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Introduction: Extracellular Vesicles (EV) recapitulate the benefits of cell therapy for heart repair. Their mechanism of action remains unsettled. Hypothesis: EV may contribute to heart repair by de novo cardiogenesis. Methods: To answer this question, we used 2 bi-transgenic mouse models: the fate-mapping MerCreMer/ZEG and the Mosaic Analysis With Double Markers (MADM). Myocardial infarction was induced by permanent coronary artery ligation. Those with a LVEF ≤ 45% were treated 3 weeks later with EV (from human iPS-derived cardiovascular progenitor cells; 10x10 9 particles) or PBS, injected under echo guidance in the peri-infarcted area (MerCreMer/ZEG: n=15/group and MADM: n=6/group). To track endogenous cardiomyocyte (CM) proliferation, we used EdU labeling in MerCreMer/ZEG delivered by osmotic pumps implanted for 7-10 days post-injection and biphoton microscopy in MADM models. Cardiac function was assessed 4-6 weeks after injection by echocardiography and MRI, blinded to treatment group. Hearts were then subjected to histological and transcriptomic analyses (qPCR and genome-wide microarray). Results: In PBS controls, EF remained stable over time in MerCreMer/ZEG mice and decreased from 34.5% ± 6.0% to 30.7% ± 7.5% in MADM mice by the end of the study. Conversely, EV injections increased EF from 32.1% ± 9.5% to 36.1% ± 7.45 % in MerCreMer/ZEG and from 36.2 %± 8.7% to 40.5% ± 8.9% in MADM mice. A significant difference in the change from baseline was found between EV and controls: 20.7% ± 10.5 % (p=0.048) and 28.0% ± 11.0 %, (p=0.045) for MerCreMer/ZEG and MADM groups, respectively. This improvement was confirmed by MRI in MerCreMer/ZEG mice (p=0.05). Improvement in EF was unrelated to the appearance of new CM, as shown by the absence of difference in TnT+/EdU+/GFP+ cell numbers and the lack of activation of the YAP/TAZ pathway between control and EV groups. However, EV reduced infarct size by 11.9% ± 5.75% (p=0.04), which was accompanied by decreased expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2 by qPCR) in heart tissue and a 2.13X overexpression of the anti-fibrotic miRNA 133a-1 compared to controls (n=3/group; p=0.001). Conclusions: EV likely improve cardiac function by modulation of fibrosis rather than by de novo cardiogenesis.

Aims We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P

Introduction Extracellular Vesicles (EV) seem to mediate the benefits of cell therapy for ischemic heart failure. Although their mechanism of action remains poorly understood, one hypothesis is that they might trigger the generation of new cardiomyocytes. The doubly transgenic fate-mapping MerCreMer/ZEG mice model was thus used to distinguish whether these putative new cardiomyocytes originated from the division of preexisting ones (GFP+, Troponin T [TnT+], EdU+) or differentiated from endogenous progenitors, in which case they would stain positive for TnT+/EdU+ but negative for GFP. Methods Myocardial infarction was induced in 35 MerCreMer/ZEG mice by permanent occlusion of the left anterior descending coronary artery. Three weeks later, the surviving mice (n=18) with a left ventricular ejection fraction (LVEF) ≤45% received transcutaneous echo-guided injections in the peri-infarct myocardium of either EV (from 1.4 million human iPS-derived cardiovascular progenitor cells; 10 billion particles, n=9) or PBS (n=9); osmotic pumps were implanted to deliver EdU for 7 days in order to track the proliferation of new and native cardiomyocytes. Four-6 weeks after treatment all mice were evaluated by echocardiography (n=9 per group) and MRI (7 in each group), and then sacrificed for histological assessment, blindly. Results Based on echocardiography (MRI data pending), EV improved LVEF by 16% relative to baseline while a decrease of 4% was observed in the PBS group (p=0.46). The number of new cardiomyocytes (TnT+/EdU+/GFP+) did not significantly differ between the EV-treated hearts and the controls, and averaged 0.54% of the total heart cell content in infarct, peri-infarct and remote areas. However, EV treatment better preserved preexisting GFP+/WGA+/TnT+ cardiomyocytes in the peri-infarct area as their number was greater by 5.15% compared to PBS (32 sections analyzed for each mouse). Compared to the PBS control group, EV delivery was also associated with a 2.5% decrease in fibrosis, a reduction of infarct size by 14.9%, and an increase in angiogenesis in the peri-infarct area (with a between-group absolute difference of 71 capillaries, on the basis of isolectin staining). Conclusions EV secreted by iPS-derived cardiovascular progenitors improve the function of chronically infarcted hearts. Preservation of the existing cardiomyocyte pool and limitation of adverse remodeling and scarred tissue, likely favored by increased neoangiogenesis, are the main mechanisms mediated by the EV, while fate mapping allowed to exclude the generation of new cardiomyocytes.

Background Cell-based therapies are being explored as a therapeutic option for patients with chronic heart failure following myocardial infarction. Extracellular vesicles (EV), including exosomes and microparticles, secreted by transplanted cells may orchestrate their paracrine therapeutic effects. We assessed whether post-infarction administration of EV released by human embryonic stem cell–derived cardiovascular progenitors (hESC-Pg) can provide equivalent benefits to administered hESC-Pg and whether hESC-Pg and EV treatments activate similar endogenous pathways. Methods Mice underwent surgical occlusion of their left coronary arteries. After 2–3 weeks, 95 mice included in the study were treated with hESC-Pg, EV, or Minimal Essential Medium Alpha Medium (alpha-MEM; vehicle control) delivered by percutaneous injections under echocardiographic guidance into the peri-infarct myocardium. functional and histologic end-points were blindly assessed 6 weeks later, and hearts were processed for gene profiling. Genes differentially expressed between control hearts and hESC-Pg–treated and EV-treated hearts were clustered into functionally relevant pathways. Results At 6 weeks after hESC-Pg administration, treated mice had significantly reduced left ventricular end-systolic (−4.20 ± 0.96 µl or −7.5%, p = 0.0007) and end-diastolic (−4.48 ± 1.47 µl or −4.4%, p = 0.009) volumes compared with baseline values despite the absence of any transplanted hESC-Pg or human embryonic stem cell–derived cardiomyocytes in the treated mouse hearts. Equal benefits were seen with the injection of hESC-Pg–derived EV, whereas animals injected with alpha-MEM (vehicle control) did not improve significantly. Histologic examination suggested a slight reduction in infarct size in hESC-Pg–treated animals and EV-treated animals compared with alpha-MEM–treated control animals. In the hESC-Pg–treated and EV-treated groups, heart gene profiling identified 927 genes that were similarly upregulated compared with the control group. Among the 49 enriched pathways associated with these up-regulated genes that could be related to cardiac function or regeneration, 78% were predicted to improve cardiac function through increased cell survival and/or proliferation or DNA repair as well as pathways related to decreased fibrosis and heart failure. Conclusions In this post-infarct heart failure model, either hESC-Pg or their secreted EV enhance recovery of cardiac function and similarly affect cardiac gene expression patterns that could be related to this recovery. Although the mechanisms by which EV improve cardiac function remain to be determined, these results support the idea that a paracrine mechanism is sufficient to effect functional recovery in cell-based therapies for post-infarction–related chronic heart failure.

Background & Aim Introduction Cell therapy for chronic heart failure has demonstrated that Cardiac Progenitor Cells (CPCs) delivered to the heart exert their beneficial effects through a paracrine mechanism harnessing endogenous repair pathways largely mediated by extracellular vesicles (EVs). We used a bi-transgenic fate-mapping MerCreMer/ZEG mouse model to assess whether the cardioprotection could be partly explained by de novo cardiogenesis. Methods, Results & Conclusion Methods Myocardial infarction (MI) was induced by permanent left anterior descending coronary artery ligation in MerCreMer/ZEG mice. Surviving animals (n=30) with a left ventricular ejection fraction (LVEF) ≤ 45% were treated 3 weeks after MI with either EV (from 1.4 × 106 human iPS-derived CPC; 10 × 109 particles; n=15) or PBS (n=15) delivered by echo-guided intra-myocardial injections targeted to the peri-infarcted area. To track a putative proliferation of endogenous cardiomyocytes, osmotic pumps delivering EdU were implanted for 10-13 days after EV or PBS treatnent. Cardiac function was assessed 4-6 weeks after injections by echocardiography (n=15 p/group) and MRI (n=6-7 p/group), blinded to treatment group. Hearts were harvested and subjected to histological and transcriptomic analyses by qRT-PCR and genome-wide microarrays (Affymetrix). Results In controls, echocardiographically assessed EF slightly decreased over time whereas in the EV-treated group, it increased so that at the end-study time point, the between-group difference in changes from baseline averaged 20.7 % ± 10.5 % (p=0.048). This improvement was confirmed by MRI (11%, p=0.05). EV-treated hearts did not show a different number of new cardiomyocytes (TnT+/EdU+/GFP+) compared to controls (32 analyzed sections/mouse). However, EV-treatment was associated with a reduction in infarct size (-11.9% ± 5.75%), and reduced expression of 4 pro-fibrotic genes (Col1a2, Col3a1, Lox, Col1a2) as assessed by qRT-PCR (n=5-6 p/group). Further, the EV-treatment group showed an over-expression of the anti-fibrotic miRNA 133a-1 (2.13 fold) as compared to controls (n=3 p/group; p=0.0015). Conclusions EV secreted by iPSC-CPC improve cardiac function in chronic heart failure by mechanisms which seem to be largely related to the modulation of fibrosis without participation of a de novo cardiogenesis. Funding Labex Revive, Fondation pour la Recherche Medicale, Fonds Marion Elizabeth BRANCHER

Previous discussions with haemophilia A (HA) carriers suggested that carriers may experience inappropriate care, resulting in poor relationships with healthcare providers (HCPs; principally physicians and nurses), and unfortunate and extreme emotional and behavioural responses. This was a qualitative study to explore medical experiences of HA carriers and their emotional and behavioural responses. Eleven HA carriers and five Haemophilia Treatment Centre nurses were interviewed. Themes were identified using QSR NVivo 8.0. Carriers and nurses reported HA-related bleeding symptoms in carriers, including life-threatening haemorrhage following injury or medical intervention. Menorrhagia was common and distressing. Negative carrier experiences were related in the determination of genotypic and phenotypic status, management, precautions and HCP attitude, including dismissing carriers' symptoms, concerns or requests for care. Carriers responded with mistrust, lost confidence, disappointment, fear, anxiety, doubt of self or child, discussing experiences, avoidance of healthcare and self-treatment. Dismissive HCP attitudes, ignorance about bleeding disorders in women and unique aspects of the carrier population appear to make errors more likely. This study indicates that carriers experience inappropriate care and encounter dismissive attitudes, and respond emotionally and behaviourally. Our model suggests that systematic medical errors aggravate a negative feedback loop leading to negative emotional and behavioural responses and worsening carrier care. Improved carrier care policies and increased awareness of women's bleeding disorders may improve this situation. Further research is needed to determine whether the themes identified in this study accurately reflect the experiences of carriers in general.