Growth differentiation factor 11 promotes differentiation of MSCs into endothelial‐like cells for angiogenesis

Abstract Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor‐β super family. It has multiple effects on development, physiology and diseases. However, the role of GDF11 in the development of mesenchymal stem cells (MSCs) is not clear. To explore the effects of GDF11 on the differentiation and pro‐angiogenic activities of MSCs, mouse bone marrow–derived MSCs were engineered to overexpress GDF11 (MSCGDF11) and their capacity for differentiation and paracrine actions were examined both in vitro and in vivo. Expression of endothelial markers CD31 and VEGFR2 at the levels of both mRNA and protein was significantly higher in MSCGDF11 than control MSCs (MSCVector) during differentiation. More tube formation was observed in MSCGDF11 as compared with controls. In an in vivo angiogenesis assay with Matrigel plug, MSCGDF11 showed more differentiation into CD31+ endothelial‐like cells and better pro‐angiogenic activity as compared with MSCVector. Mechanistically, the enhanced differentiation by GDF11 involved activation of extracellular‐signal‐related kinase (ERK) and eukaryotic translation initiation factor 4E (EIF4E). Inhibition of either TGF‐β receptor or ERK diminished the effect of GDF11 on MSC differentiation. In summary, our study unveils the function of GDF11 in the pro‐angiogenic activities of MSCs by enhancing endothelial differentiation via the TGFβ‐R/ERK/EIF4E pathway.

and can secrete various trophic factors to promote cardiovascular regeneration. 4,5 Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β (TGF-β) superfamily. It is also known as bone morphogenetic protein 11 (BMP11). 6 GDF11 is expressed in many tissues, including pancreas, intestine, kidney, skeletal muscle, heart, developing nervous system, olfactory system and retina. 7 GDF11 plays an important role in early embryonic development and regulates the development of many organs. 8 GDF11 signals through binding with activin type II/I receptors (ActRII/I) on cellular membrane and activates the canonical SMAD2/3 signalling pathway 9 to realize its various biological functions. [10][11][12] The activated SMAD2/3 forms complexes with universal SMAD4, then is transferred to the nucleus and regulates gene transcription. In addition to the canonical Smad signalling pathway, the TGF-β superfamily members can also activate other non-Smad signalling pathways. 13,14 It has been reported that GDF11 activates p38-MAPK to regulate the size and function of the nucleolus, affects c-Jun N-terminal kinase (JNK) in ECs, as well as cross talking with AMPK and NF-κB. 8 And the extracellular regulated protein kinases (ERK) pathway was reported to be involved in the differentiation process of multi-potent adult progenitor cells. 15 Bone marrow-derived MSCs have been shown to be able to promote angiogenesis by direct differentiation into ECs both in vivo and in vitro. 16,17 However, little is known about how GDF11 affects MSC differentiation and weather the effects of GDF11 on MSCs are through TGF-β/ERK pathway.

Angiogenesis can be modulated by a number of cytokines and
growth factors, among which vascular endothelial growth factor (VEGF) and TGF-β1 play prominent roles. 18,19 VEGF and TGF-β1 are often co-expressed in tissues in which angiogenesis occurs, notably in a variety of tumours. 20 TGF-β is a multifunctional growth factor with effects on cell growth, differentiation, fibroblast activation, myofibroblast formation 21 and ECM accumulation. 22 Several recent studies demonstrated that TGF-β can also induce differentiation of stem cells or progenitor cells towards smooth muscle cells or myofibroblast lineage. 23 The plasma level of GDF11 is closely related to the formation and development of appendage skeleton 24 and has been shown to be involved in cardiovascular disease. 10 A recent study confirmed that higher concentration of GDF11 in the circulation was associated with a lower risk of vascular events and death, indicating that GDF11 may be a protective factor for essential in the setting of vascular events. 25 Other studies have found that GDF11 plays an important role in angiogenesis in different organ system. [26][27][28] However, there are only a few reports showing the effect of GDF11 in stem cell differentiation 29,30 and the role of GDF11 in MSCs remains to be determined.
In this study, we hypothesized that GDF11 can enhance MSCmediated angiogenesis by increasing the ability of MSCs to differentiate into endothelial-like cells, as well as through its anti-apoptosis and paracrine functions. We found that GDF11 promoted MSC differentiation into endothelial-like cells and enhanced their pro-angiogenic activities via activation of TGF-β receptor and its downstream ERK/EIF4E pathway.

| Culture of mouse BM-MSCs
Animal protocol was approved by Zhejiang University according to Chinese guidelines for laboratory animal care and use. Bone marrow was isolated from femurs and tibias of 8-week-old C57BL/6J male mice, and BM-MSCs were obtained as described previously 31 and cultured in DMEM medium (Hyclone, USA) supplemented with 10% foetal bovine serum (FBS) (Bioind, USA), 10 U/mL penicillin, and 10 U/mL streptomycin (Hyclone,Los Angeles, CA,USA). Cells were sub-cultured to 80%-90% confluence and passed after dissociation with 0.25% Trypsin & 0.02% EDTA (Genom, China) ( Figure S1A). For normal oxygen conditions (21% O2, 5% CO2, 37℃), cells were incubated in a standard humidified CO 2 incubator. All experiments were performed using cells at passage between 3 and 5.

| Characterization of MSCs
MSCs were characterized by flow cytometric analysis of surface markers and were positive for: CD44, CD105, CD90; negative for: CD31, CD45( Figure S1B). At room temperature, cells were dissociated, re-suspended in phosphate-buffered saline (PBS) and incubated with antibodies against following markers in dark for Non-specific mouse IgG-APC and IgG-APC (Ebioscience,USA) were used as controls. After incubation, the cells were washed twice with PBS and analysed by flow cytometry (BD Biosciences,New Jersey, USA). The data were analysed by Flowjo software.

| Lentiviral vector transduction
Lentiviral vectors carrying genes for GDF11 and GFP (LV-GDF11-GFP) or control vectors (LV-GFP) and Luciferase (LV-Luc) were prepared by Genechem (Shanghai, China). MSCs were seeded at 1x10 5 cell per well onto 12-well plates one day before transduction. Medium was changed with fresh serum-free DMEM medium (500 μL/well), and viral vectors (~2.5 μL) premixed with 20 μL HiTransG P transfection reagent (Genechem) were added to each well to reach a multiplicity of infection (MOI) at 50 for all transduction. Culture medium was changed 12 hours after transfection with DMEM containing 10% FBS.

After 48 hours, cells were observed under fluorescent microscope
for GFP + cells ( Figure S2A). Then, the successfully transduced cells were selected by culturing the cells in the presence of purinomycin.
Expression of GDF11 at levels of mRNA and protein was detected by RT-PCR ( Figure S2B) and Western blot ( Figure S2C), respectively. The cells transducted LV-GDF11-GFP/ Luc were abbreviated as MSC GDF11 , and control vectors (LV-GFP/ Luc) were abbreviated as MSC vector . This abbreviation is used the following text.

| Endothelial cell differentiation
MSCs were cultured in 12-well plates (1 × 10 5 cells/well) with DMEM medium for 24 hours at 37°C and then cultured in M199 medium

| Western blot assay
Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China, P0013B). Total protein was quantified by BCA protein assay (Bio-Rad, Berkery). Each sample was adjusted to equal amount of protein using 5X load-  Fold expression relative to the reference ACTIN gene was calculated using the comparative method 2 −ΔCt . The sequences of PCR primers were listed in Table S1.

| Immunohistochemistry staining
Cells were washed with PBS containing 3% bovine serum albumin (BSA) and fixed with 4% paraformaldehyde for 15 minutes. The cells were then permeabilized with 0.5% Triton X-100 for 10 minutes, blocked with 3% BSA in PBS for 30 minutes at room temperature and then incubated with primary antibody CD31 (#77699, Cell Signalling Technology) and VEGFR2 (#9698, Cell Signalling Technology), overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at 37°C. Nuclei were stained with Hoechst (Thermo Fisher, 33342) for 5 minutes. The cells were then washed three times and viewed using a fluorescence microscope (Leica, Germany).

| Tube formation assay
Tube formation assay was performed according to the manufacturer's protocol. Matrigel (50 µL) (Corning, New York, USA, #356231#) was added to each well of a 96-well plate and allowed to polymerize.
After culture for 2-12 hours, images were taken using a fluorescence microscope (Leica,Germany). The tube formation was quantified by analysing the total tube length in each well with Image-Pro Plus (MediaCybernetics, USA).

| Cell viability assay
For in vitro cell viability assay, GDF11-overexpressed and negative control-MSCs were plated on collagen-coated 96-well plates (2 × 10 3 cells/well) and cultured in serum-free medium under hypoxia (0.1% O 2 , 5% CO 2 ) at 37°C for 48 hours. Then, 10 μL of Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was added and incubated for 2 hours at 37°C, and the absorbance was determined at a wavelength of 450 nm. MSC viability was evaluated using OD value as described above.

| Matrigel Plug Assay in vivo
Male C57BL/6 mice (8-week-old, weighting 22-25 g) were used. MSCs After 10 days, Matrigel tissues were isolated and half were dissolved with Cell Recovery Solution (Corning, #354253#) and then analysed by Flow Cytometry, and the rest of plug was fixed with 10% formalin overnight and embedded in paraffin for histological analysis.

| Histological analyses
To examine the capillary and arteriole densities, paraffin sections of Matrigel were stained with following antibodies: rat anti-mouse CD31 (562939, BD Bioscience), rat anti-rabbit luciferase (ab185924, Abcam) and rat anti-rabbit GFP (ab290, Abcam). Alexa Fluor 488 or 550 conjugated antibody (Invitrogen) were used for secondary staining. After being mounted with Hochest mounting medium, the samples were analysed using a fluorescence confocal microscope (Leica).
For morphometric analysis, sections were stained with haematoxylin and eosin (H & E). Images were taken under 200/400× magnification.
After 48 hours, cell apoptosis was analysed using a TUNEL Cell Apoptosis Assay Kit (Beyotime,Shanghai, China) according to the manufacturer's instructions. Briefly, cells on plates were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes, followed with PBS wash twice. Then, cells were incubated with TUNEL reagent for 1 hour. Nuclei were stained with Hoechst for 5 mins. The cells were then washed three times and viewed using a fluorescence microscope.

| Statistics analysis
Results were expressed as means ± SD (standard deviation).
Continuous variables were compared by Student's t test, and multiple comparisons were performed by one-way ANOVA with a Bonferroni correction. Statistical analyses were performed using Prism 6 (GraphPad Software Inc,San Diego, CA, USA). A value of P < 0.05 was accepted as statistically significant.

| Lower expression of GDF11 in MSCs reduces their differentiation into endothelial-like cells
To confirm the effect of GDF11 on MSCs, siRNA specific for GDF11 gene was transfected into MSCs to knock down GDF11 expression.

| GDF11 enhances survival rate of MSCs and protects MSCs from hypoxia-induced apoptosis in vitro
To mimic the ischaemic environment in vivo, MSCs were exposed in hypoxic environment for 48 hours to induce apoptosis in vitro.
Higher viability of MSC GDF11 was observed as compared with control MSC Vector by using CCK-8 assay ( Figure 3A). Less apoptotic cells in MSC GDF11 than in control MSC Vector were detected by flow cytometry ( Figure 3B,C) and TUNEL assay ( Figure 3D,E) after treatment with hypoxia, which was confirmed with Western Blot analysis of apoptosis-related proteins ( Figure 3F,G). more bright detaching cells were observed after the down-regulation of GDF11 and exposed to hypoxic condition ( Figure 3K). Cell viability was also lowered in GDF11-siRNA transfected MSCs than the control ( Figure 3L) The effect of GDF11 on apoptosis-related proteins Bax and Bcl-2 was also reversed by the siRNA (Figure 3M,N).

| GDF11 facilitated MSC-mediate angiogenesis in vivo
In order to verify the pro-angiogenic activity of GDF11 in vivo, Matrigel plugs containing MSC Vector or MSC GDF11 were implanted into mice ( Figure 4A). The recovered Matrigel plugs containing MSC GDF11 appeared more reddish indicating more vessel formation in the plug allowing more red blood cells come in as compared with those treated with MSC Vector (Figure 4B and Figure S6A). Indeed, more blood vessels were observed in sections of MSCs GDF11 plugs as compared to those from MSC Vector controls ( Figure 4C,D). In addition, more CD31 + endothelial-like cells were detected by flow cytometry from MSC GDF11plugs (21.40 ± 2.059%) as compared to than that of MSC Vector controls (7.478 ± 4.323%, n = 5) ( Figure 4E,F). Capillary density as detected by immunostaining for CD31 was also significantly higher in plugs with MSCs GDF11 as compared with MSC Vector controls ( Figure 4G,H).

| GDF11 promoted MSC differentiation into endothelial-like cells in vivo
To track the fate of implanted MSCs in the Matrigel plug, both MSCs GDF11 and MSC Vector were transduced with genes for GFP or luciferase before implantation. Immunofluorescence co-localization analysis ( Figure 5A) was performed to examine the implanted MSCs in the plugs recovered 10 days after implantation. There were more cells that were positive for both GFP and CD31 in plugs containing MSCs GDF11 as compared to those with MSC Vector (Figure 5B). This was further confirmed by colocalization of luciferase + with CD31 + cells ( Figure S6B,C). And we did the double-staining with antibodies against GDF11 and CD31. More CD31 + cells in the MSC GDF11 group were observed ( Figure 5C). These results indicate that the presence of GDF11 in MSCs GDF11 augments their ability to differentiate into CD31 + endothelial-like cells.
Furthermore, there were more MSCs GDF11 than control MSCs Vector in the plugs recovered 10 days after implantation ( Figure 5D). The retention rate was higher for MSCs GDF11 than MSCs Vector ( Figure 5E) concomitant with less apoptosis ( Figure 5F). The enhanced survival rate of MSCs GDF11 versus MSCs Vector was further confirmed in vitro when MSCs were placed in a hypoxic environment for 48 hours to induce apoptosis ( Figure S7A); in addition, fewer TUNEL + cells were observed in the MSCs GDF11 as compared to MSCs Vector ( Figure S7B).

| Effects of GDF11 involve TGF-β receptormediated ERK/EIF4E signalling pathway
To determine the molecular mechanism underlying in the effects of GDF11 on MSCs, we examined ERK1/2 signalling pathway, which was reported to be involved in the differentiation process of progenitor cells. 15 When GDF11 was overexpressed in MSCs, phosphorylation of both ERK and EIF4E was significantly increased ( Figure 6A

| D ISCUSS I ON
In this study, we showed that GDF11 and VEGF had mutual ef- It has been almost two decades since BM-MSCs were first used to promote angiogenesis for ischaemic diseases. 32 BM-MSCs were shown to be able to differentiate into ECs both in vitro 16 and in vivo. 33 However, the differentiation rate is low and newly formed capillaries were unstable. 34 There are several factors influencing differentiation of stem cells into ECs. For example, endothelial growth supplements, 35,36 shear forces 37,38 and composition of extracellular matrix 39 are important factors in EC differentiation. Some members in the TGF-β family have been reported to promote endothelial differentiation. Bone morphogenetic protein 4 (BMP4) accelerated F I G U R E 5 Effect of GDF11 on MSC differentiation, retention and apoptosis in vivo. MSC GDF11 or MSCs Vector expressing GFP or luciferase were mixed with Matrigel and implanted into mice. Paraffin sections of the recovered plugs 10 d after implantation were stained with DAPI for nuclei (blue) and specified antibodies (n = 5). A, Antibodies against GFP (green) and CD31 (red) were used for detection of endotheliallike cells. White arrows point at the MSCs (orange) whose GFP was colocalized with CD31. The pictures in upper panel were taken at 400× magnification and the lower panel are magnified 2×. Scale bars: 50 μm. B, Rate of MSC differentiation into endothelial-like cells was quantified by dividing number of double positive cells by number of total retained MSCs in an image. C, Antibodies against GDF11 (red) and CD31 (green) for endothelial-like cells were used. White arrows point at the double positive cells (orange). The pictures were taken at 600× magnification. Scale bars: 50 μm. D, Sections were stained for TUNEL to identify apoptotic cells (red) and retained MSCs (GFP). Scale bars: 100 μm. E, Retention rates were quantified as the number of GFP+ cells out of the total number of cells. F, Apoptosis rate was quantified by the percentage of cells positive for TUNEL staining. Data are presented as the mean ± SD for at 2 independent experiments and were analysed. *P < 0.05; **P < 0.01 and ***P < 0.001 F I G U R E 6 Analysis of the molecular pathways underlying GDF11-induced MSC differentiation. A, MSCs Vector and MSC GDF11 were cultured in the presence or absence of VEGF165 for 14days. Total (T) and phosphorylated (P) ERK and EIF4E were examined by Western blot (n = 3). B, Quantitative analysis of A. The density of each band was calibrated with its corresponding Actin band. Relative phosphorylation levels were quantified by dividing P over T, which was then compared with the level in control MSCs Vector to set as 1. C, MSCs were transfected with control (NC)-or GDF11-specific siRNA. Total (T) and phosphorylated (P) ERK and EIF4E were examined by Western blot (n = 3). D, Quantitative analysis of C in the same way as B. E, MSCs Vector and MSC GDF11 were cultured with VEGF165 for EC differentiation in the presence or absence of inhibitors for TGF-β receptor (LY2109761) or for ERK (SD5978) for 14days. T-and P-ERK and EIF4E were examined by Western blot. F, Quantitative analysis of E. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ***P < 0.001. Each WB was repeated for at least 2 times the commitment of human embryonic stem cells to the endothelial lineage. 40 Treatment of iPSCs with TGF-β2 can induce EC marker expression and in vitro tube formation. 41 GDF11, as a member of TGF-β family, has a similar function as other family members, such as TGF-β1, to promote angiogenesis. 42,43 GDF11 was reported to promote migration and sprouting of endothelial progenitor cells 44 and other angiogenic activities. 45 Here, we show for the first time that GDF11 has direct effect on MSC differentiation into endothelial-like cells. Great efforts have made to improve the efficiency of MSC differentiation 33,36 and to augment the therapeutic efficacy of stem cells. 46,47 We demonstrated that MSC GDF11 had a greater ability to differentiate into endothelial-like cells, as evidenced by significantly higher expression of EC-related markers at both mRNA and protein levels ( Figures 1D,F and 2 However, it is worthy to point out that some of TGF-β family members may not have similar effect on MSCs. It has been reported that some of TGF-β family promoted MSCs towards fibrosis, endothelial-to-mesenchymal transition (EndMT) 48,49 and osteoblastogenesis. 50 As a fact, the role of GDF11 in ageing, cardiovascular diseases and function of ECs is still not fully understood, or sometimes controversial. 6,7,50,51 The cause of contradictory may be due to the difference in materials and methods used, objects of study and genetic background etc. 52,53 In our case, further study is needed to confirm that the differentiated EC-like cells have real function in pro-angiogenesis in an ischaemic disease model, and the newly formed vessels are stable in long term.
Multiple intracellular signalling pathways have been implicated in stem cell differentiation. These signalling pathways include MAP kinase signalling pathways (such as p38, ERK and JNK), 15 Figure S8). This agrees with the previous report that roles of Smad2/3 signalling are stage-dependent during endothelial differentiation of MSCs induced by TGF-β1 or BMPs. 54,55 Indeed, the downstream pathways of TGF-β receptors could be either Smad-dependent or Smad-independent. 13,14 It has been reported that GDF11 activates p38 MAPK to regulate the size and function of the nucleolus, affects JNK in ECs, as well as cross talking with AMPK, eNOS and NF-κB. 8 On the other hand, we found greater ERK phosphorylation when MSC GDF11 were induced for endothelial differentiation by VEGF165, as compared with the control group ( Figure 6).
In addition, we also detected more phosphorylated EIF4E, the downstream target of ERK pathway. These activations can be blocked by both inhibitors for TGF-β receptor and ERK ( Figure 6E). It is known that EIF4E is an important component for initiation of translation and its phosphorylation activates the translation of cellular proteins. 56 When EIF4E was knocked down by siRNA, the translation of its downstream targets including c-Myc, VEGF, CyclinD1 and Bcl-2 were diminished. 57 Our data show that activation of the ERK/EIF4E pathway was also inhibited when GDF11 was knocked down. The anti-apoptotic effect of GDF11 on MSCs was blocked by inhibition of TGFβ-R or ERK ( Figure 7E). Together, these results suggest that the TGFβ-R/ ERK/EIF4E pathway is crucial for the actions of GDF11 in this system.
It is well established that most of growth factors usually regulate their effect via activation of the TGFβ-RAS-RAF-MEK1/2-ERK1/2 signalling cascade. 58 We would infer that GDF11 binds to the TGF-β receptor and subsequent activates RAS-RAF-MEK-ERK/EIF4E pathway to induce the endothelial differentiation of MSC. Our results are consistent with previous reports showing that the ERK/EIF4E pathway is involved in cell growth, regulating cell cycle and apoptosis. 59 Furthermore, we also showed that GDF11 and VEGF had mutual effects on the expression and differentiation of MSCs into endothelial-like cells. VEGF binding with its VEGFR receptors can also activate multiple downstream pathways, like PKC-ERK, which will F I G U R E 7 Effect of GDF11 on the differentiation of MSCs into endothelial-like cells was blocked by ERK inhibitor. MSC Vector and MSC GDF11 were treated with inhibitors for ERK (SD5978) or for TGF-β receptor (LY2109761) or with DMSO as a control during the differentiation of MSCs into endothelial-like cells for 14 d. A, Representative results of flow cytometry analysis of MSC Vector (Vector) and MSC GDF11 (GDF11) for their expression of CD31 and VEGFR2. B, Quantitative analysis of A (n = 3). C, Immunofluorescent staining of MSC Vector and MSC GDF11 with antibody against CD31 (red) after culture with or without ERK inhibitor. Nucleus was stained with Hochest. Scale bars = 100 μm. D, Quantitative analysis of C (n = 3). CD31 + rate was obtained by dividing CD31 + cells (red) by total number of cells (blue) in a picture. E, Western blot analysis of proteins in MSC Vector and MSC GDF11 in the presence of absence of inhibitors LY2109761 or SD5978during the differentiation of MSCs into endothelial-like cells. F, Quantitative analysis of E (n = 2). Each WB was repeated for at least 2 times. G, Schematic diagram of the proposed mechanisms by which GDF11 promotes MSC differentiation into endothelial-like cells. GDF11 interacts with TGF-β receptor, resulting in activation of the ERK/EIF4E pathway, which enhances the expression of proteins for angiogenesis and anti-apoptosis, and augment of EC markers during cultured with VEGFA. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ***P < 0.001. BCL-2 for B-cell lymphoma-2 protein; BAX for Bcl2 Associated X Protein enhance the expression of genes, for example GDF11, for cell differentiation and proliferation ( Figure 7G). However, the specific molecular mechanism of the interaction between GDF11 and VEGFA needs further study to be reviewed.
In summary, we found that GDF11 can significantly enhance the potential of MSCs for endothelial differentiation, increase their viability, and augment the therapeutic efficacy of MSCs to promote angiogenesis. These actions of GDF11 may involve its binding to the TGF-β receptor and subsequent activation of ERK/EIF4E pathway.
This novel function of GDF11 in MSCs could be useful for stem cell therapy for ischaemic cardiovascular diseases.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.