Localized injection of miRNA-21-enriched extracellular vesicles effectively restores cardiac function after myocardial infarction

Abstract

Myocardial infarction (MI), a main cause of heart failure, leads to irreversible cardiomyocytes loss and cardiac function impairment. Current clinical treatments for MI are largely ineffective as they mostly aim to alleviate symptoms rather than repairing the injured myocardium. Thus, development of more effective therapies is compelling. This study aims to investigate whether the extracellular vesicles (EVs) carrying specific anti-apoptotic miRNA can be efficiently internalized into myocardium to achieve desired therapeutic outcomes. Methods : EVs were isolated from HEK293T cells overexpressing miRNA-21 (miR21-EVs) and identified. The RNase resistant rate of miR21-EVs was calculated by real-time PCR and compared with liposomes and polyethylenimine (PEI). Confocal laser scanning microscopy was used for visualizing the cellular internalization of miR21-EVs in primary cultured mouse neonatal cardiomyocytes (CMs), H9c2 rat cardiomyoblasts, and human umbilical vein endothelial cells (HUVECs). The effect of miR21-EVs on the expression of PDCD4, a pro-apoptotic protein that plays an important role in regulating myocardial apoptosis, was also evaluated in these three cell types by real-time PCR and Western blot analysis. In vivo , miR21-EVs was directly injected into the infarct zone following ligation of the left anterior descending of coronary artery in mice. The miR21-EVs distribution and blood vessel (capillary and arteriole) density were evaluated by immunofluorescence staining. Fluorescence in situ hybridization of miRNA-21 was also carried out to confirm the miR21-EVs distribution in vitro and in vivo . The protein level of PDCD4 in myocardium was assessed by immunohistochemical staining. The anti-apoptotic effect of miR21-EVs in cardiomyocytes and endothelial cells were measured using TUNEL staining. Four weeks after injection, the cardiac histological and functional recovery was evaluated by histochemistry staining and echocardiography, respectively. Results : In contrast to liposomes and PEI, EVs significantly inhibited miRNA-21 degradation. MiR21-EVs efficiently delivered miRNA-21 into cardiomyocytes and endothelial cells within 4 hours. Exogenous miRNA-21 in turn significantly reduced PDCD4 expression and attenuated cell apoptosis in vitro . Consistently and importantly, in a preclinical MI animal model, miRNA-21-loaded EVs effectively sent miRNA-21 into cardiomyocytes and endothelial cells, drastically inhibited cell apoptosis and led to significant cardiac function improvement. Conclusion : Our results suggest the cell-derived, genetically engineered EVs may be used therapeutically for the delivery of miRNAs for the rescue of MI and may benefit patients in the future.

Extracellular Endothelial Cell-Derived Vesicles: Emerging Role in Cardiac and Vascular Remodeling in Heart Failure

“…Additionally, CPC-derived EVs contain a distinct repertoire of biologically active miRNAs, such as microRNA-373 and microRNA-21, that have strongly yielded anti-fibrotic effects and ameliorated fibrosis in the infarcted area targeting key pro-fibrogenic genes, i.e., TGF-β, GDF-11, and ROCK-2 (51,52). Interestingly, EVs significantly inhibited microRNA-21 degradation and thereby mediate the anti-apoptotic effect in cardiac myocytes and endothelial cells (53). It has been demonstrated that the paracrine inhibitory impact of CPC on both cardiac fibroblast activation and collagen synthesis continues through cross-talk between cardiac fibroblasts and CPC-derived EVs (54).…”

“…For instance, mi-RNA-26a,−103, -and 107 have been shown to predominantly be regulators for insulin receptor function and free fatty acid metabolism (53,(110)(111)(112)(113)(114)(115)(116). Additionally, recent pre-clinical studies have revealed that mi-RNA-378 and mi-RNA-451 may play a crucial role in energy metabolism control through interacting with carnitine O-acetyltransferase, the peroxisome proliferator-activated receptor γ coactivator 1β, and LKB1/AMPK-signaling (117)(118)(119).…”

“…Additionally, EVs accumulate in the ischemic myocardium and regulate local inflammatory responses and vascular function through Erk1/2 STAT, Akt/MAPK-NF-κB signaling pathway (69,112). Therefore, NLRP3 inflammasomes and endothelial cell-derived EVs act as cargo for a wide spectrum There is evidence showing that mi-RNA-21, after a delivery into cardiac myocytes and endothelial cells, have reduced apoptosis through decreases in Programmed Cell Death gene-4 expression, inhibition of the extracellular inhibitor of the sprout regulated kinase 1 (Spry1), and stimulation of the expression of VEGF (53,67,113). In animal models, the protective effect of microRNA-21 against ischemia-induced myocardial damage was confirmed by diminished cell apoptosis around the infarcted areas after treatment with antibody vs. miRNA-21 (114).…”

Potential roles of extracellular vesicles in the pathophysiology, diagnosis, and treatment of autoimmune diseases

“…For studying EVs size and morphology, reliable methods are electron microscopy (EM) techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), Cryo-electron microscopy (Cryo-EM), and atomic force microscopy (AFM). Confocal fluorescence microscopy (CFM) is another advantageous technique for studying the interaction between EVs and cells [41][42][43]. This technique has resulted very convenient to study the internalization of EVs by recipient cells [42][43][44][45][46].…”

“…Confocal fluorescence microscopy (CFM) is another advantageous technique for studying the interaction between EVs and cells [41][42][43]. This technique has resulted very convenient to study the internalization of EVs by recipient cells [42][43][44][45][46]. Mrinali et al developed an innovative method that combines formalin with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to improving EV imaging in situ.…”