Identification of mouse BMSCs and there of derived EVs

To isolate BMSC-EVs, primary mouse BMSCs were first separated using Ficoll-based density gradient centrifugation, followed by differential attachment methods (Supplementary Fig. 1A). Cells were then examined under a light microscope. On the second culture day, smaller colonies with uneven morphology began to form (Supplementary Fig. 1B, P0-2 day). By the fifth day, the colonies had expanded, merged, and showed central cell stacking (Supplementary Fig. 1B, P0-5 day). After passage, the cells displayed typical polygonal fibroblast-like morphology, including more uniform appearances (Supplementary Fig. 1B, P1-7-day).

To assess the cells multi-lineage differentiation potentials, adipogenic, osteogenic, and chondrogenic differentiation was induced. Oil Red O staining demonstrated the formation of lipid droplets after two weeks of adipogenic induction (Supplementary Fig. 1C, Adipogenic). Alizarin Red staining showed significant ossification nodules after two weeks of osteogenic induction (Supplementary Fig. 1C, Osteogenic). Alicin Blue staining, performed after three weeks of chondrogenic induction, revealed blue-stained cytoplasm components within the formed cell clumps, indicating chondrogenic differentiation (Supplementary Fig. 1C, Chondrogenic).

Flow cytometry analysis was also conducted to detect the expression of specific BMSC cell surface markers. The results showed that the cellular population was positive for several mesenchymal stem cell markers, including CD44 (99.7%), CD29 (98.8%), and Sca-1 (98.8%), but was negative for CD31 (0.3%), CD117 (0.5%), and CD34 (27.3%) (Supplementary Fig. 1D).

After BMSCs were passaged and expanded, cell culture supernatants were collected. EVs were isolated using ultrafiltration and low-temperature ultracentrifugation (Fig. 1A). EVs were identified based on their morphologies, particle size distribution, and surface molecule expression using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blots.

Fig. 1

Isolation and Characterization of BMSC-EV. A Schematic representation of BMSC-EV isolation from cellular supernatants using a combination of ultrafiltration and ultracentrifugation techniques. B TEM image showing the distinctive cup-shaped morphology of the isolated BMSC-EV. C NTA depicting the size distribution profile of the BMSC-EV population. D Western blots showing the presence of surface protein markers specific to BMSC-EV. Abbreviations: BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; TEM, transmission electron microscope; NTA, nanoparticle tracking analysis

TEM images revealed scattered "saucer-like" structures with convex peripheries and concave centers, all of which are typical EV characteristics (Fig. 1B). NTA results indicated that the vesicles had an average particle size of 134.4 ± 75.1 nm, with the main peak (at 114.2 nm) accounting for 99.3% of the particles and a concentration of 5.8 × 1010 particles/mL (Fig. 1C). Western blots showed that EVs expressed TSG101, CD9, and CD63, but not calnexin. In contrast, calnexin was detected in BMSC lysates (Fig. 1D).

High glucose states exacerbate atherosclerosis in vivo and foam cell formation in vitro

To construct a diabetes-exacerbated atherosclerosis model, STZ was used to induce diabetes in ApoE−/− mice on an HFD, resulting in diabetic ApoE−/− mice (Fig. 2A–C). Blood lipid analysis showed that, compared to non-diabetic ApoE−/− mice on an HFD, the DA group had significantly increased levels of total cholesterol (TC), triglycerides (TG), and LDL (P < 0.05) (Fig. 2D–F).

Fig. 2

Effects of Different Interventions on Blood Lipid Profiles and Aortic Atherosclerosis in Diabetic ApoE−/− Mice. A Flowchart depicting the establishment of animal models and group dosing. B–G After 19 weeks of intervention, blood glucose levels were assessed with a rapid blood glucose meter, and serum insulin and blood lipid profiles were determined via biochemical methods (n = 6). H Oil Red O staining illustrates the distribution of atherosclerotic plaques in intact mouse aortas, along with plaque area quantification (n = 3). I H&E staining shows the distribution of atherosclerotic plaques in the transvalvular transverse section of the mouse aortic root, alongside quantification of plaque area. J Masson staining demonstrates the collagen content and ratio of collagen/plaque area in atherosclerotic plaques in the transverse axial section of the mouse aortic root (n = 6). (Pairwise comparisons between experimental groups were conducted using one-way ANOVA, * P < 0.05). Abbreviations: H&E staining, hematoxylin–eosin staining; WT, wild type; OD, ordinary diet; HFD, high-fat diet; DA, diabetes-accelerated atherosclerosis; RAPA, rapamycin; PCSK9i, proprotein convertase subtilisin/kexin type 9 inhibitor; BMSC, bone marrow mesenchymal stem cells; BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells

Histological analysis of aortic atherosclerosis using Oil Red O and hematoxylin and eosin (H&E) staining showed a progressive increase in aortic plaque areas from the OD group to the HFD group, and then to the DA-aggravated atherosclerosis group (all P < 0.05). Additionally, Masson staining showed that collagen content, which is indicative of plaque stability, was significantly reduced in DA group aortic plaques compared to the OD and HFD groups (all P < 0.05) (Fig. 2H–I).

In vitro experiments were conducted to determine the optimal concentration and exposure time for HG combined with ox-LDL in RAW264.7 cells using CCK-8 assays (Supplementary Fig. 2A-B). Oil Red O staining demonstrated that exposure to 75 µg/mL ox-LDL for 24 h induced significant foam cell formation in RAW264.7 cells. The presence of HG led to an additional increase in the foam cell proportions (Supplementary Fig. 2C).

BMSC-EVs reduce diabetes-exacerbated atherosclerosis

Serological testing revealed that the BMSC-EV group had significantly lower glucose, TC, TG, and LDL levels compared to the DA group (all P < 0.05). Serum insulin levels were also significantly higher in the BMSC-EV group (P < 0.05), but high-density lipoprotein (HDL) levels did not significantly differ between the two groups. No significant reduction in TC and TG levels was observed in the RAPA group, but the RAPA group did have higher LDL levels (P < 0.05). There were also no statistically significant differences in TC, TG, or LDL levels between the PCSK9i and BMSC-EV groups (P > 0.05) (Fig. 2A–G).

To determine the effects of BMSC-EVs on diabetes-exacerbated atherosclerosis, we administered BMSC-EVs to diabetic ApoE−/− mice and evaluated their arteries using Oil Red O staining of the aorta, as well as H&E and Masson staining of the aortic roots. We found a significant reduction in aortic plaque area and a notable increase in plaque collagen content in the BMSC-EV group compared to the DA group (all P < 0.05) (Fig. 2H–J).

Taken together, these findings suggest that BMSC-EVs can effectively reduce the severity of atherosclerosis and enhance plaque stability in diabetic conditions.

BMSC-EVs inhibit RAW264.7 cellular migration, proliferation, and foam cell formation

To understand the cellular mechanisms underlying BMSC-EVs’ alleviation of diabetes-accelerated atherosclerosis, we first established a co-culture system of BMSCs and RAW264.7 cells. Oil Red O staining revealed a significant reduction in the proportion of macrophages forming foam cells in the co-culture group compared to the model group (P < 0.05) (Fig. 3A). The proportion of foam cells significantly increased when cells were treated with the EV secretion inhibitor GW4869 (P < 0.05) (Fig. 3A).

Fig. 3

Effects of BMSC-EVs on HG-ox-LDL-Induced Macrophage Proliferation, Migration, Foam Cell Formation, and Cholesterol Efflux. A A co-culture system of BMSC and RAW264.7 cells in Transwell chambers was constructed to evaluate the effect of BMSC-EVs on the formation of RAW264.7 foam cells using Oil Red O staining (n = 3). B Transwell migration assays were used to assess the effects of BMSC-EVs on RAW264.7 cell migration. C CCK-8 assays were used to evaluate the effects of BMSC-EVs on RAW264.7 cell proliferation (n = 9). D Oil Red O staining was used to evaluate the effect of BMSC-EVs on RAW264.7 foam cell formation. (Pairwise comparisons between experimental groups were performed using one-way ANOVA, * P < 0.05). Abbreviations: WT, wild type; OD, ordinary diet; HFD, high-fat diet; DA, diabetes-accelerated atherosclerosis; RAPA, rapamycin; PCSK9i, proprotein convertase subtilisin/kexin type 9 inhibitor; BMSC, bone marrow mesenchymal stem cells; BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; HG, high glucose; ox-LDL, oxidized low-density lipoprotein; CCK-8, Cell Counting Kit-8; BMSC, bone marrow mesenchymal stem cells; Baf A1, Bafilomycin A1

We next used Transwell chambers, CCK-8 assays, and Oil Red O staining to assess the effects of BMSC-EVs on macrophage migration, proliferation, and foam cell formation. Results indicated that macrophage migration, proliferation activity, and foam cell formation were all significantly elevated in the model group compared to the control group (all P < 0.05). However, these parameters were significantly reduced in the BMSC-EVs treatment group compared to the model group (P < 0.05) (Fig. 3B, C). Notably, reductions in foam cell formation were reversed following the administration of the autophagy blocker bafilomycin A1 (Baf A1) in the respective groups (P < 0.05) (Fig. 3D).

BMSC-EV regulates macrophage autophagy

To further explore BMSC-EVs’ effects on autophagy, we used TEM to evaluate autophagosomes and autophagolysosomes in mouse aortas. Autophagosomes and autophagolysosomes were seen in the visual fields in the DA, OD, RAPA, PCSK9i, BMSC, and BMSC-EV groups. Typical autophagosomes and autophagolysosomes were rarely observed in the HFD and DA groups, suggesting inhibited autophagy. In the RAPA group, in contrast, multiple autophagosomes (Fig. 4A, red arrow) and autophagolysosomes (Fig. 4A, yellow arrow) were evident, indicating active autophagy. We also performed immunohistochemical (IHC) staining to assess the expression of autophagy-related proteins LC3B and P62 in mouse aortic root tissue. Compared with the OD group, the DA model group showed a significant increase in P62 expression and a decrease in LC3B II expression (P < 0.05). The BMSC and BMSC-EV groups, in contrast, showed reduced P62 but increased LC3B II expression compared to the DA group (both P < 0.05). No significant difference was observed between the BMSC and BMSC-EV groups (P > 0.05). These findings suggest that BMSC-EV can upregulate autophagy in diabetes-related atherosclerotic tissue, exhibiting effects comparable to those of BMSC (Fig. 4B–D).

Fig. 4

BMSC-EV Regulates Autophagy In Vivo. A Transmission electron microscopy reveals autophagosomes (red arrows) and autolysosomes (yellow arrows) in mouse arteries, which were used to evaluate autophagy status. B–D Immunohistochemical staining demonstrated the effect of BMSC-EVs on the expression of autophagy-related proteins LC3BII and P62 in cells of the mouse aortic root (n = 6). (Pairwise comparisons between experimental groups were conducted using one-way ANOVA, * P < 0.05). Abbreviations: WT, wild type; OD, ordinary diet; HFD, high-fat diet; DA, diabetes-accelerated atherosclerosis; RAPA, rapamycin; PCSK9i, proprotein convertase subtilisin/kexin type 9 inhibitor; BMSC, bone marrow mesenchymal stem cells; BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; P62, prostacyclin; LC3B, microtubule-associated protein. AOD, Average Optical Density

In vitro, we used the mRFP-GFP-LC3B viral reporter-based autophagy flux detection tool in RAW264.7 cells and then observed and quantified autophagosomes (red dots) and autophagolysosomes (yellow dots). There were significantly fewer autophagosomes and autophagolysosomes in the model group compared to the control group, indicating blocked autophagy flux (P < 0.05). In contrast, the number of autophagosomes and autophagolysosomes was significantly increased in the BMSC-EV group compared to the model group, indicating autophagy flux activation (P < 0.05) (Fig. 5A). In the BMSC-EV plus Baf A1 group, cells were predominantly characterized by increased proportions of yellow granular autophagosomes and decreased red autophagy lysosomes compared to the BMSC-EV group. However, the proportion of red autophagy lysosomes in the combined BMSC-EV-Baf A1 group was higher than the proportion in the Baf A1 group alone (all P < 0.05). This suggest that Baf A1 partially blocked BMSC-EV-induced activation of autophagy flux (Fig. 5A).

Fig. 5

BMSC-EV Regulates Autophagy In Vitro. A After transfection of mRFP-GFP-LC3 autophagy double-labeled lentivirus into RAW264.7 cells, laser confocal microscopy was used to assess the effect of BMSC-EV on autophagy flow. (Red dots, autolysosomes; Yellow dots, autophagosomes; *Statistical difference between the sum of red points and yellow points. # Statistical difference in the proportion of red points to the total number of points.) B Using a Transwell-based co-culture system of BMSC and RAW264.7 cells, Western blots were used to evaluate the effect of BMSC on macrophage autophagy (n = 3). C Western blots showed the effect of BMSC-EV on macrophage autophagy (n = 3). (Pairwise comparisons between experimental groups were conducted using one-way ANOVA, * P < 0.05, # P < 0.05). Abbreviations: BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; HG, high glucose; ox-LDL, oxidized low-density lipoprotein; Baf A1, Bafilomycin A1; P62, prostacyclin; LC3B, microtubule-associated protein

To indirectly demonstrate that BMSC-EVs are key mediators of the therapeutic effects of BMSCs, we established a transwell co-culture system of BMSCs and RAW264.7 macrophages (Fig. 3A). Compared with the control group, the model group (e.g., ox-LDL and HG-induced-RAW264.7 cells) had significantly increased expression of the autophagy-related protein P62, but significant reductions in the LC3B II/I ratio (all P < 0.05). In the BMSC co-culture group, P62 expression was significantly reduced, and the LC3B II/I ratio was significantly increased (all P < 0.05), suggesting that BMSCs upregulated autophagy processes in co-cultured RAW264.7 cells. However, after the addition of GW4869 to the BMSC culture system, there was no significant increase in autophagy in RAW264.7 cells (P < 0.05) (Fig. 5B).

We also directly treated RAW264.7 cells with BMSC-EVs and detected autophagy-related proteins via western blot analysis. Results indicated that, compared to the control group, P62 protein expression was significantly increased, but the LC3B II/I ratio was significantly decreased, in the model group (induced by ox-LDL and HG) (all P < 0.05). In the Baf A1 group, both P62 and the LC3B II/I ratio were further increased compared with the model group, indicating inhibited autophagy in the model group. However, in the BMSC-EV group, P62 protein expression was significantly decreased compared to the model group, while the LC3B II/I ratio was significantly increased (all P < 0.05). Administration of BMSC-EVs also reversed Baf-A1-induced increases in P62 and the LC3B II/I ratio (P < 0.05) (Fig. 5C).

BMSC-EVs modulate macrophage polarization phenotypes

We evaluated the proportion of M1 macrophages in atherosclerotic plaques in mouse aortic tissue using multiplex immunofluorescence (IF) staining. We found cells co-stained green (CD68-positive, macrophage marker) and red (iNOS-positive, M1 macrophage marker) in the ApoE−/− OD, HFD, and DA groups. The OD, HFD, and DA groups also showed an increasing trend in M1 macrophage numbers, consistent with the severity of atherosclerotic lesions. In contrast, the RAPA, PCSK9, BMSC, and BMSC-EV groups, despite showing obvious aortic root plaques, had very few iNOS-positive cells (Fig. 6A).

Fig. 6

BMSC-EV Modulates Macrophage Polarization Phenotype In Vivo and In Vitro. A Immunofluorescence (IF) co-staining illustrates M1 macrophages (CD68 & iNOS positive) within atherosclerotic plaques in the transvalvular section of the mouse aortic root. (Blue, DAPI; Green, CD68; Red, iNOS). B IF shows the effects of BMSC-EV on M1 polarization of RAW264.7 macrophages induced by HG combined with ox-LDL. Green fluorescence intensity indicates the expression level of the M1 polarization marker molecule CD86. C Western blots showing the effects of BMSC-EV on the relative expression of the M1-type polarization marker molecule iNOS and M2-type polarization marker molecule Arg-1 in RAW264.7 macrophages induced by HG combined with ox-LDL (n = 3). (Pairwise comparisons between experimental groups were conducted using one-way ANOVA, *P < 0.05). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; iNOS, inducible nitric oxide synthase; WT, wild type; OD, ordinary diet; HFD, high-fat diet; DA, diabetes-accelerated atherosclerosis; RAPA, Rapamycin; PCSK9i, Proprotein Convertase Subtilisin/Kexin Type 9 inhibitor; BMSC, bone marrow mesenchymal stem cells; BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; Arg-1, Arginase-1; IF, Immunofluorescence

To further explore the impacts of BMSC-EVs on macrophage polarization phenotypes, we first performed multiple IF analyses using RAW264.7 cells. The results indicated that, compared to the control group, the fluorescence intensity of the M1 macrophage marker CD86 was significantly increased in the model group, but was decreased in the BMSC-EVs group (P < 0.05) (Fig. 6B). We also detected M1 polarization marker iNOS and M2 polarization marker Arg-1 expression in RAW264.7 cells using western blots. Compared to the control group, the model group had significant increases in iNOS expression, along with significant reductions in Arg-1 expression (all P < 0.05). BMSC-EV treatment reversed iNOS and Arg-1 expression in the model group (both P < 0.05) (Fig. 6C).

BMSC-EVs regulate macrophage autophagy and polarization via the AMPK/mTOR Pathway

To understand the mechanism by which BMSC-EVs regulate autophagy and polarization phenotypes RAW264.7 cells, we focused on the AMPK/mTOR pathway, which is known for its role in both autophagy and macrophage polarization. We validated the presence of key molecules in this pathway using western blots. Initially, compared with the untreated group, combination treatment with HG and ox-LDL increased the p-mTOR/t-mTOR ratio, decreased the p-AMPKα/t-AMPKα ratio, elevated iNOS and P62 expression, and reduced Arg-1 and LC3B II/I expression (Blank Control vs. HG+ ox-LDL Control, P < 0.05). Subsequently, administration of BMSC-EVs in the induction group decreased the p-mTOR/t-mTOR ratio, increased the p-AMPKα/t-AMPKα ratio, reduced iNOS and P62 expression, and increased Arg-1 and LC3B II/I expression compared with the HG + ox-LDL control group (HG + ox-LDL-BMSC-EV group vs. HG + ox-LDL-Control group, P < 0.05). Additionally, the AMPK inhibitor Compound C partially blocked BMSC-EV-mediated effects. Specifically, introducing Compound C after BMSC-EV treatment increased the p-mTOR/t-mTOR ratio and decreased the p-AMPKα/t-AMPKα ratio. These changes were also accompanied by increased P62 expression, as well as decreased Arg-1 and LC3B II/I expression (HG + ox-LDL-BMSC-EV group vs. HG + ox-LDL-BMSC-EV + CC group, P < 0.05). Unfortunately, no statistically significant changes in iNOS were observed(HG + ox-LDL-BMSC-EV group vs. HG + ox-LDL-BMSC-EV + CC group, P > 0.05) (Fig. 7A–G).

Fig. 7

BMSC-EV Regulates Macrophage Autophagy and Polarization Phenotype Through the AMPK/mTOR Pathway. A–G Western blots showing the relative expression and phosphorylation of AMPK/mTOR in RAW264.7 cells induced by HG and ox-LDL, as well as autophagy and polarization marker molecule expression (n = 5). H BMSC-EV alleviates diabetes-aggravated atherosclerosis by modulating autophagy-related macrophage polarization via the AMPK/mTOR pathway (Pairwise comparisons between experimental groups were conducted using one-way ANOVA, * P < 0.05). Abbreviations: HG, high glucose; ox-LDL, oxidized low-density lipoprotein; BMSC, bone marrow mesenchymal stem cells; BMSC-EV, extracellular vesicles derived from bone marrow mesenchymal stem cells; CC, compound C; p-mTOR, phosphorylated mammalian target of rapamycin; t-mTOR, total mammalian target of rapamycin; p-AMPK α, phosphorylated adenosine monophosphate-activated protein kinase α; t-AMPK, total adenosine monophosphate-activated protein kinase α; iNOS, inducible nitric oxide synthase; Arg-1, Arginase-1; P62, prostacyclin; LC3B, microtubule-associated protein

Effects of BMSC-EV on liver histopathology

HE staining of mouse livers after 12 weeks of continuous administration showed that, compared with the WT group, the DA group had widely distributed white lipid droplets of varying sizes within hepatocyte cytoplasm. In contrast, BMSC-EV-treated mouse livers showed a reduced number of lipid droplets, and these droplets had a more limited distribution. There were also no apparent structural abnormalities in BMSC-EV group liver tissue (Supplementary Fig. 3).