Growth differentiation factor 11 alleviates oxidative stress-induced senescence of endothelial progenitor cells via activating autophagy

Abstract

Background

Stem cell transplantation has been regarded as a promising therapeutic strategy for myocardial regeneration after myocardial infarction (MI). However, the survival and differentiation of the transplanted stem cells in the hostile ischaemic and inflammatory microenvironment are poor. Recent studies have focused on enhancing the survival and differentiation of the stem cells, while strategies to suppress the senescence of the transplanted stem cells is unknown. Therefore, we investigated the effect of growth differentiation factor 11 (GDF11) on attenuating oxidative stress-induced senescence in the engrafted endothelial progenitor cells (EPCs).

Methods

Rat models of oxidative stress were established by hydrogen peroxide conditioning. Oxidative stress-induced senescence was assessed through senescence-associated β-galactosidase expression and lipofuscin accumulation. The effects of GDF11 treatment on senescence and autophagy of EPCs were evaluated 345, while improvement of myocardial regeneration, neovascularization and cardiac function were examined following transplantation of the self-assembling peptide (SAP) loaded EPCs and GDF11 in the rat MI models.

Results

Following hydrogen peroxide conditioning, the level of ROS in EPCs decreased significantly upon treatment with GDF11. This resulted in reduction in the senescent cells and lipofuscin particles, as well as the damaged mitochondria and rough endoplasmic reticula. Concurrently, there was a significant increase in LC3-II expression, LC3-positive puncta and the presence of autophagic ultrastructures were increased significantly. The formulated SAP effectively adhered to EPCs and sustained the release of GDF11. Transplantation of SAP-loaded EPCs and GDF11 into the ischaemic abdominal pouch or myocardium resulted in a decreased number of the senescent EPCs. At four weeks after transplantation into the myocardium, neovascularization and myocardial regeneration were enhanced, reverse myocardial remodeling was attenuated, and cardiac function was improved effectively.

Conclusions

This study provides novel evidence suggesting that oxidative stress could induce senescence of the transplanted EPCs in the ischemic myocardium. GDF11 demonstrates the ability to mitigate oxidative stress-induced senescence in the transplanted EPCs within the myocardium by activating autophagy.

Introduction

Cardiovascular diseases, such as myocardial infarction (MI), have the highest morbidity and mortality worldwide. According to the estimation of American Heart Association in 2016, about 45.1% of the population in America would be diagnosed with some form of cardiovascular diseases by 2035 [1]. Patients with MI often develop heart failure that accounts for an approximate 50% death rate within five years after diagnosis [2]. After occlusion of the coronary arteries or their major branches, myocardial necrosis triggers local inflammation and adverse myocardial remodeling, and progressively leading to scar formation and left ventricle (LV) dilation [3]. In recent years, stem cell transplantation has emerged as a promising therapeutic strategy for myocardial regeneration [4, 5]. Both differentiation of stem cells into cardiovascular cells and paracrine of stem cells contribute to the repair of the infarcted myocardium following transplantation.

Endothelial progenitor cells (EPCs) are selected as the seed cells for promoting neovascularization in the ischaemic diseases such as MI, peripheral arterial disease, ischemic retinopathy, or stroke [6, 7]. Specific types of neovascularization may be classified into vasculogenesis, the de novo formation of new blood vessels from EPCs; angiogenesis, the formation of new capillaries from existing ones; and arteriogenesis, the remodelling of pre-existing collateral vessels or de novo arteriogenesis [8, 9]. EPCs can be isolated from peripheral blood, umbilical cord blood or bone marrow. These cells express CD34 and VEGFR-2 (vascular endothelial growth factor receptor-2), with a potential to differentiate towards endothelial cells [10]. After transplantation into the ischaemic tissue, EPCs may incorporate into the damaged microvessels and participate in neovascularization. However, poor engraftment, survival and differentiation of the transplanted stem cells in hostile ischaemic and inflammatory microenvironment have hindered success of preclinical or clinical trials involving stem cell transplantation [11, 12]. In recent years, recognizing the crucial role of the survival of the transplanted stem cells in repair of ischaemic tissues, the optimized transplantation strategies such as priming stem cells with pretreatment and improving the local microenvironment have been developed to protect the engrafted cells from apoptosis and necrosis [13, 14]. However, the strategies to alleviate senescence of the transplanted stem cells remain elusive.

Cellular senescence represents a cell state triggered by stressful insults and certain physiological processes. It is characterized by a prolonged and typically irreversible cell-cycle arrest, along with secretory features, macromolecular damage, and altered metabolism. Cellular senescence serves as a response to numerous stressors, including nutrient deprivation, hypoxia and mitochondrial dysfunction [15]. Oxidative stress is an important modulator of telomere loss, while telomere-driven replicative senescence is primarily a stress response [16]. Oxidative stress-induced senescence results in a decline of tissue-repair capacity due to cell cycle arrest in progenitor cells. Moreover, the senescent cells produce proinflammatory and matrix-degrading molecules [17]. Therefore, attenuating or delaying oxidative stress-induced senescence is beneficial for effective stem cell engraftment. Growth differentiation factor 11 (GDF11), a member of transforming growth factor-β (TGF-β) superfamily, binds to type I activin receptor-like kinase (ALK) receptors ALK-4 and ALK-5, thereby activating the SMAD2/3 pathway. Although GDF11 is expressed in the myocardium, its level is lower than that in spleen, kidney, and skeletal muscle [18]. As a circulating factor, GDF11 declines with age [18, 19]. Restoring systemic GDF11 levels with heterochonic parabiosis and systemic administration of young blood plasma or GDF11 can reverse age-related cardiac hypertrophy [18], neurodegenerative disorders [20, 21] and skeletal muscle dysfunction [22]. Activating proliferation and differentiation of the stem cells contributes to efficacy of GDF11 in reversing the age-related disorders [20, 22]. Additionally, deletion of GDF11 in the excitatory neurons accelerates the senescence of these neurons [23]. Therefore, restoring cardiac GDF11 level might be beneficial for alleviating oxidative stress-induced senescence of the cardiomyocytes, vascular cells and recruited stem cells in the myocardium after MI. Considering that the transplanted stem cells in the ischaemic and hypoxic microenvironment are prone to oxidative stress, effect of GDF11 on mitigating oxidative stress-induced senescence in stem cells needs to be investigated.

This study was designed to examine efficacy of self-assembling peptide (SAP)-released GDF11 in mitigating oxidative stress-induced senescence of the transplanted EPCs in the infarcted myocardium. Oxidative stress-induced senescence was determined by senescence-associated β-galactosidase (SA-β-gal) expression and lipofuscin accumulation. The changes in senescence and autophagy were examined after GDF11 treatment in vitro, while the improvement of myocardial regeneration, neovascularization and cardiac function was assessed after transplantation of the SAP loaded EPCs and GDF11 in rat MI models. Our findings demonstrate that SAP-released GDF11 can effectively attenuate oxidative stress-induced senescence of EPCs by activating autophagy. Thus, local release of GDF11 with SAP presents a promising strategy to attenuate senescence of the engrafted stem cells in the ischaemic tissue.

Materials and methods

The work has been reported in line with the ARRIVE guidelines 2.0.

Isolation of EPCs

Isolation of EPCs from bone marrow of Sprague-Dawley (SD) rats was performed as previously described [24] with minor modifications. In brief, young (3 − 6 weeks old) and old (64 − 68 weeks old) rats were anaesthetized via intraperitoneal injection of ketamine (80 mg/kg), and then sacrificed by cervical dislocation. The femurs and tibias were removed from the rats, and bone marrow was flushed out of the bones using PBS supplemented with 5 mM ethylene diamine tetraacetic acid (EDTA). The mononuclear cells in bone marrow were isolated using density-gradient centrifugation with Percoll solution (GE Healthcare, Leics, UK), and then incubated with Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 15% fetal bovine serum (FBS; Invitrogen, Grand Island, NY, USA), 100 U/mL penicillin and 100 µg/mL streptomycin for 24 h. The non-adherent cells were collected and seeded into gelatin-coated dishes. Upon being grown to 80% confluence, the cells were harvested by digestion with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then incubated with mouse anti-rat CD34 and rabbit anti-rat VEGFR-2 antibodies (1:100; Santa Cruz Biotech, Dallas, TX, USA) at 4 ^oC for 50 min, followed by incubation with Alexa Fluor 647-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson, West Grove, PA, USA) at 4 ^oC for 30 min. CD34⁺VEGFR-2⁺ cells in the mononuclear cells were sorted using a Beckman MoFlo™ XDP FACS (fluorescence-activated cell sorter; Beckman Coulter, Fullerton, CA, USA). To assess the differentiation potential of EPCs, the sorted cells were seeded in gelatin-coated dishes and incubated in the medium supplemented with 10 ng/mL VEGF (Peprotech, Rocky Hill, NJ, USA) and 15% FBS. Differentiation of the cells into the endothelial cells was determined by detecting of CD31 expression through immunostaining after 2 weeks of induction.

Detection of ALK-4 (activin-like kinase-4) expression

ALK-4, a member of Type-I TGF-β receptors, is expressed in EPCs [25]. ALK-4 expression in EPCs derived from young and old rats was detected using immunostaining and Western blotting respectively. For immunostaining, the cells were incubated with rabbit anti-rat ALK-4 antibody (1:250; Abcam, Cambridge, UK) at 4 ^oC overnight, followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:200; Sigma-Aldrich, Saint Louis, MO, USA) at room temperature for 1 h. For Western blotting, the cells were lysed with RIPA buffer (Beyotime, Beijing, China). After quantifying with BCA protein assay kit (Beyotime), equal amounts of proteins were separated on 15% SDS-PAGE and then transferred onto PVDF membranes. The membranes were incubated with rabbit anti-rat ALK-4 antibody (1:1000; Abcam) and mouse anti-rat β-actin antibody (1:4000; Sigma-Aldrich) at 4 ^oC overnight, followed by incubation with HRP-linked goat anti-rabbit IgG (1:4000) and HRP-linked goat anti-mouse IgG (1:4000; Cell Signaling, Danvers, MA, USA) at room temperature for 1 h. Western blot bands were visualized by a Bio-Rad’s ChemiDoc System (BIO-RAD, Hercules, CA, USA). ALK-4/β-actin ratios were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin. The experiment was repeated for three times.

Assessment of intracellular ROS (reactive oxygen species) generation

Cellular senescence can be induced with hydrogen peroxide (H₂O₂; Sinopharm, China) conditioning [26]. To determine the optimal concentration of H₂O₂ for inducing senescence without affecting the viability of the cells derived from young rats, the cells were treated with 100 µM, 250 µM and 500 µM H₂O₂ for 6 h respectively. Cell viability after H₂O₂ treatment was assessed using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), and OD₄₅₀ was measured by an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland). 250 µM H₂O₂ was used for induction of senescence of the cells in the following experiments. The level of intracellular ROS was determined by ROS assay kit (Beyotime, Jiangsu, China). The cells were divided control, GDF11, H₂O₂ and H₂O₂ + GDF11 groups. In GDF11 group and H₂O₂ group, the cells were treated with 40 ng/mL GDF11 (Sigma-Aldrich) and 250 µM H₂O₂ respectively. In the H₂O₂ + GDF11 group, the cells were treated with both H₂O₂ (250 µM) and GDF11 (40 ng/mL). After treatment for 6 h, the cells were incubated with 10 µM peroxide-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime) at 37°C for 20 min. After permeating through cell membrane, DCFH-DA is hydrolyzed by intracellular esterases to DCFH. In the presence of ROS, DCFH is rapidly oxidized to fluorescent compound 2’,7’-dichlorofluorescein (DCF) [27]. DCF fluorescence was detected by microplate reader (Tecan). The experiment was repeated for three times.

SA-β-gal staining

SA-β-gal is a biomarker associated with the senescent phenotype. It catalyzes hydrolysis of artificial substrate X-gal, which produces a blue color in senescent cells [28]. The cells were divided into control, H₂O₂, H₂O₂ + GDF11 and H₂O₂ + GDF11 + SB-431542 groups. SB-431542 (MCE, Monmouth Junction, NJ, USA) is a small molecule and selective inhibitor to ALK4 and ALK5 [29]. At 6 h after incubation, the supernate was replaced with complete medium. In H₂O₂ + GDF11 and H₂O₂ + GDF11 + SB-431542 groups, GDF11 and SB-431542 (10 µM) were added, and the cells continued to be incubated for 48 h. Expression of SA-β-gal in the cells was examined with a SA-β-gal staining kit (Genmed Scientifics, Shanghai, China). The cells were fixed in 4% (v/v) formaldehyde for 8 min and then incubated with SA-β-gal staining solution at pH 6.0 for 12 h. The positive cells were counted using a phase contrast microscope. Three fields (20 ×) were randomly selected in each experiment. The experiment was repeated for three times.

Sudan black B staining

Sudan black B staining is used for examining lipofuscin accumulation in senescent cells [30]. The cells were divided into control, H₂O₂ and H₂O₂ + GDF11 groups. After treatment for 6 h, the cells were immersed in 70% ethanol for 2 min, and incubated with freshly prepared Sudan black B solution (0.7% in 70% ethanol; Sangon Biotech, Shanghai, China) for 8 min. Subsequently, the cells were immersed in 70% ethanol for a few times and counterstained with 0.1% nuclear fast red (Servicebio, Wuhan, China) for 10 min. Distribution of lipofuscin in the cells was viewed using a microscope. The experiment was repeated for three times.

Measurement of lipofuscin content

Intracellular lipofuscin content was measured as the autofluorescence intensity of the cells with flow cytometry as described previously [31]. Grouping of the cells is same as above described. 1 × 10⁴ EPCs were harvested from each group and then washed twice in PBS by centrifugation. After the cells were resuspended in serum-free medium, the autofluorescence of the intracellular lipofuscin was analyzed using a flow cytometer (Beckman Coulter) with excitation wavelength of 488 nm and emission wavelength of 661 nm. The experiment was repeated for four times.

Detection of LC3 (microtubule-associated protein 1 light chain 3) expression

LC3 is mainly expressed on autophagosome and is a specific marker for autophagic structures [32]. For assessing LC3 expression on the autophagic structures, the cells were incubated with rabbit anti-rat LC3 antibody (1:200; Novus Biologicals, Littleton, CO, USA) at 4 ^oC overnight. Then, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Jackson) for 1 h at room temperature. LC3-positive puncta were viewed with a confocal laser scanning microscope (Leica, Mannheim, Germany) and counted in five randomly selected areas. The expression of LC3-I and LC3-II in the cells was examined by Western blotting. Briefly, proteins of the lysed cells were separated with 15% SDS-polyacrylamide gel and electrophoretically transferred to a PVDF membrane. The membrane was incubated with rabbit anti-rat LC3 antibody (1:500; Novus Biologicals) and mouse anti-rat β-actin antibody (1:4000; Sigma-Aldrich) at 4 ^oC overnight, followed by incubation with HRP-linked goat anti-rabbit IgG and HRP-linked goat anti-mouse IgG (1:4000; Cell Signaling) at room temperature for 1 h. The ratio of LC3-II/β-actin was analyzed using ImageJ (National Institutes of Health). The experiment was repeated for three times.

Transmission electron microscopy

The cells were divided into H₂O₂ and H₂O₂ + GDF11 groups. After being washed with PBS, the cells were fixed with 2.5% glutaraldehyde at 4 ^oC for 2 h, and then fixed with 1% osmium tetroxide. Subsequently, a series of dehydration was performed and ended with rinsing in 100% acetone. Then, the samples were embedded in epoxy resin, followed by being solidified in 37 ^oC, 45 ^oC and 60 ^oC for 12 h, 12 h and 24 h respectively. Ultrathin sections were prepared and then stained with 3% uranyl acetate and lead citrate. The autophagic structures in the cells were viewed using a CM120 transmission electron microscope (TEM; Philips, Eindhoven, Netherlands). The ratio of the cross-section area of the autophagic structures to that of the cytoplasm was calculated. The autophagic structures were examined in 200 cells for each group.

Atomic force and scanning electron microscopies

The SAP (AcN-RADARADARADARADARGDS-CONH₂) was synthesized by Top-peptide Biotechnology (Shanghai, China). RGDS was designed as a cell adhesion motif. The SAP powder was dissolved in distilled water at 10 mg/mL. Then, the solution was diluted to a working concentration of 0.1 mg/mL and sonicated for 30 min. The sample were dropped on a mica, and left for 5 s at room temperature. Subsequently, the mica was gently rinsed twice with distilled water. SAP sample was air-dried at room temperature for 30 min. The nanofibers assembled by SAP were viewed using an atomic force microscope (AFM; Dimension Icon, Bruker, USA). In scanning electron microscopy, 10 µL SAP-PBS mixtures were coated on a cover slide and incubated at 37 ^oC for 30 min to allow the SAP to assembly into nanofibrous scaffolds. The cells were seeded on the SAP hydrogel and incubated at 37 ^oC for 2 h. The hydrogel containing the cells was treated as follows: fixed in 5% glutaraldehyde, dehydrated by gradual ethanol, dried in vacuum and coated with platinum. The SAP scaffolds and cells were viewed using a scanning electron microscope (SEM; Su8010, Hitachi, Tokyo, Japan).

Assessment of cytoprotective effect of SAP

EPCs isolated from young rats were seeded on the SAP hydrogel coated in 96-well plate, and incubated with DMEM supplemented 2% FBS in a sealed anoxia chamber (containing 1% O₂, 5% CO₂ and 94% N₂) for 2 h. Viability of the cells was detected with CCK-8. The apoptotic cells and necrotic cells were stained with ethidium bromide and acridine orange (EB/AO), and counted using a fluorescence microscope. Concentration of VEGF in the supernate was detected with an enzyme-linked immunosorbent assay (ELISA) kit (Lichen, Shanghai, China). The experiments were repeated for six times.

Examination of the sustained release of GDF11 from SAP

10 µL GDF11 solution (10 µg/mL) was mixed with 10 µL SAP solution. Then, the mixed solution was added into 96-well plate and incubated at 37 ^oC for 30 min to allow the SAP hydrogel to encapsule GDF11. Subsequently, 200 µL PBS was added on SAP hydrogel. At 1 d, 3 d, 7 d, 14 d, 21 d and 28 d after incubation, the supernatants were drawn from the samples and replaced with PBS respectively. The concentration of GDF11 in the supernatant was measured with ELISA. The cumulative profile of GDF11 release was plotted. The experiments were repeated for six times.

Abdominal pouch assay

The abdominal pouch mimics the ischaemic and inflammatory tissue, and is convenient for examining oxidative stress-induced senescence of the implanted EPCs. The abdominal pouches of six SD rats (male, 200–250 g) was performed as previously described [33]. Briefly, after the rats were anesthetized with ketamine (80 mg/kg) and xylazine (5–10 mg/kg), the skin of the anterior abdominal wall at the median line was dissected, then the superficial fascia at both sides was bluntly dissected with a forceps to create two pouches. The blood vessels of the pouches were ligated carefully. The rats were divided into SAP and SAP + GDF11 groups. In SAP group, the cells derived from young rats were suspended with the SAP solution. In SAP + GDF11 group, 40 ng/mL GDF11 was mixed with SAP solution. Then, the cells were suspended with the mixed solution. The cells were seeded on poriferous polyethylene terephthalate membranes removed from the transwells (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 12 h. The cell-loaded membranes were implanted into the pouches, the cell side of the membranes was towards abdominal muscles. At 24 h after implantation, the membranes were harvested gently, and the senescent cells were examined with SA-β-gal staining and counted using a phase contrast microscope. Three fields (20 ×) were selected randomly in each membrane of each experiment. The experiment was repeated for three times.

Establishment of MI models and cell transplantation

Rat MI models were established as described previously [34]. Briefly, 44 female SD rats (200–250 g) were anesthetized with ketamine (80 mg/kg) and xylazine (5–10 mg/kg). After endotracheal intubation and ventilation using a rodent ventilator (Harvard Apparatus, Holliston, MA, USA), the heart was exposed through a 2-cm left lateral thoracotomy. The left anterior descending coronary artery (LADCA) was ligated. Successful infarction was determined by observing a pale discoloration in the anterior wall of the left ventricle (LV) and an obvious elevation of ST segment on electrocardiograms. Two rats died during and after LADCA ligation respectively. At 1 week after operation, 42 survived rats were randomly divided into 7 groups: sham, control, SAP, SAP + GDF11, EPCs, SAP + EPCs, SAP + GDF11 + EPCs. 10 µL SAP, 0.1 µg/mL GDF11 and 1 × 10⁶ EPCs derived from young rats were used. Except for sham and control groups, the solutions in the other groups were diluted with PBS to 80 µL. The solutions were injected at the border of the infarcted myocardium through a 30-guage needle at 4 points (20 µL per point). In sham and control groups, the same volume of PBS was injected. For tracing the engrafted cells, the cells were transfected with green fluorescence protein (GFP)-lentivirus (Sangon Biotech, Shanghai, China) at a MOI of 30 for 72 h before transplantation. For maintaining body temperature, the rat was lain on an electric blanket in supine position during surgery.

Echocardiography

To evaluate the cardiac function, echocardiographic examination was performed before MI, and at 1 week after MI and 4 weeks post-transplantation. Echocardiograms of the rats were recorded by an ultrasonocardiograph (Visual Sonics, Toronto, Canada). The M-mode cursor was positioned to the parasternal line at the level of the papillary muscles. LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were measured at least in 3 consecutive cardiac cycles. To evaluate LV systolic function, the ejection fraction (EF) and the fractional shortening (FS) were calculated as following formulae: EF (%) = (LVEDV – LVESV)/LVEDV × 100 and FS (%) = (LVEDD – LVESD)/LVEDD × 100. Two echocardiographers, blinded to the experimental treatment, acquired the images.

Histological examination

At 4 weeks after transplantation, the hearts were harvested and fixed with 4% paraformaldehyde overnight. After gradually dehydrated with 15% and 30% sucrose, the hearts were embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA). Frozen sections were obtained from upper, middle and lower part ofthe hearts, and stained with Masson’s trichrome. The myocardial tissue was stained red, while the scar tissue was stained blue. The scar area was calculated as percentage of the infarct area in LV wall with ImageJ (Wayne Rasband, NIH, USA). The wall thickness of the infarct region was measured at the thinnest part of the infarcted LV.

The microvessels were examined by CD31 immunostaining. Densities of the microvessels in infarct and peri-infarct regions were measured by counting CD31-positive structures. At least 3 independent sections and 5 fields (20 x) on one section were selected randomly. To assess myocardial regeneration after transplantation, the infarct region was examined by cTnT and Cx43 immunostaining. The antibodies used for immunostaining were rabbit anti-GFP antibody (1:100; Santa Cruz Biotech), mouse anti-rat CD31 antibody (1:200; Abcam, Cambridge, MA, USA), mouse anti-rat cTnT antibody (1:200; Santa Cruz Biotech), rabbit anti-rat Cx43 antibody (1:100; Abcam), Alexa Fluor 594-conjugated goat anti-mouse IgG (1:400) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400; Jackson). The antibodies used for immunostaining and Western blotting were listed in Table S1.

Senescence of the cardiomyocytes at peri-infarct region was assessed using SA-β-Gal staining and Sudan black B staining. Following SA-β-Gal staining, the sections were immersed in 70% ethanol for 1 min, and incubated in Sudan black B solution for 30 min at room temperature. Subsequently, the sections were immersed in 70% ethanol for a few times and counterstained with 0.1% nuclear fast red. SA-β-Gal-positive cardiomyocytes and lipofuscin granules were examined using a microscope.

The ultrastructures in the cardiomyocytes at peri-infarct region were examined using a CM120 TEM (Philips).

Identification of the senescent cells in the engrafted EPCs

Senescence of EPCs in the peri-infarct region at 4 weeks after transplantation was determined by combination of GFP immunostaining and SA-β-gal staining. Briefly, the frozen sections of the myocardium were treated with UV irradiation for 15 min, and then SA-β-gal staining of the sections was performed with SA-β-gal staining kit (Genmed Scientifics). GFP immunostaining was performed as above. The GFP⁺SA-β-gal⁺ cells in the survived cells (GFP⁺ cells) were counted using a fluorescence microscope. The experiment was repeated for six times.

Statistical analysis

Statistical analyses were performed with SPSS Statistics 23.0 (SPSS, Chicago, IL, USA), and graphical representations were generated using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). All data were presented as mean values ± standard deviation. Statistical differences between groups were evaluated using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. A P value of < 0.05 was considered statistically significant.

Results

Characteristics and ALK-4 expression of CD34⁺VEGFR-2⁺ EPCs

Flow cytometric analysis revealed that the frequency of CD34⁺VEGFR-2⁺ cells was 2.07% of the mononuclear cells isolated from bone marrow of SD rats (Fig. 1A). The freshly sorted CD34⁺VEGFR-2⁺ EPCs exhibited a round or oval morphology (Fig. 1B). Upon induction with VEGF, the cells demonstrated proliferation and differentiation toward endothelial cells. At 2 weeks after induction with VEGF, the cells displayed a spindle-like or polygon shape, and expressed CD31 (Fig. 1C). Moreover, for clarifying the effect of GDF11 on attenuating EPC senescence, ALK-4 expression in EPCs was detected using immunostaining and Western blotting. The results demonstrated that the levels of ALK-4 expression in EPCs derived from old rats was significantly decreased compared to those from young rats (Fig. 1D–F).

Fig. 1

Rat bone marrow-derived CD34⁺VEGFR-2⁺ EPCs. (A) Analysis of CD34⁺VEGFR-2⁺ EPCs by flow cytometry. (B) CD34⁺VEGFR-2⁺ EPCs (arrows) in the mononuclear cells isolated from bone marrow. Immunostaining. Scale bar = 25 μm. (C) CD31 expression in CD34⁺VEGFR-2⁺ EPCs at 2 weeks after induction with VEGF. Immunostaining. Scale bar = 10 μm. (D, E) ALK-4 expression in EPCs demonstrated with immunostaining and Western blotting respectively. Scale bar = 25 μm. (F) The statistical result of ALK-4 expression. *P < 0.01 versus young group. n = 3

Changes in intracellular ROS production after treatment with GDF11

Senescence of EPCs was induced with H₂O₂. There was no significant difference in cell viability between control group and 100 µM group or 250 µM group. However, compared to 100 µM group or 250 µM group, cell viability in 500 µM group was decreased significantly (Fig. S1). Therefore, 250 µM was selected as the optimal concentration of H₂O₂ for induction of senescence in the cells. At 6 h after induction with 250 µM H₂O₂, ROS production in the cells was increased remarkably. After treatment with GDF11, H₂O₂-induced ROS production was significantly suppressed (Fig. 2A).

Fig. 2

Reduction of ROS production, lipofuscin accumulation and SA-β-gal expression in H₂O₂-induced cells after treatment with GDF11. (A) The levels of ROS. *P < 0.05 and **P < 0.01 versus control group, ^#P < 0.01 versus H₂O₂ group. n = 3. (B) Lipofuscin granules. Sudan black B staining. Scale bar = 10 μm. (C) Autofluorescence intensities of lipofuscin in the cells of young and old groups. Analysis by flow cytometry. The abscissa indicates the number of the cells, while the ordinate indicates autofluorescence intensity of lipofuscin. (D) The statistical result of autofluorescence intensities of lipofuscin. *P < 0.05 versus control group, #P < 0.05 versus H₂O₂ group. n = 4. (E) SA-β-gal-positive cells. SA-β-gal staining. Scale bar = 20 μm. (F) The statistical result of the numbers of SA-β-gal-positive cells. *P < 0.05 versus control group, #P < 0.01 versus H₂O₂ group, &P < 0.05 versus H₂O₂ + GDF11 group of young rats. †P < 0.05 versus H₂O₂ + GDF11 group. n = 3

Changes in lipofuscin accumulation after treatment with GDF11

There were more lipofuscin granules observed in the cells from the old rats in control group. After treatment with H₂O₂, the lipofuscin granules in the cells of from both young and old rats were notably increased. Compared with H₂O₂ group, the presence of the lipofuscin granules in H₂O₂ + GDF11 group were markedly decreased. These lipofuscin granules were primarily located around the nucleus (Fig. 2B). Flow cytometric analysis revealed a significant reduction in H₂O₂-induced lipofuscin accumulation after treatment with GDF 11 (Fig. 2C, D).

Changes of SA-β-gal expression after treatment with GDF11

H₂O₂-induced oxidative stress can cause senescence in the cells, characterized by a flattened and enlarged appearance of SA-β-gal-positive cells. In H₂O₂ + GDF11 group, there were fewer SA-β-gal-positive cells compared to H₂O₂ group. The suppressive effect of GDF11 on senescence in the cells derived from old rats was weaker than that in the cells from young rats (Fig. 2E, F). To assess mediating effect of ALK signalling on GDF 11-induced alleviation of EPC senescence, the cells were treated with SB-431542. Blocking ALK-4 and ALK-5 with their antagonist SB-431542 inhibited the effect of GDF11 on reducing senescence in the cells. This result indicates that ALK-4 and ALK-5 signalling pathways mediate the effect of GDF11.

Enhancement of LC3 expression after treatment with GDF11

The autophagic structures labeled with LC3 immunostaining were round or oval puncta and located around the nucleus (Fig. 3A). In the control group, the number of LC3-posive puncta in the cells from old rats was increased compared to the cells from young rats. However, in the cells of H₂O₂ + GDF11 group, the number of LC3-posive puncta was greater than in H₂O₂ group (Fig. 3B). Furthermore, Western blotting revealed that LC3-II expression in the cells of H₂O₂ + GDF11 group was enhanced compared with H₂O₂ group (Fig. 3C–F). This result shows that GDF11 can enhance autophagy of the oxidative stress-conditioned cells.

Fig. 3

LC3 expression and autophagic structures in GDF11-treated cells. (A) LC3-positive puncta. Immunostaining. Scale bar = 10 μm. (B) The statistical result of LC3-positive puncta. *P < 0.05 versus control group, †P < 0.05 versus H₂O₂ group, #P < 0.05 versus young group. n = 3. (C, D) Western blotting of LC3 in the cells of young and old groups. (E, F) The statistical result of LC3 expression in the cells of young and old groups. *P < 0.05 and **P < 0.01 versus control group, #P < 0.01 versus H₂O₂ group. n = 3

Increase of autophagic ultrastructures in GDF11-treated cells

Representative autophagic ultrastructures in the cells of H₂O₂ group and H₂O₂₊ GDF11 group were shown in Fig. 4. After treatment with GDF11, there was a notable increase in the autophagosomes and autolysosomes, accompanied by a significant reduction in the swollen mitochondria and rough endoplasmic reticula. Compared with H₂O₂ group, the ratios of cross-section areas of the autophagic structures to that of the cytoplasm in H₂O₂₊GDF11 group were significantly greater (Table S2). GDF11-enhanced autophagy can reduce oxidative stress of the cells.

Fig. 4

Representative autophagic ultrastructures in GDF11-treated cells. Arrows indicate the autophagosomes with double membranes or multilayer membranes. Arrowheads indicate the autolysosomes containing an autophagosome with single membrane. In H₂O₂ group, some mitochondria (M) and rough endoplasmic reticula (rER) are swollen. TEM images. N, nucleus. Scale bar = 0.5 μm

Characteristics and cytoprotective effect of the SAP

At 30 min after sonication, the synthesized SAP assembled into nanofibers with a diameter of 5–10 nm and a length of 20–200 nm (Fig. 5A). By 2 h after sonication, the nanofibers had formed scaffolds with apertures ranging from 20 to 400 nm (Fig. 5B). The cells adhered to the nanofibrous SAP scaffolds and exhibited robust spreading (Fig. 5C). In the hypoxia condition, the presence of the apoptotic cells and necrotic cells in SAP hydrogel-incubated cells were significantly decreased compared with control group (Fig. 5D, E). Moreover, cell viability and VEGF production were increased in SAP group (Fig. 5F, G).

Fig. 5

Features and properties of the SAP. (A) The nanofibers assembled by SAP. AFM image. (B) The scaffolds formed from the SAP nanofibers. SEM image. Scale bar = 1 μm. (C) A cell spreading on the SAP nanofibers. Numerous filopodia protrude from the lamellipodium. SEM image. Scale bar = 2 μm. (D) The apoptotic cells (arrows) and necrotic cells (arrowheads) in the cells treated with hypoxia for 2 h. EB/AO staining. Scale bar = 25 μm. (E) The statistical result of the apoptotic cells and necrotic cells. *p < 0.05 versus control group. n = 6. (F) Viability of the hypoxic cells. CCK-8 assay. OD, optical density. *p < 0.001 versus control group. n = 6. (G) Concentration of VEGF in the supernate. *p < 0.01 versus normoxia group, #p < 0.01 versus control group. n = 6. (H) The profile of cumulative GDF11 released from the SAP hydrogel. n = 6

The sustained release of GDF11 from SAP

On the third day after incubation, the cumulative release of GDF11 from the SAP was 55%. Starting from the 7th day, the release of GDF11 slow down. By 28 days, the cumulative GDF11 released form the SAP reached 87% (Fig. 5H). This result shows that release of GDF11 from the SAP continues more than 4 weeks.

Effect of GDF11 on senescence of the cells implanted in the ischemic abdominal pouches

At 24 h after implanting the cell-loaded polyethylene terephthalate membranes into the ischemic abdominal pouches, the number of SA-β-gal-positive cells were significantly decreased in SAP + GDF11 group compared with SAP group (Fig. S2). This result indicates that GDF11 released from the SAP has a suppressive effect on the senescence of the cells in the ischaemic tissue.

Improvement of cardiac function after transplantation of SAP loading EPCs and GDF11

Representative echocardiograms of the LV free walls were shown in Fig. 6A. The echocardiograms revealed that cardiac function in all mice was severely compromised at 1 week post-MI. In the control group, the loss of cardiac function persisted for 4 weeks. At 4 weeks after transplantation, LV contraction in all experimental groups was significantly improved. Compared with SAP + GDF11 group or SAP + EPCs group, LV contraction was dramatically strengthened in SAP + GDF11 + EPCs group. After transplantation, both EF and FS were increased in all groups except the control group, while SAP + GDF11 + EPCs group showed remarkably greater EF and FS compared with SAP + GDF11 group or SAP + EPCs group (Fig. 6B, C).

Fig. 6

The changes in cardiac function at 4 weeks after transplantation. (A) Representative echocardiograms of the LV free wall. (B) Ejection fractions (EF) of LV. (C) Fractional shortening (FS) of LV. *p < 0.05 versus control group, #p < 0.05 versus SAP group, †p < 0.05 versus SAP + GDF11 group, &p < 0.05 versus EPCs group, $p < 0.05 versus SAP + EPCs group. n = 6

Alleviation of adverse remodeling of LV wall after transplantation

The morphological changes of the hearts were shown in Fig. 7A, B. In control group, the myocardium of the infarct region was replaced by fibrous tissue at 4 weeks post-transplantation. There was more myocardium presenting at the infarct region in the other groups. The scar size was significantly smaller in SAP + GDF11 + EPCs group compared with SAP + GDF11 group or SAP + EPCs group. Additionally, the thickness of the LV wall in SAP + GDF11 + EPCs group was greater than in SAP + GDF11 group or SAP + EPCs group (Fig. 7C, D).

Fig. 7

The changes in the cardiac structures at 4 weeks after transplantation. (A, B) The morphological changes of LV walls. The panels in A show the transverse sections of the ventricles at the widest part of the infarct region. The panels in B show the transverse sections of the infarct regions. Masson’s trichrome staining. Scale bar = 2 mm. (C) The statistical result of scar size. (D) The statistical result of the thickness of the LV wall at the infarct region. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ##p < 0.01 versus SAP group, †p < 0.05 and ††p < 0.01 versus SAP + GDF11 group, &p < 0.05 and &&p < 0.01 versus EPCs group, $p < 0.05 and $$p < 0.01 versus SAP + EPCs group. n = 6

Neovascularization and myocardial regeneration after transplantation

At 4 weeks after transplantation, neovascularization at the peri-infarct region was assessed by counting CD31⁺ microvessels. Compared with control group, the density of the microvessels was increased in the other groups. Notably, the number of the microvessels in SAP + GDF11 + EPCs group was significantly greater than SAP + GDF11 group or SAP + EPCs group (Fig. 8A, B). There were more GFP-positive EPCs presenting at the infarct region in SAP + GDF11 + EPCs group than SAP + EPCs group. GFP⁺CD31⁺ cells are regarded as the endothelial cells differentiated from the engrafted EPCs. We observed some GFP⁺CD31⁺ cells incorporated into the wall of the microvessels (Fig. 8C). Compared with control group, there was more myocardium at the infarct region of the other groups, especially SAP + GDF11 + EPCs group. Cx43 was expressed at the junction between cardiomyocytes. There were more Cx43⁺ junctions in SAP + GDF11 + EPCs group (Fig. S3). The junction between cardiomyocytes is structural basis for synchronous contraction of the myocardium.

Fig. 8

The microvessels at the peri-infarct region at 4 weeks after transplantation. (A) The microvessels. White dash lines indicate the epicardium. CD31 immunostaining. Scale bar = 50 μm. (B) The statistical result of the number of the microvessels. *p < 0.05 and **p < 0.01 versus control group, #p < 0.05 and ##p < 0.01 versus SAP group, †p < 0.01 versus SAP + GDF11 group, &p < 0.01 versus EPCs group, $p < 0.01 versus SAP + EPCs group. n = 6. (C) CD31-positive cells differentiated from the engrafted EPCs (GFP-positive cells) at infarct region. GFP⁺CD31⁺ cells (arrows) are located at the wall of the microvessels. In the panels of the third row, the large boxes are magnification of the small boxes. White dash lines indicate the epicardium. Immunostaing. Scale bar = 50 μm. n = 6

Effect of GDF11 on senescence of the transplanted cells in the infarcted myocardium

At 4 weeks after transplantation, the number of senescent cells (GFP⁺SA-β-gal⁺ cells) in the survived cells (GFP⁺ cells) at the peri-infarct region were significantly decreased in SAP + GDF11 + EPCs group compared with EPCs group or SAP + EPCs group (Fig. 9). This result indicates that GDF11 released from the SAP has a suppressive effect on the senescence of the transplanted cells in the infarcted myocardium.

Fig. 9

The senescent cells in the transplanted cells at peri-infarct region at 4 weeks after transplantation. (A) GFP⁺SA-β-gal⁺ cells in the survived cells (GFP⁺ cells). Immunostaining and SA-β-gal staining. Arrows indicate GFP⁺SA-β-gal⁺ cells. Scale bar = 50 μm. (B) The statistical result of the number of GFP⁺SA-β-gal⁺ cells. **p < 0.01 versus EPCs group, ##p < 0.01 versus SAP + EPCs group. n = 6

Effect of GDF11 on senescence of the cardiomyocytes after transplantation

At 4 weeks after transplantation, some mitochondria were swollen or broken in the cardiomyocytes at the peri-infarct region in control group. In SAP + GDF11 + EPCs group, swollen and broken mitochondria were decreased, the autophagic structures increased. The representative ultrastructures in the cardiomyocytes of control and SAP + GDF11 + EPCs groups were shown in Figure S4. Compared with control group, SA-β-Gal⁺ cardiomyocytes in SAP + GDF11 + EPCs group were decreased. Moreover, the lipofuscin particles were decreased (Fig. S5). These results show that GDF11 released from SAP may alleviate senescence of the cardiomyocytes via enhancing autophagy of the cel1s.

Discussion

In this study, we demonstrate that GDF11 has a suppressive effect on oxidative stress-induced senescence of bone marrow-derived EPCs, regardless of whether they are derived from young or old rats. Furthermore, we found that the effect of GDF11 on suppressing EPC senescence is mediated by ALK-4 and ALK-5 signalling. Interestingly, in EPCs derived from old rats, the decline in ALK-4 expression contributes to the weaker suppressive effect of GDF11 on the cellular senescence. In ischaemic abdominal pouch models, we found that the SAP-loaded GDF11 significantly reduces the senescence of the engrafted EPCs. After transplantation of the SAP loaded EPCs and GDF11 into the ischeamic myocardium, the sustained release of GDF11 from the SAP effectively alleviates EPC senescence. This, in turn, enhances neovascularization and myocardial regeneration, attenuates reverse myocardial remodeling and improves cardiac function. While transplantation at the border of the infarcted myocardium may avoid severe apoptosis and necrosis of the stem cells, it is possible that the transplanted cells in the ischaemic and inflammatory microenvironment still endure the implication of oxidative stress. The senescent state in age-related myocardium may also be induced by oxidative stress and metabolic dysfunction [35]. Recent research suggests that inhibition of oxidative stress with anti-oxidants can delays senescence of EPCs in a cell culture model of chronological ageing [36]. In addition to neovascularization, myocardial regeneration is enhanced in our experiment. Proliferation of the cardiomyocytes and differentiation of the recruited cardiac stem cells and bone marrow stem cells towards cardiomyocytes could be involved in myocardial regeneration post-MI [37, 38]. In our experiment, the autophagic structures in the cardiomyocytes at the peri-infarct region are increased after transplantation for four weeks. Moreover, SA-β-Gal⁺ cardiomyocytes and lipofuscin particles are decreased. Therefore, we suggest that GDF11 released from SAP may alleviate oxidative stress-induced senescence of the cardiomyocytes.

The results of this study demonstrate that cellular autophagy is involved in GDF11-mediated reduction of oxidative stress-induced senescence in EPCs. After treatment with GDF11, LC3-II expression is upregulated, and LC3-positive puncta and autophagic ultrastructures in oxidative stress-conditioned EPCs are increased significantly. Concurrently, the presence of the swollen mitochondria and rough endoplasmic reticula is notably diminished, and there is a dramatic reduction in lipofuscin accumulation. Autophagy, an evolutionarily conserved process, maintains cell survival by degrading organelles and cytoplasmic proteins in response to various forms of stress such as ischemia, reperfusion and inflammation [39]. Based on the pathways through which cargos are delivered into lysosomes, autophagy is categorized into macroautophagy (here referred to as autophagy), microautophagy and chaperone-mediated autophagy [40]. Autophagy plays important roles in maintaining the metabolism, stemness and functions of stem cells by preventing oxidative stress-induced senescence [41, 42]. Our previous study suggests that autophagy protect EPCs from apoptosis in hypoxic condition [43]. Activating autophagy by pretreatment with hypoxia or rapamycin enhances the survival of the engrafted stem cells [24, 44]. Moreover, enhancing autophagy reduces lipofuscin accumulation and mitigates cardiomyocytic senescence [45]. Interestingly, GDF11 has been shown to diminish the activity of mammalian target of rapamycin (mTOR), a key regulator of autophagy [46]. Therefore, this study suggests that activating autophagy with GDF11 represents a promising strategy for enhancing the survival, proliferation and differentiation of the engrafted stem cells.

Our experimental data indicate that the SAP, possessing dual properties of promoting EPC adhesion and sustainedly releasing GDF11, effectively promotes neovascularization in the infarcted myocardium. Acute MI inflicts massive injury to the coronary microcirculation leading to vascular disintegration and capillary rarefication in the infarct region [47]. Angiogenesis is crucial for salvaging at-risk cardiomyocytes and enhancing the survival of donor stem cells by improving the blood circulation in the infarct area [48]. Mimicking the ECM, SAP hydrogel facilitates vascular cell recruitment and vascularization [49]. In this study, the SAP hydrogel was found to protect EPCs from apoptosis and necrosis in the hypoxic condition. Moreover, the viability and VEGF production of the cells in SAP were increased. These results demonstrate that the cell-adhering motif of the SAP promotes the adhesion, proliferation and differentiation of EPCs. After transplantation into the infarcted myocardium, neovascularization was significantly enhanced in both SAP-loaded EPCs group and SAP alone group. At 4 weeks after transplantation into the myocardium, the designer SAP is mostly degraded [50]. Following degradation of SAP, GDF11 is sustainedly released. Duration of GDF11 release is approximately accorded with process of myocardial remodeling. The local sustained release of GDF11 by SAP hydrogel may offer a more effective means of delivering a higher and sustained concentration of GDF11, which is beneficial for alleviating the senescence of the engrafted stem cells in the ischaemic tissues.

In this study, the senescence of the cells was determined by SA-β-gal expression and lipofuscin accumulation. The assessments of cell-cycle, telomere length and telomerase expression may also be used in determination of the cellular senescence. Although the techniques for identifying and characterizing of the senescent transplanted stem cells in the infarcted myocardium are limited, it is promising to develop new methods for exploring the spatio-temporal changes of the senescence in the stem cells after transplantation. Moreover, oxidative stress-induced senescence in the transplanted stem cells would be investigated using the ischemia/reperfusion MI models established with the old rats in further study.

Conclusions

We provide novel evidence suggesting oxidative stress may induce senescence of the transplanted EPCs in ischemic and inflammatory tissues. Our study indicates that GDF11 can attenuate oxidative stress-induced senescence of EPCs in the myocardium via activating autophagy. Moreover, GDF11 released from SAP may alleviate senescence of the cardiomyocytes. Loading EPCs and releasing GDF11 with the SAP is effective for repairing of the infarcted myocardium after transplantation. In addition to apoptosis and necrosis, oxidative stress-induced senescence is one of the fates of the transplanted stem cells. Therefore, optimizing the approaches of stem cell transplantation is crucial for maintaining survival and preventing senescence of the engrafted cells.