Mineralocorticoid receptor deficiency improves the therapeutic effects of mesenchymal stem cells for myocardial infarction via enhanced cell survival

Abstract The poor survival of stem cells seriously limits their therapeutic efficacy for myocardial infarction (MI). Mineralocorticoid receptor (MR) activation plays an important role in the pathogenesis of multiple cardiovascular diseases. Here, we examined whether MR silencing in bone marrow derived mesenchymal stem cells (MSCs) could improve MSCs’ survival and enhance their cardioprotective effects in MI. MSCs from male Sprague‐Dawley rats were transfected with adenoviral small interfering RNA to silence MR (siRNA‐MR). MR silencing decreased hypoxia‐induced MSCs’ apoptosis, as demonstrated by Annexin V/7‐AAD staining. The mechanisms contributing to the beneficial effects of MR depletion were associated with inhibiting intracellular reactive oxygen species production and increased Bcl‐2/Bax ratio. In vivo study, 1 × 106 of MSCs with or without siRNA‐MR were injected into rat hearts immediately after MI. Depletion of MR could improve the MSCs’ survival significantly in infarcted myocardium, associated with more cardiac function improvement and smaller infarct size. Capillary density were also significantly higher in siRNA group with increased expression of vascular endothelial growth factor. Our study demonstrated that silencing MR promoted MSCs’ survival and repair efficacy in ischaemic hearts. MR might be a potential target for enhancing the efficacy of cell therapy in ischaemic heart disease.

therapy in AMI, [5][6][7][8] and its improvement in cardiac function is comparable with the results from reperfusion and pharmacological therapy. 9 Recent clinical trials observed a direct relationship between dose and efficacy in cell therapy for ischaemic heart disease. [10][11][12] However, most transplanted cells died because of the hostile microenvironment after infarction. 13 Considering a "ceiling effect" of cell therapy for heart disease, 14 it was a more attractive strategy to promote the MSCs' survival when compared with unlimited increase in MSCs' dose to enhance the therapeutic efficacy of stem cell therapy.
The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, belongs to the nuclear receptor family. MR activation played an important role in the pathogenesis of multiple cardiovascular diseases. 15,16 Pharmacological MR antagonists showed a beneficial effect in patients with heart failure. 15,17 As two important hormone ligands, aldosterone and corticosterone plasma levels were elevated after infarction, MR antagonists or MR deletion could suppress cardiomyocytes apoptosis and prevent adverse cardiac remodelling after AMI. 18,19 In addition, MR could be activated by hypoxia and involved in the pathogenesis of pulmonary hypertension. 20 Previous studies also revealed that MR activation by aldosterone could impair the function and decrease the number of endothelia progenitor cells (EPCs). 21 Thus, we hypothesized that MR might be involved in the process of MSCs' apoptosis after transplantation in AMI, and this study was conducted to verify this hypothesis.

| Isolation and culturing of rat MSCs
MSCs were isolated from the femur of 100-120 g male Sprague-Dawley rats (aged 4-5 weeks) and expanded as previously described. 22 The mesenchymal population was isolated based on its ability to adhere to the culture plate. Cultures (low-glucose Dulbecco's modified Eagle's medium containing 10% foetal bovine serum) were changed every 3-4 days. Once cultures became 80% confluent, the cells were passaged and plated out at 1:2 to 1:3 dilutions. All cells in our study were 3rd-4th passage. Animal experiments were approved by the Animal Care and Use Committee of Fudan University in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Academy Press (NIH Publication No. 85-23, revised 1996).

| Cell transfection
MSCs were seeded into 6-well plates at a density of 1 × 10 6 cells/ mL. After 24 hours of culture, the medium was removed, replaced with serum-free DMEM containing 10 μL (multiplicity of infection, MOI = 10) of AAV-MR-siRNA (siRNA group) or AAV-NC-siRNA (NC group) for a further 24 hours. After transfection with siRNA for 48 hours. Transfection efficiency was determined by fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis.
Alterations of MR mRNA was corroborated by Quantitative Realtime PCR (qRT-PCR).

| In vitro model of MSCs injury
MSCs injury was induced under hypoxia for 6 hours in serum-and glucose-free DMEM, which had been described previously, 25

| Measurement of intracellular reactive oxygen species (ROS)
The production of intracellular ROS was measured using an oxidation-sensitive fluorescent dihydroethidium (DHE) probe (Vigorous, Beijing, China).
The assay was based on the principle that DHE could cross cell membranes and be rapidly oxidized in the presence of ROS, which resulted in the formation of a highly fluorescent form of oxidative ethidium. MSCs were incubated with 10 μmol L −1 DHE for 15 minutes at 37°C in the dark, and then washed with PBS. The fluorescence level, as an indicator of ROS production, was detected using fluorescence microscopy. The excitation and emission filters were 488 nm and 590 nm, respectively.

| Cell extract preparation and Western Blot analysis
For analysis of protein levels, cells were lysed in lysis buffer and proteins were separated by SDS-PAGE, followed by blocking in PBS/Tween-20 containing 2% BSA. The blots were then incubated overnight at 4°C with antibodies specific to Bcl-2, Bax, vascular endothelial growth factor (VEGF) (Bioworld Technology, Inc., St. Louis Park, MN, USA). These membranes were incubated for 2-hour with HRP-conjugated secondary antibodies. After washing in PBST-buffer, bands were visualized by SuperSignal West Femto and exposed to radiography film (Bio Rad, Hercules, CA, USA). GAPDH or beta tubulin was used as a loading control.

| Rat AMI model and MSCs transplantation
AMI model was developed in Sprague-Dawley rats (200-250 g, aged 6-8 weeks), as previously described. 26 The male MSCs were used for F I G U R E 1 Flow cytometric analysis of surface antigen expression on MSCs. The rat MSCs were positive for CD29,CD44, and CD90 but negative for myeloid/hematopoietic cell-specific antigens of CD34 and CD45 the following two-part experiments as previously described 27

| Measurement of infarct size
Fixed hearts were embedded in paraffin and LV cross-sections were cut into 5-μm slices and processed. Myocardial infarct size was determined from three mid-LV sections after staining with Masson's Staining. The infarct size was defined as the sum of the epicardial and endocardial infarct circumference divided by the sum of the total LV epicardial and endocardial circumferences using computerbased planimetry. In addition, bright field microscopy was performed on Masson stained histological section to identify islands of myofibres in LV infarcted tissue.

| Real-time PCR for Sry-gene
Myocardial specimens from different groups were frozen in liquid nitrogen and powdered. The genomic DNA purification was performed using Genomic DNA Isolation Kit (Tiangen, Beijing, China) and the concentration of the purified DNA was determined by spectrophotometry. Real-time PCR was performed using Takara SYBR ® Premix Ex Taq ™ in a Bio-Rad iQ5 optical module. Primers for amplification of rat Y-chromosome Sry and β-actin are listed below.

| Tracking of the GFP + cells injected in infarcted hearts
Quantification of GFP + MSCs was performed on serial sections as described previously. 28 Rat hearts were harvested at 4 days after MI. 26

| Statistical analysis
Experiments were repeated at least three times. All data were described as mean ± SEM. To analysis the data statistically, we performed one-way ANOVA with post hoc analysis and considered a value of P < 0.05 as statistically significant.

| Adenoviral vector transfection of MSCs
The MSCs displayed a homogenous spindle-shaped population and maintained a similar morphology during the subsequent passages.
Characterization by flow cytometric analysis showed that most MSCs expressed CD29, CD44 and CD90, but not the hematopoietic markers of CD34 and CD45 (Figure 1). A representative GFP expression was shown in Figure 2. We observed more than 90% of MSCs were transfected by adenoviral vector having 10 MOI (Figure 2A). The transduction efficiency, as determined by the percentage of GFP positive cells was 82.7% based on FACS analysis ( Figure 2B). To evaluate the expression of MR mRNA in the genetically modified MSCs in vitro, qRT-PCR was performed on cell samples at 48 hours after transfection. The results showed that the relative expression of MR mRNA in the siRNA group was significantly lower than those in MSCs and NC group (Figure 2C). Cell viability was comparable among three groups ( Figure 2D).

| Depletion of MR protected MSCs against apoptosis in vitro and enhanced the survival in infarcted heart
To analyse the effect of MR on apoptosis of cultured MSCs, the

| Depletion of MR inhibited ROS generation and up-regulated anti-apoptotic proteins
Ischaemia could lead to ROS formation, which might intensify the anoikis signals in transplanted MSCs. 32  The expression of anti-apoptotic protein Bcl-2 was significantly decreased after hypoxia, which was markedly increased after MR depletion in hypoxic condition. Moreover, the increased level of Bax induced by hypoxia was attenuated when the MR expression was down-regulated by siRNA ( Figure 5C and D).

| Depletion of MR improved the left ventricular function and reduced infarct size
M-mode echocardiography were examined 4 weeks after cell transplantation. LV function indices were summarized in Table 1.
Both LVEF and LVFS were improved significantly in siRNA group and NC group when compared with PBS group, and the improvement of LVEF and LVFS was greater in siRNA group than that in   (Figure 6A and C). Besides, more surviving myocyte islands were observed after MSCs transplantation within the centre of infarcted tissue when compared with PBS group, and the amount of myocyte islands was even higher in siRNA group than that in NC group though it did not reach statistical significance ( Figure 6B and D, P = 0.057).

| Depletion of MR promoted angiogenesis
Capillary density was examined by immunostaining for CD31 in the peri-infarcted areas 4 weeks after AMI. Representative images were shown in Figure 7A. Quantitative analyses revealed that capillary density was significantly higher in siRNA group (13.34 ± 2.23 vessels/HPF) and NC group (7.45 ± 2.13 vessels/HPF) when compared with PBS group (4.51 ± 1.21 vessels/HPF), and capillary density in siRNA group was even higher than that in NC group ( Figure 7B).
Accordingly, MSCs transplantation could up-regulate VEGF expression in ischaemic myocardium at 4 weeks after AMI, which was significantly higher in siRNA group when compared with NC group ( Figure 7C and D). proven to mediate the ROS production in many pathological conditions, [38][39][40] Consistent with the previous reports, the present study showed hypoxia led to the accumulation of ROS in MSCs, MR silencing could decrease the ROS production and inhibit hypoxiainduced apoptosis. As known, Bcl-2 and Bax are two members of the Bcl-2 family, and the Bcl-2/Bax ratio was important in determining susceptibility to apoptosis. Our results suggested that MR depletion led to an increase in Bcl-2/Bax ratio in hypoxic condition, which could avoid cell apoptosis. Taken together, these data indicated that the protective effects of MR depletion were associated with decreased oxidative stress reaction and increased ratio of Bcl-2/Bax.
Conflicting data about the influence of MR activation on stem cells had been reported. MR antagonists such as eplerenone or spironolactone could increase the number and improve the function of EPCs. 21,41 Instead, MR activation could also improve migration and modify the secretory activity of cardiac progenitor cells. 42,43 The discrepancy might be because of different stem cell types and study design (in vitro vs in vivo). Furthermore, short-term as opposed to chronic exposure to MR activation might also explain the inconsistency among studies. For instance, in previous studies, short-term exposure to aldosterone most often led to vasodilation mediated by endothelium-derived nitric oxide, while chronic exposure to aldosterone impaired endothelial functions. 44,45 In summary, MR activation was involved in MSCs' death after transplantation, MR depletion could promote MSCs' survival by alleviating hypoxia-induced oxidative stress and increasing Bcl-2/Bax ratio.
As a treatment, transplantation of siRNA-MR modified MSCs was associated with improvement of LV function and reduced infarct size.
Thus, our study provided evidence that MR might be a potential target for enhancing the efficacy of cell therapy in ischaemic diseases.

CONF LICTS OF INTEREST
The authors confirm that there are no conflicts of interest.