Promoting Angiogenesis in Bioartificial Grafts towards enhanced myocardial restoration

Abstract

Myocardial restoration via cell therapy and cardiac tissue engineering is limited by impaired graft survival. To limit the sequelae of myocardial ischemia it is crucial to counteract oxidative stress while promoting neovascularization in the area of injury and within the bioengineered tissue. We hypothesized that: (1) supplementation with ascorbic acid (AA) improves donor cell viability in vitro and in vivo, as well as angiogenesis and remodeling of thick myocardial artificial grafts (MAG), suitable for implantation and myocardial repair; and (2) epicardial implantation of an ascorbic acid- enriched myocardial artificial graft, which has been pre-vascularized in the recipients¿ own body, promotes restoration of the ischemic heart. In experiments conducted to test our first hypothesis, MAG were generated in vitro by populating an FDA-approved gelatin scaffold with GFP-Luciferase- expressing rat cardiomyoblasts (H9C2-GFP-Fluc). We found that MAG supplementation with ascorbic acid (5 or 50 µmol/L) improved cell survival significantly, reduced apoptosis, and enhanced H9C2 cell myogenic differentiation in vitro. Furthermore, a novel model of graft prevascularization ¿ a renal pouch model, which provides the graft with blood vessels of autologous origin-, was developed in healthy Wistar rats. In our in vivo model using the renal pouch, AA enrichment improved donor cell survival and promoted neovascularization within the MAG. To test our second hypothesis, an ascorbic acid- enriched pre-vascularized MAG was implanted as a patch into the same rat¿s ischemic heart following myocardial infarction (MI). MAG-treated animals (MAG group, n=6) were compared to untreated infarcted animals as injury controls (MI group, n=6) and sham operated rats as healthy controls (healthy group, n=7). Echocardiographic, hemodynamic, and histological assessments 4 weeks after implantation indicated that AA-enriched pre-vascularized- grafts induced a robust angiogenic response in ischemic rat hearts, attenuated left ventricular (LV) remodeling and preserved LV function. In conclusion, in the current study we have indentified ascorbic acid as a suitable supplement to enhance cell survival and neovascularization in tissue engineering based therapies. Furthermore, we demonstrated the importance of promoting angiogenesis for successful post-ischemic myocardial repair using three-dimensional cardiac patches. With our approach, viability support (cell therapy and antioxidant effects), structural support (prevention of remodeling) and revascularization (stimulation of angiogenesis) have been addressed in the same setting in an acute model of myocardial repair. Also, the utilization of FDA approved compounds, as well as MAG vascularization with blood vessels of autologous origin, makes this strategy plausible for use in the clinical arena and applicable to various donor cell types (ideally of autologous origin), other organs and regenerative interventions.