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. 2014 Dec 20;21(18):2543–2557. doi: 10.1089/ars.2013.5683

Activation of Liver X Receptor Improves Viability of Adipose-Derived Mesenchymal Stem Cells to Attenuate Myocardial Ischemia Injury Through TLR4/NF-κB and Keap-1/Nrf-2 Signaling Pathways

Yabin Wang 1,,2,,*, Chunhong Li 2,,*, Kang Cheng 2,,*, Ran Zhang 1, Kazim Narsinh 3, Shuang Li 2, Xiujuan Li 2, Xing Qin 2, Rongqing Zhang 2, Congye Li 2, Tao Su 2, Jiangwei Chen 2, Feng Cao 1,,2,✉
PMCID: PMC4245883  PMID: 24915051

Abstract

Aims: Clinical application of cellular therapy for cardiac regeneration is significantly hampered by the low retention of engrafted cells, mainly attributable to the poor microenvironment dominated by inflammation and oxidative stress in the host's infarcted myocardium. This study aims at investigating whether liver X receptor (LXR) agonist T0901317 will improve survival of adipose-derived mesenchymal stem cells (AD-MSCs) after transplantation into infarcted hearts. Results: Noninvasive in vivo bioluminescence imaging and histological staining showed that LXR agonist T0901317 improved the retention and survival of intramyocardially injected AD-MSCs. Moreover, combined therapy of LXR agonist and AD-MSCs inhibited host cardiomyocyte apoptosis, reduced fibrosis, and improved cardiac function, while it concomitantly decreased inflammatory cytokines (e.g., tumor necrosis factor-α and interleukin-6) and increased growth factor (e.g., vascular endothelial growth factor and basic fibroblast growth factor) expression in infarct myocardium. To reveal possible mechanisms, AD-MSCs were subjected to hypoxia/serum deprivation (H/SD) injury to simulate ischemic conditions in vivo. The LXR agonist (10−7 mM) improved AD-MSC survival under H/SD condition. Western blot revealed that the LXR agonist reduced TLR4, TRAF-6, and MyD88 protein expression, inhibited IκBα phosphorylation and NF-κB-p65 nuclear translocation, which resulted in accelerated Keap-1 protein degradation, enhanced Nrf-2 nuclear translocation, and increased HO-1 protein expression. Innovation and Conclusion: LXR agonist can enhance the functional survival of transplanted AD-MSCs in infarcted myocardium, at least partially, via modulation of the TLR4/NF-κB and Keap-1/Nrf-2 signaling pathways. Moreover, combined therapy of LXR agonist and AD-MSCs has a synergetic effect on cardiac repair and functional improvement after infarction. Antioxid. Redox Signal. 21, 2543–2557.

Introduction

Stem cell transplantation has emerged as a promising therapeutic strategy for ischemic heart disease (11, 13, 28). Among all cell candidates for cardiac regeneration, adipose-derived mesenchymal stem cells (AD-MSCs) are considered an optimal cell type for clinical application, as they can be easily obtained from adipose tissue (26). However, low retention and poor survival of injected stem cells are major obstacles to achieve intended therapeutic effect. Our previous studies revealed that nearly 90% of the stem cells might not survive within the first 4 days after cell delivery because of ischemia and local inflammation (6, 41). Other studies demonstrated that improving the conditions of this unfavorable niche in infarcted myocardium could increase the survival of injected stem cells (31, 39). Our group also found that regulating local inflammation in ischemic tissue with sarpogrelate could enhance engrafted AD-MSCs viability and antiapoptotic/proangiogenic efficacy in vivo (7). In addition to microenvironmental regulation, increasing the stem cells' ability to resist local stress, such as local inflammation and oxidative stress, is also crucial for stem cell survival and efficacy (22, 37). Therefore, it is imperative to improve the engraftment microenvironment as well as reinforce stem cell biological function to withstand the inhospitable ischemic milieu of the infarcted heart.

Innovation.

Most donor cells do not survive for prolonged periods due to detrimental microenvironment of ischemic myocardium, which limits clinical application of cell-based therapies. Our research suggest for the first time that the liver X receptor (LXR) agonist may serve as a potent agent for protecting transplanted adipose-derived mesenchymal stem cells from inflammation and oxidative stress injury in the infarcted myocardium, and promote their functional survival, thereby enhancing cardiac repair and function. LXR agonist is a promising agent for cell-based therapy clinical translation.

Liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily that have been recently recognized as novel pharmacological targets for cardiovascular disease therapy (15). LXRα is expressed predominantly in the liver, kidney, intestine, macrophages, and adipose tissue; whereas LXRβ is expressed ubiquitously. LXR and LXR ligands have been previously reported to regulate inflammation and immune response (16, 17). Lei et al. demonstrated that LXR agonists could attenuate ischemia and reperfusion injury (19). Interestingly, LXR and LXR ligands are also found to modulate the balance between cell survival and death. It is revealed that LXR ligand 24(S), 25-epoxycholesterol (24, 25-EC) promotes embryonic stem cell proliferation, differentiation, and neurogenesis (27, 32). However, the effect of LXR agonists on AD-MSCs survival and biological behavior remains unclear. The ability of LXR agonist to enhance AD-MSCs functional survival after transplantation into infarct myocardium warrants further investigation.

In this study, we assessed the role of LXR agonist T0901317 in the survival of intramyocardially engrafted mAD-MSCsFluc+-eGFP+ isolated from Fluc+-eGFP+ transgenic mice by tracking viable cells longitudinally in vivo with noninvasive bioluminescence imaging (BLI). We also further elucidated the mechanism of the LXR agonist's effects through using a hypoxia/serum deprivation (H/SD) cell model in vitro. We sought to assess the ability of an LXR agonist to promote the survival of transplanted AD-MSCs in vivo and improve cardiac function after myocardial infarction (MI) injury.

Results

Characterization and LXRs expression of AD-MSCs

mAD-MSCsFluc+-eGFP+ could be abundantly isolated from adipose samples (∼106 cells per gram raw tissues) and exhibited a fibroblastoid morphology in vitro (Fig. 1C). Fluorescence microscopy manifested bright enhanced green fluorescence protein (eGFP) expression of mADSCsFluc+GFP+ (Fig. 1C). mADSCsFluc+GFP+ were positive for MSC markers CD90, CD44 and exhibited low expression of endothelial (CD34) and leukocyte markers (CD45), which represented the potential of differentiating into adipocytes and osteogenic cell lineages, as shown in our previous work (7). Furthermore, real-time quantitative PCR (RT-qPCR) and Western blot analysis revealed that the mRNA and protein expression of LXR α and LXR β were present in mAD-MSCs (Fig. 1D).

FIG. 1.

FIG. 1.

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Morphology and BLI of AD-MSCs in vitro. (A) BLI of different numbers of AD-MSCs; (B) The linear relationship between AD-MSCs numbers and BLI signal strength; (C) Morphology of third-passage AD-MSCs cultured in vitro (×400) (left); AD-MSCs is GFP positive (right); (D) mRNA and protein expression of LXRα and LXRβ in AD-MSCs (n=5). Scale bar: 10 μm. AD-MSCs, adipose-derived mesenchymal stem cells; BLI, bioluminescence imaging; LXR, liver X receptor.

Linear correlation between mAD-MSCsFluc+-eGFP+ number and BLI signal

mAD-MSCsFluc+-eGFP+ isolated from Fluc+-eGFP+ transgenic mice uniformly expressed firefly luciferase (Fluc) and GFP. Ex vivo bioluminescence images of mAD-MSCsFluc+-eGFP+ showed a linear relationship between the cell number and BLI signals (R2=0.9888) (Fig. 1A, B). These data indicated that BLI could be used to monitor and quantify transplanted mAD-MSCsFluc+GFP+ in living small animals.

LXR agonist improves the survival of transplanted mAD-MSCsFluc+-eGFP+

To detect the effect of the LXR agonist on the viability of transplanted mAD-MSCsFluc+-eGFP+, BLI was performed during the 4 weeks after mAD-MSCsFluc+-eGFP+ transplantation. As shown in Figure 2A and B, there was no significant difference in BLI signal intensity between groups on postoperative day (POD) 2 (p>0.05). The signals in both groups decreased gradually in the next few days. However, the BLI signal in the AD-MSCs group declined much more than that in the AD-MSCs+LXR group (on POD 7, AD-MSCs: 1.28±0.08×105 average photons per second per cm square per steridian [photons/s/cm2/sr] vs. AD-MSCs+LXR: 1.60±0.11×105 photons/s/cm2/sr, p<0.05; on POD 28, AD-MSCs: 0.05±0.03×105 photons/s/cm2/sr vs. AD-MSCs+LXR: 0.27±0.05×105 photons/s/cm2/sr, p<0.05).

FIG. 2.

FIG. 2.

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BLI of transplanted AD-MSCs. (A) To assess longitudinal transplanted AD-MSCs survival, animals were imaged for 4 weeks. Bioluminescence signals were detected in AD-MSCs+LXR group mice at day 2, 7, 14, 21, and 28, although in the AD-MSCs group bioluminescence signal is not detected at day 28; (B) Quantification of imaging signals showed that bioluminescence signals in the AD-MSCs+LXR group are much higher than those in AD-MSCs group (n=5). *p<0.05; (C) GFP-positive cells (white arrow) were hardly found in the heart of the AD-MSCs group mice, but in AD-MSCs+LXR group mice, GFP-positive cells were occasionally found (n=3). *p<0.05, Scale bar: 10 μm.

Further ex vivo confocal fluorescence microscopy confirmed that more GFP positive engrafted cells could be found in the heart tissue of mice in the AD-MSCs+LXR group than in the AD-MSCs group (ratio of GFP+/DAPI: 10.56%±1.38% vs. 3.94%±0.61%, p<0.05) (Fig. 2C).

Combined therapy of AD-MSCs and LXR agonist improves cardiac function after MI

Echocardiography analysis indicated that there was no significant difference in left ventricular ejection fraction (LVEF) and fraction shortening (FS) between all groups at baseline (p>0.05). On POD 2, LVEF significantly decreased in all groups. However, combined therapy of AD-MSCs and LXR improved cardiac function significantly more than expected. Specifically, by POD 7, 14, and 28, LVEF was improved in the combined therapy group compared with MI groups (p<0.05). Similarly, FS was significantly improved in the combined therapy group on POD 7, 14, and 28 in contrast to MI groups (p<0.05). However, pretreated AD-MSCs with LXR agonist before transplantation could not ameliorate LVEF and FS of MI mice on POD 7, 14, and 28, compared with the MI group (p>0.05). Furthermore, combined therapy of LXR agonist and AD-MSCs transfected with Nrf-2 siRNA markedly decreased LVEF and FS on POD 7, 14, and 28, in contrast to the AD-MSCs+LXR group (p<0.05) (Fig. 3A, B).

FIG. 3.

FIG. 3.

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Cardiac function and cardiomyocyte apoptosis after infarction. (A) Representative M-mode echocardiographic data of infarcted hearts receiving phosphate-buffered saline, AD-MSCs, AD-MSCs+LXR Pre, AD-MSCs with Nrf-2 siRNA+LXR, and AD-MSCs+LXR at POD 2, 7, 14, and 28; (B) Comparison of fractional shortening and ejection fraction between all groups, respectively (n=5). *p<0.05 versus MI group, #p<0.05 versus AD-MSCs+LXR group; (C) Representative photographs of TUNEL-stained myocardial sections from each group at POD 3. The images show TUNEL-positive cells (green fluorescence), myocardium stained with monoclonal antibody cTnI (red fluorescence), and DAPI-positive nuclei (blue fluorescence); (D) Graphs summarize the number of TUNEL-positive nuclei per 100 nuclei and present the average from five different fields randomly selected (×100 magnification) (n=5). *p<0.01 versus sham group, #p<0.05 versus MI group. Scale bar: 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; MI, myocardial infarction; POD, postoperative days.

Combined therapy of AD-MSCs and LXR agonist inhibits cardiomyocyte apoptosis and reduces infarcted area size after MI

TUNEL assay showed that the apoptotic index (AI) was significantly higher in mice undergoing MI than in mice undergoing sham operation (21.8%±1.46% vs. 3.9%±0.24%, p<0.01). Unexpectedly, AD-MSC administration alone did not significantly decrease AI compared with the MI group (p>0.05). In contrast, combination therapy of AD-MSCs+LXR significantly lowered AI compared with that in the MI group (11.0%±0.64% vs. 21.8%±1.46%, p<0.05) (Fig. 3C, D).

Masson's trichrome staining revealed that although the area of myocardial infarcted size was not significantly reduced in the AD-MSC group compared with that in the MI group (42.09%±1.30% vs. 53.15%±1.22%, p>0.05), it was substantially lower in the AD-MSCs+LXR group than in the MI group (26.30%±1.70% vs. 53.15%±1.22%, p<0.05). However, AD-MSCs engraftment with LXR agonist pretreated could not reduce myocardial infarcted area size, in contrast to the MI group (p>0.05). In addition, combined therapy of LXR agonist and AD-MSCs transfected with Nrf-2 siRNA was found to increase myocardial infarcted area size on POD 28, compared with AD-MSCs+LXR group (p<0.05) (Fig. 4A).

FIG. 4.

FIG. 4.

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Myocardial infarct size, fibrosis, and angiogenesis after infarction. (A) Masson's trichrome-stained myocardial sections and comparison of fibrosis areas in a subgroup of animals at POD 28 (n=5). *p<0.05 versus MI group, #p<0.05 versus AD-MSCs+LXR group; (B) Representative photographs of CD31-positive vessel (Red arrow) and quantification analysis from each group (×200 magnification) (n=5). *p<0.05 versus MI group, #p<0.05 versus AD-MSCs+LXR group. Scale bar: 100 μm.

Combined therapy of AD-MSCs and LXR agonist increases capillary density in the peri-infarcted area

Immunohistochemical staining of CD31-positive tubular structures showed that capillary density markedly increased in the peri-infarcted area of the AD-MSCs+LXR group than that in the MI group (p<0.05). However, almost no CD31-positive vessels were observed in sham group (Fig. 4B).

Combined therapy of AD-MSCs and LXR agonist regulates inflammatory cytokine, reactive oxygen species, and growth factor production

ELISA analysis revealed that MI led to a significant increase of tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in injured myocardium, compared with animals undergoing sham operation (TNF-α: 24.31±0.98 pg/mg protein vs. 5.20±0.45 pg/mg protein; IL-6: 19.21±1.31 pg/mg protein vs. 4.57±0.34 pg/mg protein, p<0.01). However, administration of the LXR agonist prevented the increase of TNF-α and IL-6 in infarcted myocardium (TNF-α: 17.94±1.28 pg/mg protein vs. 24.31±0.98 pg/mg protein; IL-6: 12.14±1.02 pg/mg protein vs. 19.21±1.31 pg/mg protein, p<0.05). Surprisingly, AD-MSCs alone did not exert a dramatic effect on TNF-α and IL-6 levels in injured myocardium (p>0.05) (Fig. 5A, B).

FIG. 5.

FIG. 5.

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Effects of LXR agonist and AD-MSCs on MI-induced inflammatory factors, growth factors, and ROS generation. (A, B) LXR agonists reduced cardiac levels of TNF-α and IL-6 in MI+LXR and MI+LXR+AD-MSCs groups compared with the MI group (n=5). *p<0.01 versus Sham group, **p<0.05 versus MI group, #p<0.05 versus MI group; (C, D) LXR agonists decrease serum levels of bFGF and VEGF in MI+LXR+AD-MSCs group compared with MI group (n=5). #p<0.05 versus sham group; (E) Representative images of dihydroethidium fluorescence staining that evaluated ROS generation in myocardium and bar graph summarizing fluorescence intensity (n=5). *p<0.01 versus Sham group, **p<0.05 versus MI group, #p<0.05 versus MI group, and $p<0.05 versus MI group. Scale bar: 50 μm. bFGF, basic fibroblast growth factor; IL-6, interleukin-6; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.

Previous research has demonstrated that reactive oxygen species (ROS) generated in infarcted myocardium stimulates cardiomyocyte apoptosis (21) and decreases AD-MSCs survival after engraftment (38). Therefore, ROS generation was assessed in our experimental model using dihydroethidium (DHE) fluorescence. The LXR agonist significantly lowered the MI-induced increase of DHE fluorescence intensity (p<0.05). Engrafted AD-MSCs alone did not influence DHE fluorescence intensity as compared with the MI group (p>0.05) (Fig. 5E).

ELISA analysis revealed that combined therapy with AD-MSCs and LXR significantly elevated plasma levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in contrast to the MI group (VEGF: 255.82±6.49 pg/mg protein vs. 174.24±6.08 pg/mg protein, p<0.05; bFGF: 404.03±6.98 pg/mg protein vs. 320.8±7.64 pg/mg protein, p<0.05). Nevertheless, plasma levels of VEGF and bFGF were not increased when mice received treatment with AD-MSCs or LXR alone (p>0.05) (Fig. 5C, D).

LXR agonist protects AD-MSCs from H/SD injury

To determine the protective effects of the LXR agonist on viability during H/SD injury, AD-MSCs were exposed to various doses of LXR agonist (1, 5, 10, and 15 μM) for 24 h, followed by H/SD injury for 6 h. Cell viability was analyzed by BLI. Results revealed that AD-MSC viability was significantly hindered by H/SD injury (1.7±0.085×105 photons/s/cm2/sr vs. 4.5±0.029×105 photons/s/cm2/sr, p<0.01). However, 10 μM of LXR agonist efficiently blocked the decreased AD-MSCs viability despite H/SD injury (1.7±0.085×105 photons/s/cm2/sr vs. 3.32±0.13×105 photons/s/cm2/sr, p<0.05) (Fig. 6A, B). Then, to assess the anti-apoptotic role of the LXR agonist (10 μM) on AD-MSCs after H/SD injury, flow cytometry analysis was performed. Results showed that H/SD injury greatly increased the percentage of apoptotic AD-MSCs (18.16%±0.68% vs. 4.52%±0.19%, p<0.01), whereas LXR agonist treatment partially inhibited H/SD-induced AD-MSCs apoptosis (10.60%±0.58% vs. 18.16%±0.69%, p<0.01).

FIG. 6.

FIG. 6.

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The effect of the LXR agonist on AD-MSCs survival and apoptosis after H/SD injury. (A) BLI of AD-MSCs undergoing H/SD injury with DMSO or different concentrations of LXR agonist treatment; normal condition AD-MSCs is used as control; (B) Comparison of fluorescence average radiance of AD-MSCs in different groups (n=5). *p<0.01 versus control, #p<0.05 versus H/SD group; (C) Apoptosis was analyzed by flow cytometry after staining with Annexin V and PI; (D) Quantification analysis of the apoptotic AD-MSCs was presented as the percentage of apoptotic cells (n=5). *p<0.01 versus control, **p<0.01 versus H/SD group, and #p<0.05 versus H/SD+LXR group. DMSO, dimethyl sulfoxide; H/SD, hypoxia/serum deprivation; PI, propidium iodide.

Furthermore, Nrf-2 siRNA intervention could block decreased apoptosis of AD-MSCs induced by LXR agonist (14.76%±0.58% vs. 10.60%±0.58%, p<0.05) (Fig. 6C, D).

LXR agonist modulates inflammatory cytokine and growth factor secretion in AD-MSCs injured by H/SD

Analysis of the inflammatory cytokine composition of AD-MSC supernatants showed that the concentration of TNF-α and IL-6 secreted by AD-MSCs was significantly higher in the H/SD group than in the control group (TNF-α: 21.90±0.80 pg/ml vs. 7.34±0.39 pg/ml, p<0.01; IL-6: 0.88±0.02 ng/ml vs. 0.25±0.02 ng/ml, p<0.01, respectively). Nevertheless, addition of the LXR agonist significantly decreased the secretion of TNF-α and IL-6 by AD-MSCs compared with the H/SD group (TNF-α: 21.90±0.80 pg/ml vs. 11.48±0.38 pg/ml, p<0.05; IL-6: 0.88±0.02 ng/ml vs. 0.52±0.02 ng/ml, p<0.05). Transfection with Nrf-2 siRNA could inhibit LXR agonist's effect of lowering TNF-α and IL-6 secretion (TNF-α: 18.36±0.86 pg/ml vs. 11.48±0.38 pg/ml, p<0.05; IL-6: 0.85±0.05 ng/ml vs. 0.52±0.02 ng/ml, p<0.05) (Fig. 7A). Analysis of the growth factor composition of AD-MSC supernatants indicated that the levels of VEGF and bFGF were much higher in the H/SD group than that in the control group (VEGF: 4.39±0.35 ng/ml vs. 1.98±0.22 ng/ml, p<0.05; bFGF: 18.61±2.43 ng/ml vs. 8.67±0.97 ng/ml, p<0.05), and that there was no major difference between H/SD group, LXR agonist-treated group, and Nrf-2 siRNA intervention group (p>0.05) (Fig. 7B).

FIG. 7.

FIG. 7.

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Effect of LXR agonists on inflammatory cytokine, growth factor secretion, and intracellular and mitochondrial ROS generation of AD-MSCs under hypoxic conditions. (A) Concentration of TNF-α and IL-6 released by AD-MSC was measured by ELISA (n=5). *p<0.01 versus control, **p<0.05 versus H/SD group, and #p<0.05 versus H/SD+LXR group; (B) Concentration of VEGF and bFGF secreted by AD-MSCs was also detected by ELISA (n=5). *p<0.05 versus control group; (C) Quantification analysis of intracellular DCF fluorescence intensity in AD-MSCs (n=5); (D) Quantification analysis of mitochondrial DCF fluorescence intensity in AD-MSCs (n=5). *p<0.01 versus control, **p<0.05 versus H/SD group, #p<0.05 versus H/SD+LXR group, and *#p<0.05 versus H/SD group; (E) NADPH oxidase subunits gp91pho expression was measured by Western blotting analysis (n=5). *p<0.01 versus control, **p<0.01 versus H/SD group, and #p<0.05 versus H/SD+LXR group. DCF, dichlorofluorescein.

LXR agonist attenuates intracellular and mitochondrial ROS production in AD-MSCs after H/SD injury

The fluorescence intensity of dichlorofluorescein (DCF) was increased in H/SD group compared with that in the control group (p<0.01). LXR agonist treatment significantly lowered the mean fluorescence intensity (p<0.05), suggesting that the LXR agonist decreased ROS production in H/SD-injured AD-MSCs. Furthermore, Nrf-2 siRNA could reverse this effect of LXR agonist (p<0.05) (Fig. 7C). Likewise, LXR agonist attenuated H/SD-induced mitochondrial ROS generation, as indicated by the DCF mean fluorescence intensity (p<0.05). However, Nrf-2 siRNA abolished the inhibitory effect of LXR agonist in regulating ROS generation (p<0.05) (Fig. 7D). Interestingly, NADPH oxidase subunits gp91phox expression was significantly increased under H/SD condition, which was reversed by LXR agonist treatment (p<0.01). However, Nrf-2 siRNA intervention partly abrogated the roles of LXR agonist in gp91phox expression (p<0.05) (Fig. 7E). In addition, depletion of gp91phox with siRNA could inhibit H/SD-induced mitochondrial ROS production (p<0.05) (Fig. 7D).

LXR agonist inhibits TLR4/NF-κB signaling pathway activation in AD-MSCs injured by H/SD or oxidative stress

To determine the role of the TLR4/NF-κB signaling pathway in LXR agonist protection of AD-MSCs from H/SD injury, the expression or phosphorylation levels of TLR4, MyD88, TRAF-6, IκBα, and NF-κB-p65 in AD-MSCs was detected by Western blot. The expression of TLR4, MyD88, and TRAF-6 significantly increased under H/SD conditions (p<0.01), whereas the change was reversed by pretreatment with 10 μM of LXR agonist (p<0.05). However, knockdown Nrf-2 by siRNA significantly inhibited the LXR agonist's effects in modulating TLR4, MyD88, and TRAF-6 expression (p<0.05). Consistent with these results, phosphorylation levels of IκBα were also markedly elevated in the H/SD group in comparison with the control group (p<0.01), and pretreatment with 10 μM LXR agonist inhibited IκBα phosphorylation (p<0.05). Similarly, NF-κB-p65 protein expression was significantly increased in the H/SD group compared with the control group (p<0.01), and 10 μM of the LXR agonist reversed the increase of NF-κB-p65 protein expression (p<0.01). Nevertheless, Nrf-2 siRNA treatment also reversed the LXR agonist's inhibition of IκBα phosphorylation and NF-κB-p65 protein expression (p<0.05) (Fig. 8 and Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/ars).

FIG. 8.

FIG. 8.

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The protein expression of TLR4, MyD88, TRAF-6, and NF-κB-p65; phosphorylation of IκBα, Keap-1, and Nrf-2 in AD-MSCs under H/SD condition were determined by Western blotting (n=5, *p<0.05 versus control, **p<0.05 versus H/SD group, and #p<0.05 versus H/SD+LXR group).

Western blot analysis also revealed that the expression of TLR4, MyD88, TRAF-6, and NF-κB-p65 protein as well as phosphorylation levels of IκBα were significantly elevated under oxidative stress condition (p<0.01). However, 10 μM of LXR agonist could downregulate these proteins' increased expression or phosphorylation levels induced by oxidative stress injury (p<0.01). Further, Nrf-2 siRNA knockdown partly abolished the roles of LXR agonist in regulating the TLR4/NF-kB pathway (p<0.05) (Fig. 9 and Supplementary Fig. S1).

FIG. 9.

FIG. 9.

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The protein expression of TLR4, MyD88, TRAF-6, and NF-κB-p65; phosphorylation of IκBα, Keap-1, and Nrf-2 in AD-MSCs under H2O2-stimulated oxidative stress condition were determined by Western blotting (n=5, *p<0.05 versus control, **p<0.05 versus H2O2 group, and #p<0.05 versus H2O2+LXR group).

The LXR agonist upregulates the Keap-1/Nrf-2 signaling pathway in AD-MSCs under H/SD or oxidative stress conditions

Our results showed that compared with the H/SD groups, Keap-1 protein expression was significantly decreased in the H/SD+LXR group (p<0.01). Simultaneously, Nrf-2 protein level was decreased in the cytoplasm and increased in the nucleus in the H/SD+LXR group (p<0.01), indicating that LXR administration promoted nuclear translocation of the Nrf-2 protein after H/SD injury. In addition, the level of HO-1 protein markedly rose in the LXR+H/SD group compared with the control group (p<0.01), but not in the H/SD group. Furthermore, treatment with the NF-κB-p65 siRNA also significantly increased Nrf-2 protein translocation and HO-1 protein expression in contrast to the control group (p<0.05) (Fig. 8 and Supplementary Fig. S1).

In addition, LXR agonist markedly attenuated Keap-1 protein expression after H2O2-stimulated oxidative stress injury, and it also boosted Nrf-2 protein translocation and HO-1 protein expression (p<0.05). Similarly, NF-κB-p65 siRNA pretreatment also upregulated Nrf-2 protein translocation and HO-1 protein expression after oxidative stress injury (p<0.05) (Fig. 9 and Supplementary Fig. S1).

Discussion

Enhanced survival of transplanted stem cells is crucial for current cell-based therapies. Most donor cells do not survive for prolonged periods in ischemic myocardium, which limits clinical application of cell-based therapies (25). Some recent studies show that inflammation and oxidative stress are major contributors to the acute death of engrafted stem cells (34, 36). Our previous work also reveals that inflammation in the ischemic hind limb inhibits functional survival of mADSCs in vivo (6). Thus, seeking effective targets to ameliorate inflammation in the host microenvironment after ischemic injury is an important part of promoting donor cell survival and cardiac repair.

Recently, investigators have found that LXR activation modulates inflammation and inhibits ischemic injury (23, 29). However, whether LXR activation enhances engrafted cell survival remains uncertain. In the present study, we found that combined therapy with LXR agonist T0901317 and AD-MSCs attenuated myocardial infarct size and improved myocardial function after infarction due to the protective effects of the LXR agonist on the functional survival of AD-MSCs after transplantation. Putative mechanisms may involve the anti-oxidative stress and anti-inflammatory effects of LXR on the myocardial microenvironment and AD-MSCs via modulation of the TLR4/NF-κB and Keap-1/Nrf-2 signaling pathways.

To clarify this, we used BLI to track engrafted AD-MSCs and showed that most of the implanted AD-MSCs underwent acute cell death in the infarcted myocardium within 7 days, which was similar with prevailing research (41). Our previous studies also found that a higher dosage of stem cells could not avoid acute loss and apoptosis after delivery (2). Furthermore, echocardiography demonstrated that transplantation of AD-MSCs after AMI did not significantly improve cardiac function. These data indicated that poor viability and severe apoptosis impaired the therapeutic effect of the transplanted AD-MSCs.

LXRs are known as regulators of glucose, lipid, and cholesterol metabolism. Many previous studies have suggested that LXR activation regulates inflammation and immune response (10). Recently, LXR ligands were reported to be involved in modulating the balance between embryonic stem cell survival and differentiation, as well as promoting neurogenesis (27, 32). To investigate the role of LXR in cell survival, we compared BLI signal intensity in mice receiving AD-MSCs alone and combined therapy that included an LXR agonist. The data indicate that LXR agonist treatment enhances the survival of AD-MSCs after engraftment. However, pretreatment AD-MSCs with LXR agonist before transplantation is not sufficient to enhance the therapeutic potential of engrafted cells. Previous work has already revealed that the microenvironment around stem cells plays a critical role in cell survival and function (14). High levels of pro-inflammatory factors and ROS, two strong components of host response to injury, will retard stem cell survival and decrease their regenerative capabilities (6, 18, 30, 33). When we measured inflammatory cytokine (TNF-α and IL-6) and ROS levels in tissue, we found that an LXR agonist could decrease TNF-α, IL-6, and ROS generation in ischemically injured myocardium, providing a better local microenvironment that was crucial to increased AD-MSC survival. In addition to microenvironmental modulation, important stem cell properties affecting survival and function, such as paracrine secretion and differentiation, may be altered by ischemic injury (20). ELISA and DCF fluorescence intensity assays showed that an LXR agonist could significantly decrease TNF-α, IL-6, and ROS levels produced by AD-MSCs under hypoxic condition, although it did not affect growth factors (e.g., VEGF and bFGF) levels which were elevated in response to hypoxic stimuli. In addition, it is recognized that NADPH oxidase subunit gp91phox contributes to mitochondrial ROS production through activation of redox-sensitive PKCɛ and the opening of mitoKATP (5a). Our data support the hypothesis that LXR agonist attenuates the inflammatory response in AD-MSCs under hypoxic conditions, and prevents oxidative stress induced by hypoxia injury through decreasing gp91phox expression.

As for the differentiation ability of transplanted AD-MSCs, our previous studies showed that transplanted MSCs rarely differentiated into cardiomyocytes in vivo (7, 41). Other literature put forward the debate of whether implanted MSCs possess beneficial multipotent differentiation ability in infarcted myocardium (1). Based on these findings, cardiac functional benefits of MSCs transplantation may not be mainly attributable to cells' differentiation. Therefore, we placed more emphasis on whether the LXR agonist has any effect or not on MSCs paracrine capacity in this research. Masson's staining and CD31 immunohistochemical staining also indicated that cell engraftment therapy exerts cardioprotective roles mainly through paracrine mechanism.

Furthermore, previous studies revealed that oxidative stress and inflammation played essential roles in pathogenesis of cardiac remodeling. ROS had been implicated to promote cardiac remodeling through regulating matrix metalloproteinase expression or cytokines and growth factors signaling pathway (3, 5, 24). In addition, inflammation is also reported to mediate initiation and development of cardiac fibrosis (35). In this study, we observed that LXR agonist significantly attenuated inflammation and oxidative stress in the myocardium after MI. Thus, it can be inferred that combined therapy of AD-MSCs and LXR agonist protects against cardiac remodeling through inhibiting inflammation and oxidative stress.

It is well recognized that NF-κB regulates at least three inflammation-related genes and that its activation exerts varied effects on cell apoptosis, survival, and autophagy (9). Recent data have shown that regulating inflammation in AD-MSCs can enhance their viability by inhibiting the TLR4/NF-κB signaling pathway (40). Therefore, we clarified whether the TLR4/MyD88/NF-κB signaling pathway was mechanistically implicated in the LXR agonist's cytoprotective effects. Interestingly, the AD-MSC in vitro assay results suggest that the LXR agonist promotes AD-MSC survival and inhibits proinflammatory cytokine production in AD-MSCs after ischemic injury, at least partly, by negatively regulating the TLR4/MyD88/NF-κB signaling pathway.

The Keap-1/Nrf-2 system has been recognized as a cellular endogenous defense mechanism against oxidative stress (4, 12). However, it was unknown whether the Keap-1/Nrf-2 signaling pathway influenced AD-MSCs viability, function, or ability to withstand ROS damage under stressful conditions. Western blot results suggest that the LXR agonist activates Keap-1/Nrf-2 signaling and promotes Nrf-2 nuclear translocation. Knockdown of Nrf-2 with siRNA could partly abrogate the protective roles of LXR agonist in promoting stem cell survival, reducing inflammatory cytokines and ROS generation as well as downregulating TLR4/MyD88/NF-κB signaling pathway in AD-MSCs under H/SD condition. Our research also confirmed that Nrf-2 silencing abolished protective roles of LXR in engrafted AD-MSCs in vivo. Based on the in vivo and in vitro experiment results, it can be concluded that although the LXR agonist partly ameliorated myocardial microenvironment after MI, Nrf-2 silencing of AD-MSCs would attenuate cells' ability to resist inflammation and oxidative stress, which might abrogate direct and indirect protective effects of LXR agonist on AD-MSCs in vivo. In addition, we used siRNA to inhibit NF-κBp-65 expression, and demonstrated that NF-κB-p65 siRNA-mediated inhibition significantly increased the expression of Nrf-2 and HO-1, and decreased the expression of Keap-1. Based on these results, we could infer that the endogenous Keap-1–Nrf-2 system of AD-MSCs was repressed by NF-κB activation under ischemic injury-induced oxidative stress conditions, but an LXR agonist can downregulate TLR4/MyD88/NF-κB and reactivate Keap-1/Nrf-2/HO-1 pathway of AD-MSCs to resist the oxidative stress (Fig. 10). However, other complementary mechanisms related to LXR agonist may also be in involved in activating the Keap-1–Nrf-2 signaling pathway, which is needed to be further elucidated.

FIG. 10.

FIG. 10.

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Proposed scheme for mechanisms of promoting AD-MSCs survival in ischemia myocardium by LXR agonist.

Despite the relevance of our findings, our study has many limitations. For instance, BLI is not suitable thus far for clinical application because of photon attenuation and scattering limiting detection depth. Second, bioluminescence signal in vivo reaches its peak value at ∼24 h after cell injection, and therefore any information about cell viability within the first 24 h is lost, although presumably a very significant amount of cell loss occurs during this period. Third, we did not use the LXR antagonist to further validate the protective effects of LXR agonist on AD-MSCs, for there was no commercial available LXR antagonist.

In conclusion, our in vivo studies showed that the administration of an LXR agonist might effectively enhance the viability of AD-MSCs transplanted into infarcted hearts. The combination treatment of an LXR agonist and AD-MSCs may have a synergistic effect on cardiac functional improvement. In addition, our in vitro studies demonstrated that the cytoprotective effect of LXR agonist on AD-MSCs injured by H/SD involves modulation of the TLR4/NF-κB and Keap-1/Nrf-2 signaling pathways. Therefore, the LXR agonist may serve as a potent agent for protecting transplanted AD-MSCs from a harmful microenvironment in the infarcted myocardium, thereby augmenting cardiac repair and function in future regenerative medicine strategies.

Materials and Methods

Animals

Fluc+-eGFP+ transgenic mice (Tg [Fluc-egfp]) were bred in a C57BL/6a background to constitutively express firefly luciferase (Fluc) and eGFP in all tissue and organs. C57BL/6a mice (Tg [Fluc-egfp] inbred strain, Fluc-eGFP−, n=100, male, 8 weeks old, 20–25 g) were used for the MI model. All animal procedures were conducted in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experiments were performed in accordance with the Helsinki declaration.

Isolation and culture of AD-MSCs

AD-MSCs were isolated from Fluc+-eGFP+ transgenic mice as previously described (7). In brief, adipose tissue was harvested from β-Actin-Fluc-GFP transgenic mice. Then, the tissue was washed with phosphate-buffered saline (PBS) and mechanically chopped before digestion with 0.2% collagenase I (Sigma) for 1 h at 37°C with intermittent shaking. The digested tissue was washed with Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 15% fetal bovine serum (FBS), and then centrifuged at 1000 rpm for 10 min to remove mature adipocytes. The cell pellet was resuspended in DMEM supplemented with 15% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37°C incubator with 5% CO2. AD-MSCs reaching 80%–90% confluency were detached with 0.02% ethylenediaminetetaacetic acid (EDTA)/0.25% trypsin (Sigma-Aldrich) for 5 min at room temperature and then replated. Cells between the third and fifth passage were used for experiments.

MI mice model

Male mice weighing 20–25 g were randomly allocated into the following groups with n=20 each: (i) Sham group (Sham), (ii) MI+PBS group (MI), (iii) MI+LXR agonist T0901317 group (LXR), (iv) MI+AD-MSCs group (AD-MSCs), (v) MI+AD-MSCs+LXR agonist T0901317 pretreatment before transplantation group (AD-MSCs+LXR Pre), (vi) MI+AD-MSCs transfected with Nrf-2 siRNA+LXR agonist T0901317 (AD-MSCs with Nrf-2 siRNA+LXR), and (vii) MI+AD-MSCs+LXR agonist T0901317 group (AD-MSCs+LXR).

Murine MI was induced by ligation of the left anterior descending (LAD) artery (8). In brief, mice were anesthetized with persistent inhaled 2% isoflurane during the operation. A left thoracotomy was performed, and the pericardium was opened. The LAD was permanently ligated with a 6-0 suture at the level of the left atrium. The ligation was deemed successful when the anterior wall of the left ventricle turned pale. Then, echocardiography was utilized to further confirm that the MI model in mice was successfully constructed. For sham-operated mice, open thoracotomy was performed without suturing of the LAD. AD-MSCs transplantation was performed immediately after MI performance. 1×106 AD-MSCs were injected intramyocardially into the peri-infarct areas at two different sites with a total volume of 20 μl in each animal. Control animals instead received a 20 μl PBS injection.

The chest cavity was closed in layers with a 4-0 suture. In the LXR group, T0901317 (Cayman Chemical Company) was administered (20 mg/kg/day) by gavage for successive 28 days post-AD-MSCs transplantation.

In vivo monitoring of transplanted mAD-MSCsFluc+-eGFP+ via bioluminesence imaging

Bioluminescence signal of engrafted mAD-MSCsFluc+-eGFP+ was detected using an IVIS Kinetic system (Caliper) as previously described. Ten minutes after an intraperitoneal injection of the reporter probe D-luciferin (150 mg/kg; Caliper), animals were imaged for 5 min on POD 2, 7, 14, 21, and 28 respectively. Bioluminescent signals were analyzed using Living Image® 3.1 software (Caliper) and quantified in units of photons/s/cm2/sr.

Analysis of mAD-MSCs Fluc+-eGFP+ engraftment

To further validate the results of in vivo BLI for mAD-MSCs Fluc+-eGFP+ survival, five mice were sacrificed and hearts were harvested to find GFP-positive cells at POD 14. The hearts were harvested and cut along the short axis with five pieces from the base to the apex. Serial sections were prepared at 5 μm thickness. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining (1:1000; Bioworld). Cell engraftment was confirmed by identification of GFP expression under a confocal laser scanning microscope (Olympus FV10i).

Left ventricular functional analysis with echocardiogram

Echocardiography studies were performed with Vevo® 2100 ultrasound system (VisualSonics) using a 30-MHz linear array ultrasound transducer on baseline (day-7) and POD 2, 14, and 28 by two blinded investigators. The mice were anesthetized with inhaled 2% isoflurane and placed in a supine position. Both two-dimensional and M-mode images were recorded. The left ventricular end-systolic volume and left ventricular end-diastolic volume were measured to calculate LVEF and FS.

Measurement of infarct size

Mice were euthanized, and hearts were harvested for histological staining at POD 28. A separate set of paraffin-embedded tissue sections were stained using Masson's trichrome, resulting in fibrotic (collagen-enriched) areas that appeared blue while cellular elements appeared red. For 10 randomly selected microscopic fields (×200 magnification) of each left ventricle, Masson's trichrome-stained myocardial sections were imaged, and the collagen area was calculated as a percentage of the total left ventricular myocardial area. This served as an estimate of infarct size. Measurement was performed by an observer who was blinded to the group assignment via Imaging Pro Plus software.

Evaluation of myocardial angiogenesis with immunohistochemical staining

To detect myocardial angiogenesis after MI, CD31 immunohistochemical staining was performed. Tissue sections were blocked with 2% normal goat serum for 30 min and then incubated with polyclonal rabbit antibodies: anti-CD31 (1:100; Bioworld) overnight at 4°C. The process of immunohistochemical staining was performed as previously described. Five sections from the heart of each animal were randomly selected, and images were photographed under×200 magnification in five vision fields/section. The immunoreactive areas for CD31 were analyzed with Image J software.

Determination of myocardial apoptosis

The cell apoptosis rate in the myocardium was determined on POD 3 by TUNEL staining according to the manufacturer's instructions (In Situ Cell Death Detection Kit; Roche Applied Science). Cells stained with cardiac troponin I were recognized as the myocardium, and the nucleus stained green was defined as TUNEL positive. DAPI were used to stain all cell nuclei. Six micrographs were randomly selected, and the numbers of healthy or apoptotic cardiomyocytes were counted. The percentage of cardiomyocytes apoptosis was calculated as the percentage of the total numbers of cells that were cardiac troponin I and TUNEL double immunostained positive. All of these assays were performed in a blinded manner.

The siRNA targeting of NF-κB-p65, Nrf-2, and gp91phox siRNA transfection

Nrf-2 siRNA, NF-κB-p65 siRNA, gp91phox siRNA, and control siRNA were purchased commercially from Genechemistry. The sequence of the mouse Nrf-2 siRNA (5′–3′) was as follows (RNA): 5′-UCC CGU UUG UAG AUG ACA-3′; 5′-UUGUCAUCUACAAACGGGA-3′ (Nrf-2, NCBI Reference Sequence: NM_010902.3). NF-κB-p65 siRNA: 5′-GUU UCA GCA GCU CCU GAA CTT-3′; 5′-GUU CAG GAG CUG CUG AAA CTG-3′ (NF-κB-p65, NCBI Reference Sequence: NM_009045.4). gp91phox siRNA: 5′-CGUUUUCCUCUUUGCUUGG-3′; 5′-CCAGACAAAGAGGAAGACG-3′ (gp91phox, NCBI Reference Sequence: NM_007807.5). Control siRNA: 5′-TTC AAG UCC UCG ACG ACU UUG-3′; 5′-CTC AAA GUC GUC CAG CAG UUG-3′. AD-MSCs were seeded onto 60-mm dishes at 24 h before transfection and then transiently transfected with 100 nM Nrf-2 siRNA, NF-κB-p65 siRNA, or control siRNA per dish at 90% confluence using the lipofectamine 2000 (Invitrogen Life Technology) according to the manufacturer's protocol. Successful knockdown of the target proteins was confirmed by Western blot analysis.

Measurement of myocardial, intracellular, and mitochondrial ROS generation, respectively

ROS generation in myocardium was detected using the ROS fluorescent probe-DHE as previously described. In brief, the isolated hearts were cryostat sectioned (10 μm) and incubated with DHE probe (2 μM) in PBS solution for 20 min at 37°C on POD 3. The images were obtained with a fluorescence microscope (Nikon) at 488 nm excitation and 590 nm emission.

Production of intracellular and mitochondrial ROS was assessed by measuring the fluorescence intensity of DCF (2,7-dichlorofluorescein-diacetate [DCFH-DA]; Invitrogen) as previously described. At 1 h after the different treatments, isolated mitochondria and cells were isolated or harvested, washed and re-suspended in PBS, respectively, and then incubated with 2 μg/ml RHO 123 and 10 μM DCFH-DA at 37°C in an incubator for 20 min with gentle shaking. The mitochondrial and intracellular fluorescence of DCF was determined in GloMax™ 20/20 Luminometer (Promega).

H/SD injury in vitro

AD-MSCs were stimulated with H/SD injury as previously described (41). Briefly, AD-MSCs were plated in 24-well plates (5×104 cells per well). Twenty-four hours later, AD-MSCs were administrated with PBS, cultured in Hanks buffer. Then, different doses of LXR agonist (1, 5, 10, and 15 μM) and dimethyl sulfoxide were added into their respective wells for another 24 h. Then, AD-MSCs were exposed to hypoxia (94% N2–5% CO2–1% O2) in an anaerobic system (Thermo Forma) at 37°C for 6 h. After 6 h of either hypoxia or normal conditions, mediums were removed from wells and collected for later ELISA. In the control group, AD-MSCs were maintained at normoxia (95% air–5% CO2) for equivalent periods.

BLI of mAD-MSCsFluc+-eGFP+ in vitro

To assess for protective effects of the LXR agonist on AD-MSCs, we assessed cell survival with BLI using the IVIS Kinetic system (Caliper). Briefly, mAD-MSCsFluc+-eGFP+ in plates after H/SD injury were imaged after D-luciferin was added to each well. Bioluminescent signals were analyzed using Living Image 3.1 software (Caliper) and quantified as average radiance in photons/s/cm2/sr.

ELISA for soluble inflammatory cytokines and growth factors

To examine the amount of IL-6, TNF-α, VEGF, and bFGF in the supernatant of AD-MSCs, measurements were carried out using commercially available ELISA kits (Sen-Xiong Company). In accordance with the manufacturer's instructions, all supernatant was stored at−80°C before measurement and both standards and samples were run in triplicate. OD450 was calculated by subtracting the background, and standard curves were plotted.

Quantitative real-time polymerase chain reaction

To investigate basal mRNA expression of LXRs in AD-MSCs, total RNA of cells was isolated using RNAlater (Qiagen) according to the manufacturer's instructions. To generate cDNA, 1 μg of total RNA was used with the oligo dT primer following the protocol for the First-Strand cDNA Synthesis kit (Amersham Bioscience). Real-time quantitative PCR was performed using an ABI Prism 7900 system (Applied Biosystems). The levels of LXRα and LXRβ mRNA expression were normalized to GADPH mRNA expression level. The sequences of forward primer (FP) and reverse primer (RP) were as follows: LXRα FP:5′-CGACAGAGCTTCGTCCACAA-3′, RP: 5′-GCTCGTTCCCCAGCATTTT-3′; LXRβ FP:5′-CGTGCCTGGGAATGGTTCT-3′, RP:5′-AGTCTCCTGCCCCTCTTCCTT-3′. GADPH FP:5′-GCCAAAAGGGTCATCAT CTC-3′, RP:5′-GTAGAG GCAGGGATGATGTTC-3′.

Western blot assay

AD-MSCs in the plate after H/SD treatment were washed twice with cold PBS, then lysed with RIPA buffer (1% Triton X-100, 20 mM of Tris [PH=7.5]), 10 mM of EDTA, 0.02% sodium azide, and protease-inhibitors such as leupeptin for about 1 h. Cell lysates were centrifuged at 10,000 g at 4°C for 20 min. Lysates were boiled in sample buffer for 5 min. The proteins were then subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes using a semi-dry electroblotting system. After blocking with 5% skim milk in PBS, the membranes were incubated with diluted polyclonal rabbit anti-mouse LXRα (1:1000; Abcam), LXRβ (1:1000; Abcam), NADPH oxidase anti-NOX2/gp91phox (1:500; Bioss), NF-κB-p65 (1:1000), MyD88 (1:1000), TRAF-6 (1:1000), TLR4 (1:1000), IκBα (1:1000), p-IκBα (1:1000), Keap-1 (1:1000), Nrf-2 (1:1000), HO-1 (1:1000; all from Bioworld), and a monoclonal anti-β-actin antibody (1:1000; Abcam) at 4°C overnight. After washing and further incubation with appropriate secondary antibodies conjugated with horseradish peroxidase (dilution: 1:5000 in TBST) at 37°C for 60 min, bands were visualized using an enhanced chemiluminescence system (ECL; Amersham). Densitometric analysis of Western blots was carried out using VisionWorks LS, version 6.7.1.

Statistical analysis

The results are expressed as mean±SEM. Statistical differences between different groups were evaluated with Prism 5.0 (GraphPad Software, Inc.). Groups were compared using Student's two-tailed unpaired t test or a one-way ANOVA analysis, followed by Dunnet's post hoc test as appropriate. Statistical significance was set at p<0.05.

Supplementary Material

Supplemental data
Supp_Figure1.pdf (45.5KB, pdf)

Abbreviations Used

AD-MSCs

adipose-derived mesenchymal stem cells

AI

apoptotic index

bFGF

basic fibroblast growth factor

BLI

bioluminescence imaging

DAPI

4′,6-diamidino-2-phenylindole

DCF

dichlorofluorescein

DCFH-DA

2,7-dichlorofluorescein-diacetate

DHE

dihydroethidium

DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethyl sulfoxide

EDTA

ethylenediaminetetaacetic acid

eGFP

enhanced green fluorescence protein

FBS

fetal bovine serum

FP

forward primer

FS

fraction shortening

H/SD

hypoxia/serum deprivation

IL-6

interleukin-6

LAD

left anterior descending

LVEF

left ventricular ejection fraction

LXR

liver X receptor

MI

myocardial infarction

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

PI

propidium iodide

POD

postoperative days

ROS

reactive oxygen species

RP

reverse primer

TNF-α

tumor necrosis factor-α

TUNEL

terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling

VEGF

vascular endothelial growth factor

Acknowledgments

This work was supported by the National Funds for Distinguished Young Scientists of China (81325009), the National Nature Science Foundation of China (No. 81270168, No. 81090274, No. 81227901) (FCao BWS12J037), Innovation Team Development Grant by China Department of Education (IRT1053), the National Basic Research Program of China (2012CB518101), and Shanxi Province Program (2013K12-02-03).

Author Disclosure Statement

The authors declare that no competing financial interests exist.

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