Studies have reported that paracrine signalling contributes to heart regeneration after myocardial infarction (MI). Since the epicardium is a signalling centre and cell source crucial for heart development and it is re-activated after MI, a potential therapeutic strategy for MI treatment would be to enhance its contribution to the injury response. The goal of this PhD thesis was to achieve in situ transfection of the epicardium to overexpress a combination of mitogenic growth factors (GF) that would enhance regenerative signalling pathways after Ml. To accomplish this goal, a novel transfection approach, consisting of a formulation of GF-expressing plasmids encapsulated in bioreducible pABOL (poly(cystamine bisacrylamide-co-4-amino-1-butanol) polyplexes and deposited onto flat and nanoneedle silicon surfaces by freeze drying, was developed. This approach demonstrated in vitro localised surface-mediated transfection necessary for the selective transfection of the thin epicardial layer. Moreover, preliminary results using ex vivo epicardial models demonstrated that primary embryonic epicardial cells were transfected using this formulation and that pABOL polyplexes were delivered to the surface of adult pig epicardial slices.
To select the GF genes to deliver to the epicardium, single and combinatorial proliferation assays using recombinant growth factors (rGF) were first performed in different cardiac cells (stem cell-derived cardiomyocytes, cardiac mesenchymal stromal cells, cardiac fibroblasts and endothelial cells). Neuregulin1-β1 (NRG1) consistently increased the S-phase proliferation marker in IMR90- and H7-derived cardiomyocytes and cardiomyocyte cell numbers (c.a. 30% increase). Hepatocyte Growth Factor (HGF) at 100 ng/mL induced proliferation of cardiac mesenchymal stromal cells (fold increase 2.75 ± 0.31) and angiogenesis (fold increase 1.47 ± 0.34). Follistatin-like 1, from animal and bacterial sources, was also tested but no proliferative effect was detected. After evaluating these rGFs proliferative potential, their overexpression by an epicardial cell line was achieved and the conditioned media (CdM) was collected for proliferation assays using single- and combinatorial-GF overexpressing CdM. Besides observing increased proliferation marker in cardiomyocytes and proliferation of endothelial cells when using basal epicardial CdM, relevant GF overexpressing-CdM, for each cell type, also enhanced the proliferation, supporting the previous rGF results. In both rGF and CdM proliferation assays, each of the GF maintained its positive proliferation effect when combined with other GFs.
This PhD project remains to be a first-in-field potential material-based gene delivery strategy specific to the epicardium that explores a combinatorial treatment via the delivery of multiple genes for induction of regenerative mechanisms for MI treatment.