Abstract Body: Background and Hypothesis: Diabetes impairs the reparative capacity of mesenchymal stromal cells (MSCs), limiting their therapeutic efficacy when used in cardiac repair following myocardial infarction (MI). We have previously demonstrated overexpression of Glypican 3 (GPC3) proteoglycan impairs diabetic MSC functions and that knockdown of GPC3 partially restores their therapeutic capacity of cardiac reparative functions. Because extracellular vesicles (EVs) mediate many paracrine signaling effects responsible for healing, we hypothesize that EVs derived from db/db-MSCs with a GPC3 knockdown would exhibit enhanced pro-reparative function as compared to unmodulated db/db-MSC-EVs.
Methods: Extracellular vesicles were isolated from wild type MSCs (wt-MSC), db/db-MSCs, and GPC3 knockdown db/db-MSCs using ultracentrifugation. Particle size and concentration were characterized by nanoparticle tracking analysis (NTA). Pro-angiogenic function was assessed in vitro using a tube formation assay with mouse cardiac endothelial cells (MCECs). Cell death was tested with neonatal rat ventricular myocytes (NRVM) and measured with Annexin V staining. Immunomodulation was investigated using RAW 264.7 macrophage polarization and EV treatment.
Results and Conclusions: NTA revealed comparable mean diameter across EV groups. Functionally, EVs from db/db-MSCs demonstrated significant impairment in tube formation capacity as compared to the wt-MSC EVs and GPC3-knockdown db/db-MSCs. In addition to pro-angiogenic properties, we also observed reduced apoptosis under stress conditions and exhibition of improved immune modulation properties in cells treated with GDC3-KD EVs compared to control diabetic-MSCs-EVs. These findings suggest that MSC-GPC3 knockdown restores reparative functions of diabetic MSC EVs. Ongoing studies will evaluate if these metabolically reprogrammed EVs also enhance cellular function in vitro and cardiac repair following MI, as well as characterize the functional cargo present in the EVs.