Human amniotic mesenchymal stem cells and their paracrine factors promote wound healing by inhibiting heat stress-induced skin cell apoptosis and enhancing their proliferation through activating PI3K/AKT signaling pathway
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Increasing evidence has shown that mesenchymal stem cells (MSCs) yield a favorable therapeutic benefit for thermal burn skin wounds. Human amniotic MSCs (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammatory potential which makes them suitable for treating skin wounds. However, the exact effects of hAMSCs on the healing of thermal burn skin wounds and their potential mechanisms are not explored.
hAMSCs were isolated from amniotic membrane and characterized by RT-PCR, flow cytometry, immunofluorescence, and tumorigenicity test. We assessed the effects of hAMSCs and hAMSC conditional medium (CM) on wound healing in a deep second-degree burn injury model of mice. We then investigated the biological effects of hAMSCs and hAMSC-CM on the apoptosis and proliferation of heat stress-injured human keratinocytes HaCAT and dermal fibroblasts (DFL) both in vivo and in vitro. Next, we explored the underlying mechanisms by assessing PI3K/AKT and GSK3β/β-catenin signaling pathways in heat injured HaCAT and DFL cells after hAMSCs and hAMSC-CM treatments using PI3K inhibitor LY294002 and β-catenin inhibitor ICG001. Antibody array assay was used to identify the cytokines secreted by hAMSCs that may activate PI3K/AKT signaling pathway.
Our results showed that hAMSCs expressed various markers of embryonic stem cells and mesenchymal stem cells and have low immunogenicity and no tumorigenicity. hAMSC and hAMSC-CM transplantation significantly promoted thermal burn wound healing by accelerating re-epithelialization with increased expression of CK19 and PCNA in vivo. hAMSCs and hAMSC-CM markedly inhibited heat stress-induced apoptosis in HaCAT and DFL cells in vitro through activation of PI3K/AKT signaling and promoted their proliferation by activating GSK3β/β-catenin signaling. Furthermore, we demonstrated that hAMSC-mediated activation of GSK3β/β-catenin signaling was dependent on PI3K/AKT signaling pathway. Antibody array assay showed that a panel of cytokines including PAI-1, C-GSF, periostin, and TIMP-1 delivered from hAMSCs may contribute to the improvement of the wound healing through activating PI3K/AKT signaling pathway.
Our results demonstrated that hAMSCs and hAMSC-CM efficiently cure heat stress-induced skin injury by inhibiting apoptosis of skin cells and promoting their proliferation through activating PI3K/AKT signaling pathway, suggesting that hAMSCs and hAMSC-CM may provide an alternative therapeutic approach for the treatment of skin injury.
KeywordsHuman amniotic membrane mesenchymal stem cells Conditioned medium Wound healing PI3K/AKT signaling Antibody array
Fluorescence-activated cell sorting
Human amniotic epithelial stem cells
Human amniotic mesenchymal stem cells
High-glucose Dulbecco’s modified Eagle’s medium
Mesenchymal stem cells
Reverse transcription-polymerase chain reaction
Skin is considered one of the most vital organs in the body due to its important functions such as an outer protective barrier against various external agents and a temperature regulator . The serious consequences of cutaneous wound, both acute and chronic, can be caused by many different factors. Cutaneous wound healing requires well-coordinated responses of inflammation, cell proliferation, neovascularization, extracellular matrix formation, and re-epithelialization [2, 3]. With a high rate of morbidity and mortality, skin burn is not only difficult to treat, but also poses a major public health burden worldwide . Thermal burns can be caused by dry sources (fire or flame) and wet sources (scalds) and classified based on the depth of burn . Compared to other wounds, thermal burn wounds are characterized by delayed wound healing because of edema, bacterial infection, chronic inflammation and necrosis . Lack of autologous skin sources or immunological rejection of allogeneic skin brings about the dilemma of clinic treatment of thermal burn . Therefore, developing new and effective wound healing therapies, especially for thermal burn wounds, is urgent.
Recently, stem cell therapy, especially using MSCs, has emerged as a promising new and effective therapeutic strategy for accelerating cutaneous wound healing , and this protective effect was predominantly mediated by paracrine rather than direct regenerative mechanisms . Numerous studies have shown that MSCs promote cutaneous wound healing by accelerating wound closure [9, 10], enhancing angiogenesis [11, 12], inhibiting the inflammatory response [13, 14], regulating extracellular matrix remodeling [15, 16], inhibiting cell apoptosis, and promoting cell proliferation . Meanwhile, MSCs have been studied as one of the more promising therapies for the healing of the thermal burns [7, 18, 19, 20]. Recently, human amniotic membrane-derived mesenchymal stem cells (hAMSCs) have been recognized as one of the most promising stem cells in the field of regenerative medicine. The active proliferative potential, low immunogenic profile, anti-inflammatory function , and tissue repair ability of hAMSCs can be beneficial to the burn wound healing as well as the severe systemic effects of burn, including hypermetabolic response, inflammation-related diseases, and immunosuppression . Anna et al. have previously found that conditioned medium derived from MSCs could enhance normal skin fibroblast proliferation and migration and promote wound healing in an excisional full-thickness skin murine model . Similarly, MSC-derived exosomes have been proved to contribute to thermal burn wound healing . Therefore, it is suggested that the effect of MSCs on the wound healing was mediated via a paracrine signaling mechanism. However, the exact effects of hAMSCs on the thermal burn wound healing and their potential mechanism are not explored.
In the present study, we isolated hAMSCs from human amniotic membrane and characterized their morphology, phenotypic profiles, pluripotency, tumorigenicity, and growth potency. In vivo, we investigated the role of hAMSCs in thermal burn wound healing using a mouse deep second-degree burn injury model. We found that hAMSC and hAMSC-CM transplantation promoted skin wound healing by enhancing proliferation and inhibiting apoptosis of skin cells in the wound area. In vitro, a transwell co-culture system and hAMSC-CM were used to assess the influences of hAMSC-secreted factors on the apoptosis, proliferation, and migration of heat injury skin cells. The results showed that hAMSCs or hAMSC-CM inhibited heat stress-induced apoptosis in HaCAT and DFL cells and promoted their proliferation through activation of PI3K/AKT signaling pathway with a paracrine manner. In addition, antibody array assay showed that hAMSCs could deliver many cytokines including PAI-1, C-GSF, periostin, TIMP-1, uPAR, and so on, which may activate PI3K/AKT signaling pathway.
Materials and methods
Isolation, culture, and expansion of hAMSCS
Human fetal placentas were obtained from the Department of Obstetrics and Gynecology, The First Affiliated Hospital of Nanchang University. The verbal consent was obtained from all of the volunteers prior to their participation. The research procedure was approved by the ethics committee of The First Affiliated Hospital of Nanchang University. The amnion is a thin, avascular membrane composed of human amniotic epithelial stem cells (hAESCs) and hAMSCs. For isolation of hAMSCs, hAESCs were firstly released from the amniotic membrane as previously described . Then, the amnion was washed three times with HBSS and digested with Collagenase IV(1 g/L, Thermo Fisher, Nanchang, China) on a rotator 40 min at 37 °C. Digestion was terminated by addition of medium containing 10% FBS; the single-cell suspension was filtered through a 70-μm cell strainer (BD Labware, Shanghai, China) and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were re-suspended with α-MEM medium (Thermo Fisher) containing 18% Chang B, 2% Chang C (Irvine Scientific), 10% FBS, 1% glutamine, and 1% penicillin/streptomycin (Gibco). hAMSCs were placed in cell culture dishes (Corning, NY, USA) at a density of 5 × 104 cells/cm2 at 37 °C with 5% CO2 atmosphere. Unattached cells and debris were removed after 2 days. In each experiment, the cells were grown to approximately 80% confluence, and only cells between passages 3 and 7 were used for subsequent experiments.
Collection of conditioned medium (CM) of hAMSCS
For the collection of hAMSC-CM, hAMSCs were grown in a normal culture medium. Once the cells reached 80% confluency, the medium was changed to high-glucose Dulbecco’s modified Eagle’s medium (H-DMEM, Thermo Fisher) containing 100 U/ml penicillin/streptomycin. CM was collected after 48 h and centrifuged at 1500 rpm for 5 min to ensure complete removal of cellular debris. CM was then concentrated 10-fold by using an Amicon® Ultra 3 K device (MilliporeSigma, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
Primers and conditions used for RT-PCR to detect gene transcripts in hAMSCs
Identification of hAMSCs by flow cytometry
Phenotypic analyses of cultured hAMSCs were performed using standard flow cytometry methods. Passage 3 hAMSCs were collected in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lakes, NJ) at a concentration of 1 × 106 cells/ml in stain FACS buffer (PBS containing 2% FBS) and then stained with FITC-conjugated antibodies against human CD29, CD90, CD45, HLA-DR, CD80, and CD40; phycoerythrin (PE)-conjugated antibodies against human CD73, CD105, CD34, HLA-ABC, and CD86; and their isotype controls (all from BD Biosciences) at 4 °C for 30 min in the dark. After washing twice, the cells were resuspended in 200 μl of PBS and acquired by a FACSCalibur instrument (BD Biosciences). Data were analyzed using FLOWJO TM software (TreeStar, Inc., Ashland, OR, USA).
Immunofluorescence experiments were carried out following our previously reported protocols . Briefly, cells growing on the glass slide were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 diluted in PBS for 10 min at room temperature. To block unspecific epitopes, cells were incubated with PBS containing 1% BSA and 0.1% Tween-20 for 1 h. Cells were then incubated with the following primary antibodies at 4 °C overnight: rabbit anti-OCT4 (5 μg/ml, Abcam, Nanchang, China), mouse anti-SSEA-4 (15 μg/ml, Abcam), rabbit anti-Nanog (1:200, Abcam), rabbit anti-Ki67 (1:100, Abcam), and mouse anti-PCNA (5 μg/ml, Abcam). After that, cells were incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 568 (Jackson, Nanchang, China). Nuclei were counterstained with DAPI (Thermo Fisher).
Adipogenic and osteogenic differentiation
Passage 3 hAMSCs were seeded at a density of 1.5 × 105 cells/well in a six-well plate. When the cells reached 100% confluence, OriCell™ human mesenchymal stem cell adipogenic differentiation medium (Cyagen Biosciences, Shanghai, China) was added to wells according to the manufacturer’s instruction. After 24 days of induction, Oil red O (Cyagen Biosciences) staining was performed to assess the differentiation potential of adipogenesis formation of intracellular lipid droplets. For osteogenic differentiation, hAMSCs were cultured with OriCell™ human mesenchymal stem cell osteogenic differentiation medium (Cyagen Biosciences) for 23 days to analyze the osteogenic differentiation. The differentiation potential for osteogenesis was assessed by Alizarin Red (pH 4.2, 40 mM) (Cyagen Biosciences) staining.
Soft agar tumorigenicity test
Each well of the 6-well culture plates was first coated with 0.6% soft agar (bottom layer). A 0.3% soft agar containing hAMSCS (1 × 103/well) was then layered on top of the 0.6% gel and incubated at 37 °C with 5% CO2 for 30 days. Human liver carcinoma cell HepG2 was used as the control. Colony formation was observed and imaged by phase-contrast microscopy.
In vivo tumorigenicity test
To test the tumorigenicity of hAMSCS in vivo, 5 × 106 hAMSCs (in 200 μl PBS) were injected into the left thigh muscle and the right back of NOD-SCID mice, respectively. The same number of embryonic stem cells was used as positive control. The tumor-forming was monitored every day for up to 20 weeks.
In vivo skin wound model and treatment
Adult male C57BL/6 mice (8 weeks old) were purchased from Changsha SLAC Laboratory Animal Company (Changsha, China, http://www.hnsja.com/) and housed under standard laboratory conditions with standard chow and water daily at the Laboratory Animal Center of Institute of Translational Medicine of Nanchang University. All animal procedures described here were reviewed and approved by the Animal Care and Use Committee of Nanchang University. Mice were anesthetized, and back cutaneous hair was removed by electrical shaving. The back skin of mice was injured with 80 °C water for 100 s to create a 10-mm diameter wound. Meanwhile, 200 μl PBS, 2 × 106 hAMSCs suspended in 200 μl PBS, 200 μl H-DMEM (10X), or 200 μl hAMSC-CM (10X) were injected subcutaneously near the wound at four sites. The normal group had no treatment.
Whole-body fluorescent imaging
For the purpose of cell tracking, hAMSCs were labeled with PKH26 red fluorescent dye (Sigma-Aldrich) and then injected subcutaneously near the wound at four sites. Mice were anesthetized after 0 days, 7 days, 14 days, and 21 days of cell injection and visualized with whole-body fluorescent imaging system (LB983; Berthold, Germany).
Histopathology and TUNEL assay
Skin tissue samples of all groups were excised and fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5-μm thickness, and mounted on slides. The slides were deparaffinized and stained with hematoxylin and eosin (H&E), PCNA (1:1000, mouse monoclonal, Abcam), CK19 (1:1000, mouse monoclonal, Abcam), Anti-Human Nuclei Antibody MAB1231 (1:200, mouse monoclonal, Merck), CD90 (1:250, rabbit monoclonal, Abcam), and CD31 (1:100, mouse monoclonal, Abcam).
Apoptosis was analyzed on paraffinic skin tissue sections of different group by TUNEL assay kit (Millipore, USA). Three sections were selected for each mouse and stained using the TUNEL assay kit following the manufacturer’s protocol.
Western blot analysis
Total protein was extracted from HaCAT cells, DFL cells, and skin tissues. Western blot was performed to detect the target proteins. Sixty micrograms of total protein was run on 10% denaturing SDS-PAGE gels, then transferred to nitrocellulose membranes (BioRad), which were incubated with primary antibodies anti-GAPDH (1:1000, rabbit monoclonal, Santa Cruz), anti-β-actin (1:1000, mouse polyclonal, CST), anti-Bcl-2 (1:1000, mouse monoclonal, Abcam), anti-Bax (1:1000, mouse monoclonal, Abcam), anti-PCNA (1:1000, mouse monoclonal, Abcam), anti-CK19 (1:1000, mouse monoclonal, Abcam), PI3K (1:1000, rabbit polyclonal, CST), P-PI3K (1:1000, rabbit polyclonal, CST), anti-AKT (1:1000, mouse monoclonal, Abcam), anti-P-AKT (1:1000, mouse monoclonal, Abcam), mTOR (1:1000, rabbit polyclonal, CST), P-mTOR (1:1000, rabbit polyclonal, CST), anti-β-Catenin (1:1000, mouse monoclonal, Abcam), anti-GSK3β (1:1000, mouse monoclonal, Abcam), and anti-P-GSK3β (1:1000, mouse monoclonal, Abcam) at 4 °C overnight. Blots were detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibody (Invitrogen) for 1 h at room temperature. Images were quantified using the Super Signal West Pico or Femto chemiluminescent detection system (Pierce).
Tube formation assay
Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC and cultured following the manufacturer’s instructions. Matrigel Basement Membrane Matrix (BD Biosciences, CA, USA) was added in 48-well plates (130 μl per well) and solidified at 37 °C for 1 h. Then, 2.0 × 104 HUVECs per well were seeded and cultured with normal medium or normal medium supplemented with 10% hAMSC-CM (10X). After incubating at 37 °C and 5% CO2 for 2 h, 4 h, and 6 h, the tube formation was detected under a microscope.
In vitro co-culture experiment
Keratinocytes HaCAT cells were purchased from ATCC and cultured in H-DMEM supplemented with 15% FBS and 1% penicillin/streptomycin (all from Thermo Fisher) at 37 °C in a 5% CO2 humidified atmosphere. DFL were isolated from discarded circumcised foreskin on healthy boys as previously described [23, 24] and cultured in H-DMEM containing 15% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. DFL cells between passages 3 to 5 were used for subsequent experiments.
Skin cells (HaCAT and DFL) were trypsinized and seeded in a 6-well dish at 1.5 × 105 cells/well. To mimic the burn injury model in vivo, cells were treated at 43 °C for 50 min in a water bath. For the normal medium (NM) group, cells were incubated with 3 ml H-DMEM containing 15% FBS and 1% penicillin/streptomycin. For hAMSCs group, a co-culture transwell chamber (2.4-cm diameter, 0.4-μm pore size; Corning) was used to assess the effects of hAMSCs on heat stress-injured skin cells in vitro. Skin cells were seeded into the lower chamber in 2.0 ml of H-DMEM with 15% FBS, and hAMSCs were seeded in the upper compartment at a 1:1 ratio with skin cells in 1.0 ml of the same medium. For the hAMSC-CM group, cells were cultured with 3 ml H-DMEM supplemented with 10% hAMSC-CM (10X), 15% FBS, and 1% penicillin/streptomycin. Skin cells cultured in the normal medium not treated at 43 °C for 50 min were used as the control group. Samples were collected after culturing for 24 and 48 h.
In vitro skin cells proliferation and apoptosis analysis
Cell proliferation was evaluated at indicated time points using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s protocol. CCK-8 reagent (10%) was added to each well for 3 h at 37 °C. Viability was evaluated by measuring the absorbance at a 450-nm wavelength with using a microplate spectrophotometer (BioRad).
For the apoptosis assays, 1.0 × 105 cells were collected from each sample and resuspended in 100 μl Annexin V binding solution containing 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) solution (Dojindo). After incubation for 15 min at room temperature, cells were washed in PBS, centrifuged at 1000 rpm for 5 min, and resuspended in 400 μl Annexin V Binding Buffer. The apoptosis assays were run and analyzed with BD Jazz.
In vitro scratch-wound-closure assay
HaCAT and DFL cells were seeded into 6-well dishes at a density of 1.5 × 105 cells per well. When the cells reached 100% confluency, the monolayers were scratched using a sterile 200 μl pipette tip. After disruption, monolayers were gently washed twice with PBS to remove cell debris. Subsequently, the cells were treated with PBS, hAMSCs, or hMBSC-CM. The dishes were incubated at 37 °C in a 5% CO2 air atmosphere for 48 h. Images were acquired at 48-h time points, and the migration area of HaCAT and DFL cells were measured by using Image Pro Plus 6.0 software.
Cytokine antibody array
We collected 3 hAMSC-CM samples; the profiles of cytokines secreted by hAMSCs were detected in the culture supernatants using a Human Cytokine Array (RayBiotech, Guangzhou, China) according to the manufacturer’s instructions.
The results are presented as average value ± standard deviation (SD). Student’s t test was used for analysis between two groups. One-way analysis of variance (ANOVA) was used to compare data among three or more groups. Differences with a P value of < 0.05 were considered statistically significant.
Identification and characterization of hAMSCs
Soft agar colony formation assay provides an ideal tool for identifying the tumorigenicity of hAMSCs in vitro. After 30 days of cell growth in the soft agar, many colonies were visible in Hepg2 group but no colony was observed in the hAMSCs group at the same time (Additional file 1: Figure S1A). In addition, we injected the hAMSCs and embryonic stem cells (positive control) into the left thigh muscle and right back of NOD-SCID mice. The results showed that large tumors were formed in all mice implanted with embryonic stem cells within 8 weeks (n = 5). In contrast, no tumor formation in any of the hAMSC-injected animals over a time period of 20 weeks (Additional file 1: Figure S1B).
hAMSC and hAMSC-CM transplantation accelerates skin wound healing in a mouse model
hAMSCs and hAMSC-CM inhibited heat stress-induced apoptosis and promoted proliferation of HaCAT and DFL cells in vitro
hAMSCs and hAMSC-CM promoted heat-injured skin cell migration and wound closure
hAMSCs and hAMSC-CM activate PI3K/AKT/mTOR and GSK3β/β-catenin pathway in heat-injured skin cells
Next, the role of β-catenin and AKT activation in hAMSC-mediated promotion of cell proliferation was examined. As shown in Fig. 7g, ICG001 significantly inhibited the increase of β-catenin and PCNA expression by hAMSCs. This result revealed that hAMSCs promoted the proliferation of heat-injured HaCAT and DFL cells by increasing the expression of β-catenin. Considering that PI3K/AKT and GSK3β/β-catenin were both activated by hAMSCs, we investigated the relationship between PI3K/AKT and GSK3β/β-catenin after hAMSC treatment. Inhibition of AKT by LY294002 significantly inhibited the phosphorylation of GSK3β and the increase of β-catenin and PCNA expression, suggesting that hAMSCs mediate the activation of GSK3β/β-catenin signaling dependent of PI3K/AKT signaling (Fig. 7h).
A panel of cytokines from hAMSCs might contribute to the acceleration of the thermal burn wound healing
Stem cells have been considered a promising source of seed cells for biological therapeutics and tissue engineering. However, in terms of clinical applications, the safety, immunological rejection, and ethical criteria are strictly required. The amniotic membrane, a medical waste after birth, has been reported to contain a population of multipotent stem cells exhibiting characteristics of MSCs. However, less effort has been made on hAMSCs. In the present study, we report that hAMSCs can be easily isolated from the donor’s amnion without ethical concerns and showed fibroblast-like morphology. hAMSCs express numerous markers such as the core pluripotency genes (OCT4, SSEA-4, and Nanog) and MSC-specific surface markers (CD29, CD73, CD105, and CD29), but the absence of hematopoietic markers (CD133, CD34, CD45) and HLA Class II (HLA-DR). Our results have also shown that the hAMSCs have low expression of HLA Class I (HLA-ABC) but none of its co-stimulatory molecules CD80, CD86, and CD40, suggesting that there are a weak immunogenicity and potential immune tolerance after transplantation of hAMSCs. In addition, we also found that hAMSCs have multi-lineage differentiation potentials and no tumorigenicity both in vivo and in vitro. These characteristics make hAMSCs as a promising source of stem cells for clinical application.
Several studies showed that MSCs derived from different tissues, such as bone marrow (BM) [14, 20], umbilical cord (UC) , and adipose , are capable of enhancing and improving wound healing in vivo and in vitro. Interestingly, it has been well-documented that the paracrine factors such as growth factors, cytokines, and exosome of stem cells contribute to the therapeutic effect [67, 68]. Anna et al. demonstrated that UC-derived MSCs enhanced normal skin fibroblast proliferation and migration and promoted wound healing in an excisional full-thickness skin murine model through paracrine signaling . Zhang et al. found that MSC-derived exosome promoted proliferation and inhibited apoptosis of skin cells after heat stress in vitro . In the present study, we reported that hAMSCs and hAMSCs-CM significantly promoted thermal burn wound healing. When the skin on the back of mice is injured with 80 °C water for 100 s, the skin cells will undergo apoptosis in a short time (within a few days). Our study showed that in vivo transplantation of hAMSCs and hAMSC-CM significantly enhanced re-epithelialization and accelerated wound closure by inhibiting apoptosis and enhancing proliferation of heat-injured skin cells in the wound area in vivo. However, hAMSCs and hAMSC-CM had no significant differences in the wound healing. Therefore, we believe that the critical period of hAMSCs and hAMSC-CM in the treatment of skin scald is the first few days after injury. Thus, despite the limited duration of the action of hAMSC-CM, it still has a similar therapeutic effect as hAMSCs. Although our results showed that hAMSCs could still be found in heat-injured skin wounds 14 and 21 days after cell injection, the numbers of hAMSCs on day 14 or 21 were significantly reduced compared with that of day 7 (Fig. 2c), indicating that the secretion of cytokines might be also significantly reduced and their concentration might be insufficient to significantly promote skin cell proliferation and wound injury repair. Meanwhile, we also demonstrated that hAMSCs and hAMSCs-CM inhibited heat stress-induced apoptosis in skin cells, promoted their proliferation, and increased their migration in a paracrine manner in vitro. To our knowledge, this is the first time to report that hAMSCs and hAMSC-derived factors have the ability to inhibit heat stress-induced apoptosis of HaCAT and DFL cells and promoted proliferation and accelerated wound closure in vitro and in vivo.
The serine/threonine kinase AKT is an important component of the PI3K signaling pathway; active AKT controls many cellular functions, including cell growth, survival, and cell metabolism . Wnt/β-catenin signaling plays an important role in embryonic patterning, cell proliferation, differentiation, and angiogenesis . Axin serves as a scaffold protein to recruit GSK3β and CKIα (caspase kinase alpha) along with APC to form a complex with beta-catenin, resulting in beta-catenin phosphorylation, ultimately causing its degradation . The phosphorylation of GSK3β is a classic negative regulator of Wnt signaling pathway. Many investigators have described that PI3K/AKT and GSK3β/β-catenin signaling play a key role in skin development and cutaneous wound healing [26, 33, 72]. Our results show that hAMSCs and hAMSC-CM significantly reversed heat stress-induced decline of phosphorylation of PI3K, AKT, GSK3β, and β-catenin expression levels in HaCAT and DFL cells, indicating that activations of PI3K/AKT and GSK3β/β-catenin signaling pathways might be involved in the therapeutic effects of hAMSCs and hAMSC-CM on the heat-injured wound healing in skin cells. Furthermore, we demonstrated that hAMSC- or hAMSC-CM-mediated activation of GSK3β/β-catenin signaling was dependent on PI3K/AKT signaling since the inhibition of hAMSCs or CM on apoptosis could be reversed by PI3K inhibitor LY294002, but not β-catenin inhibitor ICG001 although ICG001 significantly inhibited the hAMSC-mediated increase of PCNA expression. All of these results indicated that hAMSCs and hAMSC-CM reversed acute thermal injury-induced apoptosis and growth inhibition in skin cells through activation of PI3K/AKT signaling pathway.
To identify the hAMSC-secreted molecules involved in the activation of PI3K/AKT pathway, the cytokines of hAMSC-CM were analyzed using an antibody array. Our results showed that hAMSC-CM contained high levels of PAI-1, C-GSF, periostin, and TIMP-1. PAI-1 is the most abundant factor in the hAMSC-CM. Harman et al. found that MSC-derived PAI-1 significantly increased DFL migration in vitro and improved wound healing in vivo by decreasing time to wound closure . Lademann et al. found PAI-1 protects fibrosarcoma cells from etoposide-induced apoptosis through activation of PI3K/AKT cell survival pathway . G-CSF, a hematopoietic cytokine and potent stem cell mobilization agent, has been proved to accelerate wound healing by enhancing angiogenesis and attenuating apoptosis , and PI3K/AKT signaling pathway would be activated in response to G-CSF stimulation . Periostin, one of the matricellular proteins, is normally expressed in adult skin, which is highly upregulated during wound healing . Periostin has the ability to activate the PI3K/AKT signaling pathway in tumor cells by interacting with integrin molecules . Recently, increasing evidence suggested that periostin is capable of dramatically increasing the migratory and proliferative abilities of epithelial cells and dermal fibroblasts by upregulating AKT/mTOR signaling pathway [32, 33]. Tissue inhibitor of metalloproteinases-1 (TIMP-1) possesses actions of promoting growth and anti-apoptosis in cells. It has been reported that TIMP- 1 reduced cell apoptosis during the process of wound healing . In addition, other cytokines such as uPAR, IL-6, OPN, ANG-2, HGF, TGFb1, RBP4, ANG-1, FAP, IL-11, Follistatin, DcR3, Galectin-1, MIF, IGF-2, MCP-1, and IL-8 were moderately secreted by hAMSCs, and the cytokines have been identified to have the ability to activate PI3K/AKT pathway. However, although the acceleration of hAMSCs and hAMSC-CM on skin wound healing may be involved in activating PI3K/AKT pathway, it is hard to determine which cytokine(s) in hAMSC-CM mediate the activation of PI3K/AKT pathway due to the complexity of hAMSC-CM components.
In the present study, we demonstrated that hAMSCs and hAMSC-CM accelerate skin wound healing in vivo and inhibit acute heat stress-induced skin cells apoptosis and promoted their proliferation in vitro via activation of PI3K/AKT pathway. Antibody Array assay showed that PAI-1, C-GSF, periostin, TIMP-1, and uPAR secreted by hAMSCs might be involved in the activation of PI3K/AKT signaling pathway. Our findings suggest that the administration of hAMSCs or hAMSC-CM may be a novel therapeutic strategy for skin injury repair clinically.
The authors thank the healthy donors from the First Affiliated Hospital of Nanchang University for kindly providing placenta. We also extend special thanks to the Laboratory Animal Center of Institute of Translational Medicine, Nanchang University.
HBX and QWL contributed to the conception and design of the study, collection and/or assembly of data, data analysis and interpretation, and manuscript writing. JYL performed the in vivo and in vitro experiments. KKR participated in the hAMSC isolation and characterization by flow cytometry. WJZ performed animal experiments and were responsible for animal handling. LX, HYW, QYL, TD, WJN, YK, and KYD contributed to the experimentation. XCZ provided some of the study materials. All authors read and approved the final version of the manuscript.
This work was supported by the National Natural Science Foundation of China (81760118 to QWL, 81760140 to KYD, 81873659 and 91639106 to HBX); Jiangxi Provincial Department of Science and Technology, China (2018ACB21043 to QWL); Jiangxi provincial Department of Education (GJJ150214 to QWL); the grant for Jiangxi Provincial Collaborative Innovation Center of Biopharmaceutics and Biotechnology (2015202004 to HBX); and the grants from the Science Foundation of Nanchang University (06301204 to QWL).
Ethics approval and consent to participate
All procedures involving animals were approved by the Institutional Animal Care and Use Committees at Nanchang University and conducted in accordance with the national guidelines on animal care.
Consent for publication
The authors declare that they have no competing interests.
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