Short- and Long-term Therapeutic Efficacies of Intravenous Transplantation of Bone Marrow Stem Cells on Cardiac Function in Rats with Acute Myocardial Infarction： A Meta-analysis of Randomized Controlled Trials△

Can Jiang， Dong Zheng， Yun-lu Feng， Jun Guo*，Hai-rui Li， and Ai-dong Zhang

Department of Cardiology， the First Affiliated Hospital of Jinan University， Guangzhou 510630， China

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Short- and Long-term Therapeutic Efficacies of Intravenous Transplantation of Bone Marrow Stem Cells on Cardiac Function in Rats with Acute Myocardial Infarction： A Meta-analysis of Randomized Controlled Trials△

Can Jiang?， Dong Zheng?， Yun-lu Feng?， Jun Guo*，Hai-rui Li， and Ai-dong Zhang

Department of Cardiology， the First Affiliated Hospital of Jinan University， Guangzhou 510630， China

mesenchymal stem cells； rats； myocardial infarction； intravenous

Objective To investigate the short- and long-term therapeutic efficacies of intravenous transplantation of bone marrow stem cells （MSCs） in rats with experimental myocardial infarction by metaanalysis.

Methods Randomized controlled trials were systematically searched from PubMed， Science Citation Index （SCI）， Chinese journal full-text database （CJFD） up to December 2014. While the experimental groups（MSCs groups） were injected MSCs intravenously， the control groups were injected Delubecco’s minimum essential medium （DMEM） or phosphate buffered saline （PBS）. Subgroup analysis for each outcome measure was performed for the observing time point after the transplantation of MSCs. Weighted mean differences（WMD） and 95% confidence intervals （CI） were calculated for outcome parameters including ejection fraction （EF） and fractional shortening （FS）， which were measured by echocardiogram after intravenous injection and analyzed by RevMan 5.2 and STATA 12.0.

Results Data from 9 studies （190 rats） were included in the meta-analysis. As compared to the control groups， the cardiac function of the experimental groups were not improved at day 7 （EF： WMD=0.08，95%CI -1.32 to 1.16， P＞0.01； FS： WMD=-0.12， 95%CI -0.90 to 0.65， P＞0.01） until at day 14 after MSCs’transplantation （EF： WMD=10.79， 95%CI 9.16 to 12.42， P＜0.01； FS： WMD=11.34， 95%CI 10.44 to 12.23，P＜0.01）， and it lasted 4 weeks or more after transplantation of MSCs （EF： WMD=13.94， 95%CI 12.24 to 15.64， P＜0.01； FS： WMD=9.64， 95%CI 7.98 to 11.31， P＜0.01）.

Conclusion The therapeutic efficacies of MSCs in rats with myocardid infarction become increasing apparent as time advances since 2 weeks after injection.

Chin Med Sci J 2016； 31（3）：142-148

W ORLDWIDE， acute myocardial infarction （MI）is a kind of refractory and deadly disease and most will develop into congestive heart failure. It remains a major health problem，whose morbidity and mortality is still increasing despite guideline conforming pharmacological treatment and revascularization. Recently numerous experimental and clinical studies have suggested that the cell-based therapy with bone marrow stem cells （MSCs） enhanced and improved cardiac function post MI.1-6Mesenchymal stem cells is a type of non-hematopoietic， adult stem-like cells that can be easily isolated from the bone marrow， readily cultured and practically expanded in v itro. Some studies have found that MSCs could transdifferentiate into cardiomyocytes， significantly induce promoting activity of angiogenesis and myogenesis， promote neo-vascularization in infarcted hearts post MI，3，5，7while others suggested that MSCs preserved cardiac systolic and diastolic performances，improved the wall thickness of left ventricular （LV） and increased capillary density by paracrine.6，8-10The specific mechanisms how MSCs enhance the cardiac function are still unclear， but it behoves us to continue to investigate a bright future for patients with acute MI.

Ejection fraction （EF） and fractional shortening （FS）calculated via LV end-diastolic （LVEDd） and end-systolic dimensions （LVESd） are used to estimate cardiac function and analyze the therapeutic efficacies of MSCs on cardiac function， LV EF and FS were calculated as follows： EF=［（LVEDd3-LVESd3）/LVEDd3］×100； FS=（LVEDd－LVESd）/ LVEDd×100. The normal mean value of EF is almost 8411and the normal mean value of FS is about 35.12The less scores the rats get， the worse functions they have.

This systematic review with meta-analysis in MI models by comparing the experimental groups and control groups is expected to offer an academic support for curing MI.

MATERIALS AND METHODS

Search strategy

By searching pertinent studies in PubMed， Science Citation Index （SCI）， Chinese journal full-text database （CJFD） up to December 2014 we collected papers published with the keywords： “mesenchymal stem cells”， “mesenchymal stromal cells”， “rats”， “myocardial infarction”， “intravenous”， “intravenously”. The languages used were not restricted.

Inclusion criteria

The studies were included if they fulfilled the following criteria： （1） randomized controlled animal trials； （2）intravenous administration； （3） the parameters included EF or FS； （4） containing at least two groups： with and without intravenous transplantation of MSCs； （5） except for being injected with MSCs， the control groups got the same experimental treatment of the experimental groups；（6） acute MI models were established with rats； （7） cells used injection were extracted from rat bone marrow.

Exclusion criteria

The articles were excluded if these studies met one of the following exclusive criteria： （1） unable to get the full text；（2） the author is same with another study； （3） combined with other interventions； （4） review.

Data extraction

Two evaluators independently selected and extracted data which were collected by reading the headlines， abstracts and full texts according to the inclusion and exclusion criteria， and then reviewed those studies again before statistical analysis. We explored potential resources from the following aspects： first author's name； year of publication； the weight of rat； the number of injected cells；the route of administration； EF； FS； the observed time after MI. Disagreements were resolved by consensus.

Assessment of methodology quality

The quality of the included studies was assessed from the perspective of Cochrane Handbook for Systematic Reviews of Interventions version 5.2. There are 7 items： （1） random sequence generation； （2） allocation concealment； （3）blinding of participants and personnel； （4） blinding of outcome assessment； （5） incomplete outcome data； （6）selective reporting； （7） other bias. Every study was evaluated by 2 independent researchers， and the judgment of every item was at a low unclear or high risk. Disagreements were resolved by consensus.

Statistical analysis

After organizing and establishing a database， we chose theRevMan software package （version 5.2； the Cochrane collaboration） and STATA 12.0 software to conduct the meta-analysis. For continuous variables， we reported the results estimated as weighted mean difference （WMD）with the 95% confidence intervals （95%CI），13and statistical significance was set at the level that P was lower than 0.01， namely P＜0.01. The heterogeneity among those studies was evaluated by χ2-based Q-test（or Cochran's Q-statistic）， and I2was calculated， with suggested thresholds being low （I2： 25% to 49%），moderate （I2： 50% to 74%） or high （I2≥75%）. The fixed-effects model was employed with the absence of heterogeneity， otherwise the random-effects model was employed. Moreover the STATA 12.0 software was also used to perform the Begg's test and Egger's test to detect potential publication bias.

RESULTS

Description of studies

The detailed search procedures are summarized in Fig. 1. Eventually 9 studies met the inclusive criteria by reading the titles， abstracts and the full texts carefully.7，13-20Ma et al14clarified that therapeutic efficacies of MSCs was not same at different injection time point， the reasons why to choose the first injection time point （12 hours after MI） in the meta-analysis are as follows： （1） It has obviously been proved that MSCs was an appropriate candidate for cardiac generation therapies at other injection time points； （2） The heterogeneity of this meta-analysis about FS was apparently lower when we chose the first injection time point，comparing to the other injection time points. While MSCs was intravenously injected to vein of rats after being ligated the left anterior descending artery in the experimental groups， the control groups were intravenously injected Delubecco's minimum essential medium （DMEM） or phosphate buffered saline （PBS）. Then， we recorded EF and FS at 1， 2， 4 weeks respectively.

Assessment of risk of bias

A summary of methodological domain assessments for each study were detailed in our studies. Blinding of outcome assessments of 4 studies was unclear. All in all， the risk of bias was considered at a low level （Fig. 2）.

Figure 1. Flow chart of study selection. Articles were selected according to inclusion and exclusion criteria defined in the materials and methods section.

Figure 2. Risk of bias summary of the randomized controlled trials included in the meta-analysis. A review of the author's judgments about each risk of bias item for each included study. + is “l(fā)ow risk”，- is “high risk”， ？ is “unclear”.

EF and FS at least four weeks after transplantation of MSCs or culture medium

Both EF and FS were significantly increased in the MSCs groups than those of the control groups （EF： WMD = 13.94，95%CI 12.24 to 15.64， P＜0.01； FS： WMD=9.64， 95%CI 7.98 to 11.31， P＜0.01）. The heterogeneity was high （EF：I2=41%， FS： I2=72%）. （Fig. 3）

EF and FS at two weeks after trans plantation of MSCs or culture medium

Both EF and FS were significantly increased in the MSCs groups than those of the control groups （EF： WMD=10.79，95%CI 9.16 to 12.42， P＜0.01； FS： WMD=11.34， 95%CI 10.44 to 12.23， P＜0.01）. No heterogeneity was found about EF （I2=0）， on the contrary the heterogeneity about FS was high （I2=89%）. （Fig. 4）

EF and FS at one week a fter the tran splantation of MSCs or culture medium

There was no significance between the MSCs groups and control ones （EF： WMD=-0.08； 95%CI -1.32 to 1.16， P＞0.01； FS： WMD=-0.12， 95%CI -0.90 to 0.65，P＞0.01）， and no heterogeneity was found between the subgroups （EF： I2 =0， FS： I2=0）. （Fig. 4）

Results of sensitivity analysis and publication bias

Sensitivity analysis was performed to assess the stability of these results and demonstrated that the remaining studies yielded consistent results every time omitting any one study no significant changes existed across the studies （EF：between WMD=13.08， 95%CI： 12.00-15.06 and WMD= 14.83， 95%CI： 13.27-16.40； FS： between WMD=9.08，95%CI： 7.51-10.64 and WMD=10.20， 95%CI： 8.72-11.68，F(xiàn)ig. 5）. However， deletion of the Zhang et al's16study reduced the heterogeneity from moderate to low levels （EF：I2=0， FS： I2=45%）.

And no publication bias was found in a funnel plot， with plots visually symmetrically distributed along the vertical axis （Fig. 6）. Begg's test and Egger's test also showed no significant publication bias （EF： Begg's test， z=0.56，P=0.57； Egger's test， t=2.00， P=0.12； FS： Begg's test，z=0.15， P=0.88； Egger's test， t=1.50， P=0.20）.

Figure 3. Forest plot of EF or FS at least 4 weeks after the transplantation of MSCs. Each square denotes that WMD for that trial， and the horizontal lines showing the 95%CI. The size of the square is proportional to the amount of information contributed by the trial. The diamond gives the pooled WMD from the random effect model； the centre of this diamond denotes the WMD and the extremities of the 95%CI. EF： ejection fraction； FS： fractional shortening； MSCs： marrow stem cells； SD： standard deviation； IV： inverse variance；WMD： weighted mean differences； 95% CI： 95% confidence intervals.

Figure 4. Forest plot of EF or FS at l or 2 weeks after the transplantation of MSCs.

Figure 5. A： The sensitivity analysis of the included studies for the EF （A） and the FS （B）. The middle vertical line indicates the combined WMD， and the two vertical lines represent the corresponding 95%CI values. The middle small circle and two ends of the dotted lines indicate the pooled WMD and 95%CI values， when the study on the left was omitted respectively after each round of analysis.

DISCUSSION

A variety of cell types have been proposed as useful candidates to the cell-based therapies for MI including embryonic stem cells， skeleton myoblasts， fetal cardiomyocytes.21-25MSCs have great therapeutic potentials because of their regeneration， transdifferentiation capacity following multiple mesenchymal lineages and paracrine mechanism.9，26-28In addition， Lalu et al29found that no association between MSCs and tumour formation with a systematic review and meta-analysis. Therefore， MSCs is a kind of ideal resourcefor stem cell-based therapies to MI， although the special mechanisms remain a matter of ongoing debate.

Figure 6. Funnel plot of publication bias. SE： standard error； MD： mean differences.

Myocardial injection to the ischemic area or periinfarcted regions is conducted by most researchers， but maybe it is impossible to be practiced in clinic works with secondary damage. While intravenous injection is a safe and convenient route to the clinical works compared to MI. Wang et al30found that intravenous administration of MSCs was safe and did not cause deterioration of lung function，while the majority of intravenous infused cells were harbored in the lung. In this meta-analysis we found that EF and FS were significantly increased in the experimental groups as time progresses， it means that cardiac function of MI models is improved obviously after intravenous transplantation of MSCs， but improving cardiac function visibly may need at least 2 weeks after intravenous injection of MSCs. Additionally the therapeutic efficacies of MSCs on cardiac function is stable from second week to fourth week after intravenous transplantation， which indicates that it is significant about the long-term effect of MSCs on cardiac function after MI. Based on this meta-analysis it demonstrates that the therapeutic efficacies of MSCs on cardiac function after MI are time-dependent and MSCs is an appealing option in the long-term management of MI.

Although the sensitivity analysis of this study about EF and FS do not alter the results for other outcomes， it also has the different versions. The differences of the number of injected cells may be the main source of heterogeneity. Of course， after left coronary artery was ligated the ligation position and the injection time may also contribute to the heterogeneity. All in all， the heterogeneity is acceptable. By omitting Zhang et al's article，16low heterogeneity at four weeks after injection was observed among the studies（EF： I2=0； FS： I2=45%） ， but the changes of EF and FS at four weeks after injection had not significant difference from others （P＜0.01）. After observing and discussing about article of Zhang et al，16we found that FS in the control groups （WMD=33.5） was obviously different from others （WMD＜20）. Therefore， whether the basic cardiac function before treatment affects the long-term therapeutic efficacies of MSCs on cardiac function after intravenous transplantation of MSCs deserves to be further researched. Overall， MSCs treatment is effective to enhance cardiac function.

Our systematic review has several limitations. First， it is always possible that some eligible studies may have not been identified through our searches. We used comprehensive search methods to get as many eligible studies as possible and conducted careful study to avoid excluding any eligible studies. Second， the samples of some articles may be not enough. In addition， as articles only in Chinese and English were included， the review unavoidably suffers from language bias.

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for publication November 9， 2015.

?These authors contributed equally to this work.

Tel： 86-20-38688620， Fax： 86-20-38688000， E-mail： dr.guojun@163.com

△Supported by the Youth Project of National Natural Science Foundation （81100078） ， the Key Project of Chinese Ministry of Education （211207），Guangzhou Pearl River science and technology new star pr oject plan （2012J2200063） and Project of Guangdong Science and Technology Department （S2011040001392）.