Combined meta-analysis of preclinical cell therapy studies shows overlapping effect modifiers for multiple diseases.

INTRODUCTION: Cell therapy has been studied in many different research domains. Cellular replacement of damaged solid tissues is at an early stage of development, with much still to be understood. Systematic reviews and meta-analyses are widely used to aggregate data and find important patterns of results within research domains.We set out to find common biological denominators affecting efficacy in preclinical cell therapy studies for renal, neurological and cardiac disease. METHODS: We used datasets of five previously published meta-analyses investigating cell therapy in preclinical models of chronic kidney disease, spinal cord injury, stroke and ischaemic heart disease. We transformed primary outcomes to ratios of means to permit direct comparison across disease areas. Prespecified variables of interest were species, immunosuppression, cell type, cell origin, dose, delivery and timing of the cell therapy. RESULTS: The five datasets from 506 publications yielded data from 13 638 animals. Animal size affects therapeutic efficacy in an inverse manner. Cell type influenced efficacy in multiple datasets differently, with no clear trend for specific cell types being superior. Immunosuppression showed a negative effect in spinal cord injury and a positive effect in cardiac ischaemic models. There was a dose-dependent relationship across the different models. Pretreatment seems to be superior compared with administration after the onset of disease. CONCLUSIONS: Preclinical cell therapy studies are affected by multiple variables, including species, immunosuppression, dose and treatment timing. These data are important when designing preclinical studies before commencing clinical trials.

A systematic comparison of cardiovascular magnetic resonance and high resolution histological fibrosis quantification in a chronic porcine infarct model.

The noninvasive reference standard for myocardial fibrosis detection on cardiovascular magnetic resonance imaging (CMR) is late gadolinium enhancement (LGE). Currently there is no consensus on the preferred method for LGE quantification. Moreover myocardial wall thickening (WT) and strain are measures of regional deformation and function. The aim of this research was to systematically compare in vivo CMR parameters, such as LGE, WT and strain, with histological fibrosis quantification. Eight weeks after 90 min ischemia/reperfusion of the LAD artery, 16 pigs underwent in vivo Cine and LGE CMR. Histological sections from transverse heart slices were digitally analysed for fibrosis quantification. Mean fibrosis percentage of analysed sections was related to the different CMR techniques (using segmentation or feature tracking software) for each slice using a linear mixed model analysis. The full width at half maximum (FWHM) technique for quantification of LGE yielded the highest R2 of 60%. Cine derived myocardial WT explained 16-36% of the histological myocardial fibrosis. The peak circumferential and radial strain measured by feature tracking could explain 15 and 10% of the variance of myocardial fibrosis, respectively. The used method to systematically compare CMR image data with digital histological images is novel and feasible. Myocardial WT and strain were only modestly related with the amount of fibrosis. The fully automatic FWHM analysis technique is the preferred method to detect myocardial fibrosis.

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury.

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

Cardiac Stem Cell Treatment in Myocardial Infarction: A Systematic Review and Meta-Analysis of Preclinical Studies.

RATIONALE: Cardiac stem cells (CSC) therapy has been clinically introduced for cardiac repair after myocardial infarction (MI). To date, there has been no systematic overview and meta-analysis of studies using CSC therapy for MI. OBJECTIVE: Here, we used meta-analysis to establish the overall effect of CSCs in preclinical studies and assessed translational differences between and within large and small animals in the CSC therapy field. In addition, we explored the effect of CSC type and other clinically relevant parameters on functional outcome to better predict and design future (pre)clinical studies using CSCs for MI. METHODS AND RESULTS: A systematic search was performed, yielding 80 studies. We determined the overall effect of CSC therapy on left ventricular ejection fraction and performed meta-regression to investigate clinically relevant parameters. We also assessed the quality of included studies and possible bias. The overall effect observed in CSC-treated animals was 10.7% (95% confidence interval 9.4-12.1; P<0.001) improvement in ejection fraction compared with placebo controls. Interestingly, CSC therapy had a greater effect in small animals compared with large animals (P<0.001). Meta-regression indicated that cell type was a significant predictor for ejection fraction improvement in small animals. Minor publication bias was observed in small animal studies. CONCLUSIONS: CSC treatment resulted in significant improvement of ejection fraction in preclinical animal models of MI compared with placebo. There was a reduction in the magnitude of effect in large compared with small animal models. Although different CSC types have overlapping culture characteristics, we observed a significant difference in their effect in post-MI animal studies.

Translational failure of anti-inflammatory compounds for myocardial infarction: a meta-analysis of large animal models.

AIMS: Numerous anti-inflammatory drugs have been tested in large animal studies of myocardial infarction (MI). Despite positive results, translation of anti-inflammatory strategies into clinical practice has proved to be difficult. Critical disparities between preclinical and clinical study design that influence efficacy may partly be responsible for this translational failure. The aim of the present systematic review was to better understand which factors underlie the failure of transition towards the clinic. METHODS AND RESULTS: Meta-analysis and regression of large animal studies were performed to identify sources that influenced effect size of anti-inflammatory compounds in large animal models of MI. We included 183 studies, containing 3331 large animals. Infarct size (IS) as a ratio of the area at risk (12.7%; 95% confidence interval, CI 11.1-14.4%, P < 0.001) and IS as a ratio of the left ventricle (3.9%; 95% CI 3.1-4.7%, P < 0.001) were reduced in treatment compared with control groups. Effect size was higher when outcome was assessed early after MI (P = 0.013) and where studies included only male animals (P < 0.001). Mortality in treated animals was higher in studies that blinded the investigator during the experiment (P = 0.041) and depended on the type of drug used (P < 0.001). CONCLUSIONS: As expected, treatment with anti-inflammatory drugs leads to smaller infarct size in large animal MI models. Timing of outcome assessment, sex, and study quality are significantly associated with outcome and may explain part of the translational failure in clinical settings. Effect size depends on the type of drug used, enabling identification of compounds for future clinical testing.

Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial.

BACKGROUND: Recently cardiomyocyte progenitor cells (CMPCs) were successfully isolated from fetal and adult human hearts. Direct intramyocardial injection of human CMPCs (hCMPCs) in experimental mouse models of acute myocardial infarction significantly improved cardiac function compared to controls. AIM: Here, our aim was to investigate whether xenotransplantation via intracoronary infusion of fetal hCMPCs in a pig model of chronic myocardial infarction is safe and efficacious, in view of translation purposes. METHODS & RESULTS: We performed a randomized, blinded, placebo controlled trial. Four weeks after ischemia/reperfusion injury by 90 minutes of percutaneous left anterior descending artery occlusion, pigs (n = 16, 68.5 ± 5.4 kg) received intracoronary infusion of 10 million fetal hCMPCs or placebo. All animals were immunosuppressed by cyclosporin (CsA). Four weeks after infusion, endpoint analysis by MRI displayed no difference in left ventricular ejection fraction, left ventricular end diastolic and left ventricular end systolic volumes between both groups. Serial pressure volume (PV-)loop and echocardiography showed no differences in functional parameters between groups at any timepoint. Infarct size at follow-up, measured by late gadolinium enhancement MRI showed no difference between groups. Intracoronary pressure and flow measurements showed no signs of coronary obstruction 30 minutes after cell infusion. No premature death occurred in cell treated animals. CONCLUSION: Xenotransplantation via intracoronary infusion of hCMPCs is feasible and safe, but not associated with improved left ventricular performance and infarct size compared to placebo in a porcine model of chronic myocardial infarction.

Endogenous contrast MRI of cardiac fibrosis: beyond late gadolinium enhancement.

The aim of this review is to provide an overview of detection of cardiac fibrosis with MRI using current standards and novel endogenous MRI techniques. Assessment of cardiac fibrosis is important for diagnosis, prediction of prognosis and follow-up after therapy. During the past years, progress has been made in fibrosis detection using MRI. Cardiac infarct size can be assessed noninvasively with late gadolinium enhancement. Several methods for fibrosis detection using endogenous contrast have been developed, such as native T1 -mapping, T1ρ -mapping, Magnetization transfer imaging, and T2 *-mapping. Each of these methods will be described, providing the basic methodology, showing potential applications from applied studies, and discussing the potential and challenges or pitfalls. We will also identify future steps and developments that are needed for bringing these methods to the clinical practice.

Similar effect of autologous and allogeneic cell therapy for ischemic heart disease: systematic review and meta-analysis of large animal studies.

RATIONALE: In regenerative therapy for ischemic heart disease, use of both autologous and allogeneic stem cells has been investigated. Autologous cell can be applied without immunosuppression, but availability is restricted, and cells have been exposed to risk factors and aging. Allogeneic cell therapy enables preoperative production of potent cell lines and immediate availability of cell products, allowing off-the-shelf therapy. It is unknown which cell source is preferred with regard to improving cardiac function. OBJECTIVE: We performed a meta-analysis of preclinical data of cell therapy for ischemic heart disease. METHODS AND RESULTS: We conducted a systematic literature search to identify publications describing controlled preclinical trials of unmodified stem cell therapy in large animal models of myocardial ischemia. Data from 82 studies involving 1415 animals showed a significant improvement in mean left ventricular ejection fraction in treated compared with control animals (8.3%, 95% confidence interval, 7.1-9.5; P<0.001). Meta-regression revealed a similar difference in left ventricular ejection fraction in autologous (8.8%, 95% confidence interval, 7.3-10.3; n=981) and allogeneic (7.3%, 95% confidence interval, 4.4-10.2, n=331; P=0.3) cell therapies. CONCLUSIONS: Autologous and allogeneic cell therapy for ischemic heart disease show a similar improvement in left ventricular ejection fraction in large animal models of myocardial ischemia, compared with placebo. These results are important for the design of future clinical trials.

Endogenous assessment of chronic myocardial infarction with T(1ρ)-mapping in patients.

BACKGROUND: Detection of cardiac fibrosis based on endogenous magnetic resonance (MR) characteristics of the myocardium would yield a measurement that can provide quantitative information, is independent of contrast agent concentration, renal function and timing. In ex vivo myocardial infarction (MI) tissue, it has been shown that a significantly higher T(1ρ) is found in the MI region, and studies in animal models of chronic MI showed the first in vivo evidence for the ability to detect myocardial fibrosis with native T(1ρ)-mapping. In this study we aimed to translate and validate T(1ρ)-mapping for endogenous detection of chronic MI in patients. METHODS: We first performed a study in a porcine animal model of chronic MI to validate the implementation of T(1ρ)-mapping on a clinical cardiovascular MR scanner and studied the correlation with histology. Subsequently a clinical protocol was developed, to assess the feasibility of scar tissue detection with native T(1ρ)-mapping in patients (n = 21) with chronic MI, and correlated with gold standard late gadolinium enhancement (LGE) CMR. Four T1ρ-weighted images were acquired using a spin-lock preparation pulse with varying duration (0, 13, 27, 45 ms) and an amplitude of 750 Hz, and a T(1ρ)-map was calculated. The resulting T(1ρ)-maps and LGE images were scored qualitatively for the presence and extent of myocardial scarring using the 17-segment AHA model. RESULTS: In the animal model (n = 9) a significantly higher T(1ρ) relaxation time was found in the infarct region (61 ± 11 ms), compared to healthy remote myocardium (36 ± 4 ms) . In patients a higher T(1ρ) relaxation time (79 ± 11 ms) was found in the infarct region than in remote myocardium (54 ± 6 ms). Overlap in the scoring of scar tissue on LGE images and T(1ρ)-maps was 74%. CONCLUSION: We have shown the feasibility of native T(1ρ)-mapping for detection of infarct area in patients with a chronic myocardial infarction. In the near future, improvements on the T(1ρ)-mapping sequence could provide a higher sensitivity and specificity. This endogenous method could be an alternative for LGE imaging, and provide additional quantitative information on myocardial tissue characteristics.

Myocardial infarction and functional outcome assessment in pigs.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

Admittance-based pressure-volume loops versus gold standard cardiac magnetic resonance imaging in a porcine model of myocardial infarction.

Abstract A novel admittance-based pressure-volume system (AS) has recently been developed and introduced. Thus far, the new technique has been validated predominantly in small animals. In large animals it has only been compared to three-dimensional echocardiography (3DE) where the AS showed to overestimate left ventricular (LV) volumes. To fully determine the accuracy of this device, we compared the AS with gold standard cardiac magnetic resonance imaging (CMRI) in a porcine model of chronic myocardial infarction (MI). Fourteen pigs were subjected to 90 min closed chest balloon occlusion of the left anterior descending artery. After 8 weeks of follow up, pigs were consecutively subjected to LV volume measurements by the AS, CMRI, and 3DE under general anesthesia. The AS overestimated end diastolic volume (EDV; +20.9 ± 30.6 mL, P = 0.024) and end systolic volume (ESV; +17.7 ± 29.4 mL, P = 0.042) but not ejection fraction (EF; +2.46 ± 6.16%, P = NS) compared to CMRI. Good correlations of EDV (R = 0.626, P = 0.017) and EF (R = 0.704, P = 0.005) between the AS and CMRI were observed. EF measured by the AS and 3DE also correlated significantly (R = 0.624, P = 0.030). After subjection of pigs to MI, the AS very moderately overestimates LV volumes and shows accurate measurements for EF compared to CMRI. This makes the AS a useful tool to determine cardiac function and dynamic changes in large animal models of cardiac disease.

Assessment of coronary microvascular resistance in the chronic infarcted pig heart.

Pre-clinical studies aimed at treating ischemic heart disease (i.e. stem cell- and growth factor therapy) often consider restoration of the impaired microvascular circulation as an important treatment goal. However, serial in vivo measurement hereof is often lacking. The purpose of this study was to evaluate the applicability of intracoronary pressure and flow velocity as a measure of microvascular resistance in a large animal model of chronic myocardial infarction (MI). Myocardial infarction was induced in Dalland Landrace pigs (n = 13; 68.9 ± 4.1 kg) by a 75-min. balloon occlusion of the left circumflex artery (LCX). Intracoronary pressure and flow velocity parameters were measured simultaneously at rest and during adenosine-induced hyperemia, using the Combowire (Volcano) before and 4 weeks after MI. Various pressure- and/or flow-derived indices were evaluated. Hyperemic microvascular resistance (HMR) was significantly increased by 28% in the infarct-related artery, based on a significantly decreased peak average peak flow velocity (pAPV) by 20% at 4 weeks post-MI (P = 0.03). Capillary density in the infarct zone was decreased compared to the remote area (658 ± 207/mm(2) versus 1650 ± 304/mm(2) , P = 0.017). In addition, arterioles in the infarct zone showed excessive thickening of the alpha smooth muscle actin (αSMA) positive cell layer compared to the remote area (33.55 ± 4.25 µm versus 14.64 ± 1.39 µm, P = 0.002). Intracoronary measurement of HMR successfully detected increased microvascular resistance that might be caused by the loss of capillaries and arteriolar remodelling in the chronic infarcted pig heart. Thus, HMR may serve as a novel outcome measure in pre-clinical studies for serial assessment of microvascular circulation.

Cell therapy, a novel remedy for dilated cardiomyopathy? A systematic review.

BACKGROUND: Dilated cardiomyopathy (DCM) is the most common form of nonischemic cardiomyopathy worldwide and can lead to sudden cardiac death and heart failure. Despite ongoing advances made in the treatment of DCM, improvement of outcome remains problematic. Stem cell therapy has been extensively studied in preclinical and clinical models of ischemic heart disease, showing potential benefit. DCM is associated with a major health burden, and few studies have been performed on cell therapy for DCM. In this systematic review we aimed to provide an overview of preclinical and clinical studies performed on cell therapy for DCM. METHODS AND RESULTS: A systematic search, critical appraisal, and summarized outcomes are presented. In total, 29 preclinical and 15 clinical studies were included. Methodologic quality of reported studies in general was low based on the Centre for Evidence Based Medicine, Oxford University, criteria. A large heterogeneity in inclusion criteria, procedural characteristics, and outcome measures was noted. The majority of studies showed a significant increase in left ventricular ejection fraction after cell therapy during follow-up. CONCLUSIONS: Stem cell therapy has shown moderate but significant effects in clinical trials for ischemic heart disease, but it remains to be determined if we can extrapolate these results to DCM patients. There is a need for methodologically sound studies to elucidate underlying mechanisms and translate those into improved therapy for clinical practice. To validate safety and efficacy of cell therapy for DCM, adequate randomized (placebo) controlled trials using different strategies are mandatory.

Concise review: heart regeneration and the role of cardiac stem cells.

Acute myocardial infarction leads to irreversible loss of cardiac myocytes, thereby diminishing the pump function of the heart. As a result, the strenuous workload imposed on the remaining cardiac myocytes often gives rise to subsequent cell loss until the vicious circle ends in chronic heart failure (CHF). Thus, we are in need of a therapy that could ameliorate or even reverse the disease progression of CHF. Endogenous regeneration of the mammalian heart has been shown in the neonatal heart, and the discovery that it may still persist in adulthood sparked hope for novel cardioregenerative therapies. As the basis for cardiomyocyte renewal, multipotent cardiac stem/progenitor cells (CSCs) that reside in the heart have been shown to differentiate into cardiac myocytes, smooth muscle cells, and vascular endothelial cells. These CSCs do have the potential to actively regenerate the heart but clearly fail to do so after abundant and segmental loss of cells, such as what occurs with myocardial infarction. Therefore, it is vital to continue research for the most optimal therapy based on the use or in situ stimulation of these CSCs. In this review, we discuss the current status of the cardioregenerative field. In particular, we summarize the current knowledge of CSCs as the regenerative substrate in the adult heart and their use in preclinical and clinical studies to repair the injured myocardium.

Human relevance of pre-clinical studies in stem cell therapy: systematic review and meta-analysis of large animal models of ischaemic heart disease.

AIMS: Stem cell therapy is a treatment strategy for ischaemic heart disease patients. Meta-analysis of randomized human trials showed <5% improvement in left ventricular ejection fraction (LVEF). Meta-analysis of available pre-clinical data of ischaemic heart disease could provide important clues to design human clinical trials. METHODS AND RESULTS: Random-effects meta-analysis was performed on pig, dog, or sheep studies investigating the effect of cardiac stem cell therapy in ischaemic cardiomyopathy (52 studies; n = 888 animals). Endpoints were LVEF and death. Ischaemia/reperfusion infarction was performed in 23 studies and chronic occlusion in 29 studies. Pooled analysis showed a LVEF difference of 7.5% at follow-up after cell therapy vs. control (95% confidence interval, 6.2-8.9%; P < 0.001). By exploratory multivariable meta-regression, significant predictors of LVEF improvement were: cell type [bone marrow mononuclear cells (BM-MNC) showed less effect than other cell types, e.g. mesenchymal stem cells; P = 0.040] and type of infarction (left anterior descending artery 8.0 vs. left circumflex artery 5.8%; P = 0.045). Cell therapy was not associated with increased mortality (P = 0.68). Sensitivity analysis showed trends towards more improvement with higher cell number (≥10(7)), chronic occlusion models, and late injections (>1 week). After follow-up of 8 weeks, the effect of cell therapy decreased to 6%. CONCLUSION: This meta-analysis showed that large animal models are valid to predict the outcome of clinical trials. Our results showed that cell therapy is safe and leads to a preserved LVEF. Future trials should focus on cell types other than BM-MNC, large infarction, and strategies to obtain sustained effects.