1- Scientific Objectives Our main objectives are to decode the molecular and cellular mechanisms involved in cardiac repair after injury and to develop efficient therapeutic approaches to circumvent the adverse remodeling occurring in patients with cardiovascular diseases.In particular, we: i) decipher the mechanisms of cardiac repair with a specific interest on the interface between inflammatory cells and cardiac repair including cardiac regeneration and tissue remodeling; ii) develop extracellular membrane vesicles-based therapeutic approaches for patients with cardiac diseases.
2- Main Results Mechanisms of cardiac repair Defective systemic and local iron metabolism correlates with cardiac disorders. Hepcidin, a master iron sensor, actively tunes iron trafficking. We hypothesized that hepcidin could play a key role to locally regulate cardiac homeostasis after acute myocardial infarction. We found that the expression of hepcidin was elevated after acute myocardial infarction and the specific deletion of hepcidin in cardiomyocytes failed to improve cardiac repair and function. However, transplantation of bone marrow-derived cells from hepcidin-deficient mice ( Hamp-/-) or from mice with specific deletion of hepcidin in myeloid cells (LysMCRE/+/ Hampf/f) improved cardiac function. This effect was associated with a robust reduction in the infarct size and tissue fibrosis in addition to favoring cardiomyocyte renewal. Macrophages lacking hepcidin promoted cardiomyocyte proliferation in a prototypic model of apical resection-induced cardiac regeneration in neonatal mice. Interleukin (IL)-6 increased hepcidin levels in inflammatory macrophages. Hepcidin deficiency enhanced the number of CD45+/CD11b+/F4/80+/CD64+/MHCIILow/chemokine (C-C motif) receptor 2 (CCR2)+ inflammatory macrophages and fostered signal transducer and activator of transcription factor-3 (STAT3) phosphorylation, an instrumental step in the release of IL-4 and IL-13. The combined genetic suppression of hepcidin and IL-4/IL-13 in macrophages failed to improve cardiac function in both adult and neonatal injured hearts. Hepcidin refrains macrophage-induced cardiac repair and regeneration through modulation of IL-4/IL-13 pathways (Zlatanova I et al, Circulation, 2019).
Regenerative therapies for cardiac repair. The rationale for the use of embryonic stem cells (ESC) in patients with heart failure primarily stems from the assumption that regeneration of scarred myocardium likely requires the supply of cells endowed with a true cardiomyogenic differentiation potential, regardless of whether they act by generating a new myocardial tissue by themselves or by harnessing endogenous repair pathways. This approach is made possible by the intrinsic pluripotentiality of ESC which allows to drive their fate in vitro towards a cardiac lineage and, so far, our approach has been to generate early SSEA-1-positive cardiac progenitors (rather than fully mature cardiomyocytes) with the assumption that the transplanted progenitors would use local cues to instruct them to differentiate into cardiomyocytes and vascular cells. Our experimental results have documented both the safety and efficacy of these ESC-derived progenitors, including in a clinically relevant scenario of allogeneic transplantation in nonhuman primates. Asides from ethical issues, the clinical translation of this ESC-based program has entailed a stepwise approach including the following steps: (1) the expansion of a clone of pluripotent hESC to generate a master cell bank under Good Manufacturing Practice conditions (GMP); (2) a growth factor-induced cardiac specification; (3) the purification of committed cells by immunomagnetic sorting to yield a SSEA-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (4) the incorporation of these cells into a fibrin scaffold; (5) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (6) an extensive cytogenetic and viral testing; and (7) the characterization of the final cell product and its release criteria (Menasché P et al, J Am Coll Cardiol, 2018). In parallel, we have attempted to more thoroughly elucidate the mechanism of action of these cells and, in this perspective, have investigated their paracrine effects. In a mouse model of chronic post-infarction cardiac failure, we have found that the functional benefits of the hESC-derived cardiac progenitors – similar to those currently tested in humans – could actually be duplicated by the sole administration of the extracellular membrane vesicles (EV) produced by the same cells and that both the parent cells and their secreted EV activated similar pathways in the recipient hearts (Kervadec A et al, J Heart Lung transplant, 2016, Figure 2). However, in a clinically-oriented perspective, we have kept cardiovascular progenitors as the candidate cells but have changed the pluripotent stem source they are derived from by switching from ESC to induced pluripotent stem cells (iPSC). 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. Myocardial infarction and three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM, iPSC-Pg, total EV secreted or phosphate-buffered saline 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. In vivo, EV outperformed cell injections, significantly improving cardiac function. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation (El Harane N et al, Eur Heart J, 2018). We also showed that intra-myocardial delivery of EV isolated from human iPSC-derived cardiovascular progenitor cells does not trigger an allogenic immune response and seems rather to induce a systemic anti-inflammatory effect. Our results therefore suggest that human EV administration might not require immunosuppression. It remains to establish whether the systemic delivery of EV would trigger similar anti-inflammatory effects, positively impacting heart function (Lima Correa B et al, Cardiovas Res, 2021).