PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells
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Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process.
Tendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling.
TSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU− TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC.
The anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.
KeywordsPEDF Peptide Tendon stem/progenitor cell Signaling
Adipose-derived mesenchymal stem cells
Bone marrow mesenchymal stromal cells
Balance salt solution
Collagen type I
Collagen type III
Connective tissue growth factor
Dulbecco’s modified Eagle’s medium
Early growth response-1
Extracellular signal-regulated kinase
Fetal bovine serum
Fibroblast growth factor
Human anterior cruciate ligament
Medial collateral ligament
Insulin-like growth factor
Leukemia inhibitory factor
Mitogen-activated protein kinase
Octamer-binding transcription factor 4
Polymerase chain reaction
Platelet-derived growth factor
Pigment epithelial-derived factor
Patatin-like phospholipase domain-containing protein 2
Pigment epithelial-derived factor-derived short peptide
SRY-box containing gene 9
Signal transducer and activator of transcription 3
Tendon stem/progenitor cells
Ultimate tensile stress
Tendons contain dense connective tissues and mediate the transmission of muscle force to the bone, which is crucial for the control of body movement. Tendon injuries are common and often caused by overstretching the tendon. However, tendons have limited ability for self-healing following severe injury, because of their avascularity and acellularity [1, 2]. Unlike another type of connective tissue, bone marrow mesenchymal stromal cells (BM-MSCs) are difficult to mobilize into the injured sites of tendons . The repair of tendons is therefore a slow and relatively difficult process. Recently, intensive efforts have been made to use cell therapy-based approaches to accelerate tendon regeneration and repair . Adult mesenchymal stromal cells (MSCs) can be obtained to provide an adequate cellular source for tendon regeneration . However, cell transplantation is a time-consuming process. In addition, cost and technical and safety issues are obstacles to providing benefits to patients [3, 4]. Growth factors injected into and around the injured site to stimulate tendon stem/progenitor cells (TSPC) proliferation may offer an alternative option for promoting tendon repair. Connective tissue growth factor (CTGF) can promote tendon wound healing by stimulating proliferation of a TSPC population marked by CD146. The CD146+ TSPC cluster the tendon periphery, mainly near the blood vessels . Platelet-rich plasma (PRP) that harbors platelet-derived growth factor (PDGF) is another means of treating tendon injury, although the effect is limited, possibly because of a low concentration of PDGF in the preparation . It has been reported that hydrogel combinations of fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-1, and PDGF-BB can improve the survival of adipose-derived mesenchymal stem cells (ASCs) and aid tendon healing [6, 7]. The advantage of growth factor treatment is that it is readily available for acute tendon injury, avoiding the waiting period of cell therapy. In addition, several growth factors have the ability to induce TSPC proliferation in culture. CTGF can enhance the clonogenic capacity of CD146+ TSPC . FGF-2 promotes the growth of TSPC marked by expression of scleraxis (Scx) and SRY-box containing gene 9 (Sox9) . However, the mitogenic signaling stimulating TSPC proliferation remains largely unknown.
Pigment epithelial-derived factor (PEDF) has been reported to mediate the proliferation of several stem/progenitor cell populations. PEDF is effective in stimulating the proliferation of neuronal progenitor cells and human embryonic stem cells [9, 10]. Recent studies demonstrated further that PEDF and pigment epithelial-derived factor-derived short peptide (PSP) (29-mer; residues Ser93-Thr121) can stimulate the proliferation of limbal stem cells, muscle satellite cells, and hepatic stem cells [11, 12, 13]. Moreover, PSP has been suggested to have a potential for treating several types of tissue injury [11, 12, 13]. In this study, we investigated the potential application of the 29-mer to the healing of Achilles tendon ruptures in animals. The mitogenic activity of the 29-mer on TSPC in vitro was also explored.
The PSP 29-mer (Ser93-Thr121; SLGAEQRTESIIHRALYYDLISSPDIHGT) and 34-mer (Asp44-Asn77; DPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTN) were synthesized, modified by acetylation at the NH2 termini and amidation at the COOH termini for stability, and characterized by mass spectrometry (> 90% purity) at GenScript (Piscataway, NJ). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotic–antimicotic solutions, and trypsin were purchased from Invitrogen (Carlsbad, CA). 5-Bromo-2′-deoxyuridine (BrdU), insulin–transferrin–sodium selenite (ITSE) media supplement, Hoechst 33258 dye, Trichrome Stain (Masson) Kit, and all chemicals were from Sigma-Aldrich (St. Louis, MO). Dispase II and collagenase I were obtained from Roche (Indianapolis, IN). Anti-BrdU antibody (GTX42641) was from GeneTex (Taipei, Taiwan). Anti-CD146 (ab75769), anti-Oct4 (ab18976), anti-nestin (ab6142), anti-CD31 (ab24590), anti-nucleostemin (ab70346), anti-collagen I (ab6308), and anti-collagen III antibody (ab6310) antibodies were from Abcam (Cambridge, MA). Phospho-Stat3 (Tyr705), STAT3, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and ERK antibody were purchased from Cell Signaling Technology (Danvers, MA). SB203580, PD98059, and STAT3 inhibitor (No. 573096) were purchased from Calbiochem (La Jolla, CA, USA).
All animals were housed in an animal room under temperature control (24–25 °C) and a 12:12 light-dark cycle. Standard laboratory chow and tap water were available ad libitum. Experimental procedures were approved by the Mackay Memorial Hospital Review Board (New Taipei City, Taiwan) and were performed in compliance with national animal welfare regulations.
Isolation and culture of TSPC
New Zealand white rabbits (6–8 months old, 3.0–4.0 kg) were used for the isolation of tendon cells. Achilles tendons were removed from the rabbits, by cutting through their bony attachments, and washed two times in sterile phosphate-buffered saline (PBS) containing 50 μg/ml gentamicin. The tendon and tendon sheath were cut into small pieces (1–2 mm3). Each 100 mg of tissue was then digested in a solution containing 3 mg/ml of type I collagenase and 4 mg/ml of dispase II in 1 ml balanced salt solution (BSS; Alcone) at 37 °C for 4 h. The digested tissues were washed three times in PBS, collected by centrifugation (800g for 10 min), placed into tissue culture dishes (Falcon Labware, NJ, USA) and resuspended in high-glucose DMEM, supplemented with 10% FBS and 50 μg/ml gentamycin, and maintained at 37 °C with 5% CO2. After 5 days, the medium was changed to remove the loosened tissue residues. For passaging, the tendon cells were harvested with 0.25% trypsin/EDTA and counted using a hemocytometer.
Approximately 2 × 103 primary tendon cells were seeded into 25 T cell culture flasks (Corning), incubated with 10% FBS medium for 2 days, and their clonogenic capacity was determined in DMEM basal medium (2% FBS, 1% ITSE, 300 μg/ml l-glutamine, 1% antibiotic-antimicotic solutions), supplemented with 10 μM 29-mer, 34-mer, or peptide solvent (DMSO; dimethyl sulfoxide), for a further 10 days. The culture medium was changed every 3 days. Expression of TSPC markers by these expanded tendon cells was determined by immunostaining and western blot analysis.
Cells cultured on slides were fixed with 4% paraformaldehyde, treated at 4 °C with methanol for 1 min, and then blocked with 1% goat serum and 5% BSA for 1 h. The cells were stained with antibodies to CD146 (1:50 dilution), nucleostemin (1:100 dilution), or BrdU (1:100 dilution) at room temperature (RT) for 3 h. The slides were subsequently incubated with FITC-donkey anti-rabbit IgG or Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution; BioLegend, San Diego, CA) for 20 min and then counterstained with Hoechst 33258 for 6 min. The slides were rinsed with PBS with Triton X100 (0.5%) three times, mounted with FluorSave™ reagent (Calbiochem) and viewed with a Zeiss epifluorescence microscope.
After 3 weeks, the repaired Achilles tendon (n = 16) and the contralateral control tendons (n = 16) were harvested for mechanical evaluation. The specimens were dissected to remove the gastrocnemius/soleus muscle complex and soft tissues, leaving the intramuscular tendinous fibers, Achilles tendon, and calcaneal bone intact. The specimens were kept moist with normal saline during the entire testing procedure. The sagittal and transverse diameters of the mid-part of the callus were measured using a caliper to estimate the cross-sectional area . Tendon fibers were fixed in a metal clamp by fine sandpaper. The calcaneal bone was fixed in a custom-made clamp at 30° dorsiflexion, relative to the direction of traction, as described previously [14, 15]. The mechanical testing machine (MTS Systems Corp., 14000 Technology Drive, Eden Prairie, MN) pulled the mounted tendon at a constant speed (0.1 mm/s) until failure, after a preload of 0.8 N had been applied. The data acquisition rate was 1/0.03 s. The ultimate tensile stress at tendon failure, expressed in newtons (N), was recorded during the process of failure testing. Young’s modulus (MPa) was calculated from the linear slope of the axial force–axial displacement curve.
BrdU labeling in vitro
BrdU (final concentration, 10 μM) was added to the culture for 4 h. After fixing with 4% paraformaldehyde, the cells were exposed to cold methanol for 1 min and then treated with 1 N HCl at RT for 1 h before performing immunocytochemistry. The phenotype of TSPC was determined by immunostaining of nucleostemin.
Western blot analysis
Cell lysis, SDS–PAGE, and antibodies used for immunoblotting were as described in a previous study . The band intensity in immunoblots was evaluated with a Model GS-700 imaging densitometer (Bio-Rad Laboratories, Hercules, CA) and analyzed using Labworks 4.0 software.
Quantitative real-time RT-PCR
Primers used in the real-time qPCR
Surgical procedure for rat Achilles tendon injury
To investigate the effects of the 29-mer peptide on tendon healing, a rat model of Achilles tendon injury was established, as reported previously . Ten-week-old adult male Sprague-Dawley rats (initial body weight = 312 ± 11 g) were anesthetized by an intraperitoneal injection of xylazine (10 mg/kg). The left tendo-Achilles injury was created by full-thickness insertion of an 18-G needle through the tendo-Achilles, 1 cm proximal to the calcaneum attachment site. This created a horizontal wound which was flanked by intact tendon tissue to prevent the retraction of the severed ends. The skin incision was closed after the wound was irrigated with sterile saline. Treatments were applied to the area around the tendon lesion by subcutaneous injection with 150 μl of alginate gel mixed with 100 μM 29-mer or DMSO vehicle.
Formalin-fixed, paraffin-embedded tendon specimens were cut into 5-μm longitudinal sections, deparaffinized in xylene, and rehydrated in a graded series of ethanol concentrations. Slides were blocked with 10% goat serum for 60 min and then incubated with primary antibody against CD146 (1:50 dilution), nucleostemin (1:50 dilution), and CD31 (1:100 dilution) at room temperature (RT) for 2 h. The slides were subsequently incubated with the FITC-donkey anti-rabbit IgG and Alexa Fluor® 647 Goat anti-mouse IgG (1:500 dilution) for 20 min and then counterstained with Hoechst 33258 for 6 min and viewed with Zeiss epifluorescence microscope.
Deparaffinized tendon specimens were also blocked with 10% goat serum for 60 min and then incubated with antibody against CD146, collagen I, and collagen III (1:100 dilution, 37 °C for 3 h). The slides were subsequently incubated with the appropriate peroxidase-labeled goat immunoglobulin (1:500 dilution; Chemicon, Temecula, CA) for 20 min and then incubated with chromogen substrate (3,3′-diaminobenzidine) for 2 min before counterstaining with hematoxylin.
In vivo detection of DNA synthesis
For the detection of cell expansion, BrdU was reconstituted in DMSO as stock (80 mM). One hundred fifty microliters of BrdU mixed with 350 μl of PBS was injected intraperitoneally into the rats at days 0, 3, and 5 after surgery. Tissue sections were treated with 1 N HCl at RT for 1 h, and DNA synthesis was assessed by anti-BrdU antibodies.
The tendon specimens were stained with hematoxylin and eosin (H&E). Photomicrographs of the tissue were captured through an Olympus IX71 light microscope and an Olympus XC10 camera (Japan). Four sections of each sample were selected and evaluated by two blinded observers to assess the tendon morphology according to a modified semi-quantitative grading score from 0 to 3. The score analyzed the fiber arrangement, fiber structure, nuclear roundness, cell density, infiltration of inflammatory cells and fibroblasts, and neovascularization [18, 19, 20]. According to this grading system, a perfectly normal tendon scored 0 and mild and moderate prevalence scored 1 and 2, whereas a score of 3 was assigned to a severely abnormal tendon.
The data were generated from three independent experiments. All numerical values are expressed as the mean ± SD. Comparisons of two groups were made using two-tailed Student’s t test. P < 0.05 was considered significant.
The 29-mer stimulates the proliferation of CD146+ TSPC in culture
Sustained release of the 29-mer peptide promotes Achilles tendon healing
Expanded TSPC induced by 29-mer retain the capacity for tenogenic differentiation
The 29-mer promotes CD146+ TSPC proliferation in response to acute Achilles tendon injury
The 29-mer causes accumulation of nucleostemin+ TSPC in the healing region of the Achilles tendon
The mitogenic effect of the 29-mer on nucleostemin+ TSPC is modulated by ERK2 and STAT3 signaling
Insufficient numbers of resident TSPC are activated in response to an acute tendon injury, and this may limit the healing process. This study is the first to show that the PSP 29-mer can promote the clonogenicity of CD146+ TSPC and proliferation of nucleostemin+ TSPC among cells isolated from the rabbit Achilles tendon. Moreover, we provide evidence that a single injection of the 29-mer hydrogel into a damaged tendon can induce expansion of CD146+ TSPC and nucleostemin+ TSPC at the early regenerative stage (2~7 days postoperative). Our study has demonstrated, in vitro and in vivo, that the 29-mer is a newly identified mitogen for TSPC.
In this study, an injectable alginate gel was used to provide sustained release of the 29-mer in the damaged tendon, leading to improved tendon repair. Alginate gel as a vehicle displays biocompatible, nontoxic, and biodegradable properties . Our previous study showed that a single injection of the 29-mer/alginate gel can stimulate muscle satellite cell proliferation significantly to promote rat soleus muscle regeneration . Despite these advantages, this previous experiment revealed that ~ 90% of the 29-mer was released from the alginate gel within 5 days in vitro . Nonetheless, herein, our day 2 and day 7 histological data indicate that the sustained release of 29-mer for a few days was sufficient to augment CD146+ TSPC and nucleostemin+ TSPC in the niche and HR. The results support the hypothesis that the growth factor stimulates a sufficient level of CD146+ TSPC at the early phase of tendon wound healing, playing a crucial role in relieving disorganized collagen formation in the HR .
Healing of the tendons begins with the inflammatory phase that shows accumulation of hemorrhages and leukocytes and local synthesis of chemotactic and angiogenic factors . The reparative phase begins later, characterized by the initiation of TSPC proliferation and migration of TSPC/tenocytes into the wound [30, 31]. Due to the limited self-healing capacity of the tendons, the healed tendon shows disorganized fiber architecture and scar tissue with disrupted collagens [3, 30]. These result in reduced elasticity, reduced mobility, and an increased propensity for the recurrence of injury . In this study, the 29-mer hydrogel treatment accelerated TSPC proliferation that initially overlapped the inflammatory phase (day 2 to day 7), thereby leading to a decrease in fibrosis and scar tissue formation. The incomplete description of the role of 29-mer in the inflammatory phase of tendon healing is a limitation of this study. In this regard, PEDF reportedly can reduce inflammation in vitro and in vivo. For example, PEDF induces expression of the anti-inflammatory cytokine interleukin 10 (IL-10) by human macrophages . PEDF treatment can reduce retinal inflammation in a rat model of diabetes . Interestingly, our previous animal study found that PSP 44-mer (a sequence containing the 29-mer) can reduce inflammatory responses in acute liver injury through its protective effect on hepatocytes . Whether the 29-mer is able to regulate the properties of inflammatory cells during tendon injury warrants additional research.
It has been reported that the nucleostemin is a TSPC marker because its expression is eliminated in tenocytes . In this study, the 29-mer increased the number of nucleostemin+ TSPC that were observed on day 7 post-injury. Nucleostemin+ TSPC have been shown to retain a high self-renewal capacity in vitro and express tenocyte-related markers, such as collagen I, collagen III, and Tnc . Because collagen I and collagen III are important structural protein in tendons, the nucleostemin+ TSPC have a direct impact on tendon repair.
The relationship between CD146+ TSPC and nucleostemin+ TSPC remains elusive. There is still no evidence that CD146+ TSPC can differentiate to nucleostemin+ TSPC. A study discovered that stem cells derived from the human anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) express nucleostemin, but not CD146 . Interestingly, a recent report indicates that rat tendon injury leads to a large number of nucleostemin+ TSPC throughout the HR at 1 and 2 weeks post-injury . The different TSPC populations have been suggested to be involved in a time-controlled tendon repair process [3, 28].
The present cell signaling study exploits nucleostemin+ TSPC rather than CD146+ TSPC. CD146+ TSPC are rare (~ 0.8%) in the rat patellar tendon . Our histological sections also revealed that CD146+ TSPC numbers were extremely low in the uninjured rabbit Achilles tendon, and there was a rapid loss of stemness during culture in basal medium. PSP, as full-length PEDF, has been shown to initiate signaling by binding to the cell surface receptor, patatin-like phospholipase domain-containing protein 2 (PNPLA2). PNPLA2 receptor is essential for PEDF/PSP to induce mitogenic signaling on human embryonic stem cells and neural stem cells, as well as antiapoptotic signaling on hepatocytes [13, 38, 39]. Further studies are warranted to determine the expression of PNPLA2 in TSPC and the involvement of PNPLA2 in mediating PEDF/29-mer mitogenic signaling. Our study found that STAT3 signaling was critical for the induction of nucleostemin+ TSPC proliferation by the 29-mer. Our previous findings show that PSP enhances the proliferation of limbal stem cells and satellite cells by activating STAT3 signaling [11, 12]. In addition, phosphorylation of STAT3 has been found to be crucial for the proliferation of myoblasts and satellite cells induced by bFGF, leukemia inhibitory factor (LIF), and IL-6 [40, 41]. STAT3 is a transcription factor that regulates several targets closely associated with cell cycle progression, including cyclin D1 and SOCS3 [12, 42]. To our knowledge, there has been no report addressing the role of STAT3 signaling on TSPC proliferation. Our finding also implies that ERK2 activation is crucial for TSPC proliferation in vitro. This multiple signaling is reminiscent of the proliferative responses induced in satellite cells by the 29-mer . ERK signaling as STAT3 is also involved in cyclin D1 expression by satellite cells .
Growth factors accelerate TSPC expansion in the early phase of tendon wound-healing as a critical mechanism for improving tendon repair. This study shows that the PSP 29-mer displays mitogenic activity and regulates the resident TSPC, in response to acute tendon rupture, and facilitates tendon recovery of a higher quality in the animal model. The 29-mer may be a novel therapeutic remedy for acute tendon injury.
We thank Dr. Tim J Harrison for kindly reading this manuscript. The authors would like to acknowledge Chu-Ping Ho and Yi-Cheng Huang for their assistance with sample collection and histological preparations.
The funding support from the Ministry of Science and Technology, Taiwan (MOST 104-2314-B-195-006-MY3) and Mackay Memorial Hospital (MMH-E-107-006).
Availability of data and materials
TCH, YPT, SHT, and SIY contributed equally to the study design, data analysis, data interpretation, and manuscript preparation. TCH, SIY, KYT, HYC, YCL, and CHH participated in the sample collection of partial animal experiments. SLC and YPT conceived and supervised the study. All authors reviewed and approved the final version of the manuscript.
Experimental procedures were approved by the Mackay Memorial Hospital Review Board (MMH-A-S-103-38 and MMH-A-S-106-47) (New Taipei City, Taiwan), which was in accordance with MOST Guide for the Care and Use of Laboratory Animals.
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