DIABETES IMPAIRS THERAPEUTIC EFFECT OF ENDOTHELIAL PROGENITOR CELL EXOSOME-MEDIATED MYOCARDIAL REPAIR

Abstract

Myocardial infarction (MI) frequently occurs in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (BM-EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Lately, emerging evidence supports that exosomes (Exo), a family of extracellular vesicles, mediate stem cell therapy by carrying cell-specific biological cargo and by inducing signaling via transferring of bioactive molecules to target cells. Despite promising results of stem cells/Exo in preclinical studies, autologous cell-based therapies yielded modest clinical results, suggesting cellular/Exo reparative function may be compromised by the presence of comorbid diseases including complications associated with diabetes. Recent studies suggest that epigenetic mechanisms, such as histone and DNA modifications for gene silencing, promote diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content that compromise their reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic (db/+) and diabetic (db/db) mice and examined their reparative effect in vitro and on permanent left anterior descending (LAD) coronary artery ligation and ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo. Our data demonstrated that compared to non-diabetic EPC-Exo, diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival. In vivo studies revealed that non-diabetic EPC-Exo treatments improved cardiac function and remodeling while diabetic EPC-Exo significantly depressed cardiac function, reduced capillary density, increased fibrosis in the permanent LAD ligation MI injury. Moreover, in the I/R MI model, we found that non-diabetic EPC-Exo mediated cardio-protection was lost compared with diabetic-EPC-Exo, and diabetic-EPC-Exo increased immune cell infiltration, infarcted area, and plasma cardiac troponin-I. Mechanistically, histone 3 lysine 9 acetylation (H3K9Ac), a gene activating histone modification, expression was decreased in mouse cardiac endothelial cells (MCECs) treated with db/db EPC-Exo compared with db/+ EPC-Exo, suggesting diabetic EPC-Exo inhibits endothelial cell gene expression. The H3K9Ac chromatin immunoprecipitation sequencing (ChIP-Seq) results further revealed that diabetic EPC-Exo reduced H3K9Ac level on angiogenic, cell survival, and proliferative genes in MCECs. Moreover, we found that a small molecular inhibitor of HDACs, valproic acid (VPA), effectively prevented diabetic EPC-Exo-medicated H3K9Ac reduction, indicating VPA may rescue the beneficial gene expression and cell function. Taken together, our results provide evidence that diabetic EPC-Exo reparative function is impaired in the ischemic heart and this may be through HDACs-mediated H3K9Ac downregulation leading to inhibition of beneficial genes in recipient cardiac endothelial cells. Reversing diabetic EPC-Exo function by treating with HDAC inhibitors may provide a new path for autologous exosome therapy for myocardial repair in diabetic patients. However, questions still remain on what the content change of stem cell-derived exosome under diabetic condition is.Emerging evidence support a key role of variety of stem /progenitor cell-secreted Exo as a pivotal paracrine entity to mitigate cardiovascular injury. Beside EPC-, cortical bone stem cell (CBSC)-, and cardiac stem/progenitor cell (CPC)- derived Exo are adequate to enhance cardiac repair and regeneration after injury. As widely acknowledged, the comorbidities such as hyperglycemia is a characteristic of diabetes and a major driving factor in CVD. The functional role of stem/progenitor cell- derived Exo and molecular signature of their secreted Exo cargo under hyperglycemic conditions remain elusive. Therefore, we hypothesize that hyperglycemic stress causes transcriptome changes in stem/progenitor cell- derived Exo that may compromise their reparative function. To identify the content change in Exo under hyperglycemia, we performed an unbiased Exo transcriptome signatures from 3 different aforementioned stem/progenitor cells by next generation exosome RNA sequencing (RNA-seq). The results indicated that the size and number of Exo were not changed from 3 stem/progenitor cells between normal and high glucose groups. Furthermore, analysis revealed differential expression of variety of RNA species in Exo and the portions of different RNA were change under hyperglycemia. Specifically, we identified 241 common-dysregulated mRNAs, 21 ncRNAs and 16 miRNAs in three stem cell-derived Exo. Based on mRNA data, Gene Ontology (GO) revealed that potential function of common mRNAs mostly involved in metabolism and transcriptional regulation. We also provided the detail information of these non-annotated ncRNAs and the potential mRNA targets by miRNA-mRNA prediction. This study not only provides potential candidates for individual stem cell types but also identifies common genes in response to hyperglycemia. These reference data are critical for future biological studies and application of stem/progenitor cell-derived Exo in ischemic heart or other diseases to prevent the adverse effects of hyperglycemia-induced stem/progenitor cell- derived Exo dysfunction.