M1 macrophage-derived extracellular vesicle containing tsRNA-5006c promotes osteogenic differentiation of aortic valve interstitial cells through regulating mitophagy

Cell culture and transfection

Mouse mononuclear macrophage leukemia cell line RAW 264.7 was purchased from Procell (CL-0190). Dulbecco’s modified Eagle’s medium (10-013-CVRC, Corning, NY, USA) contained 10% fetal bovine serum and 1% Penicillin-Streptomycin Solution (E607011; Sangon, China) was used to culture RAW 264.7 cells in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. To obtain M1-polarized macrophages, RAW 264.7 cells were stimulated with 10 pg/mL lipopolysaccharide (LPS) and 20 ng/mL IFN-γ for 24 h.

Human AVICs were cultured in iCell Primary Mesenchymal Cell Culture System (PriMed-iCell-025; iCell Bioscience Inc., Shanghai, China) in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. For osteogenic differentiation induction, AVICs were cultured in an osteogenic differentiation medium (PH-B002; PH Biomedicine) for 7 days at 37 °C.

To silencing of tsRNA-5006c, the tsRNA-5006c inhibitor (Table S1) purchased from GenePharm (China) was transfected into M1-polarized macrophages using Lipofectamine™ 2000 (Invitrogen, USA). A nonsense sequence was used as the inhibitor negative control (NC). The day before transfection, M1-polarized macrophages were seeded in a six-well plate with 3 ×10⁵ cells/well. When the confluence reached 80%–90%, transfection was carried out. Taken 5 µL each of tsRNA-5006c inhibitor and NC and diluted in 45 µL OPTI-MEM (Corning), and then mixed with Lipofectamine™ 2000 regent for 20 min at 25 °C. After that, the mixture was added to cell samples for another 24 h culture.

Cell immunofluorescence (IF)

After the induction of M1 polarization in RAW 264.7 cells, the expression of M1 marker CD63 was detected by IF. Briefly, cells were fixed with 4% paraformaldehyde for 30 min at 25 °C and then permeabilized by 0.5% TritonX100 for 15 min at 25 °C. After washing three times with PBS, cells were incubated in a blocking solution of 3% BSA (diluted in PBS) for 30 min at 25 °C. Next, cells were incubated with pre-cooled anti-CD63 (1:200, ab239075; Abcam) at 4 °C overnight followed by incubation with goat anti-rat Alexa Flour 488 (1:300, ab150077; Abcam). Finally, cells were stained with DAPI (C1006; Beyotime, Shanghai, China) for 10 min in the dark condition and observed the fluorescence on a fluorescence microscope.

RNA isolation

Total RNA was isolated from M1-polarized macrophage cells and EVs using the TRIzol reagent (Life Technologies). The concentration and purity of total RNA were assessed by spectrophotometer. The integrity of total RNA was assessed by agarose gel electrophoresis. Qualified total RNA was stored at 80 °C and used for subsequently RT-qPCR analysis and small RNA sequencing.

RT-qPCR analysis

For mRNA expression, RNA was reverse-transcribed into cDNA by the RevertAid™ First Strand cDNA Synthesis Kit, with DNase I (K16225; Thermo Fisher, Waltham, MA, US), using random primers. For tsRNAs expression, RNA was pretreated with aRevertAid™ First Strand cDNA Synthesis Kit, with DNase I (K16225; Thermo Fisher, Waltham, MA, USA) using stem-loop reverse transcription primers. Next, the amplification reaction was carried out with cDNA as the template using the 2PCR Master Mix (Roche, Basel, Switzerland) on the ABI Q6 Flex Real-time PCR system (Applied Biosystems, Waltham, MA, USA). All primer’s sequences were synthesized by GenePharm (China) and shown in Table S1. The 2^−ΔΔCT method was used for normalizing gene expression relative to GAPDH or U6.

ELISA assays

The production of IL-6, TNF-α, IL-1β, IL-10, IL-12, and IL-13 were detected by using Mouse IL-6 ELISA Kit (ab222503; Abcam), Mouse TNF alpha ELISA Kit (ab208348, Abcam), Mouse IL-1 beta ELISA Kit (ab197742, Abcam), Mouse IL-10 ELISA Kit (ab255729, Abcam), Mouse IL-12(p70) ELISA Kit (EK0422; ScienCell™), Mouse IL-13 ELISA Kit (BMS6015, invitrogen), respectively, according to the manufacturer’s instruction.

Isolation of EVs

EVs were isolated from M1-polarized macrophages (M1-EVs) and PBS treated-RAW 264.7 cells (NC-EVs) using ultracentrifugation methods. Briefly, cells were cultured in a 10 cm dish with DMEM containing 10% FBS and 1% P/S and maintained in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C. The culture medium was replaced by a serum-free exosome-depleted medium (10 mL) when cell growth had reached the logarithmic phase (1 ×10⁷/dish). After culture for another 48 h, the medium was sequentially centrifuged at 500 g for 5 min, 2,000 g for 5 min, and 10,000 g for 30 min at 4 °C to remove the debris. Then, the supernatant was filtered through a 0.22-µm filter tube and centrifuged at 120,000 g for 2 h at 4 °C and the pellet was resuspended in PBS. Finally, crude EVs were centrifuged again at 120,000 g for 2 h at 4 °C to discard the contaminating protein to harvest purified EVs.

Identification of EVs

The size and morphology of EVs were determined by transmission electron microscopy (TEM) and the distribution and concentration of nanoparticles were accessed by nanoparticle tracking analysis (NTA; ZetaView), according to the manufacturer’s instruction. The presence of M1 marker of CD63 was detected by western blot.

Western blot

Total protein was extracted from EVs, AVICs, and arterial tissues using RIPA reagent, and the protein concentration was analyzed by a BCA kit. Then, approximately 20 µg protein sample was separated on 10% SDS-PAGE and transferred onto PVDF membrane. After that, membranes were blocked with 5% skimmed milk in TBST for 1 h at 25 °C followed by incubated with the primary antibodies at 4 °C overnight and incubated with secondary antibodies of Goat Anti-Mouse IgG H&L(HRP) (1:1000, ab205719; Abcam) or Goat Anti-Rabbit IgG H&L (HRP) (1:20000, ab6721; Abcam) for 1 h at 25 °C. Primary antibodies included CD63 (1:10000, ab216130; Abcam), BNIP3 (1:5000, ab109362; Abcam), PGC1 α (1:1000, ab106814; Abcam), LC3-II (1:1000, 3868Y; CST), BMP2 (1:1000, ab214821; Abcam), RUNX2 (1:200, sc-390715; Santa Cruz Biotechnology), osteopontin (1:1000, 25715-1-AP; Proteintech), GAPDH (1:2000, 60004-1-Lg; Proteintech). Finally, membranes were exposed to ECL and imaged by Chemiluminometer (Clinx Science Instruments, Shanghai, China).

EVs uptake experiment

The cell membrane red fluorescent probe DiI (C1036; Beyotime, Shanghai, China) was used to label EVs. Briefly, EVs were co-cultured with DiI dye solution for 10 min at 25 °C and then filtered away the unbound dye. The DiI-labeled EVs were co-cultured with AVICs in iCell medium for 24 h. To investigate the EVs internalization, AVICs underwent routine fixation, permeabilization, and DAPI nuclear staining as described in IF after co-culture. Finally, EVs internalization was visualized by a fluorescence microscope.

Alizarin red staining

The calcium deposit in AVICs was evaluated by Alizarin red staining. Briefly, AVICs were fixed in 4% paraformaldehyde for 15 min and then stained with Alizarin red solution for 30 min. After washing three times with PBS to remove the free dye, added deionized-water to protect cells from drying. Finally, AVICs were observed and imaged using a microscope.

Small RNA sequencing

RNA extracted from M1-EVs (n = 3) and NC-EVs (n = 3) were used for small RNA sequencing. The Multiplex Small RNA Library Prep Kit was used to prepare Small RNA libraries. Briefly, a 3′ adaptor was ligated to RNA and, then hybridization of reverse transcription primers followed by ligated with a 5′adaptor. Next, RNA was reverse transcribed into first-strand cDNA with bidirectional adaptor sequences as the template. After PCR amplification, size within 135-170 bp fragment was separated from 8% gel electrophoresis and purified. The PCR-amplified libraries were accessed by a Agilent 2100 BioAnalyzer. Finally, small RNA sequencing was carried out in a single-end 150 bp run using the HiSeq2500 platform (Illumina, USA). The statistical power of this experimental design, calculated in RNASeqPower, is 0.115.

Raw data were filtered through Fast-QC software to obtain clean data. Then, clean reads were mapped onto miRBase and piRNAcluster databases to exclude contamination and interferences from the miRNA and piRNA. The remaining clean reads were mapped onto tRFdb and tRFMINTbase database to obtain tsRNA, and the read counts of tsRNA were considered as expression level. The differentially expressed tsRNAs (DEtsRNAs) was identified under the condition of log2 fold change >1 or <−1, P value <0.05 using DESeq package. All the DEtsRNAs were subjected to GO and KEGG analysis.

Mitophagy observation

Lysosomes and mitochondria were labeled with the Lyso-Tracker Green (C1048; Beyotime, Shanghai, China) and Mito-Tracker Red (C1048; Beyotime, Shanghai, China) probes, respectively, and the co-localization of lysosomes and mitochondria was visualized to characterize mitophagy flux. Briefly, Lyso-Tracker Red solution was diluted into a final concentration of 50 nM using medium (1:13,333). Mito-Tracker Red solution was diluted into a final concentration of 20 nM using a medium (1:5000). Next, cells were incubated with Lyso-Tracker Red for 5 min at 37 °C followed by incubation with Mito-Tracker Red for 15 min at 37 °C. Finally, cells were observed with a fluorescence microscope.

TMRM staining analyzed by flow cytometry

Mitochondrial membrane potential (MMP) of AVICs was measured by flow cytometry using TMRM staining. AVICs were harvested and made into a single cell suspension of density 2∼5 ×10⁵ cells/mL. Added 50 nM of prepared TMRM Perchlorate staining solution to each tube of cell samples, mixed and incubated at 37 °C for 60 min in the dark. After centrifugation, the supernatant was removed and the cell pellet was washed with PBS. Finally, the AVICs were subjected to a flow cytometer to detect the fluorescence of TMRM.

Statistical analysis

One-way analysis of variance (ANOVA) with Tukey pos t-test was used to assess the difference between three groups, t-test was used to assess the difference between two groups. The significance threshold was P value less than 0.05. Data analysis was performed with GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA).

Identification of EVs derived from M1 macrophages

To investigate whether M1 macrophages can affect the phenotypic changes of AVICs, we first prepared M1-polarized macrophages. Results of IF assays showed that LPS/IFN-γ induction significantly provoked the expression of M1 polarization marker CD86 (Fig. 1A). The expression of TNF-α, IL-1β, and iNOS of M1 polarization markers were significantly increased after LPS/IFN-γ induction (Fig. 1B). Consistently, the concentration of pro-inflammatory cytokines of IL-6, TNF-α, and IL-1β in cell supernatant fluid were elevated following LPS/IFN-γ induction (Fig. 1C). On the contrary, the levels of M2-related anti-inflammatory cytokines of IL-10, IL-12, and IL-13 were significantly decreased after LPS/IFN-γ induction (Fig. 1D). Together, these results indicate that we successfully obtained M1-polarized macrophages. Next, EVs were isolated from M1-polarized macrophages (referred to as M1-EVs) or PBS treated-RAW 264.7 cells (referred to as NC-EVs) and characterized through TEM and western blot. These EVs were 50-200 nm and exhibited oval-shaped with a slight depression in the center (Fig. 1E) and readily expressed CD9 protein (Fig. 1F). Thus, M1-EVs and NC-EVs were harvested successfully.

M1-EVs promote osteogenic differentiation of AVICs

Additionally, to investigate the function of M1-EVs on the AVICs, we first examined whether M1-EVs can be taken up into AVICs. As shown in Fig. 2A, after incubation with DiI-labeled M1-EVs, bright red fluorescent DiI internalization was present in the AVICs, demonstrating that M1-EVs were readily internalized into AVICs. Next, to explore the function of M1-EVs on the osteogenic differentiation of AVICs, we assessed the formation of calcium nodules and expression of osteogenesis-related genes. Alizarin red staining results revealed that the number of mineralized nodules was significantly elevated after M1-EVs incubation compared with NC-EVs (Fig. 2B). Similarly, M1-EVs incubation led to a significant up-regulation in the expression of osteogenesis-related genes RUNX2, BMP2, and SPP1 in both mRNA and protein level (Figs. 2C and 2D). Moreover, we also detected the effect of M1-EVs on collagen-related proteins in AVICs. The results showed that compared with the NC-EVs group, the expression of α-SMA and Collagen I in AVICs in the M1-EVs incubation group were significantly increased, suggesting that M1-EVs promote the fibrotic process of AVICs (Fig. 2E). Collectively, these results suggested that M1-EVs promote osteogenic differentiation of AVICs.

tsRNA profile alters in M1-EVs

To determine whether EVs functional delivery by transporting the tsRNA, we subjected M1-EVs and NC-EVs to small RNA sequencing. RNA-seq revealed that 17 tsRNAs were significantly up-regulated such as tsRNA-5006c, while 28 tsRNAs were significantly down-regulated in M1-EVs compared with NC-EVs (Fig. 3A). These DEtsRNAs were separated into two categories and displayed in a heatmap (Fig. 3B). We predicted the target gene for DEtsRNAs and subjected it to the function and pathway enrichment analysis. Intriguingly, among the top 20 GO enrichment terms, seven terms related-transcriptional regulation were identified (Fig. 3C), suggesting that DEtsRNAs may mediate the transcriptional process of target genes. Target genes of DEtsRNAs were also enriched in cell differentiation of GO terms (Fig. 3C). Moreover, these DEtsRNAs were mainly involved in autophagy-related signaling pathways, such as MAPK, Ras, Wnt, mTOR, and the Hippo signaling pathway (Fig. 3D), suggesting that DEtsRNAs may change autophagy in recipient cells. Therefore, these M1-EVs DEtsRNAs may alter the phenotype of recipient cells through transcriptional regulation and autophagy-related pathways.

tsRNA-5006c may be responsible for the M1-EVs effects

To further verify the reliability of the sequencing results and screen out the key functional molecules in EVs, we selected five tsRNAs with high expression abundance and up-regulation in M1-EVs for RT-qPCR validation. As evident from Fig. 4A, compared with NC-EVs, only tsRNA-5006c expression was significantly up-regulated in M1-EVs, which was consistent with sequencing results. Accordingly, we focus on the tsRNA-5006c. According to MINTbase v2.0 (http://cm.jefferson.edu/MINTbase/), the tsRNA-5006c (5′-GCCCGGCTAGCTCAGTCGGTAGAGCATGGGAC-3′) also termed as tRF-32-PSQP4PW3FJIK1 and belonged to 5′-half tsRNA. The tsRNA-5006c is derived from the mature tRNA cleavage at the site of Lys-CTT. The regulatory network of tsRNA-5006c _target genes_pathway hinted at the underlying mechanisms of tsRNA-5006c function (Fig. 4B). For example, tsRNA-5006c could target Wnt4 and Btrc to involve in Hippo signaling pathway, target Ntrk2 and Mknk1 to involve in the MAPK signaling pathway, target Ppp2r5e and Camk2b to involve in the Wnt signaling pathway, and target Akt3 and Mapk9 to involve in Ras signaling pathway (Fig. 4B). Intriguingly, these four signaling pathways were associated with autophagy (Lorzadeh et al., 2021; Schmukler, Kloog & Pinkas-Kramarski, 2014; Wang et al., 2020; Zhou et al., 2015), which plays a crucial role in osteogenic differentiation (Zhou et al., 2021). Together, tsRNA-5006c may be responsible for the M1-EVs enhanced osteogenic differentiation of AVICs, through autophagy-related pathway.

M1-EVs tsRNA-5006c inhibitor suppress osteogenic differentiation of AVICs

To interrogate the role of M1-EVs tsRNA-5006c in osteogenic differentiation of AVICs, we first treated M1 macrophages with tsRNA-5006c inhibitor and then harvested EVs. After tsRNA-5006c inhibitor administration, tsRNA-5006c expression was significantly reduced in both M1 macrophages and M1-EVs, compared to the tsRNA-5006c inhibitor NC treatment (Figs. 4C and 4D), suggesting that the interference efficiency of tsRNA-5006c was good. Then, tsRNA-5006c-deleted M1-EVs were co-cultured with AVICs. As the consequence of tsRNA-5006c interference, calcium nodule formation of AVICs was significantly diminished by Alizarin Red staining (Fig. 5A). The expression of osteogenesis-related genes RUNX2, BMP2, and SPP1 in recipient AVICs were significantly decreased in both mRNA and protein level upon tsRNA-5006c silencing (Figs. 5B and 5C). Also, the protein expression of fibrotic makers Collagen I and α-SMA were significantly decreased after tsRNA-5006c silencing (Fig. 5D). Therefore, our data suggest that M1-EVs promote osteogenic differentiation of AVICs by delivering tsRNA-5006c.

M1-EVs tsRNA-5006c enhance osteogenic differentiation of AVICs through mitophagy

Recent studies have revealed that mitochondria are abundant in the heart and mitochondrial dysfunction is associated with various cardiac diseases, including cardiomyopathy (Wang et al., 2022c). Combined with the aforementioned “tsRNA-5006c—target gene—autophagy-related pathways” network results, we speculated that an enhanced osteogenic differentiation capacity by M1-EVs tsRNA-5006c may be linked to autophagy/mitophagy. To validate this conjecture, we stained lysosomes and mitochondria of AVICs after incubation M1-EVs with tsRNA-5006c-deleted (M1-EVs tsRNA-5006c inhibitor group) or not (M1-EVs tsRNA-5006c NC group) by using Lyso-Tracker (Red) and Mito-Tracker (Green), respectively. Colocation of green and red dyes allows us to readily monitor mitophagy flux in cells. As expected, mitophagosome formation of AVICs was significantly increased in the M1-EVs group compared with the NC-EVs group, whereas tsRNA-5006c interference in M1-EVs led to a significant reduce in the mitophagosome formation of AVICs (Fig. 6A). We also measured MMP of AVICs by flow cytometry using TMRM staining. As shown in Fig. 6B, M1-EVs incubation caused a significant increase of TMRM fluorescence in AVICs relative to NC-EVs, while tsRNA-5006c-deleted M1-EVs have significantly lost this ability, suggesting that M1-EVs tsRNA-5006c can enhance MMP. Besides, the expression of LC3-II, BINP3, and PGC1 α, hallmarks of mitophagy activation, were significantly elevated in the M1-EVs group compared with the NC-EVs group, but tsRNA-5006c inhibitor significantly blocked this elevation (Fig. 6C). Thus, we surmise that M1-EVs tsRNA-5006c enhance osteogenic differentiation of AVICs through mitophagy.