Thymosin β4-Enhancing Therapeutic Efficacy of Human Adipose-Derived Stem Cells in Mouse Ischemic Hindlimb Model

Thymosin β4 (Tβ4) is a G-actin sequestering protein that contributes to diverse cellular activities, such as migration and angiogenesis. In this study, the beneficial effects of combined cell therapy with Tβ4 and human adipose-derived stem cells (hASCs) in a mouse ischemic hindlimb model were investigated. We observed that exogenous treatment with Tβ4 enhanced endogenous TMSB4X mRNA expression and promoted morphological changes (increased cell length) in hASCs. Interestingly, Tβ4 induced the active state of hASCs by up-regulating intracellular signaling pathways including the PI3K/AKT/mTOR and MAPK/ERK pathways. Treatment with Tβ4 significantly increased cell migration and sprouting from microbeads. Moreover, additional treatment with Tβ4 promoted the endothelial differentiation potential of hASCs by up-regulating various angiogenic genes. To evaluate the in vivo effects of the Tβ4-hASCs combination on vessel recruitment, dorsal window chambers were transplanted, and the co-treated mice were found to have a significantly increased number of microvessel branches. Transplantation of hASCs in combination with Tβ4 was found to improve blood flow and attenuate limb or foot loss post-ischemia compared to transplantation with hASCs alone. Taken together, the therapeutic application of hASCs combined with Tβ4 could be effective in enhancing endothelial differentiation and vascularization for treating hindlimb ischemia.


Introduction
Adipose-derived stem cells (ASCs) have several advantages due to easy accessibility via minimally invasive liposuction of the stromal vascular fraction of adipose tissues and easy in vitro expansion. ASCs are capable of differentiating into diverse types of mesenchymal cells including endothelial lineages [1,2]. Increasing evidence demonstrated that ASCs have the potential to augment neovascularization and improve functional recovery post-ischemia [3,4].
Thymosin β4 (Tβ4) is a water-soluble and highly conserved protein comprising of 43 amino acids (MW: 4964 Da) [5,6]. It is reported to regulate the actin cytoskeleton by sequestering G-actin [7]. Tβ4 is a ubiquitous protein expressed in many cells and tissues and has been shown to mediate numerous biological processes including blood vessel formation and wound healing [8]. Previous studies reported that Tβ4 increases the proliferation, migration, and adhesion of endothelial progenitor cells and also inhibits apoptosis and cellular senescence in vitro [9,10]. Moreover, Tβ4 was reported to Int. J. Mol. Sci. 2020, 21, 2166 2 of 13 promote the angiogenesis potential of endothelial cells and infarcted hearts by up-regulating vascular endothelial growth factor (VEGF) expression [11,12]. Overall, previous findings suggest that Tβ4 may have significant potential for influencing the endothelial differentiation of mesenchymal stem cells (MSCs). Several studies demonstrated MSC differentiation into an endothelial lineage using VEGF or fibroblast growth factor 2 (FGF-2); [13][14][15] however, endothelial differentiation using Tβ4 has not been reported yet.
N-terminal sequence, one of the three main functional domains of Tβ4, is composed of a tetrapeptide (ac-SDKP) that is known to have anti-inflammatory and anti-fibrotic properties. These properties of Tβ4 have been reported to promote cell survival and inhibit apoptosis [16,17]. In addition, Tβ4 has been shown to facilitate cardioprotection by attenuating cardiac fibrosis post-myocardial infarction (post-MI) and regeneration of the ischemic heart by promoting cardiomyocyte survival [18][19][20]. It has also been reported that Tβ4 modulates the inflammatory environment within the infarcted region of the heart by reducing leukocyte infiltration after MI [21,22].
The purpose of the present study was to investigate whether the endothelial differentiation potential and therapeutic efficacy of ASC transplantation could be further enhanced by Tβ4 treatment. In the present study, exogenous treatment with Tβ4 was found to not only induce morphological changes, but also enhance the angiogenic sprouting of ASCs. Additionally, we revealed that the combination of ASCs and Tβ4 further promoted microvessel recruitment and enhanced blood flow post-hindlimb ischemia.

Treatment with Tβ4 Promotes Morphological Changes in hASCs
In this study, human ASCs (hASCs) were found to be positive for CD13, CD29, CD44, CD90, CD106, Stro-1, and HLA-ABC expression ( Figure 1A). Furthermore, hASCs were treated with recombinant Tβ4 protein and 100 ng/mL of Tβ4 was observed to significantly up-regulate endogenous TMSB4X mRNA expression compared to the expression in untreated cells ( Figure 1B). Interestingly, in Figure 1C, treatment with Tβ4 was observed to increase the cell length of hASCs after 24 h compared to the cell length of hASCs without Tβ4 treatment (w/o Tβ4). A single treatment with Tβ4 significantly increased cell length after 24 and 48 h compared to that observed without Tβ4. However, we observed that cell length of Tβ4-treated hASCs at after 72 h was similar to that of hASCs without Tβ4 treatment ( Figure 1D). Consistently, TMSB4X mRNA levels after 24 and 48 h of a single treatment with Tβ4 were found to be significantly elevated compared to those in hASCs without Tβ4 treatment ( Figure 1E). Conversely, TMSB4X siRNA significantly reduced TMSB4X mRNA expression; however, it did not affect the cell length of hASCs (Figure S1A-C). Thus, considering the upholding of the endogenous Tβ4 level and the morphological effect of Tβ4 treatment, we established 24 h as a suitable Tβ4 treatment time for further experiments (within 48 h) to investigate the cellular effects of Tβ4 in hASCs.

Tβ4 Induces the Active State of hASCs
We investigated the upholding of Tβ4-induced elongated cell length every 48 h (at day 0, day 2, and day 4). Long-term treatment with Tβ4 was found to sustain the significantly increased cell length of hASCs at day 1, day 3, and day 5 (at 24 h after treatment) compared to without Tβ4 treatment (Figure 2A,B). After 24 h of treatment with Tβ4, hASCs showed an elongated cell body as shown in actin cytoskeleton (F-actin)-stained images and expression of Tβ4 (red) in cell apexes (indicated by white arrowheads in magnified images in Figure 2C). In addition, the fluorescence intensity of Tβ4 expression in hASCs was significantly higher upon Tβ4 treatment than that without Tβ4 treatment ( Figure 2D). Interestingly, treatment with Tβ4 significantly enhanced the phosphorylation of AKT, mTOR, MEK, and ERK compared to that without Tβ4 treatment ( Figure 2E and Figure S2). Moreover, the wound healing assay revealed that treating hASCs with Tβ4 promoted cell migration in a timedependent manner ( Figure 2F,G). Furthermore, we evaluated the effects of Tβ4 on cell sprouting using microbeads covered with hASCs ( Figure 2H). Compared to treatment without Tβ4, the sprout number and length were significantly increased on both day 3 and day 5 after treatment with Tβ4 ( Figure 2I,J). Collectively, Tβ4 induced the active state of hASCs by up-regulating intracellular signaling pathways and increasing the migration and sprouting of cells, which showed an elongated morphology as depicted in Figure 2K.

Tβ4 Induces the Active State of hASCs
We investigated the upholding of Tβ4-induced elongated cell length every 48 h (at day 0, day 2, and day 4). Long-term treatment with Tβ4 was found to sustain the significantly increased cell length of hASCs at day 1, day 3, and day 5 (at 24 h after treatment) compared to without Tβ4 treatment (Figure 2A,B). After 24 h of treatment with Tβ4, hASCs showed an elongated cell body as shown in actin cytoskeleton (F-actin)-stained images and expression of Tβ4 (red) in cell apexes (indicated by white arrowheads in magnified images in Figure 2C). In addition, the fluorescence intensity of Tβ4 expression in hASCs was significantly higher upon Tβ4 treatment than that without Tβ4 treatment ( Figure 2D). Interestingly, treatment with Tβ4 significantly enhanced the phosphorylation of AKT, mTOR, MEK, and ERK compared to that without Tβ4 treatment ( Figure 2E and Figure S2). Moreover, the wound healing assay revealed that treating hASCs with Tβ4 promoted cell migration in a time-dependent manner ( Figure 2F,G). Furthermore, we evaluated the effects of Tβ4 on cell sprouting using microbeads covered with hASCs ( Figure 2H). Compared to treatment without Tβ4, the sprout number and length were significantly increased on both day 3 and day 5 after treatment with Tβ4 ( Figure 2I,J). Collectively, Tβ4 induced the active state of hASCs by up-regulating intracellular signaling pathways and increasing the migration and sprouting of cells, which showed an elongated morphology as depicted in Figure 2K.

Additional Treatment with Tβ4 Enhances Endothelial Differentiation of hASCs
We next investigated whether Tβ4 could influence the endothelial differentiation of hASCs. Endothelial differentiation was induced using custom endothelial differentiation induction media (CED-M), and the medium was additionally treated with Tβ4 every 48 h ( Figure 3A). After 21 days of induction, hASCs cultured in CED-M were found to have an increased expression of PDGFβ and VE-cadherin compared to hASCs. In addition, the expression of these proteins was significantly promoted upon additional treatment with Tβ4 ( Figure 3B,C). Indeed, the mRNA expression of various angiogenic factors and receptors including ANGPT1, VWF, TIE1, CXCR4, FGFR4, IGFR2, PLAUR, and CDK5 was found to be significantly increased upon CED-M-mediated differentiation induced by Tβ4 treatment compared to that observed in normal hASCs and cells that underwent CED-M-mediated differentiation without Tβ4 treatment ( Figure 3D). Therefore, additional treatment with Tβ4 promoted endothelial differentiation of hASCs with elevated expression of angiogenesis-related genes.

Additional Treatment with Tβ4 Enhances Endothelial Differentiation of hASCs
We next investigated whether Tβ4 could influence the endothelial differentiation of hASCs. Endothelial differentiation was induced using custom endothelial differentiation induction media (CED-M), and the medium was additionally treated with Tβ4 every 48 h ( Figure 3A). After 21 days of induction, hASCs cultured in CED-M were found to have an increased expression of PDGFβ and VE-cadherin compared to hASCs. In addition, the expression of these proteins was significantly promoted upon additional treatment with Tβ4 ( Figure 3B,C). Indeed, the mRNA expression of various angiogenic factors and receptors including ANGPT1, VWF, TIE1, CXCR4, FGFR4, IGFR2, PLAUR, and CDK5 was found to be significantly increased upon CED-M-mediated differentiation induced by Tβ4 treatment compared to that observed in normal hASCs and cells that underwent CED-M-mediated differentiation without Tβ4 treatment ( Figure 3D). Therefore, additional treatment with Tβ4 promoted endothelial differentiation of hASCs with elevated expression of angiogenesisrelated genes.

Transplantation of hASCs in Combination with Tβ4 Improves Blood Vessel Recruitment and Blood Flow After Ischemia
To evaluate the in vivo angiogenic effect of combinatorial treatment with hASCs and Tβ4 on recruiting vessels, window chambers containing hASCs without or with Tβ4 were transplanted onto the dorsal skin of mice. At day 5 post-transplantation, the transplanted sites were opened and microvessel formation was evaluated ( Figure 4A). We found that mice transplanted with hASCs and Tβ4 showed a significantly increased number of microvessel branches compared to mice treated with only cell culture media (sham group) and hASCs ( Figure 4B). Moreover, hindlimb ischemia was induced by ligating the left femoral arteries of nude mice, and hASCs without or with Tβ4 were injected. Furthermore, blood flow was assessed using Laser Doppler after day 0, day 7, day 21 (data not shown), and day 49 of the procedure ( Figure 4C). Transplantation of hASCs in combination with Tβ4 caused significant blood flow in the hindlimb compared to that caused by transplantation of only cell culture media (sham group) and hASCs without Tβ4, as indicated by average flux values ( Figure 4D). In addition, treatment with hASCs in combination with Tβ4 attenuated limb or foot loss compared to the sham group ( Figure 4E). Therefore, the higher blood vessel recruitment and improvement of ischemia were observed when mice were injected with a combination of hASCs and Tβ4.

Transplantation of hASCs in Combination with Tβ4 Improves Blood Vessel Recruitment and Blood Flow After Ischemia
To evaluate the in vivo angiogenic effect of combinatorial treatment with hASCs and Tβ4 on recruiting vessels, window chambers containing hASCs without or with Tβ4 were transplanted onto the dorsal skin of mice. At day 5 post-transplantation, the transplanted sites were opened and microvessel formation was evaluated ( Figure 4A). We found that mice transplanted with hASCs and Tβ4 showed a significantly increased number of microvessel branches compared to mice treated with only cell culture media (sham group) and hASCs ( Figure 4B). Moreover, hindlimb ischemia was induced by ligating the left femoral arteries of nude mice, and hASCs without or with Tβ4 were injected. Furthermore, blood flow was assessed using Laser Doppler after day 0, day 7, day 21 (data not shown), and day 49 of the procedure ( Figure 4C). Transplantation of hASCs in combination with Tβ4 caused significant blood flow in the hindlimb compared to that caused by transplantation of only cell culture media (sham group) and hASCs without Tβ4, as indicated by average flux values ( Figure  4D). In addition, treatment with hASCs in combination with Tβ4 attenuated limb or foot loss compared to the sham group ( Figure 4E). Therefore, the higher blood vessel recruitment and improvement of ischemia were observed when mice were injected with a combination of hASCs and Tβ4.

Discussion
The novel findings of our study were as follows: (i) the effects on cell morphology and underlying mechanisms by exogenous treatment with Tβ4 to hASCs, (ii) the role of Tβ4 in endothelial differentiation, and (iii) enhanced therapeutic efficacy of hASCs combined with Tβ4 for treating mouse hindlimb ischemia.
Actin sequestration and desequestration have been reported to be dynamically controlled by various actin-binding proteins such as Tβ4 [23]. We found that TMSB4X knockdown using siRNA significantly decreased the expression of other actin polymerization-associated proteins such as Rac1/Cdc42, WAVE-2, N-WASP, and ARP3 compared to their expression in normal hASCs ( Figure S1D,E). This finding reemphasizes the significant contribution of Tβ4 in actin dynamics. In addition, actin cytoskeletal rearrangement is required for morphology, motility, and migration of adult stem cells in response to their microenvironment [24]. Indeed, our results revealed that Tβ4 treatment induced cell elongation of hASCs to begin to sprout and migrate (Figure 2A-C,K). Such alterations resulted in significant increases of cell migration and sprouting of hASCs from microbeads upon Tβ4 treatment ( Figure 2F-I). On the contrary, Xiao et al. [17] reported that the depletion of Tβ4 promotes migration, proliferation, and activation of hepatic stellate cells. We confirm that TMSB4X knockdown using siRNA significantly decreased the proliferation rate compared to normal hASCs ( Figure S1F). However, in other cell types, such as palatal cells [25], hair follicle stem cells [26], and cardiac cells [20], Tβ4 has been shown to stimulate cell migration and wound healing. Overexpression of Tβ4 has been proved to enhance cell proliferation, invasion, and expression of epithelial-to-mesenchymal transition (EMT)-inducing transcription factor, Twist-related protein 1 (Twist1) in cancer cells [27].
Furthermore, we investigated the intracellular mechanisms underlying the proliferation and survival of hASCs upon exogenous Tβ4 treatment. As shown in Figure 2D,E, Tβ4 treatment was found to significantly increase the phosphorylation of AKT/mTOR and MEK/ERK in hASCs. These results are supported by previous studies that report augmented cell migration and survival of Tβ4-overexpressing cancer cells via the Tβ4-mediated up-regulation of AKT and interleukin-linked kinase (ILK) [28,29]. Consistently, apoptosis activated by Fas ligand and hydrogen peroxide has been shown to be markedly suppressed by Tβ4 in human corneal epithelial cells, indicating the anti-apoptotic effect of Tβ4 [7]. Additionally, it has been reported that Tβ4 enhances MSC proliferation [30] and interleukin-8 (IL-8) secretion by activating the ERK and NF-κB pathways [31].
A previous study showed that Tβ4 enhances adipogenic differentiation and inhibits osteogenic differentiation of bone marrow-derived MSCs by regulating F-actin formation [32]. During the capillary-like tube formation of endothelial cells, endogenous Tβ4 levels increase [33], and exogenous Tβ4 treatment has been shown to facilitate tube formation and sprouting from coronary artery rings [12]. Here, we demonstrated that the addition of Tβ4 markedly increased the endothelial differentiation of hASCs (Figure 3). Among Tβ4-mediated up-regulated angiogenic genes ( Figure 3D), specifically, the activation of Ang-1 signaling plays an important role in enhancing the therapeutic effect of Tβ4. Wang et al. [34] reported that inactivating Ang-1 using a neutralizing antibody against Tie2 inhibited the Tβ4-induced improvement of neurovascular function and remodeling in mice with diabetic peripheral neuropathy.
Tβ4 has been reported to be involved in vascular development [35] and healing liver fibrosis [5,36,37]. Specifically, during cardiovascular development, the cardiac-specific knockdown of TMSB4X has been shown to cause various defects in heart development and has also been proved to be essential for embryonic coronary vasculogenesis [38,39]. Moreover, Tβ4 facilitates angiogenesis and neovascularization in ischemic hearts resulting in enhanced functional recovery post-MI [40]. In this study, we used ASCs, which have been shown to have therapeutic potential for treating hindlimb ischemia [41,42]. Transplantation of hASCs in combination with Tβ4 treatment was found to significantly alleviate limb salvage and blood flow after 49 days of ischemia compared to transplantation of hASCs alone ( Figure 4C-E) and treatment with Tβ4 alone (data not shown). Thus, our results suggest that combined treatment with Tβ4 enhances the therapeutic revascularization potential of hASCs. Additionally, further experiment will be required as a mechanism study of blood vessel recruitment and improvement of blood flow by transplanted-Tβ4 at host tissue, and synergistic effect of Tβ4 with transplanted cells.
Through the present study, we defined the mechanisms underlying Tβ4-mediated changes in morphological features, increase in cell migration, and sprouting of ASCs. Furthermore, we suggested that Tβ4 might be an important factor for differentiating ASCs into endothelial lineages by augmenting angiogenesis-related gene expression. Moreover, the combination of Tβ4 treatment with hASC transplantation can be developed as a more effective alternative therapeutic approach for treating peripheral arterial diseases through ASC transplantation.

Real-Time Polymerase Chain Reaction
Total RNA was extracted from each sample using TRI reagent (TR118, Molecular Research Center, Cincinnati, OH, USA), and cDNA was synthesized as described previously [43]. Real-time polymerase chain reaction (real-time PCR) was performed using the iQ TM SYBR Green Supermix and My iQ TM 2 real-time PCR detection system (both from Bio-Rad, Hercules, CA, USA). The anti-human PCR primer sequences are listed in Table S1. Relative gene expression levels were quantified using the 2 −∆∆Ct method and normalized to GAPDH expression.

Wound Healing Assay
After attachment, a wound was created across the center of the dish, detached cells were removed by washing twice, and attached cells were treated with 100 ng/mL of Tβ4. Cell migration was observed at 0 h, 12 h, 24 h, and 36 h after scratching and treatment. Number of cells that migrated across the scratch line were counted using phase-contrast images.

Microbead Sprouting Assay
Autoclaved Cytodex-3 (C3275, Sigma-Aldrich) was coated with 50 × 10 4 hASCs per 2500 microbeads. After 3 h of incubation with gentle mixing every 20 min, hASC-coated microbeads were seeded on Matrigel and treated with Tβ4 in 5% DMEM. Phase-contrast images were captured at day 0, day 3, and day 5 to observe the sprouting from the microbeads. The number and length of sprouts were quantified using the ImageJ software. At day 5, microbeads were fixed and stained with actin cytoskeleton (F-actin) and DAPI to visualize cell sprouts.

Induction of Endothelial Differentiation
hASCs were plated at a cell density of 5 × 10 4 in 12-well plates and allowed to attach. Custom endothelial differentiation induction media (CED-M) was composed of ascorbic acid (A8960), sodium pyruvate (#11360070), L-glutamine (G7513, all from Sigma-Aldrich), 50 ng/mL of FGF-2 (234-FSE, R&D Systems), 10% FBS, and P/S in RPMI-1640 (12-702F, Lonza). CED-M was replaced every 2 days without or with Tβ4. After 21 days of endothelial differentiation induction, cells were analyzed by immunofluorescence staining and real-time PCR. Male C57BL/6J mice randomly assigned to the following three groups (n = 3 mice per group). Window chambers (PR0001401) were covered by membrane filters (pore size = 0.45 µM; HAWP01300, both from Millipore, Burlington, MA, USA) and filled with hASCs (5 × 10 4 ) without or with 100 ng/mL of Tβ4 in 5% DMEM. All mice were anesthetized with a mixture of ketamine (44 mg/kg; Yuhan, Seoul, Korea) and xylazine hydrochloride (0.75 mg/kg; Rompun, Bayer, Leverkusen, Germany) and shaved. After skin incision, chambers were subcutaneously implanted into the dorsal side of mice. After 5 days of implantation, mice were anesthetized and images of dorsal sides of mice were taken. The number of subcutaneous microvessels per mouse was quantified.

Mouse Hindlimb Ischemia Model
Seventeen six-week-old male nude mice were obtained from Orient Experimental Animal Laboratory and anesthetized with a mixture of ketamine and xylazine hydrochloride. All mice were subjected to surgical ligation of left proximal femoral arteries. hASCs (1 × 10 6 ) were transplanted without or with Tβ4 (5 mg/kg) via intramuscular injection at three different sites around the ligated arteries. The right leg was used as a control. After induction of ischemia (day 0) and 49 days after transplantation, blood flow was evaluated using the Laser Doppler imager and software (both from Moor Instruments, Devon, UK).

Statistical Analysis
All statistical values are expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to compare normally distributed data of all groups. Significant differences between means were determined using ANOVA followed by Student-Newman-Keuls test. All P-values are two-sided, and * p < 0.05 was considered statistically significant. All statistical analyses were performed using the Sigma Stat software (Ver. 3.1, Systat Software, San Jose, CA, USA).