Introduction

Molecular Medicine Reports

1791-2997 1791-3004

D.A. Spandidos

10.3892/mmr.2018.8855

mmr-17-06-7603

Articles

Tenascin-C promotes the migration of bone marrow stem cells via toll-like receptor 4-mediated signaling pathways: MAPK, AKT and Wnt

Ding

Huaiyu

1 Jin

Mingyu

1 Liu

Dai

1 Wang

Shujing

2 Zhang

Jianing

3 Song

Xiantao

4 Huang

Rongchong

1Department of Cardiology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, P.R. China 2Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Liaoning 116044, P.R. China 3College of Life Sciences and Pharmacy, Dalian University of Technology, Dalian, Liaoning 116027, P.R. China 4Department of Cardiology, Beijing An Zhen Hospital, Capital Medical University, Beijing 100029, P.R. China

Correspondence to: Dr Rongchong Huang, Department of Cardiology, The First Affiliated Hospital of Dalian Medical University, 222 Zhongshan Road, Xigang, Dalian, Liaoning 116011, P.R. China, E-mail: rongchonghuang66@163.com

062018

05042018

17 6 7603 7610 05092017 29032018

2018

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

There are currently limitations in stem cell therapy due to the low rate of homing and proliferation of cells following transplantation. The present study was designed to investigate the effects of Tenascin-C (TN-C) on bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. BMSCs were obtained from C57BL/6 mice. The survival and proliferation of BMSCs was analyzed by Cell Counting Kit-8 assay, migration was evaluated using the Transwell method, and differentiation was assessed by immunocytochemistry and immunofluorescence. In addition, the levels of proteins were detected by western blotting. High concentrations of TN-C promoted the migration of BMSCs. H₂O₂ at concentrations of 60–90 µmol/ml induced cell death in BMSCs, and thus, it was used to simulate oxidative stress in the microenvironment of acute myocardial infarction (AMI). High concentrations of TN-C were able to protect BMSCs from cell death, and promoted the migration of BMSCs (P<0.05). However, TAK-242 [the inhibitor of Toll-like receptor 4, (TLR4)] reduced the promoting effect of TN-C (P<0.05). By contrast, TN-C had no effect on the proliferation and differentiation of BMSCs. TN-C reduced the phosphorylation levels of p38 mitogen-activated protein kinase (MAPK), and increased the phosphorylation levels of Ser473 protein kinase B (AKT) and β-catenin, all of which were inhibited by TAK-242 (P<0.05). In the simulated AMI microenvironment, TN-C promoted the migration of BMSCs via TLR4-mediated signaling pathways, including MAPK, AKT and Wnt.

bone marrow stem cells homing myocardial infarction tenascin-c

Introduction

Many experiments have confirmed that transplantation of bone marrow mesenchymal stem cells (BMSCs) can improve damaged cardiac function after acute myocardial infarction (AMI) (1). However due to local inflammation, ischemia, hypoxia, and other factors, the homing and survival rates of BMSCs after transplantation are still very low (2). Therefore, in order for stem cell therapy to be effective, it is very important to increase the homing and survival rates of BMSCs (3).

Tenascin-C (TN-C) is an extracellular matrix glycoprotein, which is closely associated with inflammation and tissue injury (4). TN-C is detected at low levels in the healthy adult heart, but is more highly expressed under various pathological conditions, such as AMI, myocardial hibernation, myocarditis, and dilated cardiomyopathy. TN-C is secreted by the interstitial cells surrounding the infarcted myocardium in response to adverse conditions, such as ischemia, hypoxia, and other factors, and is involved in injury repair and formation of myocardial fibrosis. TN-C can thus be used as an independent predictor of left ventricular remodeling and long-term prognosis (5–7).

Apoptosis is a form of cell death that is generally triggered by normal, healthy processes in the body. Many factors, such as inflammation and injury, can increase the apoptotic rate of cells, which causes harm to the repair of tissue damage. As a result, excessive apoptosis should be inhibited (8–10).

To date, 11 human and 13 mouse Toll-like receptors (TLRs) have been identified. Recent studies indicate that BMSCs express functional TLRs, including TLR4 (11). When activated, TLRs affect many downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK), AKT, and Wnt signaling pathways. These signaling pathways play important roles in the apoptosis, proliferation, and differentiation of many cells (12–17). However, it is yet unclear whether TN-C binds to TLR4 on the surface of BMSCs and activates some downstream signaling pathways, resulting in its biological effects.

We hypothesized that in the simulated AMI microenvironment, TN-C promotes the migration, proliferation, and differentiation of BMSCs via TLR4-mediated signaling pathways, such as MAPK, AKT, and Wnt. This study was designed to investigate the effects of TN-C on BMSCs and elucidate its underlying mechanisms in vitro.

Materials and methods <sec> <title>Animals and preparation of BMSCs

C57BL/6 mice were originally purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Colonies were subsequently established by in-house breeding at the Laboratory Animal Center of Dalian Medical University. The mice (the experimental mice as well as the breeding pairs) were all housed in a specific pathogen-free animal facility. Adult C57BL/6 male mice (weighing 20–30 g) were obtained from the Laboratory Animal Center of Dalian Medical University. Primary cells from C57BL/6 mice were isolated, cultured, and passaged according to a previously described method, with some modifications (18,19). Cells at passage F3-F4 were harvested at densities of 1 to 2×10⁶ cells/ml and used for subsequent experiments.

The study was reviewed and approved by the Institutional Ethics Committee on Animal Resources of Dalian Medical University, and conformed to the guiding principles of the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication no. 83-23, revised 1996).

Flow cytometry

After digestion with 2.5 g/l trypsin, the cells were washed, resuspended (1×10⁶ cells/ml), and incubated for 30 min at 37°C with monoclonal antibodies against CD29, CD44, CD34, and CD45. The cells were then centrifuged at 1,000 × g for 10 min, washed three times with phosphate buffered saline (PBS), and incubated for 30 min with the corresponding FITC-labeled secondary antibody (1 mg/ml). Homologous IgG and PBS were used as negative controls. Expression levels of the cell surface markers were analyzed by flow cytometry.

Cell Counting Kit-8 (CCK-8) assay

BMSCs were seeded at 5×10⁴ cells/ml (100 µl/well) into 96-well plates and treated with different concentrations of TN-C (0, 0.1, 1, 10, 50, 100, and 150 µg/ml) for 48 h. The cell number was measured using a CCK-8 proliferation assay kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) following the manufacturer's instructions. The absorbance (optical density, OD) at 450 nm, representing the survival/proliferation of BMSCs, was determined using a microplate reader.

BMSCs were seeded at 5×10⁴ cells/ml (100 µl/well) into 96-well plates and treated with different concentrations of H₂O₂ (60 or 90 µmol/ml) and TN-C (0, 1, 10, 50, 100, or 150 µg/ml) for 48 h. Altogether, there were 13 experimental groups that were treated with different concentrations of H₂O₂ and TN-C: H₂O₂ 0 µmol/ml, TN-C 0 µg/ml; H₂O₂ 60 µmol/ml, TN-C 0 µg/ml; H₂O₂ 60 µmol/ml, TN-C 1 µg/ml; H₂O₂ 60 µmol/ml, TN-C 10 µg/ml; H₂O₂ 60 µmol/ml, TN-C 50 µg/ml; H₂O₂ 60 µmol/ml, TN-C 100 µg/ml; H₂O₂ 60 µmol/ml, TN-C 150 µg/ml; H₂O₂ 90 µmol/ml, TN-C 0 µg/ml; H₂O₂ 90 µmol/ml, TN-C 1 µg/ml; H₂O₂ 90 µmol/ml, TN-C 10 µg/ml; H₂O₂ 90 µmol/ml, TN-C 50 µg/ml; H₂O₂ 90 µmol/ml, TN-C 100 µg/ml; and H₂O₂ 90 µmol/ml, TN-C 150 µg/ml. The cell number was analyzed as described above. BMSCs pretreated with 1 µM TAK-242 (inhibitor of TLR4) were cultured with 60–90 µmol/ml H₂O₂ and 100–150 µg/ml TN-C for 48 h in a cell culture incubator (20). The cell number was analyzed as described above.

Transwell<sup>®</sup> method

To investigate the effects of TN-C alone on BMSC migration, Transwell^® chambers with 8 µm pores were obtained from Corning Incorporated, (Corning, NY, USA). Pelleted BMSCs were resuspended in Dulbecco's modified Eagle's medium (DMEM) at a concentration of 3×10⁵ cells/ml, and then seeded into the upper chambers of the 24-well plate. The lower chambers were filled with 500 µl DMEM at different final concentrations of TN-C (0, 0.1, 1, 10, 50, 100, and 150 µg/ml). Cells were then incubated for 12 h. At the end of the experiment, cells that migrated to the reverse side of the Transwell^® membrane were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet solution, and then counted under a light microscope at magnification, ×100. An average of six visual fields was examined.

To investigate the effects of TN-C and H₂O₂ on BMSC migration, pelleted BMSCs were resuspended in DMEM as described above, and then seeded into the upper chambers of a 24-well plate. The lower chambers were filled with 500 µl DMEM supplemented with different final concentrations of H₂O₂ and TN-C (H₂O₂ 0 µmol/ml, TN-C 0 µg/ml; H₂O₂ 60 µmol/ml, TN-C 0 µg/ml; H₂O₂ 60 µmol/ml, TN-C 10 µg/ml; H₂O₂ 60 µmol/ml, TN-C 50 µg/ml; H₂O₂ 60 µmol/ml, TN-C 100 µg/ml; H₂O₂ 90 µmol/ml, TN-C 0 µg/ml; H₂O₂ 90 µmol/ml, TN-C 10 µg/ml; H₂O₂ 90 µmol/ml, TN-C 50 µg/ml; and H₂O₂ 90 µmol/ml, TN-C 100 µg/ml) and incubated for 12 h. At the end of the experiment, cells that migrated to the reverse side of the Transwell^® membrane were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet solution, and then counted under a light microscope at magnification, ×100. An average of six visual fields was examined. BMSCs pretreated with 1 µM TAK-242 for 2 h were treated with 90 µmol/ml H₂O₂ and 10–100 µg/ml TN-C for 12 h, and then treated as described above. All the above experiments were repeated at least three times independently.

ICC/IF staining

Pelleted BMSCs were resuspended in DMEM at a concentration of 1×10⁵ cells/ml. Ten pieces of 10 mm cell sheets were disinfected with 75% alcohol, air-dried, and placed into a sterile 12-well plate. Then, 100 µl of the above BMSC suspension was directly seeded onto each piece of cell sheet, and cultured for 2 h under standard cell culture conditions. Following that, 1 ml DMEM was added to each well, and the cells were cultured for another 24 h. DMEM with different final concentrations of TN-C (0, 0.1, 1, 10, 50, 100, and 150 µg/ml) was then added to the cell sheets and they were returned to culture, with the culture medium replaced every 3 days. After 3 weeks, the cell sheet slices were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 at room temperature, blocked with goat serum at 37°C for 1 h, incubated for 2 h with 50 µl 1:100 primary antibody against mouse α-actin (1 mg/ml) at 37°C, incubated for 1 h with 50 µl 1:200 fluorogenic secondary antibody (FITC-labeled, 1 mg/ml) at 37°C, and counter-stained for 1 min with 10 µl DAPI at room temperature. Cell differentiation was observed under a fluorescence microscope. The nuclei were stained blue, whereas the α-actin was green.

As described above, the BMSC suspension was directly seeded onto each piece of cell scaffold, and then returned to the incubator for 2 h. Following that, 1 ml DMEM was added to each well and they were cultured for 24 h. Next day, DMEM with different final concentrations of H₂O₂ and TN-C, as described in section 2.4.2, was added to the cell scaffolds, and they were cultured and processed as described above. Finally, cell differentiation was observed under a fluorescence microscope.

Western blotting

BMSCs were treated with different factors (60 µmol/ml H₂O₂; H₂O₂ 60 µmol/l, TN-C 100 µg/ml; H₂O₂ 60 µmol/ml, TN-C 100 µg/ml, TAK-242) for 48 h; untreated normal cells were used as control. At the end of the treatment, the cytoplasmic proteins were extracted using a cell lysis solution (RIPA: PMSF: Phosphatase inhibitor=100: 1: 20), and the total protein and phosphorylated protein levels of p38 MAPK, AKT (Ser473), and β-catenin were then analyzed by western blot analysis as described previously (21,22).

Statistical analysis

All statistical analyses were performed using GraphPad Prism v5.0 (GraphPad Software Inc., La Jolla, CA, USA) and SPSS v13.0 (SPSS Inc., Chicago, IL, USA). All data are presented as mean ± standard deviation (SD). All OD values, cell numbers, and protein levels were compared between two groups using one-way analysis of variance (ANOVA) with LSD analysis. P<0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Identification of BMSCs

The cells obtained from the mouse bone marrow were CD29⁺ (96.9%), CD34⁺ (96.3%), CD44⁺ (40.9%), and CD45⁻ (0.7%), which confirmed that these cells were BMSCs (Fig. 1).

Effect of TN-C on the survival/proliferation of BMSCs

TN-C did not promote the proliferation of BMSCs: OD values of BMSCs treated with different concentrations of TN-C (0.1, 1, 10, 50, 100, or 150 µg/ml) were no higher than those of control BMSCs (P>0.05); however, OD values of BMSCs treated with high concentrations of TN-C (100 or 150 µg/ml) were lower than those of controls (P<0.05; Fig. 2A).

H₂O₂ reduced the survival rate of BMSCs: OD values of BMSCs treated with either 60 or 90 µmol/ml H₂O₂ were lower than those of control BMSCs (P<0.05), which showed that 60–90 µmol/ml H₂O₂ could cause BMSC death (Fig. 2B).

TN-C protected BMSCs from cell death caused by H₂O₂: OD values of BMSCs treated with 60 µmol/ml H₂O₂ together with high concentrations of TN-C (100 or 150 µmol/ml) were higher than those of BMSCs treated with 60 µmol/ml H₂O₂ alone (P<0.05). OD values of BMSCs treated with 90 µmol/ml H₂O₂ together with high concentrations of TN-C (100 or 150 µmol/ml) were also higher than those of BMSCs treated with 90 µmol/ml H₂O₂ alone (P<0.05; Fig. 2C and D).

TAK-242 reduced the protective effect of TN-C: OD values of BMSCs treated with TAK-242 were less than those of normal cells treated with the same concentrations of H₂O₂ and TN-C (P<0.05; Fig. 2E).

Effect of TN-C on the migration of BMSCs

Migrated BMSCs were observed under an inverted microscope at high magnification, ×200. Long spindle-shaped or irregularly shaped cells stained purple with crystal violet were the migrated BMSCs. After treatment with different factors, including TN-C, H₂O₂, and TAK-242, the migrated BMSCs were observed and counted.

High concentrations of TN-C promoted the migration of BMSCs: High concentrations of TN-C (10–150 µg/ml) promoted the migration of BMSCs, whereas low concentrations of TN-C (0.1–1 µg/ml) showed no significant effect (Fig. 3A).

H₂O₂ promoted the migration of BMSCs: The number of migrated BMSCs in cultures treated with 60 or 90 µmol/ml H₂O₂ was greater than that in control untreated cultures (P<0.05), demonstrating that H₂O₂ promotes the migration of BMSCs. However, we found no significant difference between the groups treated with 60 or 90 µmol/ml H₂O₂ (P>0.05; Fig. 3B).

Combined treatment with TN-C and H₂O₂ further promoted the migration of BMSCs: When BMSCs were treated with either concentration of H₂O₂ (60 or 90 µmol/ml) together with different concentrations of TN-C (10, 50, or 100 µg/ml), increased numbers of migrated cells were observed compared to that in cultures treated with different concentrations of H₂O₂ alone (60 or 90 µmol/ml) (P<0.05). However, there was no significant difference among the groups treated with different concentrations of TN-C (10, 50, or 100 µg/ml) (P>0.05; Fig. 3C).

TAK-242 reduced the migration-promoting effect of TN-C: In cultures of BMSCs treated with 90 µmol/ml H₂O₂, different concentrations of TN-C (10 or 50 µg/ml), and 1 µM TAK-242, there were fewer number of migrated cells than in cultures treated with 90 µmol/ml H₂O₂ and different concentrations of TN-C (10 or 50 µg/ml) (P<0.05). However, in cultures of BMSCs treated with 90 µmol/ml H₂O₂, 100 µg/ml TN-C, and 1 µM TAK-242, the number of migrated cells was higher than that in cultures treated only with 90 µmol/ml H₂O₂ and 100 µg/ml TN-C (P<0.05; Fig. 3D).

TN-C was unable to promote differentiation of BMSCs

Staining for α-actin was negative in all culture groups, indicating that none of the conditions tested induced the BMSCs to differentiate into cardiomyocytes (Fig. 4A).

When the same experiment was performed in the presence of H₂O₂ (60 or 90 µmol/ml) to simulate oxidative stress in the microenvironment of AMI, TN-C was still unable to induce the differentiation of BMSCs into cardiomyocytes (Fig. 4B).

The effect of TN-C on MAPK, AKT, and Wnt signaling pathways

TN-C decreased the phosphorylation levels of p38 MAPK, which were inhibited byTAK-242: The phosphorylation level of p38 MAPK in the 60 µmol/ml H₂O₂ group was higher than that in the control group (P<0.05), whereas the phosphorylation level of p38 MAPK in the 100 µg/ml TN-C group was lower than that in the 60 µmol/ml H₂O₂ group (P<0.05). In contrast, the phosphorylation level of p38 MAPK in the TAK-242 group was higher than that in the 100 µg/ml TN-C group (P<0.05; Fig. 5A and B).

TN-C increased the phosphorylation levels of Ser473 AKT, which were inhibited byTAK-242: The phosphorylation level of Ser473 AKT in the 60 µmol/ml H₂O₂ group was lower than that in the control group (P<0.05), whereas the phosphorylation level of Ser473 AKT in the 100 µg/ml TN-C group was higher than that in the 60 µmol/ml H₂O₂ group (P<0.05). Furthermore, the phosphorylation level of Ser473 AKT in the TAK-242 group was lower than that in the 100 µg/ml TN-C group (P<0.05; Fig. 5C and D).

TN-C increased the phosphorylation levels of β-catenin, which were inhibited byTAK-242: The phosphorylation level of β-catenin in the 60 µmol/ml H₂O₂ group was lower than that in the control group (P<0.05), whereas the phosphorylation level of β-catenin in the 100 µg/ml TN-C group was higher than that in the 60 µmol/ml H₂O₂ group (P<0.05). Furthermore, the phosphorylation level of β-catenin in the TAK-242 group was lower than that in the 100 µg/ml TN-C group (P<0.05; Fig. 5E and F).

Discussion

Our results showed that TN-C acts in a dose-dependent manner to promote the migration of BMSCs. When H₂O₂ was added to the culture to simulate oxidative stress in the cardiac microenvironment after AMI, TN-C promoted the migration of BMSCs and protected them from cell death. However, TN-C had no effect on promoting the proliferation or differentiation of BMSCs. Investigation of possible signaling mechanisms indicated that TN-C bound to TLR4 expressed on the surface of BMSCs, and then activated the downstream signaling pathways, including MAPK, AKT, and Wnt.

Many signaling molecules and their ligands are involved in the migration of BMSCs to areas of damage. Among them, stromal cell-derived factor-1 (SDF-1) is, so far, the only known natural chemokine that can bind to and activate the CXC chemokine receptor type 4 (CXCR4) receptor (23–25). In rats, this interaction between SDF-1 and CXCR4 has been shown to play a key role in the homing of BMSCs to the infarct area (23). Here, we demonstrated that TN-C promotes the migration of BMSCs in vitro, but it is unclear whether TN-C still exerts the same effect in vivo.

Our experiments showed that 60–90 µmol/ml H₂O₂ causes apoptosis of BMSCs. Different concentrations of TN-C were able to promote migration of BMSCs in the simulated oxidative stress environment of AMI, modeled in vitro by H₂O₂. However, it is unclear whether this effect would be reproduced in vivo, where many other factors are involved. Hence, further experiments are needed.

TLR4 is the best-studied immune sensor, which detects invading microbes. It is broadly distributed on cells throughout the immune system. It has been revealed that BMSCs also express functional TLR4 (26–33). Activation of TLR4 signaling in BMSCs has diverse effects and is likely to influence their survival, differentiation, proliferation, migration, and pro-inflammatory cytokine secretion ability (26,30,31). After AMI, ischemia leads to the activation of TLR4/MyD88-dependent and independent downstream pathways. Among these, activation of the MyD88-dependent signaling pathway is thought to mediate the response of the innate immune system, promote the rapid release of cytokines and inflammatory mediators in the heart tissue, and recruit inflammatory cells to the lesion sites for repair (34–36). Lipopolysaccharide serves as a specific ligand for TLR4, activating the TLR-4/MyD88 pathway to promote BMSC proliferation and reduce BMSC apoptosis, which suggests that the TLR4 pathway is involved in promoting the survival and proliferation of BMSCs (34–36).

In this study, we showed that TN-C (10 or 50 µg/ml) promotes BMSC migration, and this effect can be reduced by treatment with the TLR4 inhibitor, TAK-242. These results suggest that TN-C binds to TLR4 through which it exerts its effects. However, when the concentration of TN-C was 100 µg/ml, the migration of BMSCs was not reduced, but was promoted. The possible mechanism was that TN-C (100 µg/ml) could activate other receptors on the surface of BMSCs when TLR4 was inhibited by TAK-242, which promoted the migration of BMSCs. Further analysis of the downstream signaling pathways showed that TN-C reduced the phosphorylation levels of p38 MAPK, but increased the phosphorylation of both Ser473 AKT and β-catenin, and all of these effects could be inhibited by TAK-242. Taken together, these results demonstrate the possible mechanism of action of TN-C, wherein TN-C binds to TLR4 expressed on the surface of BMSCs, and activates the MAPK, AKT, and Wnt signaling pathways to exert its biological effects. This result is consistent with that reported in a previous study (28–33). There are many signaling pathways and proteins involved in the action of TN-C, but in this study, only the major signaling pathways and proteins were investigated. To further elucidate whether TN-C exerts its effects through the TLR4-mediated signal transduction pathways, more research is required.

There is a bottleneck in stem cell therapy due to the low homing and survival rates of stem cells after transplantation. Our results showed that TN-C promoted the migration of BMSCs as well as protected them from cell death. This study provides a new theoretical basis for improving the homing and survival rates of transplanted cells, which is very important for effective stem cell therapy.

When AMI occurs, a series of complex changes take place in the microenvironment of the infarct area, and hence the effect of H₂O₂ alone cannot reflect the real situation in the body following AMI. As a result, in vivo experiments are crucial.

When AMI occurs, a series of complex changes take place in the microenvironment of the infarct area. Consequently, the effect of H₂O₂ alone cannot reflect the real situation in the body, and as a result, in vivo experiments are necessary. However, addition of extracorporeal H₂O₂ to simulate oxidative stress in the microenvironment following AMI showed that TN-C reduces BMSC apoptosis and promotes the migration of BMSCs under these conditions. This finding provides a new theoretical basis for animal experiments.

In summary, TN-C acts in a dose-dependent manner to promote the migration of BMSCs in vitro. In the simulated AMI microenvironment, TN-C promoted the migration of BMSCs and protected them from cell death, but did not promote BMSC proliferation or differentiation. The possible mechanism suggested was that TN-C binds to TLR4 expressed on the surface of BMSCs, and then activates the downstream signaling pathways, such as MAPK, AKT, and Wnt. This study provides a new theoretical basis for improving the homing and survival rates of transplanted cells, which is very important for effective stem cell therapy.

Acknowledgements

The authors would like to thank Dr. Ming Tian, Dr. Zhishuai Ye and Dr. Shengnan Zhu in the Department of Cardiology of The First Affiliated Hospital of Dalian Medical University (Liaoning, P.R. China) for cell isolation and culture.

Funding

The present study was supported by National Natural Science Foundation of China (grant nos. 81100220 and 81670324) and Liaoning Provincial Natural Science Foundation of China (grant no. 2015020295).

Availability of data and materials

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

Authors' contributions

Conception: HD, MJ and RH. Data curation: HD, MJ, DL, SW, JZ and RH. Experiments: HD, MJ, DL and RH. Analysis: HD, DL, XS and RH. Validation: HD, MJ, SW, XS and RH. Funding: RH. Project administration: HD, MJ, DL and RH. Original draft of manuscript: HD and MJ. Reviewing and editing of manuscript: HD and RH.

Ethics approval and consent to participate

The present study was reviewed and approved by the Institutional Ethics Committee on Animal Resources of Dalian Medical University (Liaoning, China).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Tomita

Weisel

Mickle

Kim

Sakai

Jia

Autologous transplantation of bone marrow cells improves damaged heart function

Circulation10019 SupplII247II2561999

10.1161/01.CIR.100.suppl_2.II-247

10567312

Geng

Molecular mechanisms for cardiovascular stem cell apoptosis and growth in the hearts with atherosclerotic coronary disease and ischemic heart failure

Ann N Y Acad Sci10106876972003

10.1196/annals.1299.126

15033813

Przybyt

Harmsen

Mesenchymal stem cells: Promising for myocardial regeneration?

Curr Stem Cell Res Ther82702772013

10.2174/1574888X11308040002

23547963

Sato

Aonuma

Imanaka-Yoshida

Yoshida

Isobe

Kawase

Kinoshita

Yazaki

Hiroe

Serum tenascin-C might be a novel predictor of left ventricular remodeling and prognosis after acute myocardial infarction

J Am Coll Cardiol47231923252006

10.1016/j.jacc.2006.03.033

16750702

Jones

The tenascin family of ECM glycoproteins: Structure, function, and regulation during embryonic development and tissue remodeling

Dev Dyn2182352592000

10.1002/(SICI)1097-0177(200006)218:2<235::AID-DVDY2>3.0.CO;2-G

10842355

Niebroj-Dobosz

Tenascin-C in human cardiac pathology

Clin Chim Acta413151615182012

10.1016/j.cca.2012.06.011

22687648

Sato

Hiroe

Akiyama

Hikita

Nozato

Hoshi

Kimura

Wang

Sakai

Imanaka-Yoshida

Prognostic value of serum tenascin-C levels on long-term outcome after acute myocardial infarction

J Card Fail184804862012

10.1016/j.cardfail.2012.02.009

22633306

Loreto

Musumeci

Leonardi

Chondrocyte-like apoptosis in temporomandibular joint disc internal derangement as a repair-limiting mechanism

Histol Histopathol242932982009

19130398

Musumeci

Castrogiovanni

Loreto

Castorina

Pichler

Weinberg

Post-traumatic caspase-3 expression in the adjacent areas of growth plate injury site: A morphological study

Int J Mol Sci1415767157842013

10.3390/ijms140815767

23899790

3759885

Puzzo

Loreto

Giunta

Musumeci

Frasca

Podda

Arancio

Palmeri

Effect of phosphodiesterase-5 inhibition on apoptosis and beta amyloid load in aged mice

Neurobiol Aging355205312014

10.1016/j.neurobiolaging.2013.09.002

24112792

Pevsner-Fischer

Morad

Cohen-Sfady

Rousso-Noori

Zanin-Zhorov

Cohen

Zipori

Toll-like receptors and their ligands control mesenchymal stem cell functions

Blood109142214322007

10.1182/blood-2006-06-028704

17038530

Gutiérrez-Uzquiza

Arechederra

Bragado

Aguirre-Ghiso

Porras

p38α mediates cell survival in response to oxidative stress via induction of antioxidant genes: Effect on the p70S6K pathway

J Biol Chem287263226422012

10.1074/jbc.M111.323709

22139847

Peti

Page

Molecular basis of MAP kinase regulation

Protein Sci22169817102013

10.1002/pro.2374

24115095

3843625

Wee

Aguda

Akt versus p53 in a network of oncogenes and tumor suppressor genes regulating cell survival and death

Biophys J918578652006

10.1529/biophysj.105.077693

16648169

1563780

Pillai

Sundaresan

Gupta

Regulation of Akt signaling by sirtuins: Its implication in cardiac hypertrophy and aging

Circ Res1143683782014

10.1161/CIRCRESAHA.113.300536

24436432

4228987

Ling

Nurcombe

Cool

Wnt signaling controls the fate of mesenchymal stem cells

Gene433172009

10.1016/j.gene.2008.12.008

19135507

Kim

Jho

Wnt/β-catenin signalling: From plasma membrane to nucleus

Biochem J4509212013

10.1042/BJ20121284

23343194

Wakitani

Saito

Caplan

Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine

Muscle Nerve18141714261995

10.1002/mus.880181212

7477065

Deng

Bivalacqua

Chattergoon

Jeter

JrKadowitz

Engineering ex vivo-expanded marrow stromal cells to secrete calcitonin gene-related peptide using adenoviral vector

Stem Cells22127912912004

10.1634/stemcells.2004-0032

15579646

Hussey

Liang

Costford

Klip

DeFronzo

Sanchez-Avila

Ely

Musi

TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipid-induced inflammation and insulin resistance in muscle cells

Biosci Rep3337472012

10.1042/BSR20120098

23050932

Ouyang

Tang

Liao

Wang

Tian

Zhao

Huang

Yao

MicroRNA-590 attenuates lipid accumulation and pro-inflammatory cytokine secretion by targeting lipoprotein lipase gene in human THP-1 macrophages

Biochimie10681902014

10.1016/j.biochi.2014.08.003

25149060

Chen

Zhu

Zheng

Yang

Pan

Yan

Zhu

MicroRNA-29a regulates pro-inflammatory cytokine secretion and scavenger receptor expression by targeting LPL in oxLDL-stimulated dendritic cells

FEBS Lett5856576632011

10.1016/j.febslet.2011.01.027

21276447

Abbott

Huang

Liu

Hickey

Krause

Giordano

Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury

Circulation110330033052004

10.1161/01.CIR.0000147780.30124.CF

15533866

Sterlacci

Saker

Huber

Fiegl

Tzankov

Expression of the CXCR4 ligand SDF-1/CXCL12 is prognostically important for adenocarcinoma and large cell carcinoma of the lung

Virchows Arch4684634712016

10.1007/s00428-015-1900-y

26818832

Wang

Zhang

Fan

Jia

The SDF-1/CXCR4 chemokine axis in uveal melanoma cell proliferation and migration

Tumour Biol37417541822016

10.1007/s13277-015-4259-4

26490988

Raicevic

Rouas

Najar

Stordeur

Boufker

Bron

Martiat

Goldman

Nevessignsky

Lagneaux

Inflammation modifies the pattern and the function of Toll-like receptors expressed by human mesenchymal stromal cells

Hum Immunol712352442010

10.1016/j.humimm.2009.12.005

20034529

Liotta

Angeli

Cosmi

Filì

Manuelli

Frosali

Mazzinghi

Maggi

Pasini

Lisi

Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling

Stem Cells262792892008

10.1634/stemcells.2007-0454

17962701

Wang

Zhang

Wang

Yao

Zhao

Gao

Lipopolysaccharides can protect mesenchymal stem cells (MSCs) from oxidative stress-induced apoptosis and enhance proliferation of MSCs via Toll-like receptor (TLR)-4 and PI3K/Akt

Cell Biol Int336656742009

10.1016/j.cellbi.2009.03.006

19376254

Brewster

Rouch

Wang

Meldrum

Toll-like receptor 4 ablation improves stem cell survival after hypoxic injury

J Surg Res1773303332012

10.1016/j.jss.2012.04.042

22703984

Raicevic

Najar

Pieters

De Bruyn

Meuleman

Bron

Toungouz

Lagneaux

Inflammation and Toll-like receptor ligation differentially affect the osteogenic potential of human mesenchymal stromal cells depending on their tissue origin

Tissue Eng Part A18141014182012

10.1089/ten.tea.2011.0434

22429150

Fiedler

Salamon

Adam

Herzmann

Taubenheim

Peters

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Exp Cell Res319288328922013

10.1016/j.yexcr.2013.08.020

23988607

Alvarado

Thiagarajan

Mulkearns-Hubert

Silver

Hale

Alban

Turaga

Jarrar

Reizes

Longworth

Glioblastoma cancer stem cells evade innate immune suppression of self-Renewal through reduced TLR4 expression

Cell Stem Cell20450461.e42017

10.1016/j.stem.2016.12.001

28089910

5822422

Goloviznina

Verghese

Yoon

Taratula

Marks

Kurre

Mesenchymal stromal cell-derived extracellular vesicles promote myeloid-biased multipotent hematopoietic progenitor expansion via toll-like receptor engagement

J Biol Chem29124607246172016

10.1074/jbc.M116.745653

27758863

5114412

Yao

Zhang

Wang

Zhang

Wang

Chen

Gao

Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction

J Biomed Sci16742009

10.1186/1423-0127-16-74

19691857

2739513

Huang

Pan

Lin

Dai

Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling

Int J Mol Med396937002017

10.3892/ijmm.2017.2873

28204830

Sindhu

Al-Roub

Koshy

Thomas

Ahmad

Palmitate-induced MMP-9 expression in the human monocytic cells is mediated through the TLR4-MyD88 dependent mechanism

Cell Physiol Biochem398899002016

10.1159/000447798

27497609

Figure 1.

Cells obtained from mouse bone marrow were (A) CD29+ (96.9%), (B) CD34+ (96.3%), (C) CD44+ (40.9%) and (D) CD45- (0.7%), which suggested that these cells were bone marrow mesenchymal stem cells. CD, cluster of differentiation; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Figure 2.

Effect of TN-C on the survival/proliferation of BMSCs. (A) TN-C did not promote the proliferation of BMSCs; (B) H₂O₂ caused BMSC death; (C) TN-C protected BMSCs from cell death caused by 60 µmol/ml H₂O₂; (D) TN-C protected BMSCs from cell death caused by 90 µmol/ml H₂O₂; and (E) TAK-242 reduced the protective effect of TN-C. *P<0.05, as indicated. TN-C, Tenascin-C; BMSCs, bone marrow mesenchymal stem cells.

Figure 3.

Effect of TN-C on the migration of BMSCs. (A) High concentrations of TN-C promoted the migration of BMSCs; (B) H₂O₂ promoted the migration of BMSCs; (C) TN-C in combination with H₂O₂ further promoted the migration of BMSCs; and (D) TAK-242 reduced the migration-promoting effect of TN-C (magnification, ×200; scale bar, 100 µm). *P<0.05, as indicated. TN-C, Tenascin-C; BMSCs, bone marrow mesenchymal stem cells.

Figure 4.

TN-C was unable to promote the differentiation of BMSCs. (A) TN-C did not promote the differentiation of BMSCs in vitro. (B) TN-C in combination with H₂O₂ did not promote the differentiation of BMSCs in vitro (magnification, ×200; scale bar, 100 µm). TN-C, Tenascin-C; BMSCs, bone marrow mesenchymal stem cells.

Figure 5.

Effect of TN-C on MAPK, AKT, and Wnt signaling pathways. (A and B) TN-C reduced the phosphorylation levels of p38 MAPK, and this effect could be inhibited by TAK-242. (C and D) TN-C increased the phosphorylation levels of Ser473 AKT, and this effect could be inhibited by TAK-242. (E and F) TN-C increased the phosphorylation levels of β-catenin, and this effect could be inhibited by TAK-242. *P<0.05, as indicated. TN-C, Tenascin-C; BMSCs, bone marrow mesenchymal stem cells; MAPK, mitogen-activated protein kinase; AKT, protein kinase B; p-, phosphorylated; t-, total.