 signaling axis, offering a promising strategy for the treatment and prevention of DVCs.