Improvement of therapeutic effects of mesenchymal stem cells in myocardial infarction through genetic suppression of microRNA-142

Transplanted mesenchymal stem cells (MSCs) have been shown to contribute to myocardial repair after myocardial infarction (MI), primarily through production and secretion some growth factors and cytokines related to cell survival and regeneration. Further improvement of the therapeutic potential of MSCs appears to be an attractive strategy for MI treatment. CXC chemokine receptor (CXCR) 7 is the receptor for stromal cell-derived factor-1 (SDF-1), an important chemokine that is essential for tissue repair and angiogenesis. SDF-1/CXCR7 axis plays a critical role in the mobilization, recruitment and function of MSCs during tissue regeneration. Here, we depleted miR-142 that targets CXCR7 in MSCs cells through expression of antisense of miR-142, resulting in enhanced expression of CXCR7 in these miR-142-depleted MSCs (md-MSCs). In vitro, presence of md-MSCs reduced hypoxia-induced cardiac muscle cell apoptosis in a more pronounced manner than MSCs. In vivo, compared to transplantation of MSCs, transplantation of md-MSCs further enhanced cardiac re-vascularization and further improved cardiac functions after MI in mice. Together, our data suggest that depletion of miR-142 in MSCs may improve their therapeutic effects on MI.


INTRODUCTION
Myocardial infarction (MI) is a hallmark for all coronaryartery diseases (CAD), and effective prevention from the loss and effective enhancement of the regeneration of cardiomyocytes after MI are critical for a successful therapy for CAD [1].
Recent evidence has demonstrated the effectiveness of mesenchymal stem cells (MSCs)based therapy for MI [2][3][4][5]. Bone-marrow-derived mesenchymal stem cells (MSCs) are characterized by specific surface antigen expression for Sca-1, CD90 and CD105, and absent expression for CD34, CD45 and HLA-DR [6][7][8]. Moreover, MSCs have multipotent differential potential, and could be induced to differentiate into cells of mesodermal origin, including adipocytes, osteocytes and chondrocytes [9]. MSCs have been applied to promote cardiac muscle repair and regeneration after MI. But the effects are not optimized yet, largely due to non-optimized expression of the genes associated with therapeutic potentials of MSCs. Further improvement of the therapeutic potential of MSCs appears to be an attractive strategy for MI treatment.
Research Paper www.impactjournals.com/oncotarget CXC chemokine receptor (CXCR) 7 is the receptor for stromal cell-derived factor-1 (SDF-1), an important chemokine that is essential for tissue repair and angiogenesis [10]. SDF-1 functions through binding to two G-protein-coupled receptors, CXCR4 and CXCR7, to activate signaling cascades to control the mobilization, recruitment and function of MSCs during tissue regeneration [10]. The interaction of SDF-1 with CXCR4 and CXCR7 is critical in maintaining the viability and proper function of MSCs in harsh environments, e.g. after cell transplantation [11]. However, whether enhancement of CXCR7 expression may augment the therapeutic effects of MSCs after MI is unknown.
MicroRNAs (miRNAs) are non-coding small RNAs controlling gene expression, and Watson-Crick pairing to the 3′-untranslated region (3′-UTR) of the mRNA of a specific gene by miRNAs is one of the most important posttranscriptional regulators for cellular protein levels [12]. Strong evidence supports the importance of miRNAs in the pathogenesis of MI [13]. In the past studies on miRNAs, miR-142 has been shown to be a major regulator of cell fate decision in the hematopoietic system [14]. Moreover, missexpression of miR-142 has been implicated in the initiation and development of a panel of diseases, supported by clinical evidence and animal models [14]. Although many targets of miR-142 have been determined, CXCR7 as a target gene for miR-142 has not been reported.
Here, we depleted miR-142 in MSCs cells through expression of antisense of miR-142, resulting in enhanced expression of CXCR7 in these miR-142-depleted MSCs (md-MSCs). The effects of miR-142 depletion in MSCs on cardiac muscle cell apoptosis were tested in vitro and in vivo in an MI model in mice.

CXCR7 is targeted and suppressed by miR-142 in MSCs
Since CXCR7 is critical for the therapeutic effects of MSCs in treating different diseases including MI, we aimed to enhance its expression in MSCs through modulation of its protein translation via miRNA. We performed bioinformatics analyses to search for miRNAs that have Watson-Crick pairing to the 3′-UTR of CXCR7 mRNA. We only found one cross-species conserved miRNA, miR-142, in the library, which binds to 3′-UTR of CXCR7 mRNA at 389th-395th base site ( Figure 1A). To figure out if the binding of miR-142 on CXCR7 may be functional, we transfected mouse MSCs with miR-142, or antisense for miR-142 (as-miR-142) or a null plasmid as a control. RT-qPCR was performed in these transfected cells and confirmed the alteration of miR-142 levels in these cells ( Figure 1B). Next, awild type 3′-UTR of CXCR7 mRNA (CXCR7 3′-UTRwt) plasmid, or a 3′-UTR of CXCR7 mRNA plasmid with a mutant across miR-142-binding site (389th-395th; CXCR7 3′-UTR mut), was applied to co-transfect MSCs together with miR-142, as-miR-142 or null plasmids in a dual luciferase reporter assay. We found that miR-142 markedly inhibited the luciferase activity of CXCR7 3′-UTR wt, whereas the as-miR-142 increased the luciferase activity in MSCs ( Figure  1C). On the other hand, transfection of either miR-142 or as-miR-142 did not alter the luciferase activity of the reporter for CXCR7 3′-UTR mut in MSCs ( Figure 1D). These data suggest that the binding of miR-142 on 3′-UTR of CXCR7 mRNA is functional, which suppresses CXCR7 protein levels. Moreover, although transfection by as-miR-142 did not alter CXCR7 mRNA in MSCs ( Figure  1E), it significantly increased CXCR7 protein levels in MSCs, determined by Western blotting (Figure 1F), and by immunocytochemistry ( Figure 1G).However, transfection by miR-142 did not significantly alter either mRNA or protein of CXCR7, suggesting a relative high expression level of miR-142 in the primary MSCs.

Md-MSCs maintain MSC properties
In order to examine the effects of miR-142-depletion in MSCs on their potential in treating MI, we prepared adeno-associated viruses (AAVs) carrying as-miR-142 or null as a control. The transduced cells were termed as md-MSCs (for AAV-as-miR-142) or MSCs (for AAVnull), respectively. We checked the surface markers of md-MSCs and found that they were positive for Sca-1, CD90 and CD105, and negative for CD34, CD45 and HLA-DR, consistent with an MSC phenotype ( Figure 2A). Moreover, md-MSCs were able to differentiate into chondrocytes, osteocytes or adipocytes in corresponding differentiation media ( Figure 2B). Thus, after transduction, md-MSCs kept MSC properties. Finally, we examined the miR-142 and CXCR7 levels in md-MSCs. By RT-qPCR, we found that the levels of miR-142 significantly decreased in md-MSCs ( Figure 2C). By Western blotting, we found that the protein levels of CXCR7 significantly increased in md-MSCs ( Figure 2D). Together, these data suggest that miR-142-depletion in MSCs increases CXCR7 levels.

Enhanced protective potential of md-MSCs against hypoxia in cardiac muscle cells
In order to examine the cardio-protective effects of md-MSCs vs MSCs, we used a transwell system to co-culture isolated primary cardiac muscle cells (CMC) with MSCs under hypoxia challenge for 48 hours and then performed the Annexin V-apoptosis assay to determine the apoptosis levels of CMC. For the negative control group (CTL), CMC were cultured without hypoxia or any MSCs. For the positive control group (Hypoxia), CMC were cultured under hypoxia but not with any MSCs. For the group Hypoxia+MSCs, CMC were cultured under hypoxia and with control MSCs. For the group Hypoxia+md-MSCs, CMC were www.impactjournals.com/oncotarget cultured under hypoxia and with md-MSCs. For the group Hypoxia+MSCs-C7, CMC were cultured under hypoxia and with MSCs overexpressing CXCR7 ( Figure 3A).
Next, we analyzed the apoptosis of CMC. We found that hypoxia induced apoptosis in CMC, which was attenuated by co-culture with MSCs. Interestingly, the attenuated apoptosis of CMC was more pronounced when CMC were co-cultured with md-MSCs or MSCs-C7, compared to MSCs, shown by representative flow charts ( Figure 3B), and by quantification ( Figure 3C). Similar results were obtained in TUNUL staining ( Figure 3D). Hence, in vitro, md-MSCs, like direct overexpression of CXCR7 in MSCs,appears to have enhanced cardioprotective effects against hypoxia.

Better heart function is detected in MI-mice transplanted with md-MSCs
The heart function of the mice from 4 groups was assessed using ventricular catheterization. End systolic pressure-volume relationship (ESPVR, Figure 4A), left ventricular end systolic pressure (LVESP, Figure 4B) and positive maximal pressure derivative (+dP/dt, Figure 4C) were measured. The data showed that transplantation with MSCs resulted in a significant increase in ESPVR slope  Figure 4A), a significant increase in LVESP ( Figure 4B) and a significant increase in +dP/dt ( Figure 4C). All 3 values in these measurements were further improved by transplantation with md-MSCs ( Figure 4A-4C). Next, we used Masson's trichrome staining to determine the collagen deposition in the heart tissue of each group. We found that the infraction size after MI was significantly reduced by transplantation with MSCs, shown by quantification ( Figure 4D), and by gross images ( Figure 4E). Moreover, the infraction size after MI was further reduced by transplantation with md-MSCs, compared to transplantation with MSCs ( Figure 4D-4E). Together, these data suggest that transplantation of md-MSCs were superior to MSCs in recovery of cardiac function after MI.

Enhanced angiogenesis is detected in MI-mice by transplantation of md-MSCs than MSCs
The vessel density of the mouse hearts from 4 groups was assessed by CD31 staining. The data showed that transplantation with MSCs after MI resulted in a significant increase in CD31 density, shown by representative images (Figure 5A), and by quantification ( Figure 5B). Moreover, the CD31 density was further improved by transplantation with md-MSCs, compared to transplantation with MSCs ( Figure  5A-5B). Thus, these data suggest that transplantation of md-MSCs induces greater angiogenesis than MSCs in hearts after MI.

DISCUSSION
MSCs are great players in promoting tissue regeneration in adulthood due to high competency and self-renewal potentials [15]. Specifically, MSCs have been studied extensively in post-MI cardiac muscle recovery and regeneration [16][17][18]. Interestingly, Cheng et al. showed that transplanted MSCs expressing CXCR4 increased their potential in promoting myocardial repair following MI [19]. Further decreases in anterior wall thinning, better preservation of left ventricular chamber dimensions and decreases in collagen I/III ratio in the infarcted heart wall were detected in mice that received these CXCR4-overexpressing MSCs [19]. Compared to this study, our work has several novelties. First, CXCR7 is another potent receptor for SDF-1 yet much less investigated. Second, compared to CXCR4, CXCR7 functions more consistently. The function of CXCR4 in MSCs is rather complicated and diverse, and could be detrimental to recovery of MI under certain conditions and environments [20]. Finally, direct editing of a gene expression may result in exaggerated expression of the target gene, which may lead to side effects, off-target effects and dysregulation of gene expression network in cells. Our way of modification of CXCR7 levels indirectly by miRNA is very modest, and the expression level alteration of CXCR7 appears to be more physiological rather than artificial in cells.
Here, we used as-miR-142 to neutralize the endogenous expression of miR-142 in MSCs. The depletion of miR-142 resulted in loss of the suppression of miR-142 on CXCR7 and then increases in CXCR7 protein levels. Interestingly, overexpression of miR-142 in MSCs did not have significant effects on CXCR7 mRNA and protein. This data may reflect the endogenous high expression of miR-142 in MSCs, and further increases in miR-142 could not further increase its effects on CXCR7, which may be saturated already. Previous studies have reported several targets of miR-142 in different cells, e.g. sirtuin1 [21,22] and Bach2 [23]. However, to the best of our knowledge, this is the first report to show that CXCR7 is a target for miR-142, especially in MSCs.Compared to trophic factor administration (injection of SDF-1), modification of the receptor in MSCs (in a modest way through miRNA) has a lot of merits and is possibly more appropriate for further translational research. Moreover, it might be interesting to check the effects of miR-142 inhibition on downstream signaling of SDF-1/CXCR7 to further confirm that the effects of miR-142 inhibition is primarily affecting this signaling pathway. Furthermore, one miRNA has many targets, it is necessary to study the effects of targets other than CXCR7 for miR-142 in MSCs. These questions will be addressed in future studies. To summarize, the data from the current study encourages a study on genetic modification in human MSCs in a similar setting.

Protocol approval
All the experimental methods in the current study have been approved by the research committee at the Huai'an Hospital Affiliated to Xuzhou Medical College and Huai'an Second People's Hospital. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Huai'an Hospital Affiliated to Xuzhou Medical College and Huai'an Second People's Hospital. Mouse surgeries were performed under supervision of a qualified veterinarian.

Isolation of CMC and co-culture system
The CMC were isolated from male C57/BL6 mice of 10 weeks of age (Shanghai Laboratory Animal Center). Briefly, mouse hearts was dissected out, washed with PBS and then cut on ice into small pieces to be kept in a water bath with a magnetic bar at 37°C for 10 minutes, before the minced heart tissues were digested with fresh pre-warmed 0.2% trypsin for at 37°C for 5 minutes. The dissociated cells were suspended and plated into the transwell co-culture system.

Preparation of MSCs and ms-MSCs
The null or as-miR-142 or CXCR7 construct was cloned into a pAAV-CAG-GFP plasmid (Clontech, Mountain View, CA, USA). To generate AAVs, HEK293T cells were co-transfected with 10 µg of the prepared plasmids and 5 µg each of packaging plasmids using Lipofectamine 3000 (Invitrogen, St. Louis, MO, USA). The viruses were purified using CsCl density centrifugation and the titrated with a quantitative densitometric dot-blot assay.

MI model and MSC transplantation
MI was induced in male C57/BL6 mice at 12 weeks of age by ligation of the left anterior descending artery. Briefly, after anesthetization and setup of proper ventilation, mouse heart was exposed by left thoracotomy with the assistance of chest retractor. The left anterior descending artery was ligated with 8-0 nylon sutures in the middle. The coronary occlusion was confirmed by the blanching of the apex. One hour after ligation, mice received injection of saline or 3 × 10 5 MSCs at the three sites in the border the ligation. The CTL group received left thoracotomy only. The chest was closed with 4-0 sutures. The mice were kept for 1 month before invasive hemodynamic assessment and histology studies.

Hemodynamic assessment
After anesthetization, ventricular catheterization was performed on mice. A 1.2F pressure volume catheter was connected to an Advantage PV-loop system, and then inserted into the left ventricle via right carotid artery. The hemodynamic parameters, LV pressure and positive maximal pressure derivative (+dP/dt) were measured. The end systolic pressure-volume relationship (ESPVR) was obtained after and before inferior vena cava occlusion.

Quantitative real-time PCR (RT-qPCR)
Total RNA were extracted using the miRNeasy mini kit (Qiagen, Hilden, Germany). Complementary DNA preparation and quantitative real-time PCR (RT-qPCR) were performed. All primers were purchased from Qiagen. Data were collected and analyzed using 2-∆∆Ct method. Values of genes were first normalized against α-tubulin, and then compared to experimental controls.

Flow cytometry
For characterization of MSC phenotype or purification of transfected MSCs, flow cytometry was performed. The antibodies are FITC-conjugated anti-Sca-1, CD90, CD105, CD34, CD45 and HLA-DR (Becton-Dickinson Biosciences, San Jose, CA, USA). For determination of cell apoptosis, CMC were labeled with annexin V-FITC and propidium iodide (PI), using an apoptosis detecting kit (KeyGEN Biotech, Nanjing, China), and analyzed by flow cytometry. Data were analyzed using FlowJo software (Flowjo LLC, Ashland, OR, USA).

Vessel density
For vessel density measurement, CD31 staining was performed with rat anti-CD31 (550274; Becton-Dickinson Biosciences) and CD31 density (CD31-positive area versus total tissue area) was determined at a magnification ×20 in 5 random fields on 3 tissue slides in each animal.

Masson's trichrome staining
For Masson's trichrome staining, the heart slides were deparaffinized, rehydrated and then subjected to the staining with a Masson's trichrome staining kit (HT15; Sigma-Aldrich). The infarction size was calculated by the percentage of infarcted area to the left ventricle area.