An ApoE^−/− atherosclerotic mouse model was established. The male ApoE^−/− mice on a C57BL/6J background (Vital River Laboratory Animal Technology, Beijing, China) were fed Western diet for 11 weeks. At 8 weeks, the mice were randomly divided into four groups and received the corresponding treatments for 3 weeks: adenovirus negative control group (Ad-NC) (n = 8), adenovirus miR-216a group (Ad-miR-216a) (n = 7), Ad-NC + Rb2 group (n = 8), and Ad-miR-216a + Rb2 group (n = 8). The mice in the Ad-miR-216a and Ad-miR-216a + Rb2 groups were injected with miR-216a recombinant adenovirus (Weizhen Biosciences, Jinan, China) at the dose of 2 × 10⁹ units via the tail vein. After 3 days, the mice in the Ad-NC + Rb2 and Ad-miR-216a + Rb2 groups were intraperitoneally injected with 20 mg/kg of Rb2 (MedChemExpress, Monmouth, NJ, United States) once every 2 days. The mice in the Ad-NC and Ad-miR-216a groups received saline of the same volume.

At the end of the study, the mice were euthanized by a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then, the aortas and peripheral blood were collected. The plasma was separated by centrifugation 2000 rpm and stored at −80°C until use. The plasma levels of cholesterol and triglyceride were measured by the Labospect 008 AS Automatic Biochemical Analyzer (Hitachi, Tokyo, Japan). All animal handling and procedures were approved by the Institutional Animal Care and Use Committee at the FuWai Hospital.

The aortas of ApoE^−/− mice were collected to analyze the status of atherosclerosis plaque. The Oil red O (Sigma-Aldrich, Saint Louis, MO, United States) staining method was used to assess en face lesions of the aortas. The images were captured with a stereomicroscope-dedicated camera (Zeiss, Jena, Germany) and analyzed with Image-pro Plus 6.0. The extent of the lesion area is the percentage of the total area of the aorta that was covered by lesions.

Hematoxylin–eosin (HE) staining was performed on 8-μm-thick frozen sections for histological analysis to observe the plaque lesion area. Masson’s trichrome staining (Leagene Biotechnology, Beijing, China) was used to examine collagen content of plaque. Oil red O staining was used to examine the accumulation of neutral lipids in the plaque area. Immunohistochemical staining was used to assess plaque macrophages and matrix metalloproteinase 9 (MMP-9) expression. The details are described in the Supplementary Materials.

The human acute monocytic leukemia cell line, THP-1 cells (China Infrastructure of Cell Line Resources, Beijing, China), were cultured in the RPMI 1640 medium (HyClone, Logan, UT, United States) with 10% fetal bovine serum (Sigma-Aldrich, Saint Louis, MO, United States) in a 5% CO₂ incubator at 37°C. The THP-1 cells were differentiated to macrophages under the stimulation of 100 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, Saint Louis, MO, United States) for 48 h. The miR-216a mimics or negative control were transfected into macrophages at a final concentration of 50 nM using lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, United States) for 8 h. Then, the cells were treated with 10 μM Rb2 (MedChemExpress, Monmouth, NJ, United States) for 48 h. The sequence of miR-216a mimics was designed as the following: sense 5′-UAA​UCU​CAG​CUG​GCA​ACU​GUG​A-3′ and antisense 5′-ACA​GUU​GCC​AGC​UGA​GAU​UAU​U-3′.

The human peripheral blood was obtained from healthy volunteers with informed consent. The peripheral blood mononuclear cells (PBMCs) were isolated using a Histopaque®-1077 density gradient (Sigma-Aldrich, Saint Louis, MO, United States) as described previously (Hirose et al., 2011). The monocytes were differentiated into macrophages by incubation with RPMI 1640 medium containing 20% fetal bovine serum and 100 ng/mL M-CSF (PeproTech, Suzhou, China) for 7 days. Subsequently, macrophages were transfected with miR-216a mimics for 8 h and treated with 10 μM Rb2 for 36 h, then transformed to foam cells under the stimulation of 50 μg/ml ox-LDLs for 12 h.

The Dual-Luciferase Reporter Assay was used to assess the effects of Rb2 on the expression of Smad3, the direct target of miR-216a. The pMIR-REPORT-Smad3-3′UTR luciferase plasmid was constructed according to our previous study (Yang et al., 2018). The details are shown in the Supplementary Materials.

The telomerase repeat amplification protocol assay was performed to assess the telomerase activity during macrophages differentiation and polarization (Hou et al., 2001; Yang et al., 2019), with details as shown in the Supplementary Materials.

The mRNAs and miRNAs expression were analyzed by real-time quantitative (RT-q) PCR on the ABI 7500 System (Applied Biosystems, Foster City, CA, United States), and the gene primers are shown in Supplementary Table S1. The effects of Rb2 on protein expression of Smad3 and nuclear factor kappa B inhibitor alpha (IκBα) in macrophages were determined by the western blot assay. The details are shown in the Supplementary Materials.

The effects of Rb2 on lipid uptake ability of THP-1–derived foam cells or PBMC-derived foam cells were examined using Oil red O staining. The cholesterol efflux assay was performed in macrophages incubated with [³H]-cholesterol to explore the effect of Rb2 in cholesterol transport. The details are shown in the Supplementary Materials.

The effects of Rb2 on miR-216a–mediated polarization of M1 macrophages were assessed by flow cytometry, and the surface markers CD86 of M1 macrophages and CD206 of M2 macrophages were examined. Cell apoptosis and death were detected by flow cytometry with the Annexin V/PI assay with the FITC Annexin V/PI Apoptosis kit (BD Biosciences, New Jersey, United States). The details are shown in the Supplementary Materials.

Macrophages were transfected with miR-216a mimics and treated with Rb2 as described above. The senescent status of the macrophages was assessed by in situ staining for senescence-associated β-galactosidase (SA-β-gal). The details are described in the Supplementary Materials.

Data analysis was performed using SPSS 20.0 statistical software (SPSS Inc., Chicago, United States). Quantitative variables are presented as mean ± SEM. The statistical significance of differences was calculated using one-way ANOVA, and a p value < 0.05 was considered statistically significant.

To explore the effect of Rb2 on atherosclerosis plaque induced by miR-216a in vivo, an ApoE^−/− atherosclerotic mice model was established. The analyses of atherosclerosis lesion formation on the aortas cut lengthwise revealed that miR-216a increased en face the aortic lesion area of the thoracic aorta by 200% compared with the control group, whereas the effect of miR-216a on atherosclerosis burden was significantly decreased by 45% with Rb2 treatment (Figures 1A,B). Therefore, the thoracic aorta was chosen for further experiments to assess the stability of plaque. HE staining of the frozen sections in the thoracic aorta showed that miR-216a increased the lesion area by 300% compared with the control group, and this effect also diminished by 74% with Rb2 treatment (Figures 1C,D).

Next, we investigated whether macrophages polarization was involved in the role of Rb2 in atherosclerosis progression. The immunohistochemistry staining showed that intima macrophages stained with surface marker CD68 were markedly increased by 200% in atherosclerotic plaque of the Ad-miR-216a group compared with the control group, while the number of infiltrated macrophages was reduced in the Ad-miR-216a + Rb2 group (Figure 2A). Moreover, staining of M1 marker CD16/CD32 showed that pro-inflammatory M1 subsets were dramatically increased by 150% in plaque areas in the Ad-miR-216a group, which was also suppressed by Rb2 treatment (Figure 2B).

The miR-216a led to an increase of lipid accumulation by 50% in plaques of the thoracic aorta when compared with the control group, whereas Rb2 treatment alleviated lipid accumulation by 55% in the Ad-miR-216a + Rb2 group (Figures 3A,B). In addition, the decreased mRNA expression of cholesterol efflux–related genes ABCG1 and ABCA1 induced by miR-216a were restored by Rb2 treatment in peritoneal macrophages in the ApoE^−/− atherosclerotic mice model (Figure 3C). However, either miR-216a or Rb2 has no significant effect on the serum lipid profiles of mice (Supplementary Table S2).

MMPs are mainly secreted by M1 macrophages and MMP-9 is a crucial factor to degrade collagen in plaques (Newby, 2014). To further explore the impacts of Rb2 on atherosclerosis plaque stability, the collagen deposition of plaque was assessed by Masson’s trichrome staining. The collagen content of thoracic aorta lesion was decreased by 62% in the Ad-miR-216a group compared with the control group, while it was increased by 250% after Rb2 treatment (Figure 4A). The percentage of MMP-9–positive area in the thoracic aorta plaques was significantly reduced by 50% in the Ad-miR-216a + Rb2 group when compared with the Ad-miR-216a group (Figure 4B).

Our previous study reported that miR-216a can activate telomerase activity and promote the polarization of pro-inflammatory M1 phenotype via Smad3/IκBα signaling (Yang et al., 2019). In this study, Rb2 treatment with concentrations of 0.1, 1, or 10 μM significantly decreased endogenous miR-216a expression in macrophages by 53, 63 and 62%, respectively (Figure 5A). Given that the Kd value of Rb2 binding to miR-216a was 17.6 μM in the microscale thermophoresis experiment from our previous study (Chen et al., 2021), the concentration of 10 μM of Rb2 was chosen as the optimal dose for experiments.

In the luciferase assay in HEK293T cells, miR-216a reduced the luciferase activity of pMIR-Smad3-3′UTR reporter by targeting the Smad3, and this effect was counteracted by Rb2 treatment (Figure 5B). As for the pMIR-Smad3-3′UTR mutant reporter, unsurprisingly, there were no changes in luciferase activity when treating with miR-216a or Rb2 as compared to the control group. Moreover, western blot showed that Rb2 treatment restored the Smad3 and IκBα protein levels in macrophages with miR-216a overexpression (Figure 5C).

Next, the telomerase activity in macrophages was assessed with or without Rb2 treatment. miR-216a increased the telomerase activity by 61%, and Rb2 suppressed the effect of miR-216a on telomerase activity by 75% (Figure 5D). Consistently, Rb2 downregulated the mRNA expression of human telomerase reverse transcriptase (hTERT) induced by miR-216a (Figure 5E).

The effect of Rb2 on macrophages polarization was assessed. miR-216a promoted M1 macrophages polarization characterized by the expression of inflammatory markers including monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor alpha (TNFα), while this effect was ameliorated by Rb2 treatment (Figure 5F). Furthermore, flow cytometry experiment showed that Rb2 treatment inhibited the miR-216a–mediated polarization of M1 macrophages characterized by the surface marker CD86 expression but had no effects on M2 polarization characterized by the surface marker CD206 expression (Figure 5G).

Finally, the flow cytometry assay showed that the percentages of dead cells in THP-1 PMA stimulation and miR-216a transfection were all less than 5% (Supplementary Figure S1), indicating a low toxic effect of PMA and lipofectamine reagents on THP-1 cells. Moreover, the cytotoxic effect of Rb2 on foam cells was assessed, and there was no significant impact on cell death in THP-1–derived foam cells (Supplementary Figure S2).

We tested the effect of Rb2 on lipid accumulation in foam cells derived from THP-1 cells and human PBMC. The Oil red O staining showed that the lipid uptake ability of THP-1–derived foam cells was significantly increased in the miR-216a overexpression group but was decreased by 34% when treated with Rb2 (Figures 6A,B). In addition, the elevated expression of CD36 and macrophage scavenger receptor 1 (SR-A1) induced by miR-216a were also reduced after Rb2 treatment (Figure 6C).

By contrast, the cholesterol efflux of macrophages was reduced by 13% in the miR-216a overexpression group but was increased when treated with Rb2 (Figure 6D). The expression of cholesterol efflux–related gene ABCG1 induced by miR-216a was reversed by 30% after Rb2 treatment (Figure 6E).

Given that primary monocytes or macrophages are an adequate model to study plaque macrophages, the human PBMCs were cultured and then transformed to foam cells by ox-LDL. The lipid accumulation in foam cells was increased by miR-216a, while this effect was suppressed by Rb2 treatment, indicating that Rb2 alleviated miR-216a–mediated lipid deposition in foam cells (Figures 6F,G).

The SA-β-gal staining showed that miR-216a promoted the macrophage senescence, and this effect was reduced by 75% after Rb2 treatment (Figures 7A,B). Consistently, the elevated expression of aging-related genes p21 and p16 induced by miR-216a was restored with Rb2 treatment (Figure 7C).

In addition, cell apoptosis and necrosis were examined. MiR-216a inhibited apoptosis in macrophages by 31%, while Rb2 had no effect on apoptosis (Supplementary Figure S3A). Cell necrosis of macrophages was not affected by miR-216a or Rb2 (Supplementary Figure S3B).