N‐acetylcysteine prevents oxidized low‐density lipoprotein‐induced reduction of MG53 and enhances MG53 protective effect on bone marrow stem cells

Abstract MG53 is an important membrane repair protein and partially protects bone marrow multipotent adult progenitor cells (MAPCs) against oxidized low‐density lipoprotein (ox‐LDL). The present study was to test the hypothesis that the limited protective effect of MG53 on MAPCs was due to ox‐LDL‐induced reduction of MG53. MAPCs were cultured with and without ox‐LDL (0‐20 μg/mL) for up to 48 hours with or without MG53 and antioxidant N‐acetylcysteine (NAC). Serum MG53 level was measured in ox‐LDL‐treated mice with or without NAC treatment. Ox‐LDL induced significant membrane damage and substantially impaired MAPC survival with selective inhibition of Akt phosphorylation. NAC treatment effectively prevented ox‐LDL‐induced reduction of Akt phosphorylation without protecting MAPCs against ox‐LDL. While having no effect on Akt phosphorylation, MG53 significantly decreased ox‐LDL‐induced membrane damage and partially improved the survival, proliferation and apoptosis of MAPCs in vitro. Ox‐LDL significantly decreased MG53 level in vitro and serum MG53 level in vivo without changing MG53 clearance. NAC treatment prevented ox‐LDL‐induced MG53 reduction both in vitro and in vivo. Combined NAC and MG53 treatment significantly improved MAPC survival against ox‐LDL. These data suggested that NAC enhanced the protective effect of MG53 on MAPCs against ox‐LDL through preventing ox‐LDL‐induced reduction of MG53.


| INTRODUC TI ON
Bone marrow stem cells (BMSCs) are important sources for cellbased therapy that remains a viable and attractive option for tissue repair and regeneration. [1][2][3][4][5] However, one of the major challenges for cell-based therapy with stem cells is the poor in vivo survival after delivery into target areas. 4,5 It has been shown that the number of mesenchymal stem cells (MSCs) in the cremaster decreased to 14% of the initial number 3 days after bolus injection into the ipsilateral common iliac artery in rats. 6 MSCs were initially accumulated in the lungs and did not reach the target sites after intravenous infusion, and many cells disappeared 2 hours after delivery in mice. 7 When the stem cells were injected into the left ventricular myocardium of mice, less than 1% of the delivered cells survived in the target areas 4 days after injection. 4 The mechanisms for the poor survival of transplanted BMSCs have not been fully understood and are very likely multifactorial including inflammation, oxidative stress, mechanical stress and hypoxia. Oxidized low-density lipoproteins (ox-LDLs) are naturally present in serum and an important source for reactive oxygen species (ROS) and oxidative stress. 8,9 Ox-LDL has been shown to inhibit proliferation and endothelial differentiation of BMSCs, and induce apoptosis of BMSCs with both ROS-dependent and ROS-independent mechanisms. 10,11 Our previous study showed that ox-LDL impaired the survival of BMSCs in vitro partially through direct cell membrane damage independent of ROS formation. 12 MG53 (also known as TRIM72) is an important membrane-repairing protein that is produced in striated muscle cells and present in circulation. 13 Systemic delivery of MG53 or muscle-specific overexpression of human MG53 gene enhanced membrane repair and improved muscle and heart functions in a hamster model of muscular dystrophies and congestive heart failure. MG53 could protect muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2) and glycogen synthase kinase-3β, and inhibiting proapoptotic protein Bax. 14 Extracellular MG53 protein and recombinant human MG53 protein (rhMG53) could increase membrane repair capacity in isolated muscle or non-muscle cells in a dose-dependent manner. 15,16 In our previous study, we observed that rhMG53 treatment protected BMSCs against ox-LDL-induced membrane damage and enhanced their survival. However, the protective effect of rhMG53 on BMSCs against ox-LDL was limited with undefined mechanisms.
It is known that ischaemia/reperfusion or hypoxia/oxidative stress leads to down-regulation of MG53 in rodent cardiomyocytes. 17 Hypercholesterolaemia could block sevoflurane-induced cardioprotection against ischaemia-reperfusion injury by alteration of MG53-mediated pathway. 18 Little is known on the metabolism of MG53 in vivo. However, it has been shown that serum MG53 levels in mice with metabolic syndrome induced by a 6-month high-fat diet were significantly reduced. 19 S-nitrosylation of MG53 at C144 (cysteine 144) prevented oxidation-induced degradation of MG53 following oxidative insult, therefore enhancing cardiomyocyte survival. 20 In the present study, we tested the hypothesis that the limited protective effect of MG53 on BMSCs against ox-LDL was due to ox-LDL-induced reduction of MG53. Both in vitro and in vivo experiments were conducted to test the hypothesis. The objectives of the present study were (a) to determine the effect of ox-LDL on MG53 levels both in vitro and in vivo and (b) to define the role of ROS in mediating the effect of ox-LDL-induced reduction of MG53 by blocking ROS production with antioxidant N-acetylcysteine (NAC).

| Animals
All the animal experiments were performed in accordance with the 'Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health'. The experimental protocols for the present study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Missouri School of Medicine. Male C57 BL/6 and LDL receptor deficiency (LDLR −/− ) mice (6-8 weeks old) were obtained from Jackson Lab.

| Preparation of LDL and ox-LDL
Both native LDL and ox-LDL were prepared for the experiments as described. 10,21,22 Briefly, plasma was obtained from healthy human volunteers for the preparation of native LDL using sodium bromide stepwise density gradient centrifugation. To prepare ox-LDL, native LDL was exposed to copper sulphate (5 μmol/L) at 37°C for 3 hours, followed by the addition of EDTA (final concentration of 0.25 mmol/L) to terminate the reaction. The degree of LDL oxidation was monitored using thiobarbituric acid reactive substances (TBARS) as described. 10,21,22 To ensure product quality and reproducibility, the TBARS value for ox-LDL was maintained in the range of 40-50 nmol malondialdehyde/mg protein. There were no detectable TBARS for native LDL.

| Preparation of rhMG53
High-quality (>97% purity) rhMG53 protein was prepared using E. coli fermentation as described. 16 The efficacy of rhMG53 on membrane protection was determined as EC50 for each preparation to ensure product quality and reproducibility with our established micro-glass bead injury assay as described. 16,23 The amount of rhMG53 protein for each experiment was determined to achieve its EC50 concentration as established by micro-glass bead injury assay.
Culture dishes were coated with 100 ng/mL fibronectin (FN; Sigma) to accelerate cell adherence. Cells were strictly kept at a density of 100-200 cells/cm 2 to avoid cell-cell contact at 37°C with humidified gas mixtures of 5% O 2 , 5% CO 2 and 90% N 2 .
To investigate the effect of ox-LDL on the growth and survival of MAPCs, the cells were cultured at a density of 500 cells/cm 2 (1000 cells/well in 24-well plate) in the presence of ox-LDL (10 μg/mL) for 12, 24 and 48 hours, or cultured with 20 μg/mL ox-LDL for 24 hours at a density of 1 × 10 4 cells/cm 2 (as the majority of cells could die out within 24 hours of exposure to ox-LDL at this concentration).
Native LDL and saturated LDL were used as the controls. To determine whether rhMG53 could protect the cells, purified rhMG53 (1 mmol/L) was mixed with the culture medium 5 minutes before exposure to ox-LDL. Bovine serum albumin (BSA) (1 mmol/L) was used as control. To determine the involvement of ROS in the protection of rhMG53 on MAPCs, experiments were repeated when the antioxidant NAC (1 mmol/L, final concentration) was added into the culture system 1 minute before rhMG53 was mixed with the cells. The cells were counted in each group at each time-point, and each experiment was repeated for at least three times.

| Measurement of rhMG53 in culture system with Western blot
rhMG53 was added into culture plates with PBS at the same concentration as with cell culture (final concentration of 1 mmol/L).
To investigate the effect of ox-LDL on rhMG53, ox-LDL (final concentration of 10 μg/mL) was mixed with rhMG53 in PBS in culture plates. To determine whether NAC could protect rhMG53 against ox-LDL-induced reduction, experiments were repeated when NAC (1 mmol/L) was mixed with ox-LDL with or without rhMG53 in PBS.
The preparations were in duplicate for each sample in one 96-well cell culture plate (100 μL total volume per well) and incubated at 37°C with 5% O 2 , 5% CO 2 and 90% N 2 as for cell culture. To determine whether phospholipid-containing liposome could have an important impact on MG53 level in vitro, experiments were conducted to incubate MG53 with native LDL (10 μg/mL). After 12 hours, 50 μL sample was removed from each well and mixed with 450 μL PBS in a 1.5 mL tube; then, 10 μL sample from each tube was mixed with 10 μL protein loading buffer (Bio-Rad Laemmli sample buffer without B-mercaptoethanol). Without heating at 95°C, the samples (a total of 10 μL for each sample) were loaded on 10% SDS-polyacrylamides gels (Bio-Rad) for electrophoresis and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore). After blocking with milk (Bio-Rad), the membranes were incubated overnight at 4°C with the primary antibody for MG53. After washing with TBST, the preparations were incubated with peroxidase-conjugated secondary antibody for 1 hour. The protein bands on the membranes were visualized using the enhanced chemiluminescence reagents (Thermo Fisher Scientific Inc) and analysed with Fiji image software.

| Cell proliferation assay
Rat MAPCs were seeded on a 96-well plate at a density of 1000 cells/ well in the presence of ox-LDL (5-10 μg/mL) for 24 hours. Each treatment was in triplicate, and three independent experiments were performed. To evaluate the effect of NAC (1 mmol/L) and/or rhMG53 (50 μg/mL) on cell proliferation and survival, NAC and/or rhMG53 were added to the culture medium 30 minutes before exposure to ox-LDL. After 24 hours of incubation, the cells were prepared for proliferation assay using BrdU Proliferation Assay Kit (Calbiochem) as per manufacturer's instruction.

| Cell apoptosis assay
Rat MAPCs were plated on 6-well plates with a density of 2000 cells/cm 2 for apoptosis assay. After 24 hours of culture, the cells were treated with ox-LDL (5-10 μmol/L) for an additional 24 hours with or without NAC (1 mmol/L) and/or MG53 (50 μg/ mL). Each treatment was in triplicate, and three independent experiments were performed. The cells were then prepared for apoptosis assay using FITC Annexin V Apoptosis Detection Kit (Calbiochem) as per manufacturer's protocol. The proportion of apoptotic cells was expressed as a percentage of total cell number acquired (excluding debris) and analysed using BD FACS Diva and Flow Jo software.

| Cell cycle assay
Rat MAPCs were plated on 6-well plates with a density of 2000 cells/ cm 2 for cell cycle analysis. After 24 hours of culture, the cells were treated with ox-LDL (5-10 μmol/L) for an additional 24 hours with or without NAC (1 mmol/L) and/or MG53 (50 μg/mL). Each treatment was in triplicate, and three independent experiments were performed. The cells were then prepared for cell cycle analysis using BrdU/7-AAD kit (Biolegend) according to manufacturer's protocol.

| Statistical analysis
The data from all experiments were presented as mean ± SD (standard deviation). Statistical analyses were performed with unpaired Student's t test (two-sided) for two group of data or one-way ANOVA (analysis of variance) (Sigma Stat 2.03; Aspire Software International) followed by post hoc conservative Tukey's test for three or more groups of data with multiple comparisons to minimize the type I error as appropriate. The difference was considered statistically significant when a two-tailed P value was equal to or less than .05.

| NAC significantly enhanced the protective effect of rhMG53 on MAPCs against ox-LDL-induced inhibition
Healthy growth of MAPCs was observed under the standard conditions. In the presence of ox-LDL (from 1 to 5 μg/mL), the number of MAPCs in the culture system was significantly decreased as expected. Treatment with NAC (1 mmol/L) effectively prevented ox-LDL-induced reduction of the cell number (data not shown).
However, when ox-LDL concentration was increased to 10 μg/mL, NAC treatment did not prevent the reduction of cell number that was consistent with our previous observations. 10

| NAC effectively prevented ox-LDL-induced reduction of MG53 protein in vitro and in vivo
It is known that the protective effect of MG53 on cell membrane is dose-dependent. 16 To test the hypothesis that NAC enhanced the protective effect of MG53 on MAPCs against ox-LDL through the prevention of ox-LDL-induced reduction of MG53, MG53 level was determined in the culture system in the presence of ox-LDL or native LDL when ROS production was blocked with NAC. As expected, after 12 hours of incubation in the MAPCs culture environment, a detectable amount of MG53 protein was present in the media. Ox-LDL (10 μg/mL) significantly reduced MG53 level in the culture system with or without MAPCs (Figure 2A

| Ox-LDL had no effect on MG53 clearance in vivo
Next, we determined whether ox-LDL could have a significant im- After 2 hours, only 10% of the injected MG53 protein remained in the blood. By 6 hours, the injected protein was completely removed from the blood. Treating the mice with ox-LDL (once a day for 3 days via tail vein) had no significant effect on MG53 clearance from circulation ( Figure 3B). NAC treatment. As expected, a significant amount of FM1-43 dye was detected 6 hours after incubation with ox-LDL using confocal microscope. There was no significant difference in FM1-43 dye entry into the cells when exposed to ox-LDL with or without NAC treatment ( Figure 4A). Quantitative and dynamic analysis with a quantitative live cell imaging assay confirmed that exposure to ox-LDL dramatically increased FM1-43 dye accumulation inside the cells that was not significantly changed with NAC treatment ( Figure 4B). On the other hand, treatment with rhMG53 significantly reduced FM1-43 dye entry into and accumulation inside the cells ( Figure 4A,B).

| NAC treatment prevented ox-LDL-induced inhibition of Akt phosphorylation without enhancing the survival of MAPCs against ox-LDL in vitro
Akt-and STAT3-mediated signalling is important to cell survival and proliferation. To determine the potential role of Akt and STAT3 signalling in mediating the effect of ox-LDL on MAPCs, the levels of total and phosphorylated Akt and STAT3 were evaluated in the cells exposed to ox-LDL with and without NAC treatment. As shown in

| Effects of NAC on cell proliferation, apoptosis and cell cycle in the presence of ox-LDL
As expected, ox-LDL significantly inhibited the proliferation of MAPCs, induced their apoptosis and arrested the cell cycle at G0/ G1 phase ( Figure 6A-D). Treatment with MG53 partially prevented ox-LDL-induced inhibition of cell proliferation ( Figure 6A,B), effectively prevented ox-LDL-induced apoptosis ( Figure 6C) and largely reversed ox-LDL-induced cell cycle arrest ( Figure 6D). NAC treatment significantly prevented ox-LDL-induced inhibition of cell proliferation and blocked ox-LDL-induced early apoptosis when ox-LDL concentration was at 5 μg/mL. However, when ox-LDL concentration was increased to 10 μg/mL, NAC treatment further increased ox-LDL-induced inhibition of cell proliferation ( Figure 6A) while having no effect on ox-LDL-induced cell cycle arrest ( Figure 6D). No significant differences in proliferation, apoptosis and cell cycle of MAPCs were observed when the cells were treated with MG53 alone or with combination of NAC and MG53 in the presence of ox-LDL at 10 μg/mL ( Figure 6A-D).
F I G U R E 5 Effect of NAC and MG53 treatment on Akt phosphorylation in bone marrow stem cells in the presence of ox-LDL in vitro. Intracellular levels of total and phosphorylated Akt and STAT3 were evaluated in MAPCs after exposure to ox-LDL with and without NAC and/ or MG53 treatment. Western blotting analysis showed that ox-LDL selectively decreased Akt phosphorylation without change in total Akt and total or phosphorylated STAT3 in MAPCs. NAC treatment completely prevented ox-LDL-induced inhibition of Akt phosphorylation in MAPCs. On the other hand, MG53 treatment did not change the levels of Akt or STAT3 expression in

| D ISCUSS I ON
In the present study, rat MAPCs were used as the source of BMSCs as BMSCs are a mixture of heterogeneous cells with very different phenotypes and different capability of differentiating into multiple cell lineages. 28 MAPCs were isolated from bone marrow, clonally purified and well-characterized multipotent cells with differentiation potential into a variety of cell lineages including endothelial cells, smooth muscle cells, neurons, hepatocytes and cardiac myocytes. 24,25,29,30 MAPCs promote angiogenesis and improve cardiac and limb function when injected into the peri-infarct areas in ischaemic mice. 31 Clinical-grade human MAPCs are safe in human patients and effective on controlling human autoimmune disease and allograft rejection. 39,40,43,44 In addition, rat MAPCs are very stable phenotypically.
Thus, using rat MAPCs in the present study has unique advantage with significant translational and clinical values.
We demonstrated that ox-LDL at the concentrations that were compatible with serum ox-LDL levels or less than that in patients with stable coronary artery diseases 12,[45][46][47]  to cardiac ischaemia/reperfusion (I/R) injury, whereas MG53 overexpression significantly protected cardiomyocytes from I/R damage and improved cardiac function in animal models. 17,18,50,56 MG53 can be secreted into circulation and mediates the protective function of MG53 for tissues and organ systems away from striated muscles. 13 Thus, the level of circulating MG53 could be critical for tissue repair and/or regeneration and could be determined by the combined outcome of MG53 expression and secretion, degradation and clearance from circulation. However, the regulatory mechanism for serum MG53 concentration has not been well defined. It has been shown that mice with metabolic syndrome induced by 6-month highfat diet (HFD) feeding exhibited a significant increase in serum lipid levels and a significant reduction in circulating MG53 level without change in MG53 expression within skeletal and cardiac muscles. 19 In the present study, we also observed that serum MG53 level was significantly decreased in hyperlipidemic LDLR −/− mice ( Figure 2C).
One of the important components in blood in hyperlipidemic state is ox-LDL which is considered as a critical contributor to the development of cardiovascular diseases associated with hyperlipidemia (like atherosclerosis). 57 In the present study, we demonstrated that treatment of mice with ox-LDL significantly decreased serum MG53 level without change in MG53 clearance from circulation ( Figure 2B and Figure 3B). In vitro experiments showed that ox-LDL substantially decreased the level of MG53 (Figure 2A) Figure S1). Degradation or clearance of MG53 in circulation was not significantly affected with ox-LDL treatment in mice ( Figure 3B). These data suggested that binding to phospholipids might not be a dominant mechanism for decreased MG53 levels.
However, further studies are needed to define the interactions between MG53 and phospholipids. Another potential mechanism for decreased MG53 level by ox-LDL could be ox-LDL-induced reduction of MG53 secretion into the circulation that also requires further evaluation.
One of the major challenges to cell therapy with stem cells including BMSCs is the poor in vivo survival after delivery into target tissues. However, the factors that are involved in the in vivo survival of delivered stem cells have not been well defined. Our previous study showed that ox-LDL significantly impaired the survival of MAPCs in vitro partially through direct membrane damage independent of ROS production when ox-LDL was at 10 μg/mL or higher as NAC treatment completely blocked ROS production from ox-LDL and yet failed to protect ox-LDL-induced membrane damage in MAPCs. Treatment with rhMG53 effectively protected MAPCs against ox-LDL-induced membrane damage, but only partially enhanced their survival. 12 In the present study, we observed that ox-LDL significantly decreased MG53 concentration both in vitro and in vivo that was largely (although not completely) pre- In conclusion, the data from the present study suggested that ox-LDL significantly impaired the survival of MAPCs partially through direct membrane damage independent of ROS production in vitro.
NAC treatment synergistically enhanced the protective effect of MG53 on the survival of MAPCs against ox-LDL through attenuation of ox-LDL-induced reduction of MG53.

ACK N OWLED G EM ENT
This work was supported by US NIH grants to ZL (NIH HL124122 and ES026200) and by National Natural Science Foundation of China to Xin Li (Grant No. 8170020711).

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
Zhenguo Liu was responsible for the perception of the idea, involved in the experiment design, data analysis and interpretation, and

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors.