Small extracellular vesicles with LncRNA H19 "overload": YAP Regulation as a Tendon Repair Therapeutic Tactic.

Functional healing of tendon injuries remains a great challenge. Small extracellular vesicles (sEVs) have received attention as pro-regenerative agents. H19 overexpression could bring tendon regenerative ability, but the mechanism is still not fully elucidated, and reliable method for delivery of long non-coding RNAs (LncRNAs) was demanded. We identified the downstream mechanism of H19, the activation of yes-associated protein (YAP) via the H19-PP1-YAP axis. We established tendon stem/progenitor cells (TSPCs) stably overexpressing H19 with CRISPR-dCas9-based hnRNP A2/B1 activation (H19-CP-TSPCs). H19-OL-sEVs (H19 "overloading" sEVs) could be produced effectively from H19-CP-TSPCs. Only H19-OL-sEVs were able to significantly load large amounts of H19 rather than other competitors, and the potential of H19-OL-sEVs to promote tendon healing was far better than that of other competitors. Our study established a relatively reliable method for enrichment of LncRNAs into sEVs, providing new hints for modularized sEV-based therapies, and modularized sEVs represented a potential strategy for tendon regeneration.

Introduction

Tendons are specialized tissues with the primary function of transferring mechanical forces generated by musculoskeletal tissues from muscle to bone. Tendon injury is a common clinical disease that frequently occurs during sports and other rigorous activities, and patients often suffer from long-term pain and even disability (Sharma and Maffulli, 2006). Furthermore, the structural integrity and mechanical strength of damaged tendons rarely attain full recovery or regain native tendon functions after healing because tendons have the characteristics of low oxygen consumption, low metabolism, hypocellularity, and hypovascularity (Sharma and Maffulli, 2006). To date, functional healing of tendon injuries has been a great challenge, and novel therapeutic approaches for tendon regeneration are needed.

Small extracellular vesicles (sEVs), derived from mesenchymal stem cells (MSCs), have become one of the most promising potential therapeutic strategies in tendon regeneration (Zhu et al., 2020). Ongoing research shows that MSC-derived sEVs have many biological functions similar to their parent cells, and they might be the true effectors that play key roles in MSC-based tissue regeneration (Phinney and Pittenger, 2017; Tao et al., 2017b) as well as hold great promise as emerging therapeutic carriers, given their role in intercellular communication (Pi et al., 2018). Our previous study indicated that sEVs derived from native-source cells have various shortcomings (Tao et al., 2017c) and modularized sEVs hold significant promise for targeted and personalized drug delivery (Tao et al., 2018a).

Tendon stem/progenitor cells (TSPCs) were first identified by Bi et al. (2007), who found that they exhibited various common properties of stem cells. TSPCs were shown to display clonogenicity, multilineage differentiation potential, and self-renewal ability (Lee et al., 2015). Therefore, we hypothesized that modularized sEVs, derived from TSPCs, may be desirable promoters of tendon regeneration because of their advantages in accessibility and volume, similar features to tendinocytes, and phenotypic stability.

Recent studies indicated that long non-coding RNA (LncRNA) H19, which was first discovered via genetic screening in 1984 (Pachnis et al., 1984), stimulates tendon regeneration/formation in TSPCs, and that stable overexpression of LncRNA H19 significantly enhances tendon healing (Lu et al., 2017). Compared with MSC-based therapy, sEV-based therapy is an up-and-coming candidate with lower tumorigenicity and lower immunogenicity (Armstrong et al., 2017; Tao et al., 2018a). TSPCs have been chosen to produce H19-carrying sEVs; however, the efficiency of the conventional approach to load H19 into sEVs is low. In our previous study (Tao et al., 2018b), H19 “loading” was performed through a physical method, like squeezing and pressing, such as using extracellular vesicle-mimetic nanovesicles (EMNVs), which is not a native way to secrete sEVs. A method of improving the efficiency of molecular “loading” through naturally existing mechanisms during EV biogenesis is needed.

RNA content in sEVs is highly selective (Pigati et al., 2010). Human heterogeneous nuclear ribonucleoproteins (hnRNP) A2/B1 are RNA-binding proteins (RBPs) that are known to transport RNA to sEVs (Alarcon et al., 2015) and control RNA loading into sEVs by binding to a motif (Villarroya-Beltri et al., 2013b). H19 is secreted by packaging into sEVs, and this packaging process is reported to be mediated by hnRNP A2/B1 (Lei et al., 2018; Villarroya-Beltri et al., 2013b).

Yes-associated protein (YAP) is involved in the regulation of cell proliferation (Panciera et al., 2017). It is reported that YAP may act as a “switch” between the pro-survival and pro-apoptotic responses (Wu et al., 2015). However, this “switch” remains to be fully characterized. Some studies indicate that the inhibition of YAP (by phosphorylation) down-regulates the expression of tendon-related genes (Chu et al., 2019), whereas activation of YAP (by dephosphorylation) enhances tendon healing (Huang et al., 2020). Therefore, we hypothesized that YAP dephosphorylation might play a crucial role in tendon regeneration.

Proteins can be phosphorylated at serine, threonine, or tyrosine residues, but over 99% of phosphorylation events in mammalian cells involve serine and threonine residues (Mermoud et al., 1992). Protein phosphatase 1 (PP1) is a member of the Ser/Thr-specific protein phosphatase (PP) superfamily. PP1 removes phosphate groups from serine or threonine residues and regulates various cellular processes through the dephosphorylation of dozens of substrates (Grallert et al., 2015). PP1 plays a crucial role in YAP dephosphorylation and regulation (Haemmerle et al., 2017; Lv et al., 2015; Wang et al., 2011).

Many studies have indicated that H19 mediates the interaction between RNA and proteins (Chan et al., 2014; El Hajj et al., 2018). YAP-binding LncRNA screening by RNA immunoprecipitation (RIP) sequencing showed that H19 is one of the top eight candidates (Ni et al., 2019).

In this study, we investigated whether sEVs can be derived from TSPCs by utilizing the co-overexpression of H19 and hnRNP A2/B1, called H19 “overload” sEVs (H19-OL-sEVs), and whether they can promote proliferation, tendon differentiation, migration, collagen deposition, and YAP localization. Our findings will provide a therapeutic exploration of the role of H19-OL-sEVs in tendon regeneration and a better understanding of the regulatory role of H19-PP1-YAP in this process.

Results

Identification of TSPCs and sEVs

TSPC colonies appeared between days 7 and 21 of culture. As observed under the microscope, TSPCs exhibited typical pebble-shaped morphology (Figure 1A). The potential for adipogenic differentiation was studied by measuring the formation of small cytoplasmic lipid granules using oil red O staining after 2 weeks of induction (Figure 1B). Osteogenic differentiation potential was studied by measuring the formation of calcium mineral deposits identified by alizarin red staining after 3 weeks of induction (Figure 1C). The potential for chondrogenic differentiation was analyzed using Alcian blue staining after 4 weeks of induction in alginate beads (Figure 1D). Flow cytometry (FCM) analyses showed that these cells were positive for CD44, CD73, CD90, and CD105 but were negative for CD34 and CD45 (Figure 1E). All these data unequivocally confirmed that TSPCs were successfully isolated from the human tendon.

Figure 1

Identification of TSPCs and sEVs

(A) TSPCs exhibited a typical pebble-shaped morphology (scale bar, 500 μm).

(B–D) (B) Adipogenic, (C) osteogenic, and (D) chondrogenic differentiation of TSPCs under induction conditions (scale bar, 50 μm).

(E) Flow cytometry analysis of TSPC cell surface markers. The blue curves represent isotype controls, and the red curves represent measured surface markers (CD44, CD73, CD90, CD105, CD34, and CD45).

(F) Morphology of sEVs under TEM. Scale bar, 100 nm.

(G) The particle size distribution of sEVs measured by DLS. All experiments were repeated independently three times, and representative results are shown. TSPCs, tendon stem/progenitor cells; sEVs, small extracellular vesicles; TEM, transmission electron microscopy.

Morphology of sEVs was examined under a transmission electron microscope (TEM), and particle size distribution was measured using dynamic light scattering (DLS) to identify the sEVs, as shown in Figures 1F and 1G.

Identification of the interaction between H19 and YAP

To further understand the potential biological functions of H19, we investigated H19 expression and found that it was higher in musculoskeletal tissue than in other tissues when analyzed by the data visualization tool of the GTEx database, as shown in Figure 2A.

Figure 2

H19 and YAP in tendon regeneration

(A) Expression of H19 in 54 different tissues from GTEx (v8) datasets, showing that H19 is highly expressed in musculoskeletal tissue, whereas it is expressed at lower levels in other tissues.

(B) The correlation between H19 and YAP downstream genes (CTGF, Cyr61, and ANKRD1), analyzed via the ENCORI database (starBase v3.0 project).

(C and D) (C) PAGE and (D) western blot analyses were used to measure changes in gene expression during tenogenic differentiation.

(E) YAP cytoplasm-nucleus distribution measured by western blot analysis after nucleus-cytoplasm separate extraction.

(F) Gene expression changes after empty vector, H19-KD, H19-OE, S127A, YAP-KO, and verteporfin treatment.

(G) The phosphorylation level of YAP and expression levels of related proteins were measured by western blot analysis. All experiments were repeated independently three times, and representative results are shown. PAGE, polyacrylamide gel electrophoresis; KD, knockdown; KO, knockout; OE, overexpression; TSPCs, tendon stem/progenitor cells.

Moreover, the interaction between H19 and YAP was investigated using the RNA-RNA CoExpression tool of the ENCORI database (starBase v3.0 project). The results showed that YAP downstream genes, such as connective tissue growth factor (CTGF), cysteine-rich angiogenic inducer 61 (CYR61), and ankyrin repeat domain 1 (ANKRD1), were co-expressed (positive correlation) with H19 (Figure 2B).

TSPCs were incubated in the presence of tenogenic induction medium for different periods (t = 0, 3, 5, or 7 days). The expression of H19, YAP downstream genes (CYR61, CTGF, and ANKRD1), and tenogenic markers (SCX and TNMD) were assessed via polyacrylamide gel electrophoresis (PAGE) analysis immediately after reverse-transcriptase polymerase chain reaction (RT-PCR) (Figure 2C). The expression level of tenogenic markers were assessed via western blot (WB) analysis (Figure 2D).

Interestingly, there was a positive correlation among H19, YAP downstream genes, and tenogenic marker genes, measured using PAGE assay (Figure 2C). The endogenous YAP localization is associated with the transcriptional activation of target genes, such as CTGF and CYR61 (Zhao et al., 2010). WB analysis, using cytoplasmic and nuclear protein fractions isolated from TSPCs, further showed that YAP translocation from the cytoplasm to the nucleus followed tenogenic induction/differentiation (Figure 2E).

After treatment with LncRNA-H19 Smart Silencer (H19-KD), pcDNa3.1(+)_A009-H19 (H19-OE), S127A (permanently activated YAP), YAP-KO, or verteporfin (to block the YAP-TEAD interaction), the expression of H19, tenogenic marker genes, and YAP downstream genes was measured via PAGE analysis (Figure 2F), and the phosphorylation levels of YAP and the expression levels of tenogenic marker genes were detected via WB analysis (Figure 2G). Empty vector was used as a control.

The results showed that H19-KD inhibited YAP activation (by promoting YAP phosphorylation) and YAP downstream processes, as well as the expression of tendon marker genes. In contrast, H19-OE promoted YAP activation (by suppressing YAP phosphorylation) and YAP downstream processes, as well as tendon marker gene expression. S127A rescued cells from the effect of H19-KD, whereas YAP-KO reversed the effect of H19-OE. Verteporfin reversed the H19-OE-induced upregulation of YAP downstream and tendon marker genes, without interfering with YAP phosphorylation levels, which implied that H19-induced YAP activation affected tendon marker genes via YAP–TEAD interaction.

Identification of the binding region between H19 and YAP

The results showed that H19 overexpression (H19-OE) or knockdown (H19-KD) did not cause a significant change in the expression and phosphorylation of YAP upstream proteins (MST1/2 and LAST1) (Figure 3A), and this result implied that H19 might have a direct effect on YAP, instead of its upstream regulators. RIP assay was performed to confirm the interaction between H19 and YAP. The results of PAGE showed that H19 was enriched with the YAP antibody compared with the IgG control (Figure 3B). Reciprocally, RNA pull-down and subsequent WB analysis showed that YAP could bind to H19 (Figure 3C). These results suggested that H19 was physically associated with YAP.

Figure 3

Identification of YAP-H19 interaction

(A) The expression and phosphorylation level of upstream signal molecules of YAP were measured using western blot analysis, after empty vector, H19-KD, and H19-OE treatment.

(B) RNA immunoprecipitation assays for YAP were performed, and H19 was found to be co-precipitated by PAGE analysis.

(C) Western blot analysis after RNA pull-down assay showed the interaction between YAP and H19.

(D) Western blot detection of co-precipitated YAP, which was pulled down by in vitro-transcribed biotinylated RNAs corresponding to different fragments of H19 in TSPCs.

(E) Western blot detection of HA-tagged YAP or mutated YAP (wild-type versus domain truncation mutants) co-precipitated with in vitro-transcribed biotinylated-H19.

(F) Schematic diagram of the interaction between H19 and YAP. All experiments were repeated independently three times, and representative results are shown. PAGE, polyacrylamide gel electrophoresis; KD, knockdown; KO, knockout; OE, overexpression; TSPCs, tendon stem/progenitor cells.

To identify the unique binding sites, we took advantage of a series of deletion mutants of H19 to map the YAP-binding region (Figure 3D). Results showed that the H19 mutant Δ3 bound to YAP as efficiently as the full-length H19, whereas other mutants completely lost their binding ability, indicating that nucleotides 780–1210 of H19 are required for its association with YAP.

To identify the YAP regions that are responsible for binding to H19, we constructed four YAP domain-deletion mutants with a hemagglutinin (HA)-tag, and an RNA pull-down assay was performed. Results showed that deletion of the WW domain blocked the interaction, which implied that the WW domain played a critical role in the interaction between YAP and H19 (Figure 3E).

Next, we further used the SWISS-MODEL and RNAstructure software to predict and analyze the 3D structure of the YAP-WW domain and H19 RNA secondary structure, respectively. Figure 3F is a schematic diagram of the YAP–H19 interaction.

H19 regulates PP1-mediated activation of YAP

There are virtually no reports about the direct phosphorylation of LncRNA. Most of the literature suggests that LncRNA can act as a “bridge” between proteins. Hence, we speculated that H19 might form a bridge between YAP and phosphorylated regulatory proteins.

The results of co-immunoprecipitation showed that the binding between PP1 and YAP was significantly decreased when H19 was knocked down and significantly increased when H19 was overexpressed, as shown in Figure S1. However, the binding between PP2 and YAP was changed only a little, independently of the up- or downregulation of H19, and in addition, there were no significant changes in the binding between YAP and MST1/MST2/LATS1 (Figure S1). All these results indicate that H19 acts as a bridge between YAP and PP1.

The phosphorylation level of YAP and the expression level of tenogenic markers were examined via WB analysis upon overexpression or knockdown of H19, as well as PP1α activation (PP1α-ACT) or knockout (PP1α-KO) (Figure 4A). The expression level of H19 and tenogenic markers was examined via PAGE analysis (Figure 4B). The nucleo-cytoplasmic translocation of YAP was examined via WB analysis (Figure 4C). The results supported our speculation that H19 regulated the phosphorylation level of YAP by acting as a “bridge” between YAP and PP1.

Figure 4

H19 regulates YAP activation mediated by PP1

(A) Western blot analysis showed the levels of total and phosphorylated YAP and tenogenic markers after overexpression or knockdown of H19 with PP1α activation or knockout.

(B) PAGE analysis showed the levels of H19 and tenogenic genes, after overexpression or knockdown of H19 with PP1α activation or knockout.

(C) The nucleo-cytoplasmic translocation of YAP after overexpression or knockdown of H19 with PP1α activation or knockout.

(D) Gene interaction network from GeneMANIA (http://genemania.org/).

(E) Sub-network (physical interactions) from GeneMANIA database (http://genemania.org/). All experiments were repeated independently three times, and representative results are shown. PP1, protein phosphatase 1; PAGE, polyacrylamide gel electrophoresis.

To further visualize the relationship between YAP and PP1, we observed the correlation between YAP and PP1 based on the evidence and data recorded on the GeneMANIA database. Several connections between YAP and PP1 were mapped (Figure 4D). These could be divided into five major sub-clusters based on their biological functions: physical interactions, co-expression, predicted, co-localization, pathway, genetic interactions and shared protein domains. Physical interactions suggested that there were physical interactions between YAP and PP1 (Figure 4E). Thus, interactions between YAP and PP1 are reliable, and it is meaningful that H19 might be an important regulator of the affinity between YAP and PP1.

H19 regulates the biological functions of TSPCs through PP1-mediated dephosphorylation and translocation of YAP

After treatment with empty vector, H19-KD, H19-OE, H19-KD + S127A, H19-OE + YAP-KO, H19-OE + verteporfin, H19-KD + PP1α-ACT, or H19-OE + PP1α-KO, YAP localization was assessed via immunofluorescence (IF) assay (Figure 5A), proliferation was assessed using the EdU kit and FCM (Figure 5B), and migration was assessed via Transwell assay (Figure 5C).

Figure 5

H19 regulates the biological function of TSPCs through PP1-mediated phosphorylation and degradation of YAP

(A–C) (A) YAP localization was assessed by immunofluorescence assay (scale bar, 50 μm); (B) proliferation was assessed using an EdU kit and flow cytometry, and (C) migration was assessed by Transwell assay (scale bar, 50 μm) after H19-KD, H19-OE, H19-KD + S127A, H19-OE + YAP-KO, H19-OE + verteporfin, H19-KD + PP1α-ACT, or H19-OE + PP1α-KO treatment. All experiments were repeated independently three times, and representative results are shown. KD, knockdown; KO, knockout; OE, overexpression; ACT, activated; TSPCs, tendon stem/progenitor cells; PP1, protein phosphatase 1.

Collagen deposition was assessed using Sirius red staining after H19-KD, H19-OE, H19-KD + S127A, H19-OE + YAP-KO, H19-OE + verteporfin, H19-KD + PP1α-ACT, or H19-OE + PP1α-KO treatment (Figure 6). The results support our hypothesis that H19 promotes the proliferation, migration, and tendon-related gene expression by regulating YAP phosphorylation and translocation via H19-PP1-YAP interaction.

Figure 6

Collagen deposition

Collagen deposition (scale bar, 5 mm) was assessed by Sirius red staining after H19-KD, H19-OE, H19-KD + S127A, H19-OE + YAP-KO, H19-OE + verteporfin, H19-KD + PP1α-ACT, or H19-OE + PP1α-KO treatment. All experiments were repeated independently three times, and representative results are shown. KD, knockdown; KO, knockout; OE, overexpression; ACT, activated.

H19-OL-sEVs regulate the biological function of TSPCs

Expressions of surface markers, including CD9, CD63, and CD81, in cells carrying Con-sEVs (unmodified sEVs), H19-sOE-sEVs (sEVs derived from TSPCs stably over-expressing H19), hnRNP A2/B1-ACT-sEVs (sEVs derived from TSPCs with hnRNP A2/B1 activation), or H19-OL-sEVs (sEVs derived from TSPCs stably overexpressing H19 with hnRNP A2/B1 activation) were measured using WB and PAGE. The liquid obtained after the same isolation steps from medium in blank wells (without cells) was used as a negative control. This experiment was repeated three times independently, and representative results are shown in Figure 7A. The results support our view that only the overexpression of both H19 and hnRNP A2/B1 at the same time could increase the H19 content in sEVs (we call this phenomenon/technology “overload”). The sEV release curve was shown in Figure S2.

Figure 7

H19-OL-sEVs regulate YAP nuclear localization, proliferation, migration, and collagen deposition in TSPCs

(A) Expressions of surface markers, including CD9, CD63, CD81, in cells carrying Con-sEVs, H19-sOE-sEVs, hnRNP A2/B1-ACT-sEVs, and H19-OL-sEVs were measured using western blotting and PAGE.

(B–F) (B) PAGE assay of H19 expression in TSPCs treated with different groups of sEVs; (C) YAP localization was assessed by immunofluorescence assay (scale bar, 50 μm); (D) proliferation was assessed using an EdU kit and flow cytometry; (E) migration was assessed by Transwell assay (scale bar: 50 μm); (F) collagen deposition was assessed by Sirius red staining (scale bar, 5 mm) after treatment with control, Con-sEVs, H19-sOE-sEVs, hnRNP A1/B2-ACT-sEVs, or H19-OL-sEVs. sEVs, small extracellular vesicles; Con-sEVs, unmodified sEVs; H19-OL-sEVs, H19 “overloading” sEVs; H19-sOE-sEVs, H19-overexpressing sEVs; hnRNP A1/B2-ACT-sEVs, hnRNP A2/B1-activated sEVs; TSPCs, tendon stem/progenitor cells; PAGE, polyacrylamide gel electrophoresis.

The expression of H19 in TSPCs treated with different groups of sEVs was examined via PAGE assays (Figure 7B). The results further supported the hypothesis that only sEVs with H19 “overload” could significantly upregulate the expression of H19 in receptive cells (TSPCs).

After treatment with Con-sEVs, H19-sOE-sEVs, hnRNP A1/B2-ACT-sEVs, or H19-OL-sEVs, YAP localization was assessed via IF assay (Figure 7C), proliferation was assessed using an EdU kit and FCM (Figure 7D), migration was assessed via Transwell assay (Figure 7E), and tenogenic differentiation and collagen deposition (Figure 7F) were assessed via Sirius red staining. Con-sEVs had little effect on proliferation or migration. H19-sOE-sEVs and hnRNP A1/B2-ACT-sEVs exhibited better biological effects than Con-sEVs. However, H19-OL-sEVs (sEVs with H19 “overload”) had a more prominent and significant effect on promoting YAP activation, proliferation, migration, and tenogenic differentiation of TSPCs. Thus, H19-OL-sEVs hold the most promise as a therapeutic agent.

Histological and mechanical properties

In vivo, the effects of H19-OL-sEVs on tendon repair were examined using a rat tendon defect model. During the first 2 weeks post operation, all the skin incision wounds healed with no sign of infections, swelling, or suppuration. Over the 4 weeks post operation, no apparent difference was noticed in any of the animal groups.

After 4 weeks, H&E, Masson's trichrome and Safranin O & Fast green staining indicated that the H19-OL-sEVs group had more matrix and collagen formation in the wound region when compared with the other groups (Figure 8A). All experimental groups had higher cellularity compared with the control group at week 4. Tendon healing occurred extrinsically by the invasion of cells from the surrounding sheath and synovium (Gelberman et al., 1984).

Figure 8

H19-OL-sEVs promotes tendon repair in vivo

(A) Histological examination of the effect of different sEV groups on patellar tendon repair detected by H&E (scale bar, 200 μm), Masson's trichrome (scale bar: 200 μm), Safranin O & fast green (scale bar, 200 μm), and Sirius red staining (polarized image) (scale bar, 100 μm).

(B) Statistical charts show the ultimate stress and the Young's modulus in the different groups at week 4 after repair (data are presented as mean ± standard deviation). sEVs, small extracellular vesicles. Significant differences (one-way ANOVA test): ns, no significant difference (p > 0.05); ∗∗p < 0.01; ∗∗∗∗p < 0.0001.

Collagen birefringence was higher in the H19-OL-sEVs group compared with that in other groups at week 4, indicating better collagen fiber alignment (Figure 8A). The collagen birefringence was low in the control group and increased in all other groups. In the H19-OL-sEVs group, collagen fibers with a typical tendon structure were observed. No fibrocartilage or ectopic bone was observed in any of the groups.

At week 4, ultimate stress in the Con-sEVs group was not significantly different compared with the Control group. The ultimate stress in the H19-sOE-sEVs group, hnRNP A2/B1-ACT-sEVs group, and H19-OL-sEVs group was observed to be significantly higher than in the control group, but the ultimate stress in the H19-sOE-sEVs group was not significantly different compared with the Con-sEVs group. The H19-OL-sEVs group showed the most significant recovery of ultimate stress. Young's modulus in the Con-sEVs group was not significantly different compared with the Control group. Meanwhile Young's modulus in the H19-sOE-sEVs, hnRNP A2/B1-ACT-sEVs, and H19-OL-sEVs groups was observed to be significantly higher than in the Control group, but Young's modulus in the H19-sOE-sEVs and hnRNP A2/B1-ACT-sEVs groups was not significantly different compared with Con-sEVs. The H19-OL-sEVs group showed the most significant recovery of Young's modulus. However, there were still significant differences between the normal group and other groups. The statistical results are shown in Figure 8B.

In addition, we also analyzed dephosphorylation and accumulation of YAP in vivo (Figure S3). Con-sEVs group or H19-sOE-sEVs group was not significantly different compared with Control, hnRNP A2/B1-ACT-sEVs group was observed with significant dephosphorylation of YAP (but accumulation of YAP has no statistical difference) compared with the Control, and H19-OL-sEVs group showed the most significant difference compared with Control (both dephosphorylation and accumulation of YAP). The data confirmed that H19-OL-sEVs promoted tendon repair in vivo, consistent with the in vitro results. Thus H19-OL-sEVs could be a new technique/approach with great potential for tendon regeneration. A diagram showing the proposed mechanism is presented in Figure 9.

Figure 9

Diagram of the proposed molecular mechanism

(A) Schematic diagram of LncRNA H19 “overload” technology.

(B) Schematic diagram of H19/PP1/YAP interaction, and the mechanisms that enable biological functions.

Discussion

Tendon may require more than 1 year to recover from injury and may never completely heal (Sharma and Maffulli, 2005), as tendon tissue is not spontaneously repaired. It ultimately forms a mechanically inferior scar-like tissue, and frequently fails to regain the structural integrity, mechanical properties, or functionality of native tendon (Nourissat et al., 2015; Thomopoulos et al., 2003; Voleti et al., 2012). Increased synthesis of collagen is the primary requirement for tendon regeneration. Moreover, cell proliferation, migration, and differentiation at the tendon injury site are the prerequisites for tendon repair (Nourissat et al., 2015). Thus, it is important to find new drugs/molecules, more effective therapeutic targets, and more efficient drug delivery approaches for tendon repair.

Emerging evidence strongly demonstrates that LncRNAs may play important regulatory roles in cell differentiation and tissue regeneration (Loewer et al., 2010). LncRNAs exert their regulatory functions through specific interactions with proteins, including epigenetic modifiers, transcriptional factors/co-activators, and RNP complexes (Xing et al., 2014). LncRNA H19 is one of the most well-known imprinted genes, located on human chromosome 11. It is transcribed only from the maternally inherited allele (Keniry et al., 2012). Under normal physiological conditions, H19 is abundantly expressed in embryonic tissues of endodermal and mesodermal origin and is downregulated after birth in all tissues except skeletal muscle (Lustig et al., 1994), implying its regulatory role in determining the musculoskeletal cell fate.

Recent findings have shown that H19 is an active modulator of musculoskeletal development, promoting osteoblast differentiation of MSCs (Huang et al., 2015), mediating myoblast differentiation and skeletal muscle regeneration (Dey et al., 2014). As the tendon is a crucial component of the musculoskeletal system, it was not surprising that H19 overexpression was found to accelerate tenogenic differentiation in vitro and promote tendon repair through cell-based therapy in vivo (Lu et al., 2017). Results of the present study strongly suggest that H19 overexpression is an adaptive mechanism in response to a tendon injury, taking into consideration the vital role that H19 plays in cell proliferation.

Compared with cell-based therapy, sEV-based (or cell-free) therapy is an up-and-coming candidate with lower tumorigenicity and lower immunogenicity (Armstrong et al., 2017; Tao et al., 2018a). In addition, because the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators, and other bioactive molecules carried in sEVs, these structures can be isolated and used instead of cells for a novel therapeutic approach known as “stem cell-based cell-free therapy” (Bacakova et al., 2018). Future directions of research include methods to optimize the therapeutic potential of these stem cells and non-cellular alternatives using EVs.

EVs are small membranous vesicles originating from most, if not all, cells and tissues. Exosomes (or sEVs) are released by various cells (Xia et al., 2019). These membrane-limited vesicles carry multiple cargos, including DNAs, coding or non-coding RNAs, lipids, and proteins, which can be secreted from their parental cells and functionally recruited by recipient cells (Théry et al., 2009), where they can regulate or serve as templates for protein production (Montecalvo et al., 2012). sEVs carry different cargos according to the cell type and probably the physiological state. The transferred molecules are capable of eliciting changes in the function and gene expression of the recipient cell, independent of the recipient cell's location (Lai et al., 2015).

Recently, sEVs, sub-micron vectors used in intercellular communication, have been demonstrated to have exceptional potential as packaging tools for the therapeutic delivery of genetic material and drugs (Armstrong and Stevens, 2018). TSPCs are sEV-releasing cells (Wang et al., 2019), selected as the production source of sEVs because they are biologically closer to the repaired tissues, more widely available, and abundant with a broader amplification capacity, less terminal differentiation, and spontaneous tenogenic differentiation potential.

The discovery that sEVs are one of the key secretory products of MSCs, mediating cell-to-cell communication to enhance wound healing, and that cell-derived EV signaling organelles mediate the paracrine effects of stem cells, suggests that cell-free strategies could supplant cell-based therapy (Barile et al., 2017). Because of the relative probability of neoplastic transformation and abnormal differentiation in cell-based therapy, sEVs derived from native-source cells will play a beneficial role in the process of tendon injury and repair (Chamberlain et al., 2019; Shi et al., 2019; Wang et al., 2020). The sEVs derived from tendon stem cells can balance the synthesis and degradation of the tendon extracellular matrix, thus promoting tendon healing (Wang et al., 2019). However, as research progressed, it was realized that sEVs derived from native-source cells may have various shortcomings and show limited pharmaceutical acceptability, which can be corrected via modification and optimization (Tao et al., 2018a). As expected, we demonstrated that H19 participated in tendon regeneration through incorporation into sEVs, using an “overloading” approach.

Emerging evidence indicates that exploitation of EV-based cell-free therapeutics is a promising approach (Das et al., 2019). Much of the recent interest in sEVs was triggered by the discovery of the function of sEVs in transport of secreted extracellular RNAs (exRNAs), and these exRNAs remain biologically active and functional after they enter the recipient cells (Valadi et al., 2007). We then determined whether the extracellular expression of H19 mediated tenogenic differentiation of TSPCs. Our current study further showed that H19-OL-sEV, a modularized sEV, could promote tendon regeneration when applied as a cell-free therapy instead of a cell-based one. Treatment with H19-OL-sEVs promoted tenogenic differentiation and tendon regeneration, indicating that H19 packaging into sEVs promoted tendon regeneration.

However, the molecular mechanisms underlying specific loading of RNAs into sEVs remain unclear. The discovery of sEVs, as natural delivery tools of functional nucleic acids and proteins, has generated great interest in the drug delivery field, making it possible to harness these vesicles for therapeutic delivery of microRNAs (miRNAs), small interfering RNAs, mRNAs, LncRNAs, peptides, and synthetic drugs (Barile and Vassalli, 2017). Among these functional small RNAs and proteins, miRNAs can be secreted from the parent cells into sEVs through the overexpression of target miRNA to increase their levels (Tao et al., 2017a, 2017c). Overexpression of an miRNA leads to its enrichment in sEVs, and the mechanism that enables the over-representation of short sequence motifs in miRNAs that are commonly enriched in sEVs has been identified (Villarroya-Beltri et al., 2013a).

To solve the problem that larger molecules (including LncRNAs) are difficult to transport through sEVs in large quantities, many scientists hope to develop EMNVs from cells as a superior alternative to natural EVs (Jang et al., 2013). This new form of EV-mimetics is made by breaking down the cells through serial extrusion, using nano-sized filters with diminishing pore sizes. We have previously prepared EMNVs to increase the content of H19 using this method (Tao et al., 2018b). As expected, the load of H19 in these modularized EMNVs was increased, but the mechanism is yet to be clarified. These EMNVs were prepared by mechanically breaking down the cells instead of by natural secretion. This could not be regarded as a superior choice because their long-term adverse effects and precise mechanisms remain to be elucidated.

RBPs are present in sEVs and potentially function in RNA sorting (Mateescu et al., 2017). LncRNAs can be enriched in sEVs with an over-representation of RBP-binding motifs (Ahadi et al., 2016). RBPs are important regulators of many post-transcriptional events, including RNA splicing, transport, and stability. RBPs are likely involved in EV-RNA sorting mechanisms (Mateescu et al., 2017). In mammalian cells, there are over 500 types of RBPs (Gerstberger et al., 2014), and about a quarter of the protein content in EVs is made up of RBPs (Sork et al., 2018). Most of the RNA transfer to specific cellular locations relies on RBPs, which can travel along the cytoskeleton (Di Liegro et al., 2014; Eliscovich et al., 2013). RBPs, including HuR (Mukherjee et al., 2016), hnRNP A2/B1(Villarroya-Beltri et al., 2013a), hnRNPK (Leidal et al., 2020), hnRNPU (Zietzer et al., 2020), and scaffold-attachment factor B1(SAFB) (Leidal et al., 2020), have been found to be involved in RNA packaging into EVs. Most of the current research is focused on the observation of this phenomenon and exploration of the mechanism, but very little research has been done to exploit these mechanisms as a tool for nucleic acid delivery.

The hnRNP A/B proteins are among the smallest but most abundant RBPs, forming the core of the RNP complex that associates with nascent transcripts in eukaryotic cells. These diverse proteins perform a multitude of functions that involve interplays with DNA or, more commonly, RNA (He and Smith, 2009). Like many RBPs belonging to the A/B family, hnRNP A2/B1 plays a variety of key cellular roles, including RNA processing (Villarroya-Beltri et al., 2013a), export, and maintaining stability of its target genes (Goodarzi et al., 2012). HnRNP A2/B1 in sEVs is sumoylated, and this post-translational modification controls hnRNP A2/B1–RNA binding (Villarroya-Beltri et al., 2013a). In addition, hnRNP A2/B1 plays roles in the trafficking of a myriad of cellular RNAs, both from the nucleus to the cytoplasm and from the cytoplasm to EVs (Villarroya-Beltri et al., 2013b).

LncRNAs can be selectively packaged into sEVs, released, and transported to other cells, with subsequent modulation of cellular function (Kogure et al., 2013; Takahashi et al., 2014). Packaging of H19 into sEVs is selective, especially with an increase in EV-associated hnRNP A2/B1 (Lei et al., 2018). The interaction between hnRNP A2/B1 and the microprocessor may represent such selectivity (Alarcon et al., 2015). As H19 can be secreted by packaging into sEVs, mediated by hnRNP A2/B1 (Lei et al., 2018), the overexpression of hnRNP A2/B1 as a carrier is also required to “overload” H19.

To explore this macromolecule overloading technology, we focused on hnRNP A2/B1. Our results showed that sEVs overloaded with H19 with the activation of hnRNP A2/B1 could be released and transferred into TSPCs. Treatment with H19-OL-sEVs (H19-enriched sEVs) enhanced proliferation, migration, matrix formation, and differentiation of TSPCs.

Although exRNAs have attracted enormous interest, we currently have limited knowledge of the mechanisms that drive and regulate RNA incorporation into sEVs, and of how RNA-encoded messages affect signaling processes in sEV-targeted cells. The biological mechanisms underlying this RNA regulation among EVs remain to be elucidated.

In their latest study, Choi et al. developed an optogenetically engineered exosome system (EXPLOR) for loading a large number of soluble proteins into exosomes via reversible protein-protein interactions controlled by optogenetics (Choi et al., 2020). This is a very enlightening study, because they developed a controllable (by optogenetics) and highly extensible method (a variety of proteins, including RBPs, which could further carry various kinds of RNAs).

Given that tendons mainly function as load-bearing tissues in the musculoskeletal system (Nourissat et al., 2015), YAP is one of the suggested mediators between the mechanical stimulus perceived by the cytoskeleton and the corresponding cellular response (Driscoll et al., 2015). YAP, a component of the nuclear transcriptional complex (Sudol et al., 1995) which is predominantly cytoplasmatic, shuttles from the cytoplasm to the nucleus to activate gene expression (Wan et al., 2018). Importantly, an increase in nuclear YAP is indicative of cell spreading and proliferation (Aragona et al., 2013) but is also correlated with stem cell differentiation (Dupont et al., 2011). The Hippo-YAP signaling pathway is known as a pivotal regulator of organ growth and tissue regeneration (Moya and Halder, 2019). In recent years, there have been an increasing number of regenerative medicine strategies in different organs targeting this pathway (Aharonov et al., 2020; Aloia et al., 2019; Alsamman et al., 2020; Brusatin et al., 2018; Cheung et al., 2020; Gilbert-Honick et al., 2020; Hageman et al., 2020; Li et al., 2020; Sprangers et al., 2020; Zhou et al., 2020). Thus, the Hippo-YAP signaling pathway has great potential and is worthy of further study to better promote the development of regenerative medicine.

Phosphorylation provides an essential mechanism of YAP regulation (inhibition). It has been established that phosphorylation of YAP at Ser127 is involved in the inhibition of YAP by retaining it in the cytoplasm (Basu et al., 2003; Zhao et al., 2007). Phosphorylated YAP remaining in the cytoplasm is degraded and inactivated (Piccolo et al., 2014). YAP is regulated by LATS1/2 or MST1/2 (Ma et al., 2019). However, the results of the present study showed that H19 may directly regulated YAP phosphorylation without the influence of LATS1/2 or MST1/2 (Figure 3A). The de-phosphorylated YAP is localized in the cell nucleus and functions as a transcription co-activator to induce gene expression. Nuclear YAP binds to TEAD and stimulates the transcription of target genes such as CTGF and CYR61 (Zhao et al., 2008).

Our experimental findings suggest a crucial role of H19 in YAP dephosphorylation leading to tendon regeneration. Furthermore, we identified the H19 region that interacts with YAP, located between the nucleotides 780 and 1210 of H19, using deletion mapping. The WW domain of YAP was required for its binding to epigenetic modifiers, such as H19. Through the YAP-WW domain, H19 bound to YAP.

The level of phosphorylation of any protein, such as YAP, depends on the relative activities of protein kinases and PPs. PP1 specifically dephosphorylates the B subunit of phosphorylase kinase, which may link apicobasal polarity to the dephosphorylation of YAP (Ceulemans and Bollen, 2004). Recent studies showed that PP1 dephosphorylates YAP/TAZ in vitro (Liu et al., 2011; Wang et al., 2011). The dephosphorylation of YAP by PP1 results in its nuclear accumulation (Wang et al., 2011; Wu et al., 2015). Here, we investigated the connection between YAP activation and the presence of PP1 or PP2A and showed that PP1 is required for H19-mediated YAP activation. Inhibition of PP1 with CRISPR-Cas9 blocked H19-induced dephosphorylation of YAP. Additionally, we found that H19 promoted the interaction between YAP and PP1 as a “bridge.” When PP1 was downregulated using CRISPR-Cas9, an increase in H19 had almost no influence on the dephosphorylation of YAP. These results could explain why there is a close association between H19 and tendon regeneration and the ability of YAP to increase proliferation, migration, differentiation, and collagen deposition of TSPCs.

In this study, H19-OL-sEVs showed powerful efficacy to upregulate H19 in recipient cells and promoted tendon regeneration by highly packaging (or “overloading”) H19 cargo. Therefore, these modularized sEVs may be a promising therapeutic approach for the treatment of tendon injuries, and even for future regenerative and tailored medicine.

Limitations of the study

Certainly, there are still some limitations in our current study. In our previous study, we observed H19-induced excessive vascular formation in an angiogenesis model (Tao et al., 2018b). Some researchers reported that angiogenesis might be a double-edged sword—VEGF is conducive to tendon graft maturation and biomechanical strength; however, excess VEGF impedes improvements in biomechanical strength (Takayama et al., 2015). Our study did not go into depth regarding whether there is a threshold or a safe range for VEGF in tendon regeneration. In addition, a previous study on H19, reported by Lu et al., did not find excessive H19-induced vascular formation in tendon tissues (Lu et al., 2017), and we also did not find excessive vascular formation in our study. We considered that H19 might play different roles in different tissues or that the amount used in our study may just be within the safe range, and follow-up studies are needed to further explore the specific mechanism of this phenomenon. In addition, hnRNP A2/B1-ACT-sEVs also showed the second-best therapeutic potential (second only to the H19-OL-sEVs). The reason may be that hnRNP A2/B1 is not a specific carrier of H19, and some other nucleic acid components carrying hnRNP A2/B1 may also play a certain role in our study. However, to determine what other molecules it may be carrying, and what exactly is the function of the nucleic acid components, further research is still needed. In addition, it is also hoped that there will be follow-up studies based on this study to identify a specific H19 carrier in the future.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shang-Chun Guo (achuni@126.com).

Materials availability

All related information or materials generated in this study are available upon reasonable request.

Data and code availability

The published article includes all data generated in this study.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.