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Volume: 14 Issue: 4 August 2016


MicroRNAs Involved in Acute Rejection and Tolerance in Murine Cardiac Allografts

Objectives: Induction of immunologic tolerance is the ultimate goal of organ transplant. To investigate the involvement of microRNA in tolerance induction after organ transplant, murine cardiac allografts were performed and the expression of microRNA in the grafts was analyzed.

Materials and Methods: Cardiac allografts were per­formed using C57BL/10 (H2-Kb) to CBA/N (H2-Kk) fully mismatched combination with or without eico­sapentaenoic acid for tolerance induction. Ten microRNA, mir-146a, 15b, 223, 23a, 27a, 34a, 451, 101a, 101b, 148a, discovered in hepatic grafts were examined by quantitative reverse transcription polymerase chain reaction using RNA from the cardiac allografts.

Results: The administration of eicosapentaenoic acid markedly prolonged the cardiac allograft survival (median survival time > 100 days) and decreased the pathological score. Quantitative reverse transcription polymerase chain reaction revealed that mir-223 was up-regulated in accordance with pathological deterioration as compared with the expression observed in the syngeneic grafts. In contrast, the other microRNA was down-regulated. Pearson product moment correlation analysis demonstrated that the expression patterns of mir-223 and mir-146a had high or moderate positive associations between the cardiac and haptic allografts in mice.

Conclusions: The change in the microRNA expression in the allografts suggests that microRNA plays a role in the induction and/or maintenance of tolerance after allograft transplant. Our findings suggest that mir-223 may be associated with rejection while mir-146a, -15b, -23a, -27a, -34a, -451, -101a, -101b, -148a may be involved in tolerance. A superior grasp of the mec­hanism for rejection and tolerance observed in the murine heart allotransplant model may provide a better curative treatment strategy to mitigate allograft rejection.

Key words : MicroRNA, Biomarker, Heart transplant, Graft rejection, Tolerance


Heart transplant is recognized as the ultimate treatment for an affected individual with terminal stage cardiac insufficiency that is a heavy burden globally. Notwithstanding substantial progress in transplant, a major problem causing the loss of allograft and resulting in lethality is allograft rejection.

At present, endomyocardial biopsies are per­formed regularly for patients in a stationary condition to screen for subclinical rejection or acute clinical indications (heart insufficiency decline of function in left ventricle) as the criterion standard for the diagnosis and survey in acute cardiac rejection. Despite their effectiveness, endomyocardial biopsies remain associated with infrequent yet potentially life–threatening complications to the patients and financial economic burden.

An important issue in heart transplant is the identification of noninfiltrative and dependable biomarkers for screening cardiac graft rejection. However, this matter remains unresolved, although attempts have been performed to identify such biomarkers by the gene expression in the peripheral blood from low-risk cardiac transplant recipients.

Recently small and noncoding RNAs referred to as “microRNAs” (miRNAs) that regulate the gene expression have been found.1 miRNAs are recognized to participate in many biological stages (eg, cell proliferation, apoptosis, oncogenesis, differentiation, and development). The latest findings show that miRNAs might play a definitive role not only in the development of immune cells, but also in innate and adaptive immunity responses. Thus, miRNAs have gained much attention as novel targets with great potential in the field of organ transplant.2

Eicosapentaenoic acid (EPA) derived from fish oil is one of the omega-3 polyunsaturated fatty acids. Purified EPA has been applied to treat arteriosclerosis and hyperlipidemia.3 Iwami and associates demon­strated that EPA administration in the major histocompatibility complex-mismatched C57BL/10 to CBA murine cardiac allograft model achieved prolonged allograft survival accompanying gene­ration of regulatory T cells in grafts.4 They also showed that peroxisome proliferator-activated receptor that has been shown to be a nuclear receptor for EPA5,6 is indispensable for the immuno­modulatory effect of EPA. In addition Ye and associates demonstrated that cardiac allografting from BALB/c to C57BL/6 combination in mice with EPA exhibited significant prolongation of the graft survival by disrupting the balance between regulatory T cells and T-helper 17 cells.7

In the current study, we examined the possibility that miRNAs could be used as pertinent biomarkers for monitoring the cardiac allograft status. In a previous report, we identified miRNA expression profiles after hepatic allografting in mice.8 To confirm the generality of the miRNA expression in organ transplant, we designed an experiment using cardiac allografts in mice. We made efforts to verify the miRNA expression signature in cardiac allograft rejection and/or tolerance induced by EPA. Furthermore, the miRNA expression signature was compared with that from a liver transplant model to estimate the generality of the miRNA expression signature of organ transplant. The information from this study may have an effect upon the clinical management of recipients with cardiac allografts.

Materials and Methods

Male C57BL/10 (B10 H2-Kb) and CBA/N (H2-Kk) mice (weighing 25-30 g) were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan) and housed and cared for in agreement with the guidelines of the Institutional Animal Care and Use Committee and in agreement with National Research Institute for Child Health and Development guidelines on laboratory animal welfare. The experiment protocol was accepted by the Committee on the Care and Use of Laboratory Animals at the National Research Institute for Child Health and Development (Permission Number: 2002-003). All surgical procedures were conducted by anesthetization with isoflurane/oxygen, and all attempts were carried out to minimize pain.

Heterotopic cardiac allografting
The heart transplants were performed on the sex-matched B10 donor to the CBA recipient by microsurgical techniques. Intra-abdominal vascu­larized heterotopic mouse cardiac transplant was performed as previously described.9 In brief, the donor hearts were obtained and saved in cooling physiological saline during which time the recipient mice were arranged. The donor hearts were heterotopically transplanted into the recipient mice using end-to-side anastomosis of the donor’s aorta and pulmonary artery to the recipient’s abdominal aorta and inferior vena cava. Cardiac graft survival was determined using daily palpation of the recipient’s abdomen. Rejection was considered to be complete at the time of cessation of a palpable heartbeat and confirmed visually via laparotomy. To obtain a tolerance model on the day of transplant the recipients were given 1 intraperitoneal injection of 1.0 g/kg of purified eicosapentaenoic acid (EPA) (Mochida Pharmaceutical, Tokyo, Japan).4

miRNA isolation from cardiac graft tissue
miRNA was isolated from the cardiac grafts and immersed in RNAlater (Ambion Austin, TX, USA) soon after recovery using the miRNeasy kit (Qiagen Valencia, CA, USA) according to the manufacturer’s protocol as described previously.10 The miRNA quality and quantity were estimated using a NanoDrop 1000 spectrophotometer (Thermo Scientific Inc. Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies Santa Clara, CA, USA).

Quantification of miRNA by quantitative real-time reverse transcriptase-polymerase chain reaction
The quantification of mature miRNA was performed by TaqMan MicroRNA Assays (Applied Biosystems Foster City, CA, USA) with specific probes and primers.

U6 small RNA was used for an endogenous control for each reaction. Super Script reverse transcriptase (Invitrogen Carlsbad, CA, USA) and an oligo (dT) primer and 600 ng of each RNA sample were used for generating cDNA. The Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems) was used for qRT-PCR, and the data are represented by the comparative cycle threshold (Ct). The normalized Ct value of each gene was obtained by subtracting the Ct value of U6 small RNA. The fold change versus 1 sample in the control group was calculated as described previously.8

Histologic studies
Mice that received heart grafts were killed on days 0, 8, and 100 after surgical operation. Modifications of the 2004 revision of standardized cardiac biopsy grading11 were adopted to define the pathological grades of heart grafts as described in Table 1.

Statistical analysis
All data are expressed as the mean ± SD. The t test was used to compare the paired and unpaired variables. A Kaplan-Meier test was employed for comparisons of the graft survival. Statistical significance was considered to exist at P < .05.


Kinetics of cardiac allograft rejection
B10 cardiac allografts heterotopically transplanted into the abdomens of the CBA mice without immunosuppression were rejected within 11 days as indicated by the cessation of a heartbeat (mean survival time: MST = 8.9 ± 2.33 days; n = 9) while the B10 heart isografts were accepted universally at 100 days posttransplant (MST > 100 d; n = 5). On the other hand, administration of EPA to CBA mice transplanted with B10 hearts resulted in a permanent survival (MST > 100 days; n = 5) (Figure 1).

Histologic analysis of cardiac allografts
A histologic analysis was performed on cardiac allografts recovered on day 8 after grafting. Standardized cardiac biopsy grading (2004) with modification (Table) was adopted to assess the evidence of rejection. In syngeneic cardiac grafts (CBA to CBA) mild and diffuse interstitial and/or perivascular inflammation was observed while there was no obvious myocyte damage (Figure 2A; grade 0.5 ± 0.1). In contrast with syngeneic cardiac grafts and nontransplanted naive hearts with no rejection sign (data not shown; grade 0), severe rejection with an extensive perivascular accumulation of mononuclear cells and myocyte necrosis were observed in the allogeneic cardiac grafts (B10 to CBA) (Figure 2B; grade 2.5 ± 0.7). By contrast, the tolerant cardiac grafts at 100 days after transplant (B10 to CBA with EPA) preserved resembling cardiomyocyte structure and histologic features in syngeneic transplants and naive nontransplanted heart controls (Figure 2C; grade 0.5 ± 0.1).

Changes in the miRNA expression after cardiac transplant
To investigate and confirm the generality of the miRNA expression during rejection and/or tolerance after transplant, we performed an miRNA expression analysis using murine cardiac transplant models. In a previous study, we identified 10 miRNAs that were significantly expressed during the induction of transplant tolerance using murine liver transplant models and a microarray analysis.8 However, tolerance induction was not possible without the use of immunosuppressants in this model. Therefore, in the present study, EPA was used as an immuno­suppressant for tolerance induction in the cardiac allografts as described above.

The administration of EPA to CBA mice transplanted with B10 hearts exhibited a permanent survival (Figure 1). As previously reported,12 the cardiac allografts exhibited moderate to severe rejection on day 8 after transplant as assessed according to a standard cardiac biopsy grading system. Additionally, the cardiac allografts treated with EPA on day 100 after transplant did not display any signs of rejection. In the hepatic allograft as reported previously8 in agreement with the graft pathological score and function the expression levels of mir-146a, -15b, -223, -23a, -27a, -34a, and -451 were up-regulated compared with the expression levels observed in the syngeneic grafts, whereas the expression levels of mir-101a, -101b, and -148a were down-regulated. Therefore, the 10 miRNAs that showed a difference in the expression in the hepatic allografts were examined using different types of cardiac grafts (syngeneic day 8, allogeneic day 8, and tolerance-induced allogeneic day 100). The expression patterns of mir-101a, -101b, and -148a were similar to those observed in the liver allografts as described previously,8 namely they were not correlated with the inflammatory response. In contrast to that observed in the liver allograft model the expression levels of mir-34a, -23a, -451, -146a,-15b, and -27a were not correlated with the inflammatory response (ie, they exhibited different patterns from those noted in the hepatic allografts). Only mir-223 demonstrated the same pattern as that observed in the hepatic allografts (Figure 3). These data suggest that mir-223 may be associated with rejection and mir-146a, -15b, -23a, -27a, -34a, -451, -101a, -101b, and -148a may be involved in tolerance in cardiac allografts in mice.

Using Pearson product moment correlation analysis correlation analysis, we further estimated the relations between the expression levels of the 10 miRNAs in the hepatic and cardiac allografts and found that mir-223 and mir-146 exhibited high and moderate positive associations respectively (Figure 4).


In this study using a murine cardiac allograft model, we aimed to evaluate the expression patterns of miRNA, which were significantly up- or down-regulated after murine liver allotransplant as reported previously.8 In the murine cardiac allograft model unlike hepatic allografting, no permanent acceptance of the allografts without any immuno­suppressant was achieved. Therefore, we used EPA13 to achieve permanent acceptance of the cardiac allograft. Using EPA, the cardiac allografts could overcome the rejection response by host immunity, which was maximized around day 8 after transplant as similar to that observed in hepatic allografts.8 The kinetics of the allograft rejection response by the recipients showed similarities between the cardiac and hepatic transplant models. However, we found that the miRNA expression profiles of each model differed. The expression levels of mir-223, -34a, -23a, -451, -146a, -15b and -27a were correlated with the graft rejection response in the hepatic allografts, whereas in the cardiac allografts, mir-223 was solely shown to correlate with the graft rejection response (Figure 3). In addition, Pearson product moment correlation analysis correlation analysis showed a correlation in the miRNA expression between the cardiac and hepatic allograft models for only 2 genes (mir-223 and mir-146a) (Figure 4).

Regarding the miRNA expression after cardiac allografting, the expression pattern showed a different trend compared with hepatic allografting. This result suggested that immune responses against cardiac and hepatic allografts might be different. However, we must consider that several factors that may be involved in this difference: (1) the liver contains more immune cell populations compared with the heart (eg, plasmacytoid dendritic cells liver sinusoidal endothelial cells and hepatic stellate cells14; (2) in connection with the above-mentioned factor, the generation of miRNA has tissue specificity15,16; (3) different murine strains were used; we used C57BL/10 mice as the donors and CBA/N mice as the recipients for cardiac allografting in this study, while B10.BR (H2-Kk) and B10.D2 (D2 H2-Kd) mice were used in hepatic allografting8; and (4) differences in the immune response and involvement of an immunosuppressant might exist between the 2 models. It is imperative to reject a murine cardiac allograft (B10 to CBA) by the host immune response. In contrast, all murine liver allotransplants showed alloimmune responses to the allograft by the host, whereas a liver allograft could withstand acute immune rejection and immune tolerance to the allograft was established. To mitigate the allo­immune response to cardiac allografts and match the immune response to hepatic grafts, we used EPA as an immunosuppressant; however, there is the possibility that this regimen might not standardize the 2 models.

Personalized medicine has gathered momentum in the field of organ transplantation. The advanced knowledge concerning rejection pathophysiology, as well as the practice of technologies, has led to identification of novel potential candidate biomarkers for the prediction of allograft rejection.17,18 Recently, the renal transplant field has shifted to molecular-based medicine with extending proof that molecular biological techniques employing urine analyses and biopsies were able to be of use for both the exanimation of biopsies and screening of patients who have a risk of allograft rejection in a noninvasive manner.19-21

Precise definitions of noninvasive and relevant biomarkers to detect allograft rejection are needed in the field of heart transplantation, particularly under the current clinical practice. For the patient, frequent and routine endomyocardial biopsies for determining allograft rejection is a protocol that is not without inevitability consequences.22 A key report supplied evidence that the strategy of gene expression profiling from the peripheral blood could be applied in cardiac transplant, thereby eliminating increased risks of adverse effects.23

miRNAs have recently gained attention as pertinent candidates in the field of organ transplantation due to their ability to control innumerable genes that are critical components of immune responses.24-26 Previous studies have attempted to identify the miRNAs expressed in the graft after cardiac transplant in rodent models.27,28 The murine allograft model showed that 42 miRNAs were up-regulated and 32 miRNAs were down-regulated in allogeneic cardiac grafts as compared with syngeneic cardiac grafts 7 days after grafting.28 Among 74 miRNAs observed in cardiac allografting with significantly differences mir-451 and mir15b were included and both were up-regulated in the allograft compared with the isograft in contrast with our present data. Furthermore, a mouse to rat xenograft model showed that 13 miRNAs were up-regulated and 11 miRNAs were down-regulated in xenogeneic cardiac grafts, as compared with syngeneic cardiac grafts 24 hours after grafting.27

The miRNA expression profile determined in our present study varied from the previous studies as follows: Wei and associates showed that the expression levels of mir-451 and -15b in cardiac grafts were higher in allografts than they were in isografts at 7 days after transplant, and mir-146a expression in graft-infiltrating lymphocytes also was higher in allografts than in isografts.28 Li and associates demonstrated that mir-451, -27a, and -146a were expressed higher in xenografts than in isografts at 24 hours after transplant, and the expression levels of mir-146a, -451, and -15b were higher in xenografts than they were in isografts at 40 hours after transplant. One potential explanation for the discrepancy in the results of the current study compared with these proceeding studies is the difference in the immune reaction (allografts vs xenografts) and murine strain employed (B10 to CBA vs BALB/C to B6). Additionally, Anglicheau and associates demonstrated that the expression of mir-223 was highly predictive of acute rejection and its expression levels were strongly linked to the intragraft expression of CD3 mRNA.17

Taken together, our data demonstrate that in murine cardiac allografting B10 (H2-Kb) to CBA (H2-Kk) the administration of EPA markedly prolonged the cardiac allograft survival and decreased the pathological score. Among 10 miRNAs (mir-146a, -15b, -223, -23a, -27a, -34a, -451, -101a, -101b, and -148a), mir-223 was up-regulated in accordance with pathological deterioration, as compared with the expression observed in the syngeneic grafts. In contrast, the other miRNAs were down-regulated. Pearson product moment correlation analysis correlation analysis demonstrated that mir-223 and mir-146a have high or moderate positive associations with hepatic allografts. The alteration of miRNAs in the allografts may indicate the role of miRNAs in the induction of tolerance after transplant. A better understanding of the rejection and tolerance-inducing mechanism observed in murine cardiac allografts may provide a therapeutic strategy for attenuating allograft rejection.


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Volume : 14
Issue : 4
Pages : 424 - 430
DOI : 10.6002/ect.2015.0251

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From the 1Division of Transplantation Immunology National Research Institute for Child Health and Development; the 2AIDS Research Center National Institute of Infectious Diseases Tokyo Japan; and the 3Guangdong Cardiovascular Institute Guangdong General Hospital Guangdong Academy of Medical Sciences Guangzhou China
Acknowledgements: The authors have no conflicts of interest to declare. This study was supported by research grants from the Ministry of Education Culture Sports Science and Technology of Japan (Grants-in-Aid 15K10043) and the National Center for Child Health and Development (26-6, 26-27 and 27-21). Masayuki Fujino and Ping Zhu contribute equally in this work.
Corresponding author: Xiao-Kang Li, MD, PhD, Division of Transplantation Immunology National Research Institute for Child Health and Development 2-10-1 Okura Setagaya-ku Tokyo 157-8535 Japan
Phone: +81 3 3416 0181
Jian Zhuang, MD, PhD, Guangdong Cardiovascular Institute Guangdong General Hospital Guangdong Academic of Medical Sciences 106 Zhongshan Er Road Guangzhou 510100 China
Phone: +86 20 8382 7812