Meta-Analysis of the Effect of Mesenchymal Stem Cell Transplantation on Vascular Remodeling after Carotid Balloon Injury in Animal Models

Aim A meta-analysis was conducted to assess the efficacy of mesenchymal stem cell (MSC) transplantation in small animal coronary vessels after balloon injury, to provide data for the design of future pre-clinical experiments and human clinical trials. Methods The search strategy included the PubMed, EMBASE, Chinese Biomedical Literature (CBM), and China National Knowledge Infrastructure (CKNI) databases. The endpoint was the ratio of vascular neointima/media (I/M). Moreover, neointimal area, re-endothelialization, and proliferating cell nuclear antigen (PCNA) expression were analyzed. Pooled analyses were conducted using random effects models. Heterogeneity and publication bias were also explored. All data were analyzed using RevMan 5.2 and Stata 12.0. Results Fifteen studies were reviewed from 238 retrieved animal studies. Compared with controls, MSC transplantation resulted in greater I/M reduction (pooled difference, 0.39; 95% CI, 0.57–0.21; P < 0.0001), greater neointimal area reduction (pooled difference, 0.16; 95% CI, 0.22–0.10; P < 0.0001), decreased PCNA expression (pooled difference, 17.69; 95% CI, 28.94–6.44; P = 0.002), and enhanced re-endothelialization (pooled difference, 3.37; 95% CI, 1.78–4.95; P < 0.0001). The multivariable meta-regression analysis showed that a higher number of transplanted cells (>106; P = 0.017) and later time point of I/M measurement (P = 0.022) were significantly associated with I/M reduction. Subgroup analysis demonstrated a trend for a greater reduction in the ratio of I/M with late MSC transplantation (>1 day), MSCs transplanted through intravenous injection, and atherosclerotic vessels. Conclusion The meta-analysis results demonstrate that MSC transplantation might improve injured vascular remodeling. In addition to greater efficacy with a greater number of transplanted MSCs (>106), the long-term effect of MSC transplantation appears to be more significant. The findings of this meta-analysis may help to design future, effective MSC trials.


Introduction
Percutaneous coronary intervention (PCI) is an effective treatment method for coronary heart disease. However, restenosis after PCI seriously affects the long-term prognosis. Post-angioplasty restenosis is caused largely by smooth muscle cell proliferation and neointimal proliferation, as well as elastic recoil [1]. A number of studies have suggested that inward vascular neointimal remodeling is the main cause of restenosis [2]. There is great potential for stem cells to regenerate damaged tissues in cardiovascular diseases [3]; mesenchymal stem cells (MSCs) could differentiate into functional cell types that are able to repair the diseased or injured tissue in which they are localized [4]. Despite an increase in the number of animal experiments studying the effects of MSCs on the repair of vascular injury, there is variation in not only experimental design but also the results. Animal experiments provide relevant information for clinical practice, while pre-clinical studies have to anticipate if the new therapy is feasible and effective. There are still a number of unknowns regarding MSC therapy in clinical practice, including the effect of MSCs on vascular remodeling after carotid balloon injury, effective number of MSCs, appropriate route, and appropriate timing of cell delivery.
The present study includes a meta-analysis and sub-analysis of data from published animal studies investigating the effect of MSC treatment, to assess the effects of MSC transplantation after carotid balloon injury.

Eligibility criteria
Two reviewers (XXJ and LJP) independently judged the eligibility of the studies. Eligible studies were randomized controlled trials (RCTs) of carotid balloon injury animal models. Additional requirements included MSC transplantation as the only intervention in the experimental group(s) and comparison with a placebo group. The studies were also required to investigate parameters for vascular remodeling (ratio of vascular neointima/media [I/M], neointimal area, re-endothelialization, and positive expression of proliferating cell nuclear antigen [PCNA]) as final outcomes. Reviews, comments, and editorials were excluded.

Search strategy
We searched the electronic databases PubMed, EMBASE, Chinese Biomedical Literature (CBM), and China National Knowledge Infrastructure (CKNI) (last search was updated on 5 April 2014) using the following key words and search terms: (mesenchymal stem cell OR mesenchymal stromal cell OR bone marrow stromal cells OR bone marrow mesenchymal stem cells OR mesenchymal progenitor cell) AND (endothelium OR vessel OR vascular) AND (carotid balloon injury OR balloon OR angioplasty).

Data extraction
Two reviewers (XXJ and LJP) independently screened full-text articles. The following information was extracted from the complete manuscripts of each qualified study: basal characteristics, I/M, neointimal area, re-endothelialization, positive expression of PCNA, time point of I/M measurement, and timing of cell therapy after injury. If necessary, data were estimated from figures of the included studies [5]. The I/M data were all expressed as a fraction of 1 (i.e., data presented as percentage values were converted to a fraction).

Data analysis
The mean I/M ratios were different between the experimental and control groups. The heterogeneity tests resulted in significant heterogeneity (P < 0.1); therefore, a random-effect model was applied [6]. Multivariable meta-regression analysis was performed to find the source of the significant heterogeneity. Continuous variables were estimated as weighted mean differences with 95% confidence intervals (CIs) between the MSC-treated animals and control animals. In the case of multiple experimental groups compared with one control group within one study, the number of animals in the control group was divided equally by the number of experimental groups [5,7]. P-values were reported by statistical hypothesis testing at the two-sided 0.05 level.
From a clinical viewpoint, multivariate meta-analyses were performed for animal model (rabbits or mice), diet (cholesterol or normal), number of MSCs injected (10 6 or >10 6 ), the route of cell delivery (intravenous or carotid artery local transplantation), timing of cell therapy after injury (<1 day or 1 day), and time point of I/M measurement after MSC therapy (<4 weeks or 4 weeks) to find the factors associated with a reduction in I/M. For further insight, subgroup analyses were performed with the following factors: diet, number of cells injected (10 5 , 10 5 -10 6 , 10 6 -10 7 , or >10 7 ), the route of cell delivery, timing of cell therapy after injury, and time point of I/M measurement after MSC therapy.
The mean neointimal area, re-endothelialization, and positive expression of PCNA values were also different between the experimental and control groups. The heterogeneity tests resulted in significant heterogeneity (P < 0.1); therefore, a random-effect model was applied. The degree of re-endothelialization was assessed using 3 methods: the expression of CD31, vascular endothelial growth factor (VEGF), and ratio of the re-endothelialized area (defined as the area not stained with Evans blue/total injured surface area).
Publication bias was explored by funnel plot. All analyses were performed with Review Manager Version 5.2 (The Nordic Cochrane Centre, The Cochrane Collaboration, 2012) and Stata 12.0.

Study characteristics
The search strategy identified 281 articles (Fig. 1). Of the 238 animal studies, 14 were eligible for review (Table 1). Mice (7 studies) and rabbits (7 studies) were used as animal models. The studies investigated the effect of MSC on injured atherosclerotic vessels (6 studies) or injured normal vessels (8 studies). The atherosclerotic vessel models resulted primarily from animals fed a cholesterol diet, while the normal vessel models resulted primarily from animals fed a normal diet. MSCs were injected either intravenously (n = 4) or near the injured carotid artery (n = 10). The MSC doses varied, as summarized by 2 categories: low dose (10 6 ) in 4 studies and high dose (>10 6 ) in 9 studies. Timing between vessel injury and cell transplantation was <1 day (12 studies) or 1 day (2 studies). After cell transplantation, the I/M level was measured at <4 weeks (4 studies) or 4 weeks (12 studies).

Meta-analyses
The I/M data are presented as continuous variables, using mean and standard deviation. Pooled analysis showed that the I/M level was 0.39 lower in the experimental group compared with the control group after cell therapy (95% CI, 0.57-0.21; Z = 4.25; P < 0.0001) with significant heterogeneity (P < 0.001; I Square, 98%; Fig. 2).
Multivariable meta-regression analysis showed that the number of MSCs transplanted (P = 0.017) and time point of I/M measurement after cell therapy (P = 0.022) were significantly associated with a reduction in I/M.    The mean neointimal area and positive expression of PCNA are presented as continuous variables, using mean and standard deviation. Pooled analysis showed that the neointimal area was 0.16 lower in the MSC group compared with the control group (95% CI, 0.22-0.10; Z = 5.24; P < 0.0001; Fig. 4). The positive expression of PCNA was 17.69 lower in the MSC group compared with the control group (95% CI, 28.94-6.44; Z = 3.08; P = 0.002; Fig. 5). Fig. 6 shows the degree of re-endothelialization with each of the 3 assessment methods. Pooled analysis showed that MSC engraftment could enhance vascular re-endothelialization after carotid balloon injury (pooled difference, 3.37; 95% CI, 1.78-4.95; Z = 4.16; P < 0.0001).

Sensitivity analysis
The number of cells transplanted (P = 0.017) and time point of I/M measurement after MSC therapy (P = 0.022) were significant factors for I/M reduction. A trend was observed (Fig. 3) for greater improvement with MSC engraftment with a higher number of cells transplanted (>10 6 ) and later engraftment (1 day). Local MSC engraftment, around the carotid artery, showed less effect than intravenous engraftment. Animals fed with cholesterol had greater reductions in I/M than animals fed a normal diet. During the follow-up, the late effect of MSC transplantation appeared to be more significant. No trend in I/M differences was observed based on animal type (P = 0.914). The funnel plot for I/M suggests a lack of publication bias, as values were evenly distributed around the overall estimate (Fig. 7).

Discussion
The present meta-analysis revealed that MSC engraftment reduces vascular remodeling after coronary vessel balloon injury, evidenced by lower/smaller I/M, neointimal area, and positive expression of PCNA in addition to enhanced re-endothelialization after MSC transplantation. In addition, the meta-analysis revealed that that the number of transplanted MSCs and time point of I/M measurement were significant factors for vascular remodeling. Furthermore, the subgroup analysis indicated that late therapy (>1 day), the atherosclerotic vascular model, and intravenously injected MSCs were beneficial for I/M reduction. These results were not affected by the type of animal studied.
A number of studies have indicated that MSCs are candidates for cell therapy in coronary heart disease, including myocardial infarction (MI) [32] and ischemic heart disease [33]. With regards to MI, MSCs might improve angiogenesis after MI, reduce MI size, and improve heart function, as demonstrated through animal studies [34] and clinical use [35][36][37]. As a result, studies have also investigated the effect of MSCs on restenosis after PCI; however, the results have been inconclusive, and MSC use has not been applied clinically.
The present meta-analysis, which analyzed the I/M, neointimal area, positive expression of PCNA, and degree of re-endothelialization as parameters indicating vascular remodeling after carotid balloon injury, showed that MSC transplantation could improve vascular remodeling and prevent neointimal formation. Furthermore, the I/M results suggested that vascular remodeling is significantly influenced by the number of MSCs transplanted, with a higher number of cells (>10 6 ) resulting in a greater reduction in I/M. This result supports those of other meta-analyses regarding the effect of MSC transplantation on other clinical diseases [5,7]. Collectively, these findings indicate that future studies of MSC therapy for restenosis should focus on transplantation of a higher number of MSCs (>10 6 ).
I/M reduction was also influenced by the time point of measurement; a greater reduction in I/M was measured at later times (4 weeks) after MSC injection. It may be likely that the reduction in intimal remodeling with MSC therapy is only evident over time. However, the results of MSC therapy for ischemic heart disease in pre-clinical studies do not agree with the present findings [5]. The follow-up duration in these studies may not have been long enough; the majority of the studies followed the patients up for no more than 4 weeks, while only one study measured I/M at 5 weeks [10]. Therefore, the long-term effect of MSC therapy on intimal remodeling was previously unknown. Future pre-clinical studies should study the effectiveness of MSC treatment over a longer period.
The subgroup analysis indicated that a greater benefit in intimal remodeling is realized with MSC injections administered 1 day after vascular injury. In the acute setting (<24 h), cellular retention and survival are likely influenced by the local hostile microenvironment. Furthermore, MSC therapy tended to be more effective with injured atherosclerotic vessels than with injured normal vessels. Meanwhile, the effectiveness of MSC transplantation was similar between intravenous and carotid artery delivery routes as well as between animal types. Further research is required to determine if there is a best administration route, while the efficacy of MSC may be the same regardless of species (animal or human).
Meta-analyses of animal studies are not common, yet they are recommended in many settings and can often guide research, even clinical endeavors. Performing meta-analyses of animal studies may be a useful way to evaluate the effects of therapies for designing future animal experiment and pre-clinical trials. Our meta-analysis is the first systematic review and metaanalysis of animal models to evaluate the effects of MSC therapy in injured vessels, demonstrating that MSC therapy can lead to improvements in vascular remolding. Moreover, we analyzed the factors influencing vascular remodeling after balloon injury, which could form the basis for future large animal experiments and even human clinical trials. To date, trials for MSC therapy have been performed primarily in small animals. In our opinion, more animal experiments of different species, especially nonhuman primate trials, should be performed in order to assess the efficacy and safety of MSC therapy before human clinical trials.

Limitations
A limitation of the present study was the presence of obvious heterogeneity. Furthermore, the analysis was based on study outcomes; we did not have access to individual data. However, the risk of erroneous estimates was minimized through the use of random-effect analysis. The observed outcomes were influenced by many factors, such as the diversity in animal type, vascular conditions, cell delivery method, number of cells, time of injection after balloon injury, and follow-up after cell therapy. We performed multivariate analysis, as an exploratory tool, that showed the number of cells and follow-up duration after cell therapy could influence the metaanalysis results. Despite these limitations, our results are highly relevant for future clinical trials.