- Open Access
- Open Peer Review
Efficacy and safety of regenerative cell therapy for pulmonary arterial hypertension in animal models: a preclinical systematic review protocol
© Suen et al. 2016
- Received: 8 March 2016
- Accepted: 10 May 2016
- Published: 25 May 2016
Pulmonary arterial hypertension (PAH) is a rare disease (15 cases per million) that is characterized by widespread loss of the pulmonary microcirculation and elevated pulmonary vascular resistance leading to pathological right ventricular remodeling and ultimately right heart failure. Regenerative cell therapies (i.e., therapies involving cells with stem or progenitor-like properties) could potentially restore the effective lung microcirculation and provide a curative therapy for PAH. Preclinical evidence suggests that regenerative cell therapy using endothelial progenitor cells or mesenchymal stem cells may be beneficial in the treatment of PAH. These findings have led to the completion of a small number of human clinical trials, albeit with modest effect compared to animal studies. The objective of this systematic review is to compare the efficacy and safety of regenerative cell therapies in preclinical models of PAH as well as assess study quality to inform future clinical studies.
We will include preclinical studies of PAH in which a regenerative cell type was administered and outcomes compared to a disease control. The primary outcome will be pulmonary hemodynamics as assessed by measurement of right ventricular systolic pressure and/or mean pulmonary arterial pressure. Secondary outcomes will include mortality, survival, right ventricular remodeling, pulmonary vascular resistance, cardiac output, cardiac index, pulmonary acceleration time, tricuspid annular systolic excursion, and right ventricular wall thickness. Electronic searches of MEDLINE and EMBASE databases will be constructed and reviewed by the Peer Review of Electronic Search Strategies (PRESS) process. Search results will be screened independently in duplicate. Data from eligible studies will be extracted, pooled, and analyzed using random effects models. Risk of bias will be assessed using the SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE) risk of bias tool, and individual study reporting will be assessed according to an itemized checklist based on the Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines.
This systematic review will examine the efficacy and safety of regenerative cell therapy in preclinical models of PAH. As well, the literature will be assessed for study quality and risk of bias. The results will guide the design of future clinical trials and preclinical animal studies.
Systematic review registration
- Pulmonary arterial hypertension
- Systematic review
- Risk of bias
- Stem cells
- Cell therapy
- Endothelial progenitor cells
- Mesenchymal stem cells
- Animal models
Clinical classification of WHO group 1 pulmonary hypertension
1. Pulmonary arterial hypertension (PAH)
1.1 Idiopathic PAH
1.2 Heritable PAH
1.2.2 ALK-1, ENG, SMAD9, CAV1, KCNK3
1.3 Drug- and toxin-induced
1.4 Associated with:
1.4.1 Connective tissue disease
1.4.2 HIV infection
1.4.3 Portal hypertension
1.4.4 Congenital heart disease
1′ Pulmonary veno-occlusive disease and/or pulmonary capillary hemangiomatosis
1″ Persistent pulmonary hypertension of the newborn (PPHN)
The current standard of care PAH-specific therapies consists largely of pharmacological vasodilator agents, such as phosphodiesterase-5 inhibitors, prostacyclin analogs, and endothelin antagonists. These have only modest effects on pulmonary hemodynamics, and prognosis remains poor despite introduction of a number of new therapies in the last 5 years . The most recent estimate of 5-year survival of newly diagnosed PAH is 61.2 % . Thus, the development of clinically effective strategies to restore normal pulmonary structure and function in established PAH are needed.
Recent understanding of the role of adult stem and progenitor cells in the maintenance of vascular homeostasis and repair of injury has stimulated interest in the potential for regenerative cell therapies for PAH. Most of the preclinical studies of cell therapy for PAH have used two cell types in particular, early-outgrowth endothelial progenitor cells (EPCs, also known as circulating angiogenic cells, myeloid angiogenic cells) and mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells, adult stem cells) [8, 9]. EPCs and MSCs have been described as dynamic and responsive cells that can migrate to sites of vascular injury in several in vivo animal disease models , facilitating neovascularization and reducing inflammation . As a treatment for PAH, preclinical studies involving these cell types have demonstrated efficacy in improving key pathological features of PAH such as cardiopulmonary hemodynamics, restoring the degenerated microvascular area, and reducing both right ventricular and pulmonary vascular remodeling . A small number of clinical studies involving stem/progenitor cell therapy on PAH patients have been completed or are underway and show some promise in controlling the extent of the disease [3, 11]. However, to date, there has been no systematic synthesis of preclinical studies investigating stem cells therapy for the treatment of PAH.
In order to address this knowledge gap, we will conduct a preclinical systematic review of regenerative cell therapy for PAH. In contrast to narrative preclinical reviews of stem cells for PAH treatment, this systematic review will provide additional key evaluations. The meta-analysis will provide an estimation of the cumulative overall effect of stem cell treatment on pulmonary hemodynamics based on currently available data in preclinical studies. This systematic review will also attempt to evaluate the quality of currently available evidence based on risk of bias assessment and completeness of reporting, and the potential for publication bias. This data may impact the design of current preclinical testing of stem cell therapies and potentially influence the design of future clinical trials.
In preclinical studies of pulmonary arterial hypertension, do PAH animals receiving regenerative cell therapy exhibit improved pulmonary hemodynamics compared to PAH animals not receiving regenerative cell therapy and is regenerative cell therapy safe?
Protocol and registration
The protocol was developed by a research team of clinical (DS, AZ) and preclinical research scientists (CS, ML), experts in knowledge synthesis and translation (DF, LM). The protocol will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) checklist (Additional file 1: PRISMA checklist). The protocol is registered through the Collaborative Approach to Meta Analyses and Review of Animal Data from Experimental Studies (CAMARADES) website (http://www.camarades.info).
Types of studies
This review will include controlled studies (randomized, pseudo-randomized and non-randomized) that evaluate the efficacy or safety of regenerative cell therapy for PAH.
Types of preclinical animal models
We will include preclinical in vivo models of PAH that reproduce features of the pathophysiology associated and/or etiology of human PAH . Specifically, the eligible animal models are the rodent monocrotaline (MCT), chronic hypoxia (CH), and SU5416 + chronic hypoxia (SU+CH) models. These are recognized as the most representative and most widely utilized methods of induction for modeling human PAH . These models do not require invasive surgery or extensive manipulation that could be subject to technical variability and therefore provide a predictable disease phenotype. These models share in common the characteristic pathological features of human PAH: endothelial dysfunction, SMC proliferation, inflammation, and vascular narrowing and rarefaction resulting in pulmonary hypertension and right ventricular remodeling; however, only the SU+CH model reproduces the complex obliterative vascular lesions that are typical of human PAH . Mouse models will not be considered for this systematic review as currently available models (chronic hypoxia) lack significantly elevated pulmonary pressures, right ventricular hypertrophy, and pulmonary arteriolar remodeling .
Animal models of PH secondary to other causes such as left heart disease, lung disease, or thromboembolism (WHO Groups 3-5)  will not be included. Genetically modified animals and will also be excluded. Since the purpose of this study to inform future decisions for designing clinical trials for adult populations, this study will exclude neonatal animal models of pulmonary hypertension.
Types of interventions
The intervention group will include animals receiving any regenerative cell therapy (xenogeneic, syngeneic, or allogeneic cells from any tissue source). Experiments involving pretreatment of cells, co-treatment, and/or genetic manipulation (e.g., engineered to over- or under-express certain genes) will be classified as “cell modifications” for subgroup analysis. Studies using non-stem/progenitor cells (i.e., terminally differentiated cells such as mature endothelial cells, smooth muscle cells, or fibroblasts) as the therapeutic intervention will be excluded. Studies that do not include the administration of viable cells, for example, studies with only cell-free products derived from stem/progenitor cells such as conditioned media, will also be excluded.
Types of control comparisons
The preclinical comparison group will include animals from studies that have had experimentally induced PAH but have not been administered a regenerative cell (vehicle control, control cell type, or no treatment).
Timing of outcome measurements
Outcomes will be assessed at least 1 week after intervention to exclude the possibility of acute effects of cell administration.
Preclinical primary endpoints
Quantitative measures of severity of pulmonary hypertension
Surrogate index for
Type of variable
Right ventricular systolic pressure (RVSP)
Mean pulmonary arterial pressure (mPAP)
Cardiac output, cardiac index
Pulmonary vascular resistance (PVR)
Post-mortem tissue collection
Morphometric RV hypertrophy (RV/LV+S)
Pulmonary artery acceleration time (PAT)
RV free wall thickness
Tricuspid annular plane systolic excursion (TAPSE)
Ratio of deaths/total participants at experimental endpoint
Survival time relative to induction
Preclinical secondary endpoints
We will collect data on all deaths and animal mortality. Right ventricular (RV) remodeling is a forerunner of right heart failure, which is characterized by decreased function and dilatation of the RV and strongly correlates with prognosis and survival in PH patients . We will collect morphometric data on right ventricular remodeling expressed as the weight ratio of right ventricle/left ventricle + septum. Other measures of cardiac function and hemodynamics will also be collected to assess functional performance of the heart such as cardiac output and cardiac index (cardiac output/body weight) and pulmonary vascular resistance.
We will collect data from other noninvasive measures obtained by echocardiography to evaluate cardiac structure and pulmonary hemodynamics (Table 2). Mortality and survival will also be collected to evaluate the safety of the intervention.
Electronic search methods for study identification
In consultation with the review team, electronic search strategies will be developed for each database by an experienced medical information specialist. Ovid MEDLINE®, Ovid MEDLINE® In-Process & Other Non-Indexed Citations, and EMBASE Classic + EMBASE will be searched. The strategy appended was used to search in MEDLINE (Additional file 2). The search strategy will be validated using the Peer Review of Electronic Search Strategies (PRESS) template by another information specialist .
Search strategies will use a combination of controlled vocabulary (for example stem cells, pulmonary hypertension) and keywords (for example, EPC, MSC, iPSC, HSC, PAH), and parsing will be formatted accordingly to each database. We will use modified animal filters from previously published methods [16, 17] validated for PubMed/MEDLINE and EMBASE. There will be no date restrictions on any of the searches. In addition, a manual review of the bibliographies of selected articles and relevant reviews will be performed. Only articles in the English language will be included in the review.
Titles and abstracts of search results will be screened independently by two individuals. Full text of all potentially eligible studies will be reviewed based on our eligibility criteria. Disagreements between reviewers will be resolved by consensus or by a third member of the systematic review team (DJS). Reasons for exclusion of potentially eligible studies will be recorded in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines developed for proper reporting of clinical systematic reviews .
Data collection and process and data items
Data will be extracted independently by two individuals into standardized, electronic pilot-tested forms using DistillerSR software (https://distillercer.com). Specific data elements collected for this review are listed in Table 2.
Assessment of risk of bias
SYRCLE risk of bias (RoB) tool
Type of bias
Description of domain
Review authors’ judgment
Describe the methods used, if any, to generate the allocation sequence in sufficient detail to allow an assessment whether it should produce comparable groups.
Was the allocation sequence adequately generated and applied?a
Describe all the possible prognostic factors or animal characteristics, if any, that are compared in order to judge whether or not intervention and control groups were similar at the start of the experiment.
Were the groups similar at baseline or were they adjusted for confounders in the analysis?
Describe the method used to conceal the allocation sequence in sufficient detail to determine whether intervention allocations could have been foreseen before or during enrolment.
Was the allocation adequately concealed?a
Describe all measures used, if any, to house the animals randomly within the animal room.
Were the animals randomly housed during the experiment?
Describe all measures used, if any, to blind trial caregivers and researchers from knowing which intervention each animal received. Provide any information relating to whether the intended blinding was effective.
Were the caregivers and/or investigators blinded from knowledge which intervention each animal received during the experiment?
Random outcome assessment
Describe whether or not animals were selected at random for outcome assessment, and which methods to select the animals, if any, were used.
Were animals selected at random for outcome assessment?
Describe all measures used, if any, to blind outcome assessors from knowing which intervention each animal received. Provide any information relating to whether the intended blinding was effective.
Was the outcome assessor blinded?
Incomplete outcome data
Describe the completeness of outcome data for each main outcome, including attrition and exclusions from the analysis. State whether attrition and exclusions were reported, the numbers in each intervention group (compared with total randomized animals), reasons for attrition or exclusions, and any re-inclusions in analyses for the review.
Were incomplete outcome data adequately addressed?a
Selective outcome reporting
State how selective outcome reporting was examined and what was found.
Are reports of the study free of selective outcome reporting?a
Other sources of bias
State any important concerns about bias not covered by other domains in the tool.
Was the study apparently free of other problems that could result in high risk of bias?a
Assessment of external validity
Elements of external validity
e.g., Sprague-Dawley versus Fischer 344
Rat, mouse, dog, pig, etc.
Age in weeks, body weight
Male versus female versus mix of genders used
Model of PAH
MCT, CH, or SUHx
Severity of PAH
RVSP or mPAP in control group
Fresh versus fresh from previously cryopreserved versus thawed cryopreserved product
Tissue source of regenerative cell product
Bone marrow versus peripheral blood versus cord blood
Timing of cell administration following induction of PAH
Early (<2 weeks) versus late (≥2 weeks) intervention
Primary outcome assessed at 1, 2, 3, and 4+ weeks after intervention
Route of cell administration
Intravenous versus intratracheal versus intraperitoneal versus intramuscular
Dose of cells
e.g., 1 × 106 cells
Type of control
PBS versus normal saline versus fibroblasts versus heat-killed cells
Yes versus No
Number of participating study centers
Single versus multi-center
Assessment of construct validity
Construct validity in preclinical research refers to the ability of a study to generalizable to a clinical scenario . Threats to construct validity arise when experimental conditions (model, intervention, outcomes) are not representative of the clinical scenario.
Elements of construct validity
Recommendations from guidelinesa
Specific application to PAH
Model matches age of patients to clinical setting
Adult animals included
Characterization of animal properties at baseline
RVSP or mPAP assessed at baseline
Confirmation that the model successfully establishes PAH
Matching model to sex of patients in clinical setting
Both male and female animal included
Matching of measure to clinical outcome
Clinically relevant outcome reported (e.g., pulmonary hemodynamics, RV remodeling, cardiac function, mortality)
Clinically relevant outcomes may increase potential generalizability to clinical setting
Long-term follow-up (>3 weeks post-intervention)
PAH is a chronic disease; long-term assessment increases reliability of findings
Pulmonary hemodynamics assessed by direct catheterization
Right heart catheterization is the gold standard for diagnosing PAH
Assessment of multiple manifestations of disease phenotype
Study reports ≥2 types of outcome measurements (pulmonary hemodynamics, RV remodeling, cardiac function, mortality, histopathological assessment of vascular lesions)
Efficacy in multiple manifestations of PAH may increase reliability of findings
Modeling of disease
Matching model to human manifestation
PAH is induced successfully in the model
Treatment response along mechanistic pathway
A molecular or cellular mechanism of treatment is measured and reported
Ensures therapy is producing a biological effect; ensures negative effects cannot be ascribed to a lack of biological activity
Administration of intervention
Matching timing of treatment delivery to clinical setting
Intervention is given after PAH is established (>2 weeks in animal models)
PAH usually present with symptoms before diagnosis
Matching the duration/exposure of treatment to clinical setting
Evidence of cell persistence in any animal organ
Ensures presence of cells during course of treatment
Matching model to co-interventions in clinical setting
Animals are on background medical therapy for PAH (e.g., Prostacyclins endothelin receptor antagonists, PDE5 inhibitors, calcium channel blockers)
PAH patients would be on conventional pharmacotherapy
Address confounders associated with setting, experimental setting
General anesthetic is not used during outcome measurements
Anesthetics may exert effects on cardiovascular system; patients undergo right heart catheterization under local anesthetic, echocardiography performed without anesthetics in patients
Description of reporting
We will apply the Animal Research: Reporting of In vivo Experiments (ARRIVE) guidelines to evaluate the quality of reporting in preclinical studies. The ARRIVE guidelines were developed by the National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) to improve the transparent and comprehensive reporting of research methods and results for in vivo animal experiments .
Continuous endpoints will be pooled using the ratio of weighted means using the inverse variance random effects method [22, 23]. Death will be analyzed as the mortality ratio of ndeaths/ntotal at defined endpoints and by mean survival in days. Statistical heterogeneity of included studies will be measured using the I2 test with 95 % confidence intervals . An evaluation for the presence of publication bias will be conducted with funnel plots and Egger’s regression test .
Planned subgroup analyses will be examined on the primary endpoint of right heart catheterization hemodynamics (RVSP/mPAP). Heterogeneity will be analyzed for the following: PAH induction method, age, sex, strain, regenerative cell type, tissue origin of cells, timing of administration (<2 weeks post-induction, ≥2 weeks post-induction), follow-up period (0, 1, 2, 3, 4+ weeks post-intervention), route of delivery, cell dose, dose frequency, use of cell pretreatments and other enhancement strategies (e.g., gene transfection), and sample sizes of study.
The timing of this review is highly relevant, as clinical trials have been completed (NCT00257413, NCT00641836, NCT00469027) [11, 3 ,26]. So far, based on limited short-term data, the results of completed clinical trials have shown relatively modest benefits  compared to the effect sizes reported in some preclinical literature [27, 28]. Most of the evidence supporting regenerative cell therapy for PAH has been based on findings observed in prevention studies rather than treatment studies in established pulmonary hypertension , which may have contributed to the overestimation of the degree of their efficacy. Therefore, a thorough examination of the study design with attention to the timing of interventions and suitability of follow-up duration must be conducted to determine the validity of the available evidence and potential for clinical translation.
Systematic reviews should be valuable in stem cell research because of the high degree of heterogeneity of cell products used for therapy. As such, a secondary aim of this study will be to evaluate the relative efficacy between regenerative cell types. Although EPCs and MSCs have both been studied extensively in preclinical models, we anticipate that other cell products such as cells derived from embryonic stem cells or induced pluripotent stem cell may also have in vivo efficacy. Still, guidelines and/or criteria for what constitutes a particular regenerative cell type are ill-defined and when available, they are loosely defined based on an evolving understanding of stem cell biology . In the absence of such standards, this review will address the level of transparency in reporting of pertinent information regarding stem cell isolation and culture techniques and quality control for each cell type, as well as the delivery and dosing methods.
We anticipate that this comprehensive synthesis will provide valuable information in translating stem cell therapy to the clinic. In addition, this review will have an immediate impact on preclinical research by highlighting knowledge gaps and areas to improve study design for future preclinical investigations of PAH.
ALK-1, activin receptor-like kinase 1; ARRIVE, Animal Research, Reporting of In vivo Experiments; BMPR2, bone morphogenetic receptor, type 2; CAMARADES, Collaborative Approach to Meta Analysis and Review of Animal Data from Experimental Studies; CAV1, caveolin 1; ENG, endoglin; EPC, endothelial progenitor cell; KCNK3, potassium channel subfamily K member 3; mPAP, mean pulmonary arterial pressure; MSC, mesenchymal stem cell, marrow stromal cell, or mesenchymal stromal cell; PAH, pulmonary arterial hypertension; PVR, pulmonary vascular resistance; RV, right ventricle; RVSP, right ventricular systolic pressure; SMAD9, mothers against decapentaplegic homolog 9; SYRCLE, Systematic Review Centre for Laboratory animal Experimentation.
CS holds a Vanier Canada Graduate Scholarship and a Canadian Institutes of Health Research Training Program in Regenerative Medicine fellowship. We would like to thank Becky Skidmore (Information Specialist) and Risa Shorr (Librarian, The Ottawa Hospital) for providing assistance with the generation of a systematic search strategy and article retrieval.
CS, AZ, and DS conceived the study design. CS and ML were responsible for the initial drafting of the manuscript. CW and BL were responsible for the data collection. DS, ML, LM, and DF provided critical revisions for important intellectual content. All authors have reviewed and approved the final version of the manuscript.
DS is President and CEO of Northern Therapeutics (Montréal, QC, Canada).
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