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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2015 Sep 1;8(9):10555–10564.

Bone marrow mesenchymal stromal cells with CD47 high expression via the signal transducer and activators of transcription signaling pathway preventing myocardial fibrosis

Wei Deng 1, Qing-Wei Chen 1, Xing-Sheng Li 1, Zhong-Ming Yuan 1, Gui-Qiong Li 1, Da-Zhi Ke 1, Li Wang 1, Zhi-Qing Wu 1, Shi-Lan Luo 1
PMCID: PMC4637580  PMID: 26617765

Abstract

This study was initiated to investigate the efficacy of myocardial fibrosis intervention via signal transducer and activators of transcription (STAT) signaling using bone marrow (BM) mesenchymal stromal cells (MSC) in which being over-expressed with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). BiAb was prepared and combined with isolated MSC with CD47 overexpression from male mice and trans-fused into female mice with isoproterenol-induced myocardial fibrosis via the tail vein, followed by MB. This study included five groups. Five weeks after treatment, expression levels of the sex-determining region of Y-chromosome (SRY), matrix metalloproteinases (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of signal transducer and activators of transcription (STAT) 1 and STAT 3 was detected by Western blot. Results: The highest homing number of MSC was in the CD47 + MSC + BiAb + MB group, second highest in the CD47 + MSC + BiAb group, and lowest in MSC alone. Compared with the Control group, CD47 + MSC + BiAb + MB, CD47 + MSC + BiAb, CD47 + MSC and MSC groups had decreased levels of MMP-9, TIMP-1, STAT 1 and collagen deposition, and increased levels of STAT 3. Up regulated STAT 3 and down regulated TIMP-1 were significantly different in CD47 + MSC + BiAb + MB compared with CD47 + MSC or CD47 + MSC + BiAb. Conclusion: CD47 can enhance the homing rate and repairing efficacy of MSC. MSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.

Keywords: Myocardial fibrosis, mesenchymal stromal cells, CD47, signal transducer and activators of transcription

Introduction

Myocardial fibrosis is a pathological feature at the end stage of some cardiovascular diseases and characterized by the proliferation of cardiac fibroblasts in the myocardial interstitium, the transformation of these fibroblasts into myofibroblasts and the excessive deposition of extracellular matrix (ECM) [1]. Bone marrow (BM)-derived mesenchymal stem cells (MSCs) transplantation has been used in drug intervention, interventional therapy and surgery for the treatment of ischemic heart disease because they have the ability to differentiate in to cardiomyocyte or cardiomyocyte-like cells and repair myocardial infarction. However, numerous animal experiments and clinical trials have shown that the repairing efficacy of BMSCs transplantation is often limited by the migratory number and colonization amount of targeted stem cells [2].

Recent studies have demonstrated that more than 60% implanted stem cells are retentated by lung, liver, spleen and other non-target organ rejection in transplanted host body, and the number of valid stem cell homing to the damaged target tissue rarely. This directly affects the effectiveness of stem cells to repair [3]. The most commonly used three stem cell transplantation approaches have certain limitations: I Intracoronary transplanted restricted to coronary condition and special equipment; II Transendocardial and epicardial injection require sophisticated equipment and precise positioning; III Intravenous transplanted safe, easy to operate, but poor targeting and low efficiency. Therefore, developing a method, by which stem cell transplantation not only from the vein, but also increased its survival rate, to ameliorate the therapeutic effect of cell cardiomyoplasty is extremely important [4-6].

CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration [7-10]. Furthermore, it plays a key role in immune and angiogenic responses. Considerable researches have shown that CD47 has the ability to evade the immune cells phagocytosis as an exempted mark of cell death. Monocytes-macrophages removed the damaged cells in the bloodstream and exogenous foreign body by primarily monitoring cell surface CD47. CD47 molecules can be recognized by receptor signal-regulated protein α on the cell surface of macrophages, which can protect cells carry CD47 against the phagocytosis. It has been suggested that genetic modification of BMSC with CD47 can enhance the stem cell therapies. However, the molecular mechanism underling this was unclear.

In order to promote the homing of MSC and understand its repairing mechanism, we combined bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB) to guide MSC with CD47 high expression to improve the homing number of MSC and evaluate the therapeutic efficacy of this treatment on myocardial fibrosis.

Materials and methods

Animals

One-hundren male Sprague-Dawley (SD) rats (Shanghai Slac Laboratory Animal Co. Ltd., China) were used in this study. The animal protocol was in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and was approved by the Experimental Animal Care Committee of Chongqing Medical University, Chongqing, China.

Preparation of BMSCs

Flow cytometry was used to detect the markers of BMSCs (P3) that included the cells positive for CD29, CD44, and CD90 and negative for CD34 and CD45. Adipogenic induction medium and osteogenic induction medium were then added to induce differentiation into adipocytes or osteoblasts, respectively.

Preparation of BiAb

Rabbit anti-rat CD29 (Bioss, Beijing, China) was dissolved in Traut’s reagent (Pierce, Rockford, IL, USA) for 1 h according to the chemical crosslinking method. Anti-myosin light chain antibody (AMLCA) (Abcam, Cambridge, UK) was dissolved in Sulpho-SMCC reagent (Pierce) for 1 h. Both monoclonal antibodies were mixed immediately in equimolar proportions and kept overnight at 4°C. The BiAb was identified using non-reductive sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Management of ultrasound-mediated MB

MB were awarded the National Invention Patent of China in 2005 and have been widely applied in experimental studies. The ultrasound treatment meter Type UGT 1025 (10-12) was developed by the Institute of Ultrasound Imaging, Chongqing Medical University. The concentration of MB used was approximately 8.5×108 bubbles/mL. Ultrasound irradiation was performed at a frequency of 1 MHz and an intensity of 1.5 W/cm2 for 1 min.

Construction of CD47 over-erpression vector and cell transfection

The rat CD47 cDNA sequence was derived from NCBI gene bank. Recognition sites sequence of Xho1 and EcoR I restriction enzymes were added to 5’ ends of reverse and forward primers to introduce Xho I site at the 5’ and EcoR I site (Italics and bold) at the 3’ end of the PCR products. We included the sequence between Xho I site and SnaBI site (underlined) in the 5’ site of the CD47-fwd primer after the XhoI recognition site sequence. These 24 nucleotides between Xho I site and SnaBI site which encodes the KEX2 and STE13 cleavage sites must be recreated in order for efficient cleavage of the fusion protein to occur. Meanwhile the stop codon (TAA) was included after the EcoR I site in reverse primer. The sequences of the primers were 5’-ATCTCGAGAAAAGAGAGGCTGAAGCTGAAGGGATCTGCAGG-3’ and 5’-AATGAATTCTTAGGCTGCAACAGGGGGTAACATAAATGG-3’. A 1213 bp fragment including whole coding sequence of DC47 gene was amplified with these primers by PCR in a final volume 25 µL. Following Xho I/EcoR I digestion and purification of both vector and PCR product, the PCR product was ligated into the corresponding EcoR I and Xho I sites within the multi-cloning site (MCS) of PIRES2-EGFP plasmid. Subsequent the ligation reaction, bacterial transformation and Amp selection on LB agar plates, a few numbers of clones were obtained. A total of 10 bacterial clones were screened by PCR using the CD47-Fwd and CD47-Rev primers. Transfection of BMSCs by lipofection, and then RT-PCR for detecting CD47 gene expression after transfection.

Establishment of the myocardial fibrosis model

One-hundred healthy Sprague-Dawley, 6 weeks old (200-250 g; provided by the Experimental Animal Center of Chongqing Medical University), were used in this study. The rat were subjected to hypodermic injection with isoproterenol (batch number 080701; Shanghai Hefeng Pharmaceutical Co. Ltd, Shanghai, China) at 50 mg/kg. The injection was carried out twice a day for a continuous 10 days.

The prepared models were divided into the five groups, each containing 20 animals. The groups were: untreated, pure MSC transplantation (MSC group); MSC with CD47 high-expression (CD47 + MSC group); MSC with CD47 high-expression combined BiAb (CD47 + MSC + BiAb group); and CD47 + MSC + BiAb + MB group. The handling of animals during these experiments was approved by the Animal Use Ethics Committee of Chongqing Medical University.

Cellular transplantation

One day after the last isoproterenol injection, all rat in each group were subjected to tail vein transfusion. The rat in the untreated group were transfused with 0.1 mL PBS., The rats were transfused with 4×106 MSC/kg in the MSC group and MSC CD47 + MSC group respectively. The rats in CD47 + MSC + BiAb group were transfused with 4×106 MSC/kg and 50 ng BiAb mixture. The rats in the CD47 + MSC + BiAb + MB group group were first transfused with 0.1 mL MB and then ultrasound wave irradiation (frequency 1 MHz, intensity 1.5 W/cm2, duration 1 min) was performed on the precordial region. Subsequently, 4×106 MSC/kg and 50 ng BiAb mixture were transfused. The rats in the control group were transfused with 0.1 mL PBS.

Fluorescent quantitative polymerase chain reaction analysis on expression of the sex-determining region of Y-chromosome, vascular endothelial growth factor, matrix metalloproteinases-9, tissue inhibitor of metalloproteinase-1 in myocardium, signal transducer and activator transcription-1 and signal transducer and activator transcription-3.

Rats were killed 5 weeks after cell transplantation and their hearts collected. The cardiac apexes were sampled and subjected to fluorescent quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The trizol one-step method was used to extract the total RNA and its purity was verified using an ultraviolet spectrophotometer. Reverse trancription and cDNA synthesis were carried out using conventional methods. Specific primers (Table 1) were designed according to the sequences of sex-determining region of Y-chromosome (SRY), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, vascular endothelial growth factor (VEGF) and β-actin in GenBank. Primers were synthesized by Shinegene Biotechnological Co. (Shanghai, China). The TaKaRa TP (Japan) fluorescent qRT-PCR detection system was used for amplification. An SYBR green fluorescent quantitation PCR kit (Shine-gene Biotechnological Co.) was used for quantitative detection of the target genes. Each reaction system included 1 μL cDNA, 25 μL 2× SYBR Premix Ex Taq TM II buffer, 0.3 μL of each primer for the target gene (10 μM/L), and 8.4 μL RNase-free water. The expression level of β-actin was also detected as an internal control. The cycle threshold was read and the relative ration method was used for the calculation. The standard curve, amplification curves and melting curve were plotted.

Table 1.

The mRNA expression of MMP-9, TIMP-1 and VEGF in myocardium of the five groups of rat by qRT-PCR

Gene Control MSC CD47 + MSC CD47 + MSC + BiAb CD47 + MSC + BiAb + MB
MMP-9/β-actin 4.31±0.3 3.16±0.25a 2.71±0.34b 2.93±0.24c 1.83±0.16d
TIMP-1/β-actin 0.92±0.06 0.78±0.03a 0.65±0.03b 0.62±0.02c 0.41±0.08d
STAT 1 1.03±0.08 0.83±0.04a 0.67±0.04b 0.66±0.12c 0.43±0.02d
VEGF 0.35±0.05 0.54±0.03a 0.85±0.03b 0.82±0.04c 1.01±0.05d
STAT 3 0.3±0.03 0.83±0.04e 0.8±0.03 0.79±0.03 1±0.02d
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a

standing for Control Vs MSC;

b

standing for Control Vs CD + 47 + MSC;

c

standing for CD47 + MSC + BiAb Vs Control;

d

standing for CD47 + MSC + BiAb + MB Vs control;

e

standing for MSC Vs CD47 + MSC + BiAb.

Assessment of myocardial collagen with Sirius Red staining and polarized light

The transverse plane of the left ventricle with a thickness of 2 mm was collected for the preparation of successive paraffin sections to a thickness of 5 μm. This was followed by carbazotic acid-Sirius Red staining. Myocardial collagen was observed under a polarized light microscope. Image J software (version 1.43; http://rsb.info.nih.gov/ij/, 2010-01) was used for the quantitative analysis. Collagen with Sirius Red staining was analyzed using image enhancements, color processing and measuring in Image J software. Significant differences were determined by analysis of variance (ANOVA) with appropriate post-hoc testing.

Western blot analysis of signal transducer and activators of transcription 1 and 3 expression in myocardium

Fresh cardiac tissue (250-500 mg) was collected and 1 mL total protein extraction reagent containing protease inhibitor added. Total proteins were extracted after homogenization. Coomassie brilliant blue staining was used to determine the protein concentration. Subsequently, SDS-PAGE electrophoresis was used to separate the proteins, and proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then incubated with rabbit anti rat signal transducer and activators of transcription (STAT)1 or STAT 3 antibodies (Aviva Systems Biology, San Diego, CA, USA.), followed with anti-rabbit IgG (Sigma, Santa Clara, CA, USA.) staining, and then subjected to film development and further analysis.

Statistical analysis

SPSS 16.0 statistical software was used for the data analysis. The measurement data were represented by mean standard deviation. An ANOVA was used to compare differences among the seven groups. A P-value of 0.05 was used to determine statistical significance.

Result

Identification of MSC

The in vitro cultured MSC grew in adherence and most of them grew in parallel arrays or a whirlpool-like pattern. The cells had a fusiform or polygonal shap and the karyoplasmic ratio was large (Figure 1A). The MSC became polygonal 1 week after osteogenic induction, and calcium content deposition between cells were observed 2 weeks after induction (Figure 1B). Significant formation of calcium nodes with Alizarin Red staining was observed 3 weeks after induction (Figure 1C). The morphology of MSC became irregular 1 week after adipogenic induction, and small fatty granules with a relatively strong refraction rate were found in the cytosol 2 weeks after nduction (Figure 1D). Lipid droplet accumulation with Oil Red O staining was found 3 weeks after induction (Figure 1E). Flow cytometry results indicated that the positive expression rates of CD29, CD44 and CD117 on MSC were 99.91%, 98.96% and 2.01%, respectively. These results indicated that the cultured cells expressed CD29 and CD44, but not CD117, which is in accordance with the phenotypic characteristics of MSC. The above-mentioned morphologic data and induced differentiation results in conjunction with the classification of cell phenotypes indicated that these cells were indeed MSC.

Figure 1.

Figure 1

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MSC identification using morphologic classification, functional indexes and cellular phenotyping.

Identification of overexpression CD47 in MSC

After transfection, total RNA of MSC cells was detected by RT-PCR detection. It was found that, compared to empty transfection group the level of CD47 mRNA and protein expression in PIRES2-EGFP-Rat/CD47 group was significantly higher (Figure 2, P<0.05).

Figure 2.

Figure 2

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The expression of CD47 in BMS after transfection A: mRNA expression, B: Protein expression.

Homing efficiency of MSC and its functions in the treatment of myocardial fibrosis

The mRNA expression of SRY (Figure 3), MMP-9, TIMP-1 and VEGF in myocardium of the five groups of rat by qRT-PCR is shown in Table 1. Expression of SRY mRNA represented the amount of male MSC that were recruited to cardiac tissue in the receptor rat. The highest expression of SRY was found in the CD47 + MSC + BiAb + MB group, the CD47 + MSC + BiAb group ranked second, and expression in the MSC group and CD47 + MSC were relatively low (P<0.05). Almost no expression of SRY gene was found in the control group.

Figure 3.

Figure 3

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The mRNA expression of SRY in myocardium of the five groups of rat by qRT-PCR.

Differential expression of myocardial STAT 1 and STAT 3

Five weeks after treatment administration, the expression of myocardial STAT 1 and STAT 3 was determined by Western blot. The results are shown in Table 1, with separate bands for STAT 1 and STAT 3. The analysis of the optical density of each band is shown in Figure 4.

Figure 4.

Figure 4

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The protein expression of STAT 1 and STAT 3 in myocardium of the five groups of rat by WB.

STAT 1 expression in rat with myocardial fibrosis significantly increased compared with that of the control group (P<0.05). Higher levels of STAT 1 in the control was seen than in the MSC, CD47 + MSC, CD47 + MSC + BiAb, CD47 + MSC + BiAb + MB. STAT 3 expression in the MSC, MSC, CD47 + MSC, CD47 + MSC + BiAb, CD47 + MSC + BiAb + MB groups significantly increased compared with that of the control.

Discussion

We investigated the homing and therapeutic efficacy of MSC in which being CD47 over-expressed transplantation in isoproterenol-induced myocardial fibrosis mice with a combination treatment of BiAb and ultrasound-mediated MB. With the combination of BiAb and ultrasound-mediated MB, the homing number of transplanted MSC increased, MMP-9 [11] and TIMP-1 [12] decreased, and pathologic myocardial fibrosis was improved. These results may indicate a potential mechanism relevant to the STAT signaling pathway [13,14].

BMSC transplantation is generally recognized to be the most promising stem cell therapy in treatment of ischemic heart disease due to its advantages of self-replicating ability, multi-directional differentiation potential, easily obtaining, no immune rejection, and no ethical conflicts [15-17]. However, simple BMSC transplantation has limitations because of limited survival and poor differentiation or maturation of the transplanted cells. More importantly, the adverse host micro-environment in infarction foci such as hypoxia and ischemia, ischemia-reperfusion injury with subsequent excessive inflammation in acute or subacute phase, and local structural alterations (e.g. myocardial fibrosis and micro-vascular damage) in chronic phase is not favorable to trans-planted cell surviving [18,19]. Accordingly, it is desirable to explore a newly approach that modify not only the donor cells but the host micro-environment as well.

Prepared CD29× AMLCA is the connecting bridge between injured myocardium and MSC. AMLCA can specifically recognize injured myocardium, and CD29 is the molecular marker that shows a positive rate of more than 99% on the surface of MSC. Therefore CD29× AMLCA can bind to both MSC and injured myocardium, and it can act as the bridge between them. In the present study, we transfused MSC and BiAb in a free state into mice that suffered from isoproterenol-induced myocardial fibrosis and found that BiAb can increase the homing number of MSC, and the homing rate was even higher with the aid of ultrasound-mediated MB. These findings indicate that BiAb and ultrasound-mediated MB can guide the targeted migration of MSC [20-25]. Previous work has revealed that the transfusion of BiAb and effector cells via intravenous infusion into lymphoma mice and BiAb can assist the effector cells to bring tumor inhibiting activities into play.

Additionally, BiAb in a free state can guide effector cells to damaged tissues containing target antigen in animal models. This may be attributed to the maintenance of stronger biologic activity by MSC and BiAb, as they were not subjected to the assembly process in vitro.MMP-9 and TIMP-1 in untreated myocardial fibrosis mice were significantly upregulated and fibrous degeneration of hearts was expedited. MMP-9 and TIMP-1 levels decreased 5 weeks after MSC transplantation, but they were still higher than those of normal mice. Meanwhile, collagen deposition decreased and interstitial fibrosis was alleviated. Among the treatment groups, the homing number of MSC in the CD47 + MSC + BiAb + MB group was the highest, and the degree of myocardial fibrosis was moderate, while the expression of TIMP-1 andMMP-9 was relatively low, indicating that MSC can not only be recruited efficiently with the aid of BiAb and ultrasound-mediated MB, but can also prevent myocardial fibrosis at the homing site. These results indicated that the intervention of MSC on myocardial fibrosis is closely related to its homing amount.

The upregulation and imbalance in MMP-TIMP expression after myocardial damage affects the degree of fibrous degeneration of heart [26-31]. Therefore, the internal mechanism for the intervention of MSC on MMP-TIMP needs to be elucidated. First, more transplanted MSC (which can ensure a greater homing amount) can regulate a pathologic MMP-TIMP imbalance, indicating that the amount of stem cells at the local target site can affect the efficacy of MMP-TIMP. Second, some investigations have found that the culture solution of MSC (not containing MSC) can regulate the expression of MMP-TIMP and inhibit the collagen secretion of cardiac fibroblasts. These results indicate that MSC can secrete many kinds of biologically active molecules, such as various growth factors, cytokines and chemical mediators (although these mechanisms remain unclear), that help MSC to function in repair at injured sites. Some investigations also indicate that MSC have anti-inflammatory functions in the target position of the transplantation, and thus exhibit anti-inflammatory mechanisms and participate in protection of the heart in ischemic heart disease.

Our investigation shows that myocardial STAT 1 expression increases in mice that suffer from myocardial fibrosis compared with normal mice. When tail vein transplantation of MSC was performed, the high expression of STAT 1 was inhibited, while the expression of STAT 3 was stimulated and upregulated. With the aid of BiAb and ultrasound-mediated MB, STAT 3 expression increased and TIMP-1 was downregulated. At the same time, the homing amount of MSC was increased, and the improvement in myocardial fibrosis was significant. These results indicate that STAT 1 is involved in myocardial fibrosis and STAT 3 is related to anti-fibrosis of MSC.

Although many investigations have indicated the superior effects of MSC on the repair of ischemic myocardium, some investigations presume that MSC transplantation is not always optimal [32,33]. Furlani et al. found that regional injections of MSC, carried out for the treatment of rats with acute myocardial infarction, for 6 weeks did not improve heart function. These conflicting results may occur because of different treatment details and evaluation methods utilized during the early research stages in MSC transplantation therapy. In the present study, MMP-9 and TIMP-1 expression was significantly downregulated in the CD47 + MSC + BiAb + MB group, which showed the highest homing rate, and the intervention effects on myocardial fibrosis were improved; however, VEGF expression was not significantly improved. This indicates that MSC transplanted via the peripheral intravenous route can significantly improve the mesenchymal structure of the fibrotic heart to some extent, but its improvement on vascular neogenesis is not significant. Such results could be attributed to the short duration of the treatment, or intrinsic limitations of the isoproterenol-induced myocardial fibrosis model. Further treatment and investigations in long-term studies are needed.

Acknowledgements

This project was supporyed by grants from the National Nature Science Foundation of China (No. 81201101), Chongqing municipal Department of Health Foundation (No: 2012-2-057, 2015MSXM019) and Chongqing Municipal Science and Technology Foundation and cutting-edge research projects generally projects (No: cstc2015jcyjA10074). The author are thankful to all of the involved laboratories and related research members, Finally, we sincerely thank Ma ke Ph.D. student at Medical College of Qingdao University and state key laboratory of brain cognitive science in institute of biophysics, CAS for his assistance with data analysis and manuscript revision.

Disclosure of conflict of interest

None.

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