Comparative Analysis of Methods to Induce Myocardial Infarction in a Closed-Chest Rabbit Model

Objective. To develop a rabbit model of closed-chest catheter-induced myocardial infarction. Background. Limitations of rodent and large animal models justify the search for clinically relevant alternatives. Methods. Microcatheterization of the heart was performed in 47 anesthetized 3-4 kg New Zealand rabbits to test five techniques of myocardial ischemia: free coils (n=4), interlocking coils (n=4), thrombogenic gelatin sponge (n=4), balloon occlusion (n=4), and alcohol injection (n=8). In order to limit ventricular fibrillation, an antiarrhythmic protocol was implemented, with beta-blockers/amiodarone before and xylocaine infusion during the procedure. Clinical, angiographic, and echographic data were gathered. End points included demonstration of vessel occlusion (TIMI flow grades 0 and 1 on the angiogram), impairment of left ventricular function at 2 weeks after procedure (by echocardiography), and pathologically confirmed myocardial infarction. Results. The best arterial access was determined to be through the right carotid artery. The internal mammary guiding catheter 4-Fr was selected as the optimal device for selective intracoronary injection. Free coils deployed prematurely and tended to prolapse into the aorta. Interlocking coils did not deploy completely and failed to provide reliable results. Gelatin sponge was difficult to handle, adhered to the catheter, and could not be clearly visualized by fluoroscopy. Balloon occlusion yielded inconsistent results. Alcohol injection was the most efficient and reproducible method for inducing myocardial infarction (4 out of 6 animals), the extent of which could be fine-tuned by using a coaxial balloon catheter as a microcatheter (0.52 mm) to achieve a superselective injection of 0.2 mL of alcohol. This approach resulted in a 20% decrease in LVEF and infarcted myocardium was confirmed histologically. Conclusions. By following a stepwise approach, a minimally invasive, effective, and reproducible rabbit model of catheter-induced myocardial infarction has been developed which addresses the limitations of rodent experiments while avoiding the logistical and cost issues associated with large animal models

In vitro controlled release of extracellular vesicles for cardiac repair from poly(glycerol sebacate) acrylate-based polymers

International audienceCell therapy to restore cardiac function in chronic heart failure has been extensively studied. However, its therapeutic value is limited due to poor cell engraftment and survival and the therapeutic outcomes have been attributed to paracrine secretions such as extracellular vesicles (EV). The direct use of EV is an attractive therapeutic strategy and it has been shown that the kinetics of delivery of the EV to the targeted tissue may impact the outcomes. However, there are currently no technologies to deliver EV to the heart in a controlled and tunable manner. The objective of this study was to design a controlled release system, based on a photocurable adhesive polymer, to locally deliver EV to the cardiac tissue. We have first demonstrated that the adhesive polymer, PGSA-g-EG, did not impact the EV bioactivity in vitro and was biocompatible in vivo when tested in a rat model. Importantly, the polymer remained attached to the heart surface for at least 1 month. We have then evaluated and optimized the in vitro release kinetics of the EV from the PGSA-g-EG polymer. Freeze-dried EV formulations were developed to tune the release kinetics and maximize the loading in the polymeric material. Moreover, despite the instability of the EV in aqueous medium at 37°C, the PGSA-g-EG polymer was able to release bioactive EV for at least 14 days. Overall, these results suggest that the PGSA-g-EG is a suitable material to promote the controlled delivery of bioactive EV over an extended period of time. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EV) are an investigational class of therapeutics that has shown promise to restore cardiac function following an ischemic event. Furthermore, its translation to the clinics is expected to pose less regulatory challenges than cell-based therapies. However, EV therapeutic outcomes are likely to be impacted by the route of administration and the kinetics of delivery to the target tissue. Therefore, there is a need for biomaterial-based technologies to deliver, in a controlled and tunable manner, EV to the heart. The present study describes the use of PGSA-g-EG polymer as an adhesive cardiac patch with potential to enable the controlled delivery of bioactive EV over an extended period of time to the cardiac tissue

Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold

International audienceBACKGROUND: Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination.METHODS: Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) ≤55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1+ progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner.RESULTS: The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified.CONCLUSIONS: A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect

Differentiation in vivo of cardiac committed human embryonic stem cells in postmyocardial infarcted rats.: Human ES cells in postmyocardial infarction

International audienceHuman embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration

Dynamic contrast enhanced – MRI efficiency in detecting embolization-induced perfusion defects in a rabbit model of critical-limb-ischemia

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Therapeutic potential of extracellular vesicles derived from cardiac progenitor cells in rodent models of chemotherapy-induced cardiomyopathy

International audienceBackground Current treatments of chemotherapy-induced cardiomyopathy (CCM) are of limited efficacy. We assessed whether repeated intravenous injections of human extracellular vesicles from cardiac progenitor cells (EV-CPC) could represent a new therapeutic option and whether EV manufacturing according to a Good Manufacturing Practices (GMP)-compatible process did not impair their bioactivity. Methods Immuno-competent mice received intra-peritoneal injections (IP) of doxorubicin (DOX) (4 mg/kg each; cumulative dose: 12 mg/kg) and were then intravenously (IV) injected three times with EV-CPC (total dose: 30 billion). Cardiac function was assessed 9–11 weeks later by cardiac magnetic resonance imaging (CMR) using strain as the primary end point. Then, immuno-competent rats received 5 IP injections of DOX (3 mg/kg each; cumulative dose 15 mg/kg) followed by 3 equal IV injections of GMP-EV (total dose: 100 billion). Cardiac function was assessed by two dimensional-echocardiography. Results In the chronic mouse model of CCM, DOX + placebo-injected hearts incurred a significant decline in basal (global, epi- and endocardial) circumferential strain compared with sham DOX-untreated mice ( p = 0.043, p = 0.042, p = 0.048 respectively) while EV-CPC preserved these indices. Global longitudinal strain followed a similar pattern. In the rat model, IV injections of GMP-EV also preserved left ventricular end-systolic and end-diastolic volumes compared with untreated controls. Conclusions Intravenously-injected extracellular vesicles derived from CPC have cardio-protective effects which may make them an attractive user-friendly option for the treatment of CCM

Extracellular vesicles fail to trigger the generation of new cardiomyocytes in chronically infarcted hearts

Background: Extracellular vesicles (EV) mediate the therapeutic effects of stem cells but it is unclear whether this involves cardiac regeneration mediated by endogenous cardiomyocyte proliferation.Methods: Bi-transgenic MerCreMer/ZEG (n = 15/group) and Mosaic Analysis With Double Markers (MADM; n = 6/group) mouse models underwent permanent coronary artery ligation and received, 3 weeks later, 10 billion EV (from human iPS-derived cardiovascular progenitor cells [CPC]), or saline, injected percutaneously under echo guidance in the peri-infarcted myocardium. Endogenous cardiomyocyte proliferation was tracked by EdU labeling and biphoton microscopy. Other end points, including cardiac function (echocardiography and MRI), histology and transcriptomics were blindly assessed 4-6 weeks after injections.Results: There was no proliferation of cardiomyocytes in either transgenic mouse strains. Nevertheless, EV improved cardiac function in both models. In MerCreMer/ZEG mice, LVEF increased by 18.3 +/- 0.2% between baseline and the end-study time point in EV-treated hearts which contrasted with a decrease by 2.3 +/- 0.2% in the PBS group; MADM mice featured a similar pattern as intra-myocardial administration of EV improved LVEF by 13.3 +/- 0.16% from baseline whereas it decreased by 14.4 +/- 0.16% in the control PBS-injected group. This functional improvement was confirmed by MRI and associated with a reduction in infarct size, the decreased expression of several pro-fibrotic genes and an overexpression of the anti-fibrotic mi RNA 133-al compared to controls. Experiments with an anti-miR133-a demonstrated that the cardio-reparative effects of EV were partly abrogated.Conclusions: EV-CPC do not trigger cardiomyocyte proliferation but still improve cardiac function by other mechanisms which may include the regulation of fibrosis