VEGFA-Enriched Exosomes from Tendon-Derived Stem Cells Facilitate Tenocyte Differentiation, Migration, and Transition to a Fibroblastic Phenotype

Tendon-derived stem cells (TDSCs) play a vital role in repair of rotator cuff tear injuries by secreting paracrine proteins that regulate resident cell functions. Secreted exosomes may play a role in tendon injury repair by mediating intercellular communication; however, the detailed mechanisms by which TDSC-derived exosomes affect tenocyte development remain unknown. Here, we examined the effects of exosomes isolated from conditioned medium of TDSCs on tenocyte differentiation, migration, and transition to a fibroblastic phenotype in vitro. Successful isolation of exosomes from TDSCs was confirmed by high expression levels of CD81, CD63, CD9, and TSG101. Treatment with TDSC-derived exosomes promoted the growth and migration of cultured rat tenocytes, and increased the levels of the fibrosis markers collagen I, collagen III, scleraxis, tenascin C, and α-smooth muscle actin. Furthermore, vascular endothelial growth factor A (VEGFA) expression was higher in TDSC-derived exosomes than in TDSCs, and genetic knockdown of VEGFA suppressed the stimulatory effect of TDSC-derived exosomes on tenocyte development. Overall, these results demonstrate that VEGFA-enriched exosomes isolated from TDSCs promote differentiation and migration of cultured tenocytes and their transition to a fibroblastic phenotype. These data provide a new potential clinical treatment strategy for tendon injury.


Introduction
Rotator cuff tear (RCT) is one of the most common clinical injuries seen in orthopedic practice and sports medicine. The prevalence of RCT in various populations has been estimated to be approximately 20% [1,2]. RCT is particularly common in elderly patients and can result in acute or chronic joint pain or other tissue injuries [3,4]. In particular, RCT can cause substantial shoulder joint pain and dysfunction, and thereby impact an individual's quality of life and ability to work [5]. A variety of factors perturb RCT healing, including tendon degeneration, large tear size, and advanced age [6][7][8]. The incidence of RCT rerupture is relatively high because of the incomplete healing of tendon bone injury; therefore, it is of great importance to explore the mechanisms underlying tendon injury and identify new strategies for clinical treatment.
Tenocytes and immature tenoblasts are the major cell types in tendon tissue. Mature tenocytes are typically quiescent, nondividing cells that synthesize collagen, extracellular matrix molecules, and other components [9]. Degradation of these cells has been reported in rotator cuff-related diseases [10], and tenocyte proliferation, migration, and fibrosis greatly contribute to tendon repair [11,12]. Novel treatment approaches for tendon injuries that aim to induce development of tenocytes into tenoligamentocytes have been reported [13].
Exosomes mediate cellular communication and molecular transport, and are crucial regulators of biological processes [25]. MSC-derived exosomes target the NLRP3 pathway to inhibit inflammatory responses in intervertebral disc degeneration [26] and have a potential application for tissue regeneration [27]. Thus, application of exosomes isolated from embryonic stem cells may be an effective method to treat tendon injury. However, there are few reports describing the targeting and regulation of tenocytes by TDSC-derived exosomes.
Vascular endothelial growth factor A (VEGFA), a member of the VEGF family of proteins, promotes angiogenesis, matrix formation, and collagen formation, all of which are important for tendon repair [28]. VEGFA in exosomes derived from stem cells is involved in a variety of bone functions and is reportedly upregulated in an animal model of osteoarthritis of the knee [29]. Notably, a recent study reported that VEGFA-enriched exosomes from cortical bone-derived MSCs stimulated by a CTRP9 polypeptide exert proangiogenic, antifibrotic, and cardioprotective effects [30]. However, whether VEGFA is enriched in TDSCderived exosomes and its potential role in tenocyte function remain to be explored.
Here, we found that TDSC-derived exosomes are enriched with VEGFA and can regulate the migration, fibrotic activity, and proliferation of isolated rat tenocytes.

Ethics Statement and Animal
Use. Sprague-Dawley (SD) rats were purchased from the Laboratory Animal Center of Jinan University, and the experiments were approved by the Ethics Committee of the First Affiliated Hospital, Jinan University. Rats were kept in a room with a light-controlled 12 h-12 h light-dark cycle, controlled room temperature (25°C), and unlimited food and water.

Isolation of TDSC-Derived Exosomes.
To extract exosomes, after culture of TDSCs for 48 h, culture medium collected from 150 cm 2 dishes was centrifuged at 300 × g for 20 min, 2000 × g for 10 min, and 10,000 × g for 60 min at 4°C. The supernatant was filtered with a 0.22 μm filter (Merck-Millipore, MA, USA), ultracentrifuged at 100,000 × g for 1 h at 4°C, and resuspended in 200 μl phosphate-buffered saline. The morphology of isolated exosomes was assessed by transmission electron microscopy (TEM), and their size was measured using NanoSight viewer (Malvern Instruments, Malvern, UK). The concentration of exosomes (i.e., the protein concentration in exosomes) was measured using a BCA Protein Kit (#P0009; Beyotime, Shanghai, China). Western blotting with primary antibodies against the exosome markers TSG101 (ab125011), CD81 (ab109201), CD63 (ab108950), and CD9 (#ab92726, all from Abcam) was performed. Extracted exosomes (20 μg/ml) were added to the culture for treatment of tenocytes. The design of the procedures is shown in Figure 1(a).
2.6. MTT Assay. Growth and proliferation of tenocytes were determined by an MTT assay. Briefly, tenocytes were seeded at a density of 1 × 10 4 /well into 96-well plates and cultured for the indicated duration. Subsequently, cells were treated with 50 μl of 2 mg/ml MTT at 37°C for 2 h. After dissolving formazan crystals, absorbance at 570 and 620 nm was measured using a Chameleon™ multitechnology microplate reader (Hidex, Turku, Finland).

TDSC-Derived Exosomes Promote Tenocyte Differentiation, Migration, and Transition to a Fibroblastic
Phenotype. An MTT assay revealed that treatment with TDSC-derived exosomes significantly promoted growth of tenocytes (Figure 2(a)). In addition, a transwell assay revealed that the migration ability of tenocytes treated with TDSC-derived exosomes was superior to that of untreated tenocytes (Figures 2(b)-2(c)). The mRNA and protein levels of the fibrosis markers collagen I, collagen III, α-SMA, Scx, and TnC were determined in untreated and TDSC-derived exosome-treated tenocytes. The mRNA levels were significantly upregulated in tenocytes treated with TDSC-derived exosomes (Figures 2(d)). The protein levels showed the same trend as the mRNA levels (Figures 2(e) and 2(f), indicating that treatment with TDSC-derived exosomes promotes the transition of tenocytes to a fibroblastic phenotype. Taken together, these data suggest that TDSC-derived exosomes regulate the differentiation and migration of tenocytes and their transition to a fibroblastic phenotype.

VEGFA Is Upregulated in TDSC-Derived Exosomes and
Tenocytes Treated with these Exosomes. To elucidate the mechanism underlying the growth-promoting effect of TDSCderived exosomes on tenocytes, we performed qPCR analyses of various genes in TDSC-derived exosomes and tenocytes treated with these exosomes. The mRNA and protein levels of VEGFA were markedly higher in TDSC-derived exosomes than in the control group (Figures 3(a)-3(c)). Furthermore, the mRNA and protein levels of VEGFA were markedly higher in tenocytes treated with TDSC-derived exosomes than in untreated tenocytes (Figures 3(d)-3(f)). These results suggest that VEGFA is upregulated in TDSC-derived exosomes and tenocytes treated with these exosomes.

Genetic Knockdown of VEGFA Abolishes the Effects of TDSC-Derived Exosomes on Tenocyte Differentiation, Migration, and Transition to a Fibroblastic Phenotype.
To confirm the effect of exosomal VEGFA on tenocytes, we constructed a TDSC line in which VEGFA was knocked down using a lentiviral shRNA (shVEGFA, Figure 4 generated as a control. Successful knockdown of VEGFA was confirmed by western blotting (Figure 4(b)). Exosomes were extracted from both these cell lines and added to tenocyte cultures. Exogenous VEGFA was added to tenocytes as a positive control. Treatment with VEGFA or exosomes derived from shNC-TDSCs significantly promoted the growth and migration of tenocytes (Figures 4(c)-4(e)). However, knockdown of VEGFA abolished the stimulatory effect of TDSC-derived exosomes on growth (Figure 4(c)) and migration (Figures 4(d)-4(e)) of tenocytes. Furthermore, treatment of tenocytes with shNC-TDSC-derived exosomes or VEGFA significantly increased the levels of collagen I, α-SMA, collagen III, Scx, and TnC, whereas treatment with shVEGFA-TDSC-derived exosomes did not (Figures 4(g)-4(h)). Overall, these results indicate that VEGFA promotes tenocyte growth, migration, and transition to a fibroblastic phenotype, and that the stimulatory effects of TDSC-derived exosomes on these behaviors of tenocytes are mediated via upregulation of VEGFA.

Discussion
Tenocytes play an important role in the repair of tendon injuries because they are the main cell type in tendon tissue.
The results presented here demonstrate that TDSC-derived exosomes promote the growth and migration of rat tenocytes and their transition to a fibroblastic phenotype. VEGFA was upregulated in TDSC-derived exosomes and tenocytes treated with these exosomes, and silencing of VEGFA abolished the stimulatory effects of TDSC-derived exosomes on the behaviors of tenocytes. Taken together, these results suggest that VEGFA in TDSC-derived exosomes plays a novel role in regulating differentiation of tenocytes.
Exosomes isolated from various cell types can induce musculoskeletal tissue repair. For example, exosomes isolated from BMSCs promote myogenesis and muscle differentiation in a mouse injury model [37]. In addition, exosomes secreted during the transformation of myoblasts into myotubules promote myogenesis of adipose-derived stem cells and induce myofiber regeneration for injury repair [38]. Exosomes might also offer a novel way to treat osteoporosis. Exosomes derived from MSCs in a scaffold promote repair of bone defects via the PI3K/Akt pathway [39]. BMSC-derived exosomes improve osteoporosis by suppressing cell apoptosis and promoting osteoblast proliferation [40,41]. In addition, a recent study found that exosomes secreted by endothelial cells improve osteoporosis by inhibiting osteoclast activity, and that this effect is superior to that of BMSC-derived exosomes [42]. Furthermore, exosomes are related to cartilage regeneration and osteoarthritis. Embryonic MSC-derived exosomes induce cartilage repair by increasing chondrocyte proliferation, enhancing collagen production, and regulating immune reactivity [43,44]. miR-100-5p in exosomes derived from fat pad MSCs maintains cartilage homeostasis by regulating the autophagy pathway [45].
In view of their superior clonogenicity and tenogenic proliferation potential, TDSCs were selected as an ideal source of exosomes to manipulate the function of tenocytes in the current study. Exosomes isolated from TDSCs promote the tenogenesis of resident TDSCs, and regulate synthesis and degradation of the tendon matricellular matrix [46]. TDSC-derived exosomes suppress inflammation in a model of Achilles tendon injury [47]. Moreover, TDSCderived exosomes promote growth and migration by modulating the transforming growth factor signaling pathway and promote tendon repair through miR-144-3p in TDSCs [35]. Here, we found that high expression of VEGFA is responsible for the stimulatory effects of TDSC-derived exosomes on tenocyte proliferation, migration, and transition to a fibroblastic phenotype because shRNA-mediated knockdown of VEGFA abolished the ability of TDSC-derived exosomes to promote tenocyte development. VEGF is critical for bone tissue repair. Expression of VEGF is upregulated in injured leg bones of rats and cruciate ligaments of dogs [48]. In addition, during acute injury healing, BEGF and VEGF are upregulated at the bone-tendon junction [28]. Recently, high expression of VEGF in MSCs was reported to regulate repair of RCT via regulation of miR-205-5p [49]. In addition, induction of VEGFA expression by engineered nanoparticles promotes tendon healing. In our previous study, we showed that the long noncoding RNA H19 accelerates differentiation of rat tenocytes by inhibiting miR-140-5p, which leads to upregulation of VEGFA at the lesion site [31]. Expanding on this finding, we demonstrated here that upregulation of VEGFA in exosomes from TDSCs promotes tenocyte development. Taken together, these findings suggest that VEGFA plays a critical role in tendon differentiation and repair. However, the detailed mechanism by which VEGFA is upregulated in TDSC-derived exosomes and the role of these exosomes in RCT repair require further investigation.   In summary, we demonstrated that VEGFA-enriched exosomes isolated from TDSCs promote growth and migration of rat tenocytes and their transition to a fibroblastic phenotype. Further studies of TDSC-derived VEGFAexpressing exosomes may provide new insights into novel approaches for the clinical treatment of tendon injury.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
The author(s) declare(s) that they have no conflicts of interest.