Department of Medicine, Division of Cardiology, Institute for Stem Cell Biology and Regenerative Medicine & Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, California, USA
Department of Medicine, Division of Cardiology, Institute for Stem Cell Biology and Regenerative Medicine & Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, California, USA
Author contributions: W.F.: collection and assembly of data and manuscript writing; K.C., Y.W., and J.C.W.: data analysis and interpretation; X.Q. and S.H.: provision of study material; K.N.: manuscript writing and editing; S.W.: assembly of data; Y.C.: collection of data; L.X.: administrative support; F.C.: conception and design, financial support, and final approval of manuscript. W.F. and K.C. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS October 18, 2012.
Poor cell survival severely limits the beneficial effects of stem cell therapy for peripheral arterial disease (PAD). This study was designed to investigate the role of mammalian target of rapamycin (mTOR) in the survival and therapeutic function of transplanted murine adipose-derived stromal cells (mADSCs) in a murine PAD model. mADSCs (1.0 × 107) were isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein-positive transgenic mice, intramuscularly implanted into the hind limb of C57BL/6 mice after femoral artery ligation/excision, and monitored using noninvasive bioluminescence imaging (BLI). Although engrafted mADSCs produced antiapoptotic/proangiogenic effects in vivo by modulating the inflammatory and angiogenic cytokine response involving the mTOR pathway, longitudinal BLI revealed progressive death of post-transplant mADSCs within ∼4 weeks in the ischemic hind limb. Selectively targeting mTOR complex-1 (mTORC1) using low-dose rapamycin treatment with mADSCs attenuated proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor-alpha [TNF-α]) expression and neutrophil/macrophage infiltration, which overtly promoted mADSCs viability and antiapoptotic/proangiogenic efficacy in vivo. However, targeting dual mTORC1/mTORC2 using PP242 or high-dose rapamycin caused IL-1β/TNF-α upregulation and anti-inflammatory IL-10, IL-6, and vascular endothelial growth factor/vascular endothelial growth factor receptor 2 downregulation, undermining the survival and antiapoptotic/proangiogenic action of mADSCs in vivo. Furthermore, low-dose rapamycin abrogated TNF-α secretion by mADSCs and rescued the cells from hypoxia/reoxygenation-induced death in vitro, while PP242 or high-dose rapamycin exerted proinflammatory effects and promoted cell death. In conclusion, mTORC1 and mTORC2 may differentially regulate inflammation and affect transplanted mADSCs' functional survival in ischemic hind limb. These findings uncover that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the translation of cell-based PAD therapy. STEM Cells2013;31:203–214
Peripheral arterial disease (PAD) is among the leading causes of disability worldwide . Stem cell therapy provides a promising avenue for tissue rescue and functional recovery in PAD. Nonetheless, heterogeneous results have been reported by a number of cell-based PAD therapeutic investigations . One major challenge raised by a few recent reports and our previous studies is the limited survival of transplanted adult stem cells in ischemic tissues, which is associated with the cells' unconvincing therapeutic efficacy [3–5]. More importantly, the long-term outcome and mechanism of function for the surviving engrafted cells have not been fully elucidated. Accordingly, the need to improve the survival and function of transplanted stem cells via mechanism-driven strategies should be stressed. Furthermore, noninvasive cell tracking approaches are definitely pertinent to a better understanding of the longitudinal survival and behavior of donor cells in vivo .
Recent reports have shown that inflammation is a strong contributor to the mechanism of PAD onset and progression, and that the inflammatory microenvironment is unfavorable for engrafted cell survival, impairing efficacy to varying extent en route to treatment failure [2, 7, 8]. The serine/threonine kinase mammalian target of rapamycin (mTOR) is emerging in several inflammatory models . mTOR assembles into two complexes, as rapamycin(Rapa)-sensitive mTOR complex (mTORC1) and Rapa-insensitive complex (mTORC2) . mTORC1/mTORC2 contain shared and distinct (e.g., raptor and rictor) partner proteins, which are proven to orchestrate diverse cellular processes including inflammation, angiogenesis, survival, and apoptosis [9–11]. However, the effect and significance of mTOR on donor cell survival and therapeutic efficacy in ischemic model have not been well explored.
In this study, murine adipose-derived stromal cells (mADSCs) stably expressing the dual-reporter genes firefly luciferase and enhanced green fluorescent protein (Fluc+-eGFP+, mADSCsFluc+GFP+) were delivered intramuscularly in a murine PAD model and monitored longitudinally using noninvasive bioluminescence imaging (BLI). BLI has been previously applied in our studies for noninvasive cell tracking due to its high sensitivity and signal-to-noise ratio [12–14]. Rapa, a selective mTORC1 inhibitor, and PP242, an ATP-competitive dual mTORC1/mTORC2 inhibitor, were used as small molecular regulators of mTOR. We sought  to understand how mTORC1/mTORC2 influence the functional survival of mADSCs after ischemia in vivo or after hypoxia/reoxygenation (H/R) treatment in vitro and  to achieve mechanistic insight into new strategies for promoting mADSC survival in an ischemic setting.
MATERIALS AND METHODS
Fluc+-eGFP+ transgenic mice (Tg[Fluc-egfp]) were bred on a C57BL/6a background to constitutively express Fluc-eGFP in all tissues and organs (Supporting Information Fig. S1). C57BL/6a mice (Tg[Fluc-egfp] inbred strain, Fluc−-eGFP−, n = 360, female, 10-week-old, 20–25 g, specific pathogen free) were used to construct the PAD model. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health. The protocol was approved by the Fourth Military Medical University ethics review board. Animal anesthesia is described in Supporting Information Methods.
Isolation, Culture, and Identification of mADSCs
mADSCs were isolated from Tg[Fluc-egfp] mice and expanded using a previously described procedure with minor modifications . mADSCs were identified for immunophenotype and multipotency using flow cytometry and chemical induction, respectively. Osteogenesis of mADSCs was confirmed by Alk Phos and Runx2 gene expression using reverse transcription-polymerase chain reaction (RT-PCR)  and osteocalcin expression using enzyme-linked immunosorbent assay (ELISA), while adipogenesis was determined using analysis for peroxisome proliferator-activated receptor gamma (PPARγ) gene. Undifferentiated mADSC and primary murine mature adipocyte (mMA) were assayed as controls. Methods for cell culture, immunophenotype identification, multilineage differentiation, and conventional RT-PCR are described in Supporting Information. Primer sequences for RT-PCR are listed in Supporting Information Table S1.
In Vitro Reporter Gene Imaging of mADSCsFluc+GFP+
Dual-modality reporter gene imaging was performed to determine the correlation between mADSC number and Fluc-eGFP activity in vitro . For BLI, mADSCs of different quantities were, respectively, suspended in 500 μl phosphate-buffered saline (PBS), incubated with reporter probe D-luciferin (150 ng/μl, Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and then imaged using a charge-coupled device (CCD) camera within Xenogen Kinetic In Vivo Imaging System (IVIS, Caliper Life Sciences, Hopkinton, MA, http://www.caliperls.com), with the following parameters: Binning: 4, F/Stop: 1, and Exposure time: 1 minute. For fluorescence imaging (FRI), cell suspensions were directly imaged by CCD with its excitation wavelength at 465 nm/430 nm and emission filter at 560 nm. LivingImage 4.2 (Caliper) was used for imaging analysis. Peak signal intensity was expressed in average radiance unit (photons/second/cm2/steridian, P s−1 cm−2 sr−1) from a fixed-area region of interest (ROI).
PAD Model and Cell Delivery
C57BL/6a mice were randomized into nine groups (n = 40 each, matched for weight): (a) Sham, (b) PBS, (c) ADSC, (d) ADSC + low-dose rapamycin (ADSC+RapaLD), (e) ADSC + high-dose rapamycin (ADSC+RapaHD), (f) ADSC+PP242, (g) RapaLD, (h) RapaHD, and (i) PP242. For the PAD model, unilateral hind limb ischemia was induced by ligating and excising the left femoral artery with all superficial and deep branches for all groups except Sham. Surgical procedure for hind limb ischemia is described in Supporting Information Methods. Sham-operated mice received incision without artery ligation and PBS treatment. Mice in the ADSC, ADSC+RapaLD, ADSC+RapaHD, and ADSC+PP242 group were subjected to mADSCsFluc+GFP+ (1.0 × 107) delivery on postoperative day1 (POD1). Cells were suspended in 30 μl PBS and cautiously injected into the left adductor muscle using a 29-gauge insulin syringe (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). ADSC+RapaLD, ADSC+RapaHD, and ADSC+PP242 group animals were administered RapaLD (50 nM/kg per day, Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), RapaHD (10 μM/kg per day), and PP242 (200 nM/kg per day, Sigma-Aldrich), respectively, via adductor injection, while PBS, RapaLD, RapaHD, and PP242 group animals, respectively, received PBS, RapaLD, RapaHD, and PP242 only, without mADSCs.
In Vivo BLI for mADSCsFluc+GFP+ Tracking
In vivo BLI was performed to track the survival of engrafted mADSCsFluc+GFP+. Mice were anesthetized and intraperitoneally injected with 150 mg/kg D-luciferin. Using IVIS, images were acquired at 3-minute intervals until the peak signal was observed. Fixed-area ROIs were created over left hind limbs, and photons emitted from the ROIs were quantified by P s−1 cm−2 sr−1 using LivingImage 4.2. Animals were longitudinally imaged at 3-day intervals.
In Vitro and Ex Vivo Luciferase Assay
In vitro Fluc assays were performed with different amounts of mADSCsFluc+GFP+ using our previously described protocol . Cells were lysed using 1× Passive Lysis Buffer (PLB, Promega, Madison, WI, http://www.promega.com) at 4°C. For every sample, 20 μl of supernatant was added to 100 μl of luciferase assay reagent (LARII, Promega) and luminosity in relative light units (RLU) was detected by a 20/20n luminometer within the Luciferase Assay System (Promega). PBS without mADSCsFluc+GFP+ was used as control. For ex vivo luciferase assay, left adductor muscle tissues were removed from sacrificed mice on POD14, homogenized in PBS containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, http://www.roche-applied-science.com), and lysed with 1× PLB. After centrifugation at 14,000 rpm for 10 minutes at 4°C, the supernatant was collected. Luciferase activity was then measured using the Luciferase Assay System.
Serial Laser Doppler Perfusion Imaging of Hind Limb
To serially monitor the blood perfusion recovery of the ischemic hind limb, mice were placed on a 37.4–38.0°C heating pad to minimize temperature variation and then imaged using an Laser Doppler Perfusion Imaging (LDPI) analyzer (PeriScan-PIM3 Perimed AB, Järfälla, Sweden, http://www.perimed-instruments.com). The blood flux was quantified using perfusion ratio (PR, ratio of average LDPI index of ischemic to nonischemic [contralateral, self-control] hind limb) by LDPIwin3.1.3 (Perimed AB).
Confocal Microscopy for mADSCFluc+GFP+ Tracking and Angiogenesis in the Ischemic Hind Limb
Immunofluorescence assays were conducted to examine the engraftment of mADSCsFluc+GFP+ and to visualize CD31(PECAM-1)-positive vessels. Frozen sections of left adductor muscle were sequentially fixed within cold acetone for 10 minutes, washed three times with PBS containing 0.3% Triton X-100 (Sigma-Aldrich), and blocked with 10% goat serum (ab7481, Abcam, Abcam, Cambridge, MA, http://www.abcam.com) for 30 minutes at room temperature. Then the slides were incubated with rabbit anti-α-sarcomeric actin (ab52219, 1:200, Abcam) overnight at 4°C. After three washes with PBS, slides were incubated with NorthernLights-637 (NL-637) fluorochrome-labeled donkey anti-rabbit IgG (NL005, 1:200, R&D System, Minneapolis, MN, http://www.rndsystems.com) and FITC-conjugated goat anti-GFP (ab6662, 1:200) for 1 hour at 37°C. The nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). Furthermore, double immunofluorescence staining using rat monoclonal anti-CD31 (ab7388, 1:100, Abcam) and DAPI was performed on the frozen sections of adductor muscle to show CD31-positive vasculature. Sections were stained using isotype nonspecific IgG as controls. Slides were photographed using a confocal microscope (FluoView-FV1000, Olympus, Tokyo, Japan, http://www.olympusfluoview.com).
In Vitro BLI of mADSCsFluc+GFP+ Following Hypoxia/Reoxygenation
Experiments using an in vitro H/R model were performed to further evaluate the role of mTOR in mADSC survival in temporary anoxia. mADSCs were uniformly plated in 24-well plates at a density of 5.0 × 104 cells per well and then randomized into five groups: (a) Normoxia (Control), (b) H/R, (c) H/R+RapaLD (1 nM/ml), (d) H/R+RapaHD (100 nM/ml), and (e) H/R+PP242 (4 nM/ml). To mimic hypoxia/reoxygenation, the cells were incubated in an anoxic chamber (95% N2/5% CO2) at 37°C for 21 hours and subsequently moved into a normoxic incubator (95% air/5% CO2) at 37°C for 3 hours. The control group was cultured in standard conditions for 24 hours. In vitro BLI was performed as described above.
Ex Vivo and In Vitro Apoptosis Assessment
Frozen sections of left adductor muscle tissues (isolated on POD14 as described above) or cultured mADSCs following H/R treatment were collected for terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptotic assay using a TUNEL Apoptosis Assay Kit (Promega) according to the manufacturer's instructions. DAPI staining was performed for total nuclei quantification. Apoptosis Index (AI), that is, the number of TUNEL-positive cells divided by the total cells per field, was examined. Each AI was assessed in 20 randomly selected fields.
Matrigel Plug Assay for Inflammatory Cell Infiltration
A Matrigel plug assay was used to assess the infiltrating cells in the ischemic hind limb following mADSCs transplantation . mADSCs (1.0 × 107) with/without RapaLD (1 nM/ml) were embedded in a Matrigel collagen gel (500 μl, 4°C, BD Biosciences) and subcutaneously inoculated into the hind limb following femoral artery occlusion (POD1). After 72 hours, the Matrigel plugs were surgically excised, dissolved, and centrifuged in vitro using collagenase-IV (5 mg/ml, Roche Applied Science) for 30 minutes at 37°C. The cell pellet was washed three times in PBS and filtered through a 70-μm cell strainer. Single-cell suspensions were obtained, incubated with monoclonal antibodies Gr1+ (RB6-8C5), F4/80 (BM8), CD115 (CSF-1R), and Ly-6C (ER-MP20) directly conjugated to PE/Cy5.5, FITC, allophycocyanin (APC) and phycoerythrin (PE) (all from Abcam), and analyzed using the FACscan cytometer (BD Biosciences). Inflammatory cytokines in cell-free supernatant were assessed using ELISA.
Protein Assays of mTOR Signaling and Cytokines
Left adductor muscle tissue or cultured mADSCs were harvested, pulverized, and homogenized to extract protein for Western blotting using our previous protocols . Protein lysates were run on 5%–20% SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes. NC membranes were blocked with 5% milk in 1× tris buffered saline (TBS)-Tween buffer and incubated overnight at 4°C with the primary antibodies for mTOR signal pathway in 1× TBS-Tween. Immunoreactivity was detected by sequential incubation with horseradish peroxidase conjugated antibodies and enzymatic chemiluminescence. Quantitative analysis was performed using QuantiOne imaging software (Bio-Rad, Hercules, CA, http://www.bio-rad.com) to assess the integrated optical density (IOD) of each band. Western blot protocol and antibodies are described in Supporting Information Methods and Supporting Information Table S2, respectively. ELISA were performed on the protein lysates and collected mADSCs' culture supernatants using the Quantikine ELISA kit (R&D Systems) to quantify the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-10, and vascular endothelial growth factor (VEGF) according to the manufacturer's instructions.
Results are expressed as mean ± SD. Prism 5.0 (GraphPad Software, La Jolla, CA, http://www.graphpad.com) was used to perform the one-way analysis of variance (ANOVA) for evaluating the differences in bioluminescence radiance, AI, cell number, IOD, and cytokine concentration among the different experimental groups and different time points within each group. Tukey test in conjunction with ANOVA was used for pairwise multiple comparisons to identify the differences of parameters between two groups. A two-tailed p-value <.05 was considered significant. Polynomial regression analysis was performed to evaluate the correlation between cell numbers and optical radiance in vitro.
Characterization of mADSCsFluc+GFP+
mADSCsFluc+GFP+ exhibited a distinctive in vitro fibroblastoid morphology, as homogeneous mononuclear cells with a fusiform shape in the third cell passage (Fig. 1A). Fluorescence microscopy manifested bright eGFP fluorescence of mADSCsFluc+GFP+ (Fig. 1B). mADSCsFluc+GFP+ retained their “stemness” properties. Following osteogenic induction, alkaline phosphatase activity and mineralized matrix deposition assessed using alizarin red-S staining were observed within mADSCsFluc+GFP+ (Supporting Information Fig. S2A, S2B). mADSCsGFP+ were also inducible to differentiate into adipogenic and chondrogenic lineage cells, as assessed by oil red O and collagen-II staining (Supporting Information Fig. S2C, S2D). RT-PCR (Fig. 1C, 1D) demonstrated that in comparison to undifferentiated mADSCs, differentiated mADSCs exhibited upregulation of Alk Phos (Fig. 1E), Runx2 (Fig. 1F), or PPARγ (Fig. 1G), the key regulatory genes in osteogenesis and adipogenesis, respectively. Furthermore, the level of osteocalcin released from mADSCs following osteogenic induction was significantly higher than from undifferentiated mADSCs and mMAs (Fig. 1H). mADSCsFluc+GFP+ could emit dual-modality BLI/FRI signals (Fig. 1I). Importantly, BLI/FRI quantification demonstrated a robust linear correlation between the number of mADSCsFluc+GFP+ and Fluc average radiance (r2 = .992, Fig. 1J), eGFP average radiance (r2 = .969, Fig. 1J), and Fluc enzymatic activity (r2 = .974; Fig. 1K) in vitro. Flow cytometry revealed ∼50% of the mADSCsFluc+GFP+ was positive for stem cell marker Sca-1 (Fig. 1L). mADSCsFluc+GFP+ had higher expression of mesenchymal stem cell markers (CD44, CD90, and CD29) and lower expression of hematopoietic markers (CD34 and CD45). Other characterization of mADSCsFluc+GFP+ is described in Supporting Information Results.
Survival of Engrafted mADSCsFluc+GFP+ in Murine PAD Model
Noninvasive BLI longitudinally revealed the fate of mADSCsFluc+GFP+ in the ischemic adductor muscle within the 7-week post-transplant period (Fig. 2A). In the first 3 days after initial cell transplantation, the BLI signal intensity maintained at a stable level, showing no significant difference among groups (p = non-significant [NS]). However, engrafted mADSCsFluc+GFP+ in the ADSC group mice experienced a progressive death in the following 4 weeks, with a significant decay in BLI signal from (7.12 × 106 ± 8.98 × 105) P s−1 cm−2 sr−1 on POD1 to (1.02 × 106 ± 3.46 × 105) P s−1 cm−2 sr−1 on POD14, to background levels on POD35 (Fig. 2B). PP242 or RapaHD treatment reduced mADSCs' survival in vivo. In the ADSC+PP242 and ADSC+RapaHD group, donor mADSCsFluc+GFP+ exhibited an earlier trend of cell death, as evidenced by remarkably lower BLI signal intensity than the ADSC group on POD7 (p < .05, respectively). In contrast, RapaLD treatment improved the viability of donor mADSCs. In the ADSC+RapaLD group, more than 70% of the donor mADSCsFluc+GFP+ survived on POD14 (p < .001 vs. ADSC group), and the BLI signal was still detectable on POD35. Ex vivo Fluc assays (Fig. 2C) and immunofluorescence analysis (Fig. 2D) further validated our BLI findings. On POD14, significantly higher Fluc enzymatic activity was detected by luminometry in the ADSC+RapaLD group compared with the ADSC group (6.00 × 106 ± 7.23 × 105 vs. 1.49 × 106 ± 4.70 × 105, RLU, p < .001). Imaging using laser confocal microscopy clearly showed more GFP-positive mADSCs within the ischemic muscle tissue in the ADSC+RapaLD rather than the ADSC group on POD14 (p < .001, Fig. 2E). Moreover, GFP-positive mADSCs were less frequently observed in the ADSC+PP242 and ADSC+RapaHD group compared with the ADSC group (p < .01, respectively, Fig. 2E). Similar survival trend of transplanted mADSCsFluc+GFP+ was also observed in the ischemic gastrocnemius muscle (Supporting Information Results and Supporting Information Fig. S3).
Surviving mADSCs-Mediated Antiapoptotic and Proangiogenic Effects
We went on to evaluate cell apoptosis and revascularization outcome of the ischemic hind limb. The AI, as assessed by TUNEL assay (Fig. 3A), was significantly higher in the PBS group compared to the sham group on POD14 (p < .001, Fig. 3B), indicating induction of cell apoptosis by hind limb ischemia. Engrafted mADSCs mediated an antiapoptotic effect, as decreased AI was observed in cell-injected muscle compared to PBS-injected muscle (8.89% ± 1.00% vs. 16.50% ± 3.25%, p < .001). Furthermore, mADSCs remarkably improved the PR (0.70 ± 0.02 vs. 0.62 ± 0.03, p < .05, Fig. 4A, 4B) and density of CD31(PECAM-1)-positive vessels (353 ± 19 vs. 285 ± 35, p < .001, Fig. 4C; Supporting Information Fig. S4) in the ischemic hind limb when compared with PBS treatment on POD14, as assessed using serial LDPI measurements in vivo and immunofluorescence analysis ex vivo. Although RapaLD treatment alone failed to elicit any statistically significant antiapoptotic or proangiogenic effects by POD14 (p = NS vs. PBS group), a combined RapaLD treatment with mADSCs overtly promoted mADSC-mediated antiapoptosis and angiogenesis in the ADSC+RapaLD group (p < .001 vs. ADSC group). Accordingly, the enhanced antiapoptotic and proangiogenic effects may mainly derive from more surviving donor mADSCs in the ADSC+RapaLD group compared with other groups. In contrast, additional PP242 or RapaHD administration with mADSCs abrogated ADSC-mediated antiapoptotic and proangiogenic benefit (p < .001 vs. ADSC group, respectively), while single PP242 or RapaHD treatment also significantly increased AI and attenuated angiogenesis compared with the PBS group (p < .05, respectively).
Activation of mTOR Signal Pathway in the mADSC-Transplanted Ischemic Hind Limb
To further understand the mechanism of action behind the efficacy of transplanted mADSCs, we examined putative mTOR signal pathways in the ischemic hind limb using Western blotting (Fig. 5A) and ELISA. Immunoblot analysis revealed a spontaneous activation of mTOR along with its downstream targets in the postischemic hind limb compared with the sham-operated hind limb on POD7. Engrafted mADSCs significantly augmented the phosphorylation level of mTOR (Fig. 5B), including raptor (Fig. 5C) and rictor (Fig. 5D), which serve as the main component protein of mTORC1 and mTORC2, respectively . Meanwhile, within the mTOR signal pathway, p70S6K (S6K, Fig. 5E), Akt/PKB (Fig. 5F), and STAT3 (Fig. 5G) were also activated following mADSCs transplantation. Our data demonstrated that RapaLD partially suppressed mTOR phosphorylation and selectively inhibited the activation of mTORC1 (raptor) with its downstream target S6K (mTORC1-S6K). mTORC1-S6K has been proven to be involved in a series of inflammatory and immune responses [18, 19]. Nevertheless, RapaLD elicited a nonsignificant effect on mTORC2 (rictor) and its confirmed downstream factor Akt, which has been established as central in mediating cell survival and antiapoptosis . STAT3, another mTOR downstream target associated with prosurvival mechanisms , was not affected by RapaLD treatment either. In contrast, PP242 abolished both mTORC1 (raptor) and mTORC2 (rictor) activation, while it also severely attenuated S6K, Akt, and STAT3 phosphorylation. We also demonstrated that RapaHD further inhibited the phosphorylation of mTORC2 assembly with Akt/STAT3, yielding dual mTORC1/mTORC2 antagonism comparable with PP242. Moreover, activation of VEGF receptor-2 (VEGFR2, Fig. 5H) and VEGF (Fig. 5I), as potent proangiogenic signaling molecules , was permissive to mADSC-mediated angiogenesis. Dual mTORC1/mTORC2 inhibition using PP242 or RapaHD severely attenuated mADSC-induced VEGF/VEGFR2 activation, while selective mTORC1 inhibition using RapaLD treatment with mADSCs mildly retarded VEGF expression and yielded a statistically nonsignificant effect on VEGFR2.
mTOR Regulated Inflammatory Response in Ischemic Hind Limb
Analysis of inflammatory cytokines revealed that inflammation underlay the correlation between mTOR and mADSCs' functional survival. On POD7, ELISA documented ∼31-, 12-, and 8-fold elevated levels of IL-1β, TNF-α, and IL-6 in the ischemic adductor muscle compared with the sham-operated one, consistent with acute postischemic inflammation (Fig. 6A). The ischemic gastrocnemius muscle exhibited a more profound inflammatory cytokines response as compared to the ischemic adductor muscle (Supporting Information Results and Supporting Information Fig. S5). Engrafted mADSCs modulated the host inflammatory response, as evidenced by higher IL-1β, TNF-α, IL-6, and IL-10 expression in the ADSC group compared with the PBS group (p < .001, respectively). Our data demonstrated that mTOR regulated the inflammation in the ischemic hind limb. Selective mTORC1-S6K inhibition by RapaLD administration with mADSCs transplantation notably attenuated the expression of IL-1β/TNF-α, known proinflammatory cytokines that induce acute cell death and transplanted stem cell dysfunction [23–25]. Nonetheless, RapaLD yielded a nonsignificant effect on anti-inflammatory cytokine IL-10 and IL-6, which were demonstrated to promote donor cell survival in vivo [26–28]. In contrast, dual mTORC1/mTORC2 antagonism using PP242 or RapaHD remarkably upregulated IL-1β/TNF-α and downregulated IL-10/IL-6 expression. Accordingly, the suppression of IL-1β/TNF-α and upregulation of IL-10/IL-6 probably result from mTORC2 activation. Run charts depicted that RapaLD treatment counteracted the early peak of postischemic IL-1β/TNF-α expression (Fig. 6B). More surviving mADSCs in the ADSC+RapaLD group profoundly enhanced IL-10/IL-6 levels, even at a later stage following hind limb ischemia (POD49: p < .05 vs. ADSC group, respectively). Matrigel plug assays (Supporting Information Fig. S6) demonstrated that RapaLD significantly mitigated the infiltration of neutrophils (Fig. 6C) and macrophages (Fig. 6D) into the cellular graft site in ischemic hind limb. Furthermore, levels of IL-1β (Fig. 6E) and TNF-α (Fig. 6F) in the Matrigel plug mixed with mADSCs and RapaLD were remarkably reduced compared with Matrigel plugs mixed with mADSCs alone.
mTOR Signaling Regulated mADSCs' Survival Over Hypoxia/Reoxygenation In Vitro
To further characterize the role of mTOR in mADSCs' survival, we performed an in vitro H/R model of mADSCs. In vitro BLI (Fig. 7A) displayed a remarkable decline of BLI signal intensity in mADSCsFluc+GFP+ following H/R compared with normoxic controls (3.92 × 104 ± 3.06 × 103 vs. 7.96 × 104 ± 6.00 × 103, P s−1 cm−2 sr−1, p < .001, Fig. 7B). mADSCs in the H/R group also exhibited higher AI compared with the control group using the TUNEL assay (Fig. 7C; Supporting Information Fig. S7), indicating the vulnerability of mADSCs to temporary anoxia. Importantly, following H/R treatment, activation of mTORC1/mTORC2 signaling was noted within mADSCs using the Western blot analysis (Fig. 7D), while TNF-α, IL-6, IL-10, and VEGF levels also significantly increased within mADSCs' supernatant as assessed by ELISA (Fig. 7E). Interestingly, RapaLD treatment counteracted the activation of mTORC1(raptor)-S6K (Fig. 7D) and inhibited mADSC-derived TNF-α secretion during H/R (Fig. 7E). As a result, RapaLD significantly reinforced mADSCs' survival (Fig. 7B) and prevented the cells from H/R-induced apoptotic death (Fig. 7C). Contrarily, PP242 or RapaHD treatment abolished the activation of both mTORC1(raptor)-S6K and mTORC2(rictor)-Akt-STAT3, leading to the elevation of TNF-α and suppression of IL-6, IL-10, and VEGF. Accordingly, consistent with in vivo results, mTORC2-Akt-STAT3 inhibition elicited proinflammatory effects, which aggravated H/R-induced mADSC death and apoptosis.
Effective cell engraftment and survival are necessary for the success of stem cell-based PAD therapy. However, the surviving fraction of donor cells is quite variable in different studies, ranging from 0% to 90%, and uncertainty about the therapeutic efficacy of the cells continues [2, 29]. Although previous studies demonstrated that the ADSC with its secretome could augment tissue regeneration in ischemic models , the fate of transplanted ADSCs in the PAD model has not been fully elucidated using traditional tracking techniques. Previously, we demonstrated that molecular imaging strategy provided valuable insight into the in vivo kinetics of engrafted cells [12–14]. Namely, BLI is an accurate, sensitive, and high-throughput approach for noninvasive stem cell tracking of as few as 500 cells [12–14]. Relying on BLI, we longitudinally and spatiotemporally visualized the abbreviated lives of mADSCs following transplantation into murine ischemic hind limbs. BLI further showed that RapaLD treatment ameliorated the survival of mADSCs in ischemic hind limb in vivo, or following H/R treatment in vitro, which favorably provided an incremental benefit in mADSC-mediated antiapoptosis and angiogenesis. Therefore, improving the functional survival of engrafted cells is pertinent to cell transplantation therapy.
Previous studies reported that the functional survival of transplanted stem cells was limited by postischemic inflammation, insufficient perfusion, tissue dysfunction, and related causes [24, 29]. Among these, inflammation is increasingly recognized as a primary contributor to loss of engrafted cells, and inhibiting proinflammatory cytokine activity has been shown to reverse inflammation-mediated dysfunction of cell grafts [7, 24, 29]. Inflammation also plays a pivotal role in PAD progression, and the inflammatory mediators involved in this process are similar to those contributing to the development of coronary artery disease . Intriguingly, evidence shows that transplanted stem cells may speed tissue repair via regulation of inflammation . In our experiment, although in vivo functional survival of the engrafted mADSCs was impaired by postischemic inflammation, surviving mADSCs could modulate the host inflammatory response and yielded limited antiapoptotic and proangiogenic effects on the ischemic hind limb. These findings are in conformity with the putative paracrine action of ADSCs  and further verify the regulative effects of ADSC on host inflammation .
Recent investigations have associated mTOR with stem cell survival and inflammation [9, 10], while mTORC1/mTORC2, as structurally and functionally distinct complexes, may play different roles in cellular responsiveness . Our data demonstrated that mTORC1/mTORC2 were responsible for the survival of donor mADSCs and mediated mADSC-directed inflammatory modulation and angiogenesis in our PAD model. Engrafted mADSCs promoted the activation of mTORC1/mTORC2 and their respective downstream signaling pathways in the ischemic hind limb. Dual mTORC1/mTORC2 inhibition using PP242 reduced mADSC survival and offset mADSC-mediated perfusion benefits. Importantly, mTORC1 and mTORC2 might exert opposite effects on the functional survival of transplanted mADSCs, via positively and negatively regulating inflammation, respectively.
The mTORC1-S6K axis exhibits proinflammatory potential, as mTORC1-S6K upregulation in several organs of mice challenged with endotoxemia can lead to lethal inflammation [33, 34]. In this study, mTORC1-S6K activation contributed to the spontaneous and mADSC-induced IL-1β/TNF-α upregulation in the ischemic hind limb. IL-1β is known to induce a dose-dependent response of apoptosis in cardiomyocytes, while inhibition of IL-1β attenuates the acute inflammatory response and prevents loss of grafted stem cells [25, 35]. TNF-α, as an important mediator of early inflammatory events, was recently determined to mediate proapoptotic cascade and undermine graft stem cells' viability . Rapa-induced mTORC1-S6K inhibition has been reported to reduce inflammation  and mediate resistance to cell apoptosis [36, 37]. This data revealed that selective mTORC1-S6K inhibition using RapaLD effectively suppressed IL-1β/TNF-α expression, which facilitated mADSC survival and therapeutic benefit to a significant extent in vivo. In vitro cell study also showed that RapaLD treatment mitigated the TNF-α secretion of mADSCs over H/R and rescued the cells from H/R-induced cell death via mTORC1-S6K inhibition. Furthermore, we have demonstrated that Rapa administration attenuated the infiltration of neutrophils, macrophages, and CD4+ T-cells  into the cellular graft site. These cells predominate in the inflammatory response in different phases and have been showed to induce dysfunction and death of transplanted stem cells [29, 38]. These results are in accordance with the antiapoptotic effect of Rapa in other ischemic/reperfusion models . Intriguingly, Rapa has also been reported to enhance cell longevity and extend the lifespan of mice up to 14% . Since high levels of telomerase activity in ADSCs have been demonstrated to play a pivotal role in the cells' self-renewal potential, multipotency, and immunomodulatory property , it is conceivable that mTOR signaling may involve telomerase-dependent regulation of ADSC functional survival.
Engrafted mADSCs also activated IL-6, IL-10, and proangiogenic VEGF/VEGFR2 signaling in vivo. IL-6 was another early cytokine following hind limb ischemia. Previous evidence shows that IL-6 can mediate cell survival and angiogenesis [26, 27]. IL-10 is an established anti-inflammatory cytokine that enhances stem cell survival in vivo . VEGF acts as a central mediator of angiogenesis . We previously reported that VEGF enhanced the functional survival of donor cells in ischemic myocardium , suggesting VEGF secretion is a protective response of mADSCs to ischemia in vivo and hypoxic stimuli in vitro. VEGF primarily uses the VEGFR2 (KDR/Flk-1) tyrosine kinase, a key regulator of proangiogenic and antiapoptotic responses . Activation of IL-6, IL-10, and VEGF/VEGFR2 facilitated the functional survival of mADSCs, in which mTORC2 might be heavily involved. Simultaneous mTORC1/mTORC2 counteraction using PP242 retarded the activation of IL-6, IL-10, and VEGF/VEGFR2, and enhanced the expression of IL-1β and TNF-α, which tilted the balance of inflammation to the proinflammatory side and inhibited angiogenesis. Moreover, our data demonstrated that RapaHD, comparable with PP242, could further target mTORC2 assembly, inducing dual mTORC1/mTORC2 inhibition and decreasing mADSC functional survival. Other reports have concluded that dual mTORC1/mTORC2 inhibitors suppressed tumor cell survival, eliciting cell apoptosis, and even cytotoxic effects . Accordingly, activation of mTORC2 may negatively regulate the inflammatory response and apoptosis, which is pertinent to improved mADSC survival and action in vivo.
Despite a decreased understanding of mTORC2 compared to mTORC1, mTORC2 has emerged as an anti-inflammatory target with significant differences from mTORC1 based on recent evidence [44, 45]. In this study, mTORC2 also exhibited a more potent and sustained proangiogenic effect compared with the mTORC1. Accordingly, the inhibition of mTORC2 pathway may be the key to Rapa's antiangiogenic activity, and it would represent the main reason why dual mTORC1/mTORC2 inhibitors are more efficient than Rapa in antiangiogenesis. mTORC1 and mTORC2 also have different actions on Akt, the former working as an inhibitor and the latter as an activator . Our data demonstrated that engrafted mADSCs could upregulate mTORC2-Akt, a crucial pathway that necessarily mediates cell survival, angiogenesis, and antiapoptosis [20, 44, 45]. Also, mADSCs significantly activated STAT3, a cytokine-activated anti-inflammatory protein. IL-6, IL-10, and VEGF can use STAT3 in a feedforward manner, inducing cell resistance to apoptosis, angiogenesis, and cytoprotective effects [46, 47]. Thus, a favorable sequential activation loop is conceivable, in which mADSC-activated mTORC2-STAT3 stimulates IL-6, IL-10, and VEGF, which subsequently activates STAT3 and facilitates mADSC viability and angiogenesis. In vitro study further determined that mTORC2-Akt-STAT3 activation was essential for mADSC survival in response to H/R stimuli.
Although reporter gene imaging can be applied as a powerful tool for in vivo tracking of surviving stem cells, the research is still limited to the laboratory. Recently, the tremendous growth of epigenetics and translational modification potentiate the low-risk induction of prosurvival factor and/or reporter in stem cells. Previously, we also reported that these strategies facilitated the survival and noninvasive evaluation of donor stem cells [48, 49]. To this end, the DNA-free modifications would aid in promoting the telomerase activity in ADSCs through mTOR signaling, then to further potentiate the cells' therapeutic efficacy and clinical translation.
This study demonstrated that modulation of mTORC1/mTORC2 signaling can significantly contribute to the functional survival of transplanted mADSCs in a murine PAD model. Activation of mTORC1-S6K exhibited proinflammatory potential, along with subsequent impairment of mADSC viability and function in vivo. In contrast, activation of mTORC2-Akt-STAT3 induced anti-inflammation, antiapoptosis, and proangiogenesis, which enabled mADSC survival and therapeutic action. These data lead us to believe that mTOR may evolve into a promising candidate for mechanism-driven approaches to facilitate the clinical translation of cell-based PAD therapy.
This work was supported by National Basic Research Program of China (2012CB518101); National Natural Science Foundation of China (Nos. 30970845, 81090274, and 81090270); FCao (BWS12J037); Innovation Team Development Grant by China Department of Education (2010CXTD01); and China's Ministry of Science and Technology 863 Program (2012AA02A603).
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.