Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Abstract

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

Angiogenic microRNAs Linked to Incidence and Progression of Diabetic Retinopathy in Type 1 Diabetes

“…Diverse miRNAs can also act cooperatively or redundantly to regulate the effectors of the same biological process. miR-320a regulates glycolysis and represses angiogenic factors, including Flk1, VEGFc, insulin-like growth factor 1, insulin-like growth factor 1 receptor, and fibroblast growth factor (28,29). miR-320a has also been implicated in tumor angiogenesis by silencing NRP1 (30).…”

Endothelial cells and the IGF system

“…Angiogenesis is impaired in the diabetic heart, and a recent study has demonstrated that cardiomyocytes from rats with diabetes secreted exosomes containing miR320, which were subsequently taken up by endothelial cells (Wang et al 2014). This microRNA decreased expression of a number of protein targets including IGF1, and inhibited endothelial cell migration and tube formation.…”

En el Infarto agudo al miocardio los niveles plasmáticos de microvesículas extracelulares se elevan más precozmente que el aumento de la Troponina-I

“…Otros métodos se basan en el uso de matriz de polímeros que permiten su fácil y rápida obtención. Este método, por lo demás, es aplicable en clínica debido a su bajo costo, al reducido tiempo de realización y no requiere personal especializado 8,[15][16][17][18][19] . En nuestro laboratorio, hemos implementado la extracción de MVECs mediante el uso de ExoQuick®, el cual purifica una fracción enriquecida de exosomas, que además se recomienda para la caracterización de diversas moléculas como proteínas y RNA 10,16-18, 20, 21 .…”