The Addition of GFP to Exosomes Influences Cardioprotective Potential Through an RNA Binding Mechanism
Authors: Haar Lauren , Lynch Tom , Guo Amy , Wang Yang , Kuffel Gina , Jones W. Keith 
Published: June 2021

Exosomes are 40–100nm vesicles that have been extensively investigated for their therapeutic potential. Exosomes isolated from mesenchymal stem cells (MSCs) are shown to have cardioprotective potential both in models of infarction and heart failure. Our previous work supports that this cardioprotection occurs through the delivery of microRNAs (specifically, mir21a‐5p). We observed that with the addition of a green fluorescent protein (GFP), the protective potential of MSC derived exosomes decreases. In order to better understand this observed effect, we addressed the hypothesis that the modification of MSCs with GFP limits the cardioprotective potential of the exosomes by binding cardioprotective RNA and limiting its functional delivery to target cells.Extracellular vesicles (EVs) were isolated via ultracentrifugation from C57BL/6 derived MSCs that are transduced with GFP producing plasmids. Confirmation of exosomal content was done via exosomal protein marker analysis (LAMP1, CD63) with Amnis Imaging Flowcytometry. Populations in the GFP containing MSC‐derived exosomes showed detectable GFP signal and were validated to contain exosomal protein markers. GFP protein was immunoprecipitated from isolated EVs. The Qubit Fluorometer and Agilent 2100 Bioanalyzer were used to assess the integrity and concentration and indicated that the RNA is measurable in MSC derived EVs (both GFP and wild type). The isolated miRNA was prepared into libraries of template molecules and sequenced on the Illumina MiSeq. Analyses of the derived data show that miRNA components of EVs vary with the addition of GFP. However, miR21a‐5p was shown to be the most abundant in both wild type (WT) and GFP containing groups. Functionally, RNA delivery via GFP containing exosomes into H9c2 cardiomyoblasts does not show protection against cell death in a simulated ischemia/reperfusion model. In vivo murine models also indicate that exosomes isolated from GFP‐MSCs do not confer protection against left anterior descending coronary artery (LAD) occlusion‐induced myocardial infarction, as exosomes isolated from WT‐MSCs were shown to do. Further, real‐time RT‐PCR showed that miR‐21a‐5P is present in GFP‐associated RNA.Analysis of MiSeq data from WT and GFP‐MSC exosomes showed no major differences in miR‐21a‐5P levels. However, after purification of GFP‐associated RNA, we found that this RNA contains miR‐21a‐5P, suggesting that at least some amount of this miR is bound to GFP in the GFP‐containing exosomes. Future work will establish the profiles of miRNAs associated with GFP in order to develop a profile of cardioprotective exosomal content, and better understand the potential for GFP binding and transfection of miRNAs.Support or Funding InformationThis work was supported by the National Institutes of Health under award number R01HL091478 to WK Jones.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.