Twelve six-week-old male ApoE^-⁣/- mice (C57BL/6), weighing 20 to 22 g, were purchased from Huafukang Bioscience (Beijing, China) and randomly assigned into two groups: the High-fat diet (HFD) group (n = 6) and the HFD+M2 macrophage-derived exosomes (HFD+Exo^M2) group (n = 6). After 6 weeks on a high-fat diet (H10540, Beijing HFK Bioscience Co. Beijing, China , Protein:Carbohydrate:Fat = 20:40:40 [kcal%]), mice in the HFD+Exo^M2 group received tail vein injections of Exo^M2, 200 µg per mouse, twice a week for 4 weeks. Mice in the HFD+PBS group were administrated an equivalent of phosphate-buffered saline (PBS) via the tail vein. The dosing regimen was based on previous studies [16, 17]. Mice were killed by cervical dislocation under isoflurane anesthesia, followed by exsanguination and organ isolation.

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Fuwai Hospital, Chinese Academy of Medical Sciences.

The mouse macrophage cell line RAW264.7 was purchased from the Cell Resource Center of Peking Union Medical College (PCRC). When cell confluency reached 50%–60%, the cell culture medium was replaced with exosome-free Dulbecco’s Modified Eagle Medium (Gibco BRL, Carlsbad, CA, USA), supplemented with 20 ng/mL lnterleukin 4 (IL-4) (Peprotech, Rocky Hill, NJ, USA). After 24 hours, the polarization of macrophages towards the M2 phenotype was assessed using immunofluorescence staining for CD206, an M2 macrophage-specific protein marker.

Mouse aortic smooth muscle cells (MOVAs) were purchased from Fenghui Biotechnology (Beijing, China). When MOVAs reached 60% confluency, 30 ng/mL platelet-derived growth factor-BB (PDGF-BB) was added to induce atherosclerotic pathological changes [18]. After 24 hours, cells were lysed to collect proteins, and the expression levels of contractile phenotype markers of $\alpha$-SMA and SM22$\alpha$, as well as the synthetic phenotype marker of OPN, were detected using immunoblotting experiments.

All cell lines were validated by STR profiling and tested negative for mycoplasma. Cells were all cultured in a humidified incubator at 37 °C and 5% CO₂.

Serum-free M2 macrophage cell culture supernatant was collected and centrifuged at 4 °C, 2000 $\times$g for 20 minutes to remove cell pellets. Next, the supernatant was transferred to a high-speed centrifuge tube and centrifuged at 4 °C, 16,500 $\times$g for 30 minutes, followed by transfer to an ultra-speed centrifuge tube and centrifugation at 4 °C and 120,000 $\times$g for 70 minutes. After discarding the supernatant, the pellets were resuspended in pre-cooled PBS and centrifuged at 4 °C, 120,000 $\times$g for 60 minutes. After centrifugation, the exosome pellet was resuspended in PBS buffer and stored at –80 °C immediately for subsequent studies.

Exosome ultrastructure and size distribution were assessed using transmission electron microscopy (Hitachi Model H-7650 TEM, Tokyo, Japan) and Nanoparticle Tracking Analysis (Malvern, UK). Drawing from previous studies [19], the MicroBCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the protein concentration of the isolated exosomes. Immunoblotting was performed to detect specific exosome markers, including tumor susceptibility gene 10 (TSG101), ALG-2 interacting protein X (ALIX), heat shock protein 70 (HSP70), and Calnexin.

For the in vitro exosome uptake assay, purified Exo^M2 were labeled with the membrane-labeling dye PKH67 (Sigma-Aldrich, Eschenstr, Taufkirchen, Germany) according to the manufacturer’s instructions. Next, VSMCs were incubated with 100 µg/mL PKH67-labeled exosomes for 0, 1, 2, 3, and 4 hours, and stained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). The internalization of PKH67-labeled exosomes by VSMCs was visualized using confocal microscopy (Leica TCS SP8, Leica Microsystems Inc., Deerfield, MA, USA).

Purified Exo^M2 were labeled with PKH67 (Sigma-Aldrich, Eschenstr, Taufkirchen, Germany) following the manufacturer’s instructions. ApoE^-⁣/- mice in the PKH67-labeled Exo^M2 group were injected intravenously with 200 µg of exosomes every other day for 2 weeks [16]. Animals in the blank control group received an equal volume of PBS. At the endpoint, major organs (the aorta, lung, spleen, kidney, liver, and heart) were harvested to assess the in vivo distribution of PKH67-labeled Exo^M2. The fluorescence intensity was quantified using the IVIS® Spectrum system and Living Image® Software (Version 4.4, PerkinElmer, Shanghai, China). Additionally, fluorescence quantification of tissue sections was performed using fluorescence microscopy. Cell nuclei were stained with DAPI (Sigma-Aldrich, Eschenstr, Taufkirchen, Germany).

Purified exosome pellets or cultured MOVAs were harvested and lysed in RIPA (Beyotime Biotechnology, Shanghai, China) buffer supplemented with complete protease inhibitor cocktail tablets (Roche, Basel, Swiss). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to Polyvinylidene Fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), and respectively incubated with antibodies against TSG101 (ab125011), Alix (ab275377), HSP70 (ab2787), and Calnexin (ab22595) (for exosome, Abcam, London, UK), or antibodies against ɑ-SMA (55135-1-AP), SM22ɑ (10493-1-AP), OPN (22952-1-AP) and ɑ-Tubulin (11224-1-AP) (for MOVAs, Proteintech, Wuhan, Hunan, China). The Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to calculate the protein concentration. The signal intensity of primary antibody binding was quantitatively analyzed using ImageJ software (Version: 2.3.0/1.53q, LOCI, University of Wisconsin, Madison, WI, USA) and normalized to the signal intensity of GAPDH or $\alpha$-Tubulin.

The wound healing assay is a well-established method for measuring cell migration [20]. To evaluate the effects of Exo^M2 on the PDGF-BB-induced migration of VSMCs, MOVAs (2 $\times$ 10⁶/well) were seeded in a 6-well plate (CORNING, Corning, NY, USA) and cultured in a thermostat incubator (Thermo Fisher Scientific, Waltham, MA, USA). When cells reached 80–90% confluency, Exo^M0 or Exo^M2 were added. After co-culturing for 4 hours, cells in the center of the plate were gently removed using a 200 µL sterile plastic pipette tip to present a “cross” shaped scratch. Subsequently, 30 ng/mL PDGF-BB was added to the serum-free medium in the PDGF-BB group, PDGF-BB+Exo^M0 group, and PDGF-BB+Exo^M2 group. MOVA migration was observed and photographed at 0, 12, and 24 h of induction using a Leica inverted fluorescence microscope. Finally, the scratch areas of different groups were quantified using ImageJ software, with the migration area (percentage) calculated as (A₀ – A_n)/(A₀) $\times$ 100%, where A₀ is the initial wound area and A_n is the wound area at the measurement time point.

VSMCs were seeded into 96-well plates (Beyotime Biotechnology, Shanghai, China) at a density of 2 $\times$ 10³ cells per well and cultured in a cell culture incubator at 37 °C with 5% CO₂ (Thermo Fisher Scientific, Waltham, MA, USA). The cells were treated with PDGF-BB (30 ng/mL) in the presence of Exo^M0 or Exo^M2 (100 µg/mL), while control cells received the same volume of serum-free cell culture medium. After 22 hours, 10 µL of CCK-8 solution was added to each well, and the incubation was continued for 2 h. Finally, the absorbance value at 450 nm was determined.

At the endpoint of the study, the animals were euthanized by CO₂ suffocation. Arterial tissues containing plaques were collected, and a comprehensive analysis of the effects of Exo^M2 on the progression and stability of atherosclerotic plaques was performed using Oil Red O (ORO) staining, hematoxylin-eosin (H&E) staining, and Masson’s trichrome staining. To explore the cellular mechanisms underlying the atheroprotective effects of Exo^M2, immunohistochemistry analysis was used to detect the expression levels and spatial distribution of OPN, CD68, MMP-9, and $\alpha$-SMA in the plaque tissues. Quantitative analysis was performed using ImageJ software (Version: 2.3.0/1.53q, LOCI, University of Wisconsin, Madison, WI, USA).

Quantitative analyses were conducted with at least three independent experiments. Results are presented as the mean $\pm$ SD. Statistical significance was determined using Student’s t-test for two-group comparisons, one-way ANOVA for three-group comparisons, and two-way ANOVA for normalized data. Differences were considered statistically significant at p 0.05.

The Exo^M2 used in this study was derived from IL-4 (20 ng/mL)-induced RAW264.7 cells. As shown in Fig. 1A, CD206 expression in RAW264.7 cells reached 100%, indicating successful polarization into M2 macrophages. To ensure the quality and purity of the exosomes, we used transmission electron microscopy, NanoSight, and Western blotting to identify their morphological structures and protein markers, respectively. The results showed that Exo^M2 had a diameter ranging from 30 nm to 150 nm and a ‘round-shaped’ morphology (Fig. 1B,C). Western blotting revealed that the Exo^M2 expressed TSG101, Alix, and HSP70, but lacked calnexin, an endoplasmic reticulum-specific marker (Fig. 1D), consistent with typical exosome characteristics.

Fig. 1.

Isolation and identification of Exo^𝐌𝟐 in vitro. (A) Identification of M2 RAW264.7 macrophages induced by IL-4. Fluorescence staining showed that CD206 (green) was expressed in all cells. Scale bar: 50 µm. (B) Size distribution profile of Exo^M2 as determined by Nanosight. (C) Ultrastructure of Exo^M2 observed by transmission electron microscopy. Scale bar: 100 nm. (D) Western blot was used to detect the levels of exosome markers TSG101, Alix, HSP70, and Calnexin. (E) Representative fluorescence images of VSMCs loaded with PKH67-labeled Exo^M2 (green) (100 µg/mL) after 0, 1, 2, 3, and 4 h of co-incubation. Nuclei were stained with DAPI (blue). Scale bar: 25 µm. Exo^M2, exosomes derived from M2 macrophages; IL-4, lnterleukin 4; DAPI, 4${}^{\prime }$,6-Diamidino-2-phenylindole; CD206, mannose receptor; TSG101, tumor susceptibility gene 10; HSP70, heat shock protein 70.

To detect the manner of interactions between Exo^M2 and recipient VSMCs, Exo^M2 were labeled with PKH67 and co-cultured with VSMCs, and then visualized with confocal microscopy. Results revealed an increase in the number of internalized vesicles increased over time (Fig. 1E). Exo^M2 were observed around the nuclei or lining the inner surface of the cell membrane after internalization into VSMCs, suggesting that Exo^M2 can be effectively taken up by VSMCs, and membrane fusion may be the primary mechanism through which VSMCs internalize exosomes.

PDGF-BB is a commonly used inducer to replicate in vitro the pathological changes of VSMCs associated with atherosclerotic cardiovascular disease, such as proliferation, migration, and transformation from a contractile to a synthetic phenotype [21]. To investigate whether Exo^M2 regulate these atherosclerotic pathological changes in VSMCs CCK8 and wound-healing assays were conducted using mouse aortic vascular smooth muscle cells (MOVAs). CCK8 assay results showed that Exo^M2 (100 µg/mL) attenuated PDGF-BB-induced proliferation by approximately 30% (Fig. 2A,B). Compared to Exo^M0, Exo^M2 more significantly reversed the proliferation of MOVAs induced by PDGF-BB (Fig. 2C). In the wound-healing assays, neither Exo^M0 nor Exo^M2 significantly changed cell mobility at 12 h compared to the control (PDGF-BB) group. However, at 24 h of co-incubation, Exo^M2 treatment significantly inhibited PDGF-BB-induced migration of MOVAs (p 0.001) (Fig. 2D,E). The effects of Exo^M2 on the phenotypic transformation of MOVAs were determined by western blotting. As shown in Fig. 2F,G, PDGF-BB downregulated the expression levels of contractile phenotype markers ($\alpha$-SMA and SM22$\alpha$) and upregulated that of the synthetic phenotype marker OPN. In contrast to Exo^M0, which showed no effects on MOVA phenotypic transformation, Exo^M2 treatment successfully reversed the PDGF-BB-induced transformation of MOVAs to a synthetic phenotype.

Fig. 2.

The effects of Exo^𝐌𝟐 on PDGF-BB-induced proliferation, migration, and phenotypic transformation of MOVAs. CCK8 assays were used to assess cell proliferation. (A) PDGF-BB-induced MOVA proliferation in a dose-dependent manner (n = 6 per group). (B) Exo^M2 (40–100 µg/mL) effectively inhibited PDGF-BB (30 ng/mL)-induced MOVA proliferation (n = 6, per group). (C) Exo^M2 (100 µg/mL) had a more significant inhibitory effect on PDGF-BB-induced MOVA proliferation compared to the same dose of Exo^M0 (n = 6 per group). (D) MOVAs were incubated with Exo^M0 or Exo^M2 for 4 h and then were stimulated with or without PDGF-BB (30 ng/mL) for 12 or 24 h. Differences in the relative migration rate of MOVAs were detected by wound-healing assays (n = 6 per group; Scale bar: 200 µm). (E) Quantification of the VSMC migration area using ImageJ software. (F) Western blotting bonds represent the regulatory effects of Exo^M0/Exo^M2 on the expression of $\alpha$-SMA, SM22$\alpha$, and OPN (n = 3). (G) Quantification of relative protein levels normalized to $\alpha$-Tublin. Statistical significance was determined by one-way or two-way ANOVA. * p 0.05, ** p 0.01, *** p 0.001, ns, no significant difference. Exo^M2, exosomes derived from M2 macrophages; CCK8, Cell Counting Kit-8; PB indicates PDGF-BB. PDGF-BB, platelet-derived growth factor-BB; MOVAs, mouse aortic smooth muscle cells; VSMC, vascular smooth muscle cell; $\alpha$-SMA, alpha-smooth muscle actin; SM22$\alpha$, smooth muscle 22 alpha; OPN, osteopontin.

To explore the lesion-targeting ability of Exo^M2, we labeled purified Exo^M2 with PKH67 and administered it into ApoE^-⁣/- mice via tail vein injections (200 µg of exosomes per mouse) every other day for 2 weeks [16]. As shown in Fig. 3A, fluorescence-labeled Exo^M2 mainly accumulated in the liver, spleen, and aorta, consistent with the results of fluorescence imaging of tissue sections (Fig. 3B), indicating that Exo^M2 could be successfully taken up by histiocytes, including those within plaque tissue at the aortic root.

Fig. 3.

The effect of Exo^𝐌𝟐 on plaque progression in ApoE^-⁣/- mice. (A) Ex vivo fluorescence bio-imaging of major organs after ApoE^-⁣/- mice were administrated PKH67-labeled Exo^M2 (200 µg per mouse, once every other day for 2 weeks). (B) Representative images of PKH-67-labeled Exo^M2 in different tissue sections. Nuclei were stained with DAPI (blue). The arrows indicate PKH-67-labeled M2 macrophage derived exosomes. Scale Bar: 200 µm. (C) Oil Red O staining aortic tissues from atherosclerotic mice with or without Exo^M2 treatment (n = 3 per group). Scale bar: 5 mm. (D–F) Representative images of H&E staining, Oil Red O staining, and Masson staining in cross-sections of the proximal aorta. Scale Bar: 200 µm. (G) Quantitative analysis of atherosclerotic lesion size in the whole aorta (n = 3). (H) Quantification of plaque area in the aortic root in H&E-stained proximal aorta (n = 6). (I) Quantification of necrotic area in H&E-stained proximal aorta (n = 6). (J) Quantification of positive area in Oil Red O-stained proximal aorta (n = 4). (K) Quantification of collagen content in aorta root sections stained with Masson’s trichrome (n = 6). Statistical analysis was conducted using Student’s t-test. ** p 0.01, *** p 0.001. HFD, high fat diet; PBS, phosphate buffered saline; H&E, hematoxylin-eosin staining.

Exo^M2 inhibited plaque progression in ApoE^-⁣/- mice fed a high-fat diet (HFD). Oil red O staining of longitudinal aortic sections revealed that compared to the HFD+PBS group, Exo^M2 treatment significantly reduced the percentage of the plaque area relative to the total aortic area (10.94% $\pm$ 1.86% vs. 18.40% $\pm$ 1.13%, p 0.01) (Fig. 3C,G). H&E staining of the proximal aorta showed a significant reduction in the plaque area (Fig. 3D,H) and necrotic core (Fig. 3D,I) in the HFD+Exo^M2 group compared to the HFD+PBS group. The lipid accumulation in the proximal aorta was also markedly reduced in the HFD+Exo^M2 group (Fig. 3E,J). Meanwhile, Masson staining indicated that the collagen content in plaques from Exo^M2-treated mice was significantly higher than that in the HFD+PBS group (Fig. 3F,K).

To elucidate whether the atheroprotective role of Exo^M2 was associated with smooth muscle cells, further histopathological analysis of the plaques was performed. Immunohistochemical staining results showed that compared to the HFD+PBS group, the areas of $\alpha$-SMA-positive (Fig. 4A,E) and OPN-positive (Fig. 4C,G) in plaques from the HFD+Exo^M2 group were both significantly decreased, while the $\alpha$-SMA/OPN ratio (Fig. 4J) was significantly increased, indicating a significantly reduced number of smooth muscle cells in plaques and suppressed transformation from a contractile to a synthetic phenotype. The ratio of CD68-positive macrophages to $\alpha$-SMA-positive VSMCs within plaques in Exo^M2-treated mice was also notably lower (Fig. 4B,F,I). Additionally, the area of MMP9 within plaques decreased significantly in ApoE^-⁣/- mice treated with Exo^M2, indicating increased plaque stability (Fig. 4D,H). An association between the atheroprotective effect of Exo^M2 and its inhibition of the proliferation, migration, and transformation to a synthetic phenotype of VSMCs (Fig. 5), is present.

Fig. 4.

The effect of Exo^𝐌𝟐 on atherosclerotic plaque stability. (A–H) Representative images and quantitative analysis of aorta root sections stained with antibodies against $\alpha$-SMA, CD68, OPN, and MMP9 (n = 3). Scale bar: 40 µm or 200 µm. (I) Quantification of the ratio of $\alpha$-SMA to CD68. (J) Quantification of the ratio of $\alpha$-SMA to OPN. Statistical analysis was conducted using Student’s t-test. * p 0.05, ** p 0.01. $\alpha$-SMA, alpha-smooth muscle actin; OPN, osteopontin; MMP9, matrix metalloproteinase 9.

Fig. 5.

Schematic diagram of the cellular mechanism of Exo^𝐌𝟐 regulating atherosclerosis. (A) In atherosclerotic plaque progression, smooth muscle cells proliferate and migrate from the media to the intima, where they transform into a synthetic phenotype, secreting high levels of MMP9 and cytokines, which promotes plaque progression and reduces plaque stability. (B) The above process was considerably suppressed by Exo^M2. MMP9, matrix metalloproteinase 9; VSMC, Vascular smooth muscle cell.