1. Review Title

Efficacy and safety of regenerative cell therapy for pulmonary arterial hypertension in animal models: a preclinical systematic review protocol 2. Anticipated or Actual Start Date: July 2015 3. Anticipated Completion Date: July 2016 (12 months) 4. Stage of Review at Time of this Submission Review stage Preliminary searches Piloting of the study selection process Formal screening of search results against eligibility criteria Data extraction Risk of bias (quality) assessment Data analysis Prospective meta-analysis

Started X X

Principal Investigator(s) Duncan Stewart, MD, FRCPC 501 Smyth Rd Ottawa, ON K1H8L6 1 (613) 737-8899 x73491 [email protected] 5. Organizational Affiliation of Review Ottawa Hospital Research Institute (www.ohri.ca) 6. Review Team Members and their Organizational Affiliations Colin Suen1,2 Alex Zhai1 Manoj Lalu3 Christopher Welsh5 Brendan Levac5 Dean Ferguson4 Lauralyn McIntyre4,5 Duncan Stewart1,2 1Regenerative

Medicine Program, The Ottawa Hospital Research Institute of Cell and Molecular Medicine, University of Ottawa 3Department of Anesthesia, The Ottawa Hospital 4The Ottawa Hospital Research Institute, Clinical Epidemiology Program 5Department of Medicine (Division of Critical Care), University of Ottawa 2Department

7. Funding Sources / Sponsors Canadian Institutes of Health Research (CIHR) – MOP57726 8. Conflicts of Interest Duncan Stewart is President and CEO of Northern Therapeutics Shirley Mei is an employee of Northern Therapeutics 9. Collaborators Becky Skidmore, Information strategist Katrina Sullivan, Data analysis

Completed

10. Review Question In controlled 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? 11. Searches The search strategies outlined below will be developed in conjunction with an information specialist and a librarian (See Appendix A1). Ovid MEDLINE®, Ovid MEDLINE® In-Process & Other Non-Indexed Citations, and EMBASE Classic+ EMBASE will be searched. The strategy below was used to search in MEDLINE. In addition, a manual review of the bibliographies of selected articles and relevant reviews will be performed. Relevant conference proceedings and abstracts will also be searched. Only articles in the English language will be included in the review. 12. Condition or Domain Being Studied PAH is a rare but progressive disease with significant burden of mortality and morbidity. In spite of recent advances, therapy of pulmonary hypertension is still largely limited to symptomatic control rather than treatment of underlying pathophysiology. On the other hand, cell-based therapy holds great promise in the treatment of the underlying disease process of pulmonary hypertension. There is evidence to suggest that regenerative cell therapies using cell types such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) can migrate to sites of vascular injury, facilitate neovascularization and limit inflammation [1]. Preclinical studies involving these cell types have demonstrated efficacy in improving features of PAH such as cardiopulmonary hemodynamics, reducing right ventricular remodeling and pulmonary vascular remodeling. Clinical studies involving stem/progenitor cell therapy have been under way and show some promise in controlling the extent of the disease [2, 3]. However, to date, there has been no systematic review and/or meta analysis of the preclinical data on stem cells for as a therapy for the treatment of pulmonary hypertension. Thus, this systematic review will fulfill a need in the field and serve to aid the design of future clinical trials.

13. Participants / Population Inclusion: Preclinical in vivo models of pulmonary hypertension. Acceptable preclinical models that fulfill these criteria and will be included are the monocrotaline, chronic hypoxia, and SU5416 combined with chronic hypoxia models of pulmonary hypertension. Exclusion: Mouse models and any other animal models other than listed above. In vitro studies and human studies of PAH will be excluded. Also, features excluded that would exclude participants from etiologies other than Group I PAH classification such as PH due to left heart failure, PH due to lung diseases, chronic thromboembolic PH, and PH due to unclear multifactorial mechanisms (hematological, systemic, metabolic disorders). Genetically modified animals will also be excluded. 14. Interventions Inclusion: ANY regenerative cell (xenogenic; syn/allogenic) infusion following the induction of PAH. Exclusion: Treatment that does not include infusion of viable cells. Cell-free products derived from

stem/progenitor cells such as conditioned media. Terminally differentiated or mature cells (eg. Endothelial cell, smooth muscle cell, fibroblast). 15. Comparator(s)/control Comparator for Intervention: Inclusion: Animals that undergo experimentally-induced PAH but have not received cell therapy Exclusion: No exclusions based on comparators.

16. Types of study to be included initially Eligible studies include controlled comparative studies (randomized, pseudo-randomized, and non-randomized) of preclinical pulmonary arterial hypertension. 17. Outcomes Outcomes will be collected as long as they are measured at least one week (≥ 7 days) post-intervention to exclude the possibility of acute effects of cell administration. Primary endpoints Direct hemodynamic measures of pulmonary pressures: Mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RVSP) measured by direct catheterization Secondary endpoints     

Death Survival Right ventricular remodeling, for example Fulton’s index (weight ratio of right ventricle/left ventricle plus septum) Calculated cardiac index (CI), cardiac output (CO) and total pulmonary vascular resistance (PVR) Echocardiography measures such as cardiac structure and pulmonary hemodynamics (for example, pulmonary acceleration time).

18. Data Extraction Two independent reviewers will review studies and extract data into standardized, piloted forms. Discrepancies will be resolved through discussion with the principal investigator. 19. Risk of Bias (Quality) Assessment We will apply the SYRCLE Risk of Bias tool to assess the internal validity of included studies (see Appendix A2) 20. Construct Validity We will assess construct validity based on a pre-defined checklist of factors aimed evaluate how closely the study resembles clinical PAH. The domains assessed will be animal subjects, outcome measures, modeling of disease, administration of intervention, and environment (Appendix A3). These will be recorded as yes/no answers.

21. Strategy for Data Synthesis Meta-analysis including subgroup analysis performed on data aggregated from eligible studies. Continuous endpoints will be pooled using the ratio of weighted means method with inverse variance random effects modeling with Forest plots for presentation of the data [4, 5]. Statistical heterogeneity of included preclinical studies will be measured using the I2 test with 95% uncertainty intervals [6]. An evaluation for the presence of publication bias will be conducted with funnel plot techniques, and Egger’s regression test [7].

22. Analysis of Subgroups or Subsets Continuous endpoints will be pooled using the ratio of weighted means method with inverse variance random effects modeling[4, 5]. Statistical heterogeneity of included preclinical studies will be measured using the I2 test with 95% uncertainty intervals[6]. An evaluation for the presence of publication bias will be conducted with funnel plot techniques, and Egger’s regression test[7]. Dichotomous endpoints (e.g. death) from each included study will be pooled and described as odds ratios and 95% confidence intervals. An inverse variance random effects modeling approach will be used, with Forest plots for presentation of the data[5]. Planned subgroup analyses will be examined on the primary endpoint hemodynamics by direct catheterization (RVSP/mPAP). Heterogeneity will be analyzed for the following factors: Factors affecting cell treatment efficacy: a. Age of animal b. Sex c. Strain of animal d. Model of PAH e. Severity of model based on hemodynamic measures f. Cell type g. Route of delivery (intravenous vs intraperitoneal vs intratracheal vs intramuscular) h. Timing of treatment relative to induction (< 2 weeks or ≥ 2 weeks) i. Follow up period (1,2,3,4+ weeks after intervention) j. Cell dose k. Frequency of dosing l. Tissue origin of cells m. Cell modification Factors affecting study quality: n. Randomization reported (Y/N) o. Blinding reported (Y/N) p. Risk of bias items, all separately Y vs N vs NR q. Overall risk of bias (per total number of items)

References 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Suen CM, Mei SH, Kugathasan L, Stewart DJ: Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases. Compr Physiol 2013, 3:1749-1779. Wang X-X, Zhang F-R, Shang Y-P, Zhu J-H, Xie X-D, Tao Q-M, Zhu J-H, Chen J-Z: Transplantation of autologous endothelial progenitor cells may be beneficial in patients with idiopathic pulmonary arterial hypertension: a pilot randomized controlled trial. Journal of the American College of Cardiology 2007, 49:1566-1571. Jurasz P, Courtman D, Babaie S, Stewart DJ: Role of apoptosis in pulmonary hypertension: from experimental models to clinical trials. Pharmacology & therapeutics 2010, 126:1-8. Friedrich JO, Adhikari NK, Beyene J: The ratio of means method as an alternative to mean differences for analyzing continuous outcome variables in meta-analysis: a simulation study. BMC Med Res Methodol 2008, 8:32. DerSimonian R, Laird N: Meta-analysis in clinical trials. Control Clin Trials 1986, 7:177-188. Higgins JP, Thompson SG: Quantifying heterogeneity in a meta-analysis. Stat Med 2002, 21:15391558. Hayashino Y, Noguchi Y, Fukui T: Systematic evaluation and comparison of statistical tests for publication bias. J Epidemiol 2005, 15:235-243. Badesch DB, Raskob GE, Elliott CG, Krichman AM, Farber HW, Frost AE, Barst RJ, Benza RL, Liou TG, Turner M, et al: Pulmonary arterial hypertension: baseline characteristics from the REVEAL Registry. Chest 2010, 137:376-387. Gaine SP, Rubin LJ: Primary pulmonary hypertension. Lancet 1998, 352:719-725. McQuillan BM, Picard MH, Leavitt M, Weyman AE: Clinical correlates and reference intervals for pulmonary artery systolic pressure among echocardiographically normal subjects. Circulation 2001, 104:2797-2802. Ryan JJ, Marsboom G, Archer SL: Rodent models of group 1 pulmonary hypertension. Handb Exp Pharmacol 2013, 218:105-149. Henderson VC, Kimmelman J, Fergusson D, Grimshaw JM, Hackam DG: Threats to validity in the design and conduct of preclinical efficacy studies: a systematic review of guidelines for in vivo animal experiments. PLoS Med 2013, 10:e1001489.

Appendix A1 MEDLINE Search Strategy Database: Embase Classic+Embase <1947 to 2014 July 02>, Ovid MEDLINE(R) In-Process & Other Non-Indexed Citations and Ovid MEDLINE(R) <1946 to Present> Search Strategy: -------------------------------------------------------------------------------1 Hypertension, Pulmonary/ (77671) 2 (pulmonary adj2 hypertens*).tw. (76144) 3 (PAH)tw. (36107) 4 or/1-3 (123104) 5 Monocrotaline/ (3008) 6 (monocrotalin* or MCT).tw. (10828) 7 5 or 6 (11229) 8 4 or 7 (131708) 9 exp Stem Cells/ (338939) 10 ((stem or progenitor* or mother or precursor*) adj1 cell$1).tw. (455508) 11 ("colony-forming" adj (cell$1 or unit or units)).tw. (38308) 12 (CFU or CFUs).tw. (71887) 13 ((embryoid or embroid or embryonic) adj2 (body or bodies or cell$1)).tw. (66276) 14 (angioblast* or hemangioblast* or haemangioblast*).tw. (6922) 15 myoblast$1.tw. (22839) 16 ((haemogenic or hemogenic) adj2 (endothelial or endothelium)).tw. (311) 17 ((osteoprogenitor adj cell$1) or OPC or OPCs).tw. (9125) 18 (side adj population adj cell$1).tw. (842) 19 (circulating angiogenic adj cell$1).tw. (299) 20 (CAC or CACs).tw. (8858) 21 IPS cell$1.tw. (3869) 22 hiPSC$1.tw. (1124) 23 common lymphoid progenitor$1.tw. (516) 24 (("Pre-B" or "Pro-B") adj1 (cell$1 or lymphocyte*)).tw. (8285) 25 (("B-cell" or "B-cells" or B-lymphcyte$1 or B-lymphoid$1) adj1 (immatur* or precursor* or progenitor* or transitional)).tw. (8221) 26 (("T-cell" or "T-cells" or T-lymphcyte$1 or T-lymphoid$1) adj1 (immatur* or precursor* or progenitor* or transitional)).tw. (5507) 27 (thymocyte* or T-lymphocyte*).tw. (209185) 28 ((mesenchymal adj3 (stem or stroma$1 or progenitor* or precursor*)) and cell$1).tw. (55069) 29 (MSC or MSCs or ADMSC or ADMSCs or BM-MSC or BM-MSCs or BMD-MSC or BMD-MSCs or BMDMSC or BMDMSCs).tw. (35017) 30 ((multipotent or multi-potent) adj3 (stroma$1 cell$1 or stem cell$1)).tw. (6514) 31 marrow stroma$1 cell$1.tw. (12298) 32 CFU-F$1.tw. (1384) 33 exp Mesoderm/cy [Cytology] (6036) 34 mesenchymal.tw. and exp Genetic Therapy/ (1740) 35 exp Bone Marrow Cells/ (237902) 36 (bone marrow adj3 cell$1).tw. (122869) 37 (BMD-EPC or BMD-EPCs or BMDEPC or BMDEPCs).tw. (23) 38 exp Stem Cell Transplantation/ (140672) 39 ((hematopoietic or hemato-poietic or HSC or HSCs) adj10 transplant*).tw. (45247) 40 ((autologous or auto-logous or auto) adj HCT).tw. (534) 41 autoHCT.tw. (61)

42 ((allogeneic or allo-geneic or homologous or homo-logous) adj HCT).tw. (1436) 43 ((PBSC or PBSCs) adj10 transplant*).tw. (2248) 44 PBSCT.tw. (2851) 45 ((autologous or auto-logous or auto) adj (PBSC or PSC)).tw. (467) 46 (autoPBSCT or autoPBSC or autoPSC).tw. (97) 47 CBSCT.tw. (69) 48 Hematopoietic Stem Cell Mobilization/ (8427) 49 ((hematopoietic or hemato-poietic or HSC or HSCs) adj10 mobili#ation).tw. (2457) 50 Transplantation, Autologous/ (70268) 51 (transplant* adj1 (autologous or auto-logous)).tw. (8071) 52 (autograft* or auto-graft* or autotransplant* or auto-transplant*).tw. (41108) 53 exp Transplantation, Homologous/ (110758) 54 (transplant* adj1 (homologous or homo-logous or allogeneic or allo-geneic or isogeneic or iso-geneic or syngeneic)).tw. (11199) 55 (allotransplant* or allo-transplant* or allograft* or allo-graft* or homograft* or homo-graft* or isograft* or iso-graft*).tw. (143292) 56 Bone Marrow Transplantation/ (85051) 57 (bone marrow adj2 (graft* or transplant*)).tw. (70135) 58 exp "Cell- and Tissue-Based Therapy"/ (1189439) 59 ((cell or cells or cell-based or tissue$1 or tissue-based) adj (therapy or therapies or transplant*)).tw. (129614) 60 or/9-59 (2394286) 61 8 and 60 (5910) 62 exp animal experimentation/ or exp models, animal/ or animals/ or mammals/ or vertebrates/ or exp fishes/ or exp amphibia/ or exp reptiles/ or exp birds/ or exp hyraxes/ or exp marsupialia/ or exp monotremata/ or exp scandentia/ or exp chiroptera/ or exp carnivora/ or exp cetacea/ or exp Xenarthra/ or exp elephants/ or exp insectivora/ or exp lagomorpha/ or exp rodentia/ or exp sirenia/ or exp Perissodactyla/ or primates/ or exp strepsirhini/ or haplorhini/ or exp tarsii/ or exp platyrrhini/ or catarrhini/ or exp cercopithecidae/ or gorilla gorilla/ or pan paniscus/ or pan troglodytes/ or exp pongo/ or exp hylobatidae/ or hominidae/ (10738734) 63 (animal$1 or chordata or vertebrate* or fish$2 or amphibian* or amphibium* or reptile$1 or bird$1 or mammal* or dog or dogs or canine$1 or cat or cats or hyrax* or marsupial* or monotrem* or scandentia or bat or bats or carnivor* or cetacea or edentata* or elephant* or insect or insects or insectivore or lagomorph* or rodent$2 or mouse or mice or murine or murinae or muridae or rat or rats or pig or pigs or piglet$1 or swine or rabbit$1 or sheep$1 or goat$1 or horse$1 or equus or cow or cows or cattle or calf or calves or bovine or sirenia or ungulate$1 or primate$1 or prosimian* or haplorhini* or tarsiiform* or simian*or platyrrhini or catarrhini or cercopithecidae or ape or apes or hylobatidae or hominid* or chimpanzee* or gorilla* or orangutan* or monkey or monkeys or ape or apes).tw. (8554114) 64 exp Drug Evaluation, Preclinical/ (306518) 65 (preclinic* or pre-clinic*).tw. (140341) 66 or/62-65 (12502571) 67 61 and 66 (1789) 68 67 use prmz (540) 69 exp pulmonary hypertension/ (88479) 70 (pulmonary adj2 hypertens*).tw. (76144) 71 (PAH).tw. (36107) 72 or/69-71 (132392) 73 monocrotaline/ (3008) 74 (monocrotalin* or MCT).tw. (10828) 75 73 or 74 (11229)

76 72 or 75 (140985) 77 exp stem cell/ (338939) 78 ((stem or progenitor* or mother or precursor*) adj1 cell$1).tw. (455508) 79 exp bone marrow cell/ (237902) 80 (bone marrow adj3 cell$1).tw. (122869) 81 (BMD-EPC or BMD-EPCs or BMDEPC or BMDEPCs).tw. (23) 82 ("colony-forming" adj (cell$1 or unit or units)).tw. (38308) 83 (CFU or CFUs).tw. (71887) 84 ((embryoid or embroid or embryonic) adj2 (body or bodies or cell$1)).tw. (66276) 85 (angioblast* or hemangioblast* or haemangioblast*).tw. (6922) 86 myoblast$1.tw. (22839) 87 ((haemogenic or hemogenic) adj2 (endothelial or endothelium)).tw. (311) 88 ((osteoprogenitor adj cell$1) or OPC or OPCs).tw. (9125) 89 (side adj population adj cell$1).tw. (842) 90 (circulating angiogenic adj (cell or cells)).tw. (299) 91 (CAC or CACs).tw. (8858) 92 IPS cell$1.tw. (3869) 93 hiPSC$1.tw. (1124) 94 common lymphoid progenitor$1.tw. (516) 95 (("Pre-B" or "Pro-B") adj1 (cell$1 or lymphocyte*)).tw. (8285) 96 (("B-cell" or "B-cells" or B-lymphcyte$1 or B-lymphoid$1) adj1 (immatur* or precursor* or progenitor* or transitional)).tw. (8221) 97 (("T-cell" or "T-cells" or T-lymphcyte$1 or T-lymphoid$1) adj1 (immatur* or precursor* or progenitor* or transitional)).tw. (5507) 98 (thymocyte* or T-lymphocyte*).tw. (209185) 99 ((mesenchymal adj3 (stem or stroma$1 or progenitor* or precursor*)) and cell$1).tw. (55069) 100 (MSC or MSCs or ADMSC or ADMSCs or BM-MSC or BM-MSCs or BMD-MSC or BMD-MSCs or BMDMSC or BMDMSCs).tw. (35017) 101 ((multipotent or multi-potent) adj3 (stroma$1 cell$1 or stem cell$1)).tw. (6514) 102 marrow stroma$1 cell$1.tw. (12298) 103 CFU-F$1.tw. (1384) 104 mesenchymal.tw. and exp Gene Therapy/ (1740) 105 exp stem cell transplantation/ (140672) 106 ((hematopoietic or hemato-poietic or HSC or HSCs) adj10 transplant*).tw. (45247) 107 ((autologous or auto-logous or auto) adj HCT).tw. (534) 108 autoHCT.tw. (61) 109 ((allogeneic or allo-geneic or homologous or homo-logous) adj HCT).tw. (1436) 110 ((PBSC or PBSCs) adj10 transplant*).tw. (2248) 111 PBSCT.tw. (2851) 112 ((autologous or auto-logous or auto) adj (PBSC or PSC)).tw. (467) 113 (autoPBSCT or autoPBSC or autoPSC).tw. (97) 114 CBSCT.tw. (69) 115 stem cell mobilization/ (8427) 116 ((hematopoietic or hemato-poietic or HSC or HSCs) adj10 mobili#ation).tw. (2457) 117 autotransplantation/ (26878) 118 (transplant* adj1 (autologous or auto-logous)).tw. (8071) 119 allotransplantation/ (32679) 120 (transplant* adj1 (homologous or homo-logous or allogeneic or allo-geneic or isogeneic or iso-geneic or syngeneic)).tw. (11199)

121 (allotransplant* or allo-transplant* or allograft* or allo-graft* or homograft* or homo-graft* or isograft* or iso-graft*).tw. (143292) 122 exp bone marrow transplantation/ (97871) 123 (bone marrow adj2 (graft* or transplant*)).tw. (70135) 124 ((cell or cells or cell-based or tissue$1 or tissue-based) adj (therapy or therapies or transplant*)).tw. (129614) 125 or/77-124 (1349117) 126 76 and 125 (3044) 127 exp animal experiment/ or exp animal model/ or animal/ or exp invertebrate Chordata/ or exp experimental animal/ or exp transgenic animal/ or exp male animal/ or exp female animal/ or exp juvenile animal/ or vertebrate/ or exp fish/ or exp amphibia/ or exp reptile/ or exp bird/ or mammal/ or exp hyrax/ or exp marsupial/ or exp monotremate/ or exp scandentia/ or placental mammals/ or exp bat/ or exp carnivora/ or exp cetacea/ or exp edentata/ or exp elephant/ or exp insectivora/ or exp lagomorph/ or exp rodent/ or exp sirenia/ or exp ungulate/ or primate/ or exp prosimian/ or haplorhini/ or exp tarsiiform/ or simian/ or exp platyrrhini/ or catarrhini/ or exp cercopithecidae/ or ape/ or exp hylobatidae/ or hominid/ or exp chimpanzee/ or exp gorilla/ or exp orang utan/ (11140546) 128 (animal$1 or chordata or vertebrate* or fish$2 or amphibian* or amphibium* or reptile$1 or bird$1 or mammal* or dog or dogs or canine$1 or cat or cats or hyrax* or marsupial* or monotrem* or scandentia or bat or bats or carnivor* or cetacea or edentata* or elephant* or insect or insects or insectivore or lagomorph* or rodent$2 or mouse or mice or murine or murinae or muridae or rat or rats or pig or pigs or piglet$1 or swine or rabbit$1 or sheep$1 or goat$1 or horse$1 or equus or cow or cows or cattle or calf or calves or bovine or sirenia or ungulate$1 or primate$1 or prosimian* or haplorhini* or tarsiiform* or simian*or platyrrhini or catarrhini or cercopithecidae or ape or apes or hylobatidae or hominid* or chimpanzee* or gorilla* or orangutan* or monkey or monkeys or ape or apes).tw. (8554114) 129 (preclinic* or pre-clinic*).tw. (140341) 130 or/127-129 (12706717) 131 126 and 130 (1252) 132 131 use emczd (763) 133 68 or 132 (1303) 134 remove duplicates from 133 (925) [total unique records] 135 134 use prmz (514) [unique MEDLINE records] 136 134 use emczd (411) [unique Embase records]

Appendix A2: SYRCLE Risk of Bias assessment tool Domain

Description of domain

Review authors judgment

Sequence generation

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? (*)

Baseline characteristics

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?

3

Selection bias

Allocation concealment

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? (*)

4

Performance bias

Random housing

Describe all measures used, if any, to house the animals randomly within the animal room.

Were the animals randomly housed during the experiment? Were the caregivers and/or investigators blinded from knowledge which intervention each animal received during the experiment?

Item

1

2

Type of bias

Selection bias

Selection bias

5

Performance bias

Blinding

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.

6

Detection bias

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?

Blinding

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?

Were incomplete outcome data adequately addressed? (*) Are reports of the study free of selective outcome reporting? (*)

7

Detection bias

8

Attrition bias

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.

9

Reporting bias

Selective outcome reporting

State how selective outcome reporting was examined and what was found.

Incomplete

10

Other

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? (*)

*Items in agreement with the items in the Cochrane Risk of Bias tool. Hooijmans et al. BMC Medical Research Methodology 2014 14:43 doi:10.1186/1471-2288-14-43

Appendix A3: Elements of construct validity Grouping Animal Subjects

Outcome measurements

Recommendations From Guidelines*+ Model matches age of patients to clinical setting Characterization of animal properties at baseline Matching model to sex of patients in clinical setting Matching of measure to clinical outcome

Specific Application to PAH Adult animals included

Justification

RVSP or mPAP assessed at baseline

Confirmation that the model successfully establishes PAH Prevalence of PAH occurs in female vs males is 2:1 [8, 9] Clinically relevant outcomes may increase potential generalizability to clinical setting

Both male and female animal included Clinically relevant outcome reported (eg. pulmonary hemodynamics, RV remodeling, cardiac function, mortality) Long-term follow up (> 3 weeks post-intervention) Pulmonary hemodynamics assessed by direct catheterization

Assessment of multiple manifestations of disease phenotype

Modeling of disease

Matching model to human manifestation Treatment response along mechanistic pathway

Administration of intervention

Study reports ≥ 2 types of outcome measurements (pulmonary hemodynamics, RV remodeling, cardiac function, mortality, histopathological assessment of vascular lesions) Criteria for PAH are met in disease control (mPAP > 25 mm Hg; RVSP > 35 mm Hg[10, 11]) A molecular or cellular mechanism of treatment is measured and reported

Typical onset of PAH occurs in adulthood [8, 9]

PAH is a chronic disease; long-term assessment increases reliability of findings Right heart catheterization is the gold standard for diagnosing PAH Efficacy in multiple manifestations of PAH may increase reliability of findings

PAH is induced successfully in the model Ensures therapy is producing a biological effect; ensures negative effects cannot be ascribed to a lack of biological activity PAH usually present with symptoms before diagnosis

Matching timing of treatment delivery to clinical setting Matching the duration/exposure of treatment to clinical setting

Intervention is given after PAH is established (>2 weeks in animal models) Evidence of cell persistence in any animal organ

Ensures presence of cells during course of treatment

Matching model to cointerventions in clinical setting

Animals are on background medical therapy for PAH (eg. Prostacyclins endothelin

PAH patients would be on conventional pharmacotherapy

Environment

Address confounders associated with setting, experimental setting

receptor antagonists, PDE5 inhibitors, calcium channel blockers) 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

*Recommendations to reduce threats to construct validity were identified by Henderson VC, Kimmelman J, Fergusson D, et al. Threats to validity in the design and conduct of preclinical efficacy studies: a systematic review of guidelines for in vivo animal experiments. PLoS Medicine. 2013;10:e1001489.
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