INFLAMMATION ALTERS ENDOTHELIAL PROGENITOR CELL-DERIVED EXOSOME CONTENTS AND THERAPEUTIC EFFECT ON MYOCARDIAL REPAIR

Abstract

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide and Myocardial Infarction (MI) and subsequent heart failure remains the leading cause for death. Despite the improvement in prognosis and treatment of acute MI patients, the underlying causes including loss of cardiomyocytes and microvasculature remain potential risk and lack proper and efficient solutions. Stem cell-based therapies for repair and regeneration have evolved and have been applied in clinical trials. Different types of stem cells, including Endothelial progenitor cell (EPC), Mesenchymal Stem Cell (MSC), induced Pluripotent Stem Cell (iPSC) and cardiac progenitor cells etc. have been used for potential long term recovery and cardiac regeneration. However, results from the clinical trials have been largely disappointing and improvement in cardiac functions have been modest likely due to the limitations of cell therapy including low integration in myocardium, poor survival, cellular dysfunction and limited differentiation ability. It is therefore necessary and urgent to develop cell free alternatives as next generation regenerative therapies. There is a consensus that the beneficial effect of stem cell therapy is largely due to paracrine effects. Exosomes have recently emerged as important functional units mediating stem cell paracrine effects. Exosomes are the family of extracellular vesicles (EV) which are 30-150nm in size, secreted by almost all types of cells and responsible for cell-cell communication via delivering their cargo including RNAs and proteins to host cells. Studies from our and other labs have shown that exosomes mimic parental stem cell in improving post-MI functions. The essential feature of exosome is decided by their cargo including RNA and protein, which are subject to dynamic changes depending on the environment of parental cells. Our studies were focused on Endothelial Progenitor Cell (EPC)-derived exosomes. EPCs are generated in bone marrow, and home to the site of tissue injury and orchestrate neovascularization and tissue repair. Patients with ischemic heart disease, are usually accompanied with comorbidities such as systemic inflammation, aging, diabetes, etc. which are known to compromise EPC functions. We hypothesized that EPCs under inflammatory stress produce dysfunctional exosomes with altered RNA and protein content, leading to impaired cardiac reparative properties. We chose interleukin-10 knockout (IL-10KO) mice as a model of systemic inflammation. EPCs were isolated from IL-10KO and wild-type (WT) mice, and their exosomes (Exo) were compared for their reparative properties both in vitro and in vivo. Our in vitro studies showed WT-EPC-Exo treatment attenuated recipient cell apoptosis, enhanced cell mobilization and tube formation, whereas IL-10KO-EPC-Exo were functionally deficient or even had detrimental effects. We used MI mouse model to compare the in vivo function of two groups of exosomes and found WT-EPC-Exo treatment significantly improved left ventricular (LV) cardiac function, inhibited cell death, promoted angiogenesis and attenuated cardiac remodeling; while these cardioprotective effects were lost in IL-10KO-EPC-Exo treated group. Both in vitro and in vivo studies proved that even the same progenitor cell type (EPCs), under inflammatory stimulus (IL-10KO), secretes exosomes with different reparative properties. Next, we explored whether the observed difference in exosome function is caused by altered exosome content. Using Next Generation RNA Sequencing (NGS RNAseq) and mass spectrometry we found RNA and protein expression patterns were drastically different in wild type and IL-10 knockout EPC derived exosomes. This evidence leads to the conclusion that alteration in exosome content is fundamental for exosome function. We picked two candidates that are highly enriched in IL-10KO-EPC-Exo for further study, miR-375 and Integrin-Linked Kinase (ILK). We treated IL-10KO-EPC with anti-miR against miR-375 and siRNA against ILK separately, and successfully decreased the expression of miR-375 and ILK in both EPCs and EPC derived exosomes. Then we explored the function of those miR and protein ‘modified exosomes’ with similar in vitro and in vivo experiments as previously described. Compared to IL-10KO-EPC-Exo, miR-375 knockdown exosomes showed enhanced angiogenesis and inhibited cell apoptosis, while ILK knockdown in exosomes rescued functions in both in vitro and in vivo experiments. These results suggested the possibility that exosome manipulation of identified factors may partially rescue their reparative functionality. In summary, our studies revealed that stem cell derived exosomes are capable for independent cardiac repair in ischemic heart disease, however, parental stem cells under pathological stimulus secrete dysfunctional exosomes with altered RNA and protein content. Exosome function can be rescued or enhanced through RNA and protein content modification.