Small extracellular vesicle-mediated CRISPR-Cas9 RNP delivery for cardiac-specific genome editing.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo, we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease.

A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride-induced neuronal death.

Autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis. Dysfunction of autophagy is associated with the progression of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although one of the typical features of brain aging is an accumulation of redox-active metals that eventually lead to neurodegeneration, a plausible link between trace metal-induced neurodegeneration and dysregulated autophagy has not been clearly determined. Here, we used a cupric chloride-induced neurodegeneration model in MN9D dopaminergic neuronal cells along with ultrastructural and biochemical analyses to demonstrate impaired autophagic flux with accompanying lysosomal dysfunction. We found that a surge of cytosolic calcium was involved in cupric chloride-induced dysregulated autophagy. Consequently, buffering of cytosolic calcium by calbindin-D28K overexpression or co-treatment with the calcium chelator BAPTA attenuated the cupric chloride-induced impairment in autophagic flux by ameliorating dysregulation of lysosomal function. Thus, these events allowed the rescue of cells from cupric chloride-induced neuronal death. These phenomena were largely confirmed in cupric chloride-treated primary cultures of cortical neurons. Taken together, these results suggest that abnormal accumulation of trace metal elements and a resultant surge of cytosolic calcium leads to neuronal death by impairing autophagic flux at the lysosomal level.

Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy.

Small-interfering RNA (siRNA) therapy is considered a powerful therapeutic strategy for treating cardiac hypertrophy, an important risk factor for subsequent cardiac morbidity and mortality. However, the lack of safe and efficient in vivo delivery of siRNAs is a major challenge for broadening its clinical applications. Small extracellular vesicles (sEVs) are a promising delivery system for siRNAs but have limited cell/tissue-specific targeting ability. In this study, a new generation of heart-targeting sEVs (CEVs) has been developed by conjugating cardiac-targeting peptide (CTP) to human peripheral blood-derived sEVs (PB-EVs), using a simple, rapid and scalable method based on bio-orthogonal copper-free click chemistry. The experimental results show that CEVs have typical sEVs properties and excellent heart-targeting ability. Furthermore, to treat cardiac hypertrophy, CEVs are loaded with NADPH Oxidase 4 (NOX4) siRNA (siNOX4). Consequently, CEVs@siNOX4 treatment enhances the in vitro anti-hypertrophic effects by CEVs with siRNA protection and heart-targeting ability. In addition, the intravenous injection of CEVs@siNOX4 into angiotensin II (Ang II)-treated mice significantly improves cardiac function and reduces fibrosis and cardiomyocyte cross-sectional area, with limited side effects. In conclusion, the utilization of CEVs represents an efficient strategy for heart-targeted delivery of therapeutic siRNAs and holds great promise for the treatment of cardiac hypertrophy.

Asymmetric dimethylation of AMPKα1 by PRMT6 contributes to the formation of phase-separated puncta.

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase comprising α, ß, and γ subunits. AMPK is involved in intracellular energy metabolism and functions as a switch that turns various biological pathways in eukaryotes on and off. Several post-translational modifications regulating AMPK function have been demonstrated, including phosphorylation, acetylation, and ubiquitination; however, arginine methylation has not been reported in AMPKα1. We investigated whether arginine methylation occurs in AMPKα1. Screening experiments revealed arginine methylation of AMPKα1 mediated by protein arginine methyltransferase 6 (PRMT6). In vitro methylation and co-immunoprecipitation assays indicated that PRMT6 can directly interact with and methylate AMPKα1 without involvement of other intracellular components. In vitro methylation assays with truncated and point mutants of AMPKα1 revealed that Arg403 is the residue methylated by PRMT6. Immunocytochemical studies showed that the number of AMPKα1 puncta was enhanced in saponin-permeabilized cells when AMPKα1 was co-expressed with PRMT6, suggesting that PRMT6-mediated methylation of AMPKα1 at Arg403 alters the physiological characteristics of AMPKα1 and may lead to liquid-liquid phase separation.

Generation of two PITX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

PITX2 is a homeobox gene located in the human 4q25 locus and is commonly associated with atrial fibrillation (AF). Here, we generated two PITX2 knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 genome editing. The edited iPSCs maintained fullpluripotency, normal karyotype and spontaneousdifferentiation capability. This cell line provides a suitable model for investigating the physiopathologyof PITX2 mutation in atrial fibrillation.

Generation of three TTN knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system.

TTN mutations are the common genetic cause for various types of cardiomyopathies (e.g., dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy) and skeletal myopathies. Here, we generated three TTN knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 system. These cell lines, which exhibit normal karyotype, typical morphology and pluripotency, could provide useful platform for investigating the role of TTN in associated disorders.

Generation of a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell line using CRISPR/Cas9 system.

E192K missense mutation of TPM1 has been found in different types of cardiomyopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction), leading to systolic dysfunction, diastolic dysfunction, and/or tachyarrhythmias. Here, we generated a heterozygous TPM1-E192K knock-in human induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-based genome editing system. The cells exhibit normal karyotype, typical stem cell morphology, expression of pluripotency markers and differentiation ability into three germ layers. Accordingly, this cell line could provide a useful cell resource for exploring the pathogenic role of TPM1-E192K mutation in different types of cardiomyopathies.

Serum exosomal long noncoding RNAs as a diagnostic biomarker for atrial fibrillation.

BACKGROUND: Exosomal long noncoding RNAs (lncRNAs) are known as ideal diagnostic biomarkers of various diseases. However, there are no reports on the use of serum exosomal lncRNAs as diagnostic biomarkers for atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to explore serum exosomal lncRNAs as a useful tool for diagnosing AF. METHODS: Serum exosomes from patients with persistent AF and controls were isolated using a polymer-based exosome precipitation kit. We conducted a multiphase process including screening and 2 independent validation phases. In the screening phase, serum exosomal lncRNA expression profiles were examined using RNA sequencing analysis. In 2 validation phases, we evaluated the expression levels of candidate exosomal lncRNAs using quantitative reverse transcription polymerase chain reaction. Finally, we performed different statistical and functional analyses. RESULTS: After the screening phase, we identified 26 differentially expressed lncRNAs (ie, 15 upregulated and 11 downregulated lncRNAs with a |fold change| ≥2 and P <.05) in serum exosomes from patients with persistent AF compared with controls. We then screened out 6 exosomal lncRNAs as biomarker candidates following parameters: read length ≥200 nucleotides; exon number ≥2; and coding potential score <0.1. In 2 validation phases, exosomal lncRNAs LOC105377989 and LOC107986997 were consistently upregulated in the serum of patients with persistent AF compared with controls (P <.0001). Moreover, both exosomal lncRNAs exhibited significant diagnostic validity for AF. Notably, exosomal lncRNA LOC107986997 was involved in AF-related pathophysiological mechanisms. CONCLUSION: Serum-derived exosomal lncRNA LOC107986997 could serve as a potential biomarker for AF diagnosis.

Small extracellular vesicles derived from patients with persistent atrial fibrillation exacerbate arrhythmogenesis via miR-30a-5p.

Small extracellular vesicles (sEVs) are nanometer-sized membranous vesicles that contribute to the pathogenesis of atrial fibrillation (AF). Here, we investigated the role of sEVs derived from patients with persistent AF in the pathophysiology of AF. First, we evaluated the pathological effects of sEVs derived from the peripheral blood of patients with persistent AF (AF-sEVs). AF-sEVs treatment reduced cell viability, caused abnormal Ca2+ handling, induced reactive oxygen species (ROS) production and led to increased CaMKII activation of non-paced and paced atrial cardiomyocytes. Next, we analyzed the miRNA profile of AF-sEVs to investigate which components of AF-sEVs promote arrhythmias, and we selected six miRNAs that correlated with CaMKII activation. qRT-PCR experiment identified that miR-30a-5p was significantly down-regulated in AF-sEVs, paced cardiomyocytes, and atrial tissues of patients with persistent AF. CaMKII was predicted by bioinformatics analysis as a miR-30a-5p target gene and validated by a dual luciferase reporter; hence, we evaluated the effects of miR-30a-5p on paced cardiomyocytes and validated miR-30a-5p as a pro-arrhythmic signature of AF-sEVs. Consequently, AF-sEVs-loaded with miR-30a-5p attenuated pacing-induced Ca2+-handling abnormalities, whereas AF-sEVs-loaded with anti-miR-30a-5p reversed the change in paced cardiomyocytes. Taken together, the regulation of CaMKII by miR-30a-5p revealed that miR-30a-5p is a major mediator for AF-sEVs-mediated AF pathogenesis. Accordingly, these findings suggest that sEVs derived from patients with persistent AF exacerbate arrhythmogenesis via miR-30a-5p.

RNF166 plays a dual role for Lys63-linked ubiquitination and sumoylation of its target proteins.

Ubiquitination and sumoylation are two important posttranslational modifications in cells. RING (Really Interesting New Gene)-type E3 ligases play essential roles in regulating a plethora of biological processes such as cell survival and death. In our previous study, we performed a microarray using inputs from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine and identified a novel RING-type E3 ligase, RNF166. We showed that RNF166 exerts proapoptotic effects via ubiquitin-dependent degradation of X-linked inhibitor of apoptosis and subsequent overactivation of caspase-dependent neuronal death following 6-hydroxydopamine treatment. In the present study, we further expanded the list of RNF166's binding substrates using mass spectral analyses of immunoprecipitates obtained from RNF166-overexpressing HEK293 cells. Poly (ADP-ribose) polymerase 1, ATPase WRNIP1, X-ray repair cross-complementing protein 5 (Ku80), and replication protein A 70 were identified as potential binding partners of RNF166. Additionally, we confirmed that RNF166 interacts with and forms lysine 63-linked polyubiquitin chains in Ku80. Consequently, these events promoted the increased stability of Ku80. Intriguingly, we found that RNF166 also contains distinct consensus sequences termed SUMO-interacting motifs and interacts with apoptosis signal-regulating kinase 1 (ASK1). We determined that RNF166 induces the sumoylation of ASK1. Overall, our data provide novel evidence that RNF166 has a dual function of Lys63-linked ubiquitination and sumoylation of its cellular targets.

Improved cardiac-specific delivery of RAGE siRNA within small extracellular vesicles engineered to express intense cardiac targeting peptide attenuates myocarditis.

Small extracellular vesicles (sEVs) are nanometer-sized membranous vesicles secreted by cells, with important roles in physiological and pathological processes. Recent research has established the application of sEVs as therapeutic vehicles in various conditions, including heart disease. However, the high risk of off-target effects is a major barrier for their introduction into the clinic. This study evaluated the use of modified sEVs expressing high levels of cardiac-targeting peptide (CTP) for therapeutic small interfering RNA (siRNA) delivery in myocarditis, an inflammatory disease of heart. sEVs were extracted from the cell culture medium of HEK293 cells stably expressing CTP-LAMP2b (referred to as C-sEVs). The cardiac targeting ability of C-sEVs with the highest CTP-LAMP2b expression was >2-fold greater than that of normal sEVs (N-sEVs). An siRNA targeting the receptor for advanced glycation end products (RAGE) (siRAGE) was selected as a therapeutic siRNA and loaded into C-sEVs. The efficiency of cardiac-specific siRNA delivery via C-sEVs was >2-fold higher than that via N-sEVs. Furthermore, siRAGE-loaded C-sEVs attenuated inflammation in both cell culture and an in vivo model of myocarditis. Taken together, C-sEVs may be a useful drug delivery vehicle for the treatment of heart disease.

Dysregulated autophagy is linked to BAX oligomerization and subsequent cytochrome c release in 6-hydroxydopmaine-treated neuronal cells.

Autophagy and apoptosis are essential physiological pathways that are required to maintain cellular homeostasis. Therefore, it is suggested that dysregulation in both pathways is linked to several disease states. Moreover, the crosstalk between autophagy and apoptosis plays an important role in pathophysiological processes associated with several neurodegenerative disorders. We have previously reported that 6-hydroxydopamine (6-OHDA)-triggered reactive oxygen species (ROS) induces dysregulated autophagy, and that a dysregulated autophagic flux contributes to caspase-dependent neuronal apoptosis. Based on our previous findings, we specifically aimed to elucidate the molecular mechanisms underlying the potential role of dysregulated autophagy in apoptotic neurodegeneration. The disuccinimidyl suberate (DSS) cross-linking assay and immunological analyses indicated that exposure of several types of cells to 6-OHDA resulted in BAX activation and subsequent oligomerization. Pharmacological inhibition and genetic perturbation of autophagy prevented 6-OHDA-induced BAX oligomerization and subsequent release of mitochondrial cytochrome c into the cytosol and caspase activation. These events were independent of expression levels of XIAP. Taken together, our results suggest that BAX oligomerization comprises a critical step by which 6-OHDA-induced dysregulated autophagy mediates neuronal apoptosis.

Co-delivery of curcumin and miRNA-144-3p using heart-targeted extracellular vesicles enhances the therapeutic efficacy for myocardial infarction.

Curcumin exerts therapeutic effects in heart disease, but has limited bioavailability. Extracellular vesicles (EVs) have gained attention as nanovehicles; however, the poor targeting ability of systemically administered EVs still remains a crucial issue. Herein, we generated heart-targeted EVs (CTP-EVs) by functionalizing EVs surface with cardiac targeting peptide (CTP) using genetic modification of EVs-secreting cells, and further loaded curcumin into CTP-EVs (CTP-EVs-Cur). Consequently, CTP-EVs were able to specifically deliver curcumin to the heart. In addition, curcumin-loaded CTP-EVs possess improved bioavailability, and are fully functional with a high cardioprotective efficiency. Moreover, we loaded miR-144-3p in CTP-EVs-Cur following validation of miR-144-3p as a major contributor in curcumin-mediated therapeutic effects. The simultaneous packing of curcumin and miR-144-3p in CTP-EVs not only retains the active heart-targeting ability but also achieves enhanced cardioprotective effects both in vitro and in vivo, indicating the possibility of combining and sustaining their therapeutic potential by simultaneously loading in CTP-EVs. Therefore, CTP-EVs could be a potential and effective strategy for the delivery of therapeutic molecules, thereby providing a promising nanomedicine for MI therapy.

RING-finger protein 166 plays a novel pro-apoptotic role in neurotoxin-induced neurodegeneration via ubiquitination of XIAP.

The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been widely utilized to establish experimental models of Parkinson disease and to reveal the critical molecules and pathway underlying neuronal death. The profile of gene expression changes following 6-OHDA treatment of MN9D dopaminergic neuronal cells was investigated using a TwinChip Mouse-7.4K microarray. Functional clustering of altered sets of genes identified RING-finger protein 166 (RNF166). RNF166 is composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif. RNF166 localized in the cytosol and nucleus. At the tissue level, RNF166 was widely expressed in the central nervous system and peripheral organs. In the cerebral cortex, its expression decreased over time. In certain conditions, overexpression of RNF166 accelerates the naturally occurring neuronal death and 6-OHDA-induced MN9D cell death as determined by TUNEL and annexin-V staining, and caspase activation. Consequently, 6-OHDA-induced apoptotic cell death was attenuated in RNF166-knockdown cells. In an attempt to elucidate the mechanism underlying this pro-apoptotic activity, binding protein profiles were assessed using the yeast two-hybrid system. Among several potential binding candidates, RNF166 was shown to interact with the cytoplasmic X-linked inhibitor of apoptosis (XIAP), inducing ubiquitin-dependent degradation of XIAP and eventually accelerating caspase activation following 6-OHDA treatment. RNF166's interaction with and resulting inhibition of the XIAP anti-caspase activity was further enhanced by XIAP-associated factor-1 (XAF-1). Consequently, depletion of RNF166 suppressed 6-OHDA-induced caspase activation and apoptotic cell death, which was reversed by XIAP knockdown. In summary, our data suggest that RNF166, a novel E3 ligase, plays a pro-apoptotic role via caspase activation in neuronal cells.

Calpain-mediated cleavage of Fbxw7 during excitotoxicity.

Neuronal cell death induced by ischemic injury has been attributed to glutamate receptor-mediated excitotoxicity, which is known to be accompanied by Ca2+ overload in the cytoplasm with concomitant activation of calcium-dependent mechanisms. More specifically, the overactivation of calpains, calcium-dependent cysteine proteases, have been associated with neuronal cell death following glutamate treatment. Previously, we observed decreased expression levels of F-box/WD repeat domain-containing protein 7 (Fbxw7) after the hyperactivation of cyclin-dependent kinase 5 (Cdk5) in cortical neurons challenged with glutamate. As determined using in vitro calpain cleavage assays, we demonstrated that the cleavage of Fbxw7 was mediated by activated calpain and attenuated in the presence of the calpain inhibitor, calpeptin. Using the rat middle cerebral artery occlusion model, we confirmed that Fbxw7 was indeed cleaved by activated calpain in the ipsilateral cortex. Based on our data, we hypothesize that the negative regulation of Fbxw7 by calpain may contribute to neuronal cell death and that the preservation of Fbxw7 by the inhibition of calpain, Cdk5, or both composes a novel protective mechanism following excitotoxicity.

Therapeutic potential of miR-21 regulation by human peripheral blood derived-small extracellular vesicles in myocardial infarction.

Small extracellular vesicles (sEVs) as natural membranous vesicles are on the frontiers of nanomedical research, due to their ability to deliver therapeutic molecules such as microRNAs (miRNAs). The miRNA-21 (miR-21) is thought to be involved in the initiation and development of myocardial infarction (MI). Here, we examined whether miR-21 regulation using human peripheral blood-derived sEVs (PB-sEVs) could serve as a potential therapeutic strategy for MI. First, we examined miR-21 levels in hypoxic conditions and validated the ability of PB-sEVs to serve as a potential delivery system for miRNAs. Further, bioinformatics analysis and luciferase assay were performed to identify target genes of miR-21 mechanistically. Among numerous target pathways, we focused on nitrogen metabolism, which remains relatively unexplored compared with other possible miR-21-mediated pathways; hence, we aimed to determine novel target genes of miR-21 related to nitrogen metabolism. In hypoxic conditions, the expression of miR-21 was significantly up-regulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The down-regulation of miR-21 expression by PB-sEVs loaded with anti-miR-21 significantly improved survival rates, consistent with the augmentation of cardiac function. However, the up-regulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and down-regulated the mRNA and protein expression of striatin (STRN), which could regulate NOS3 expression. In conclusion, we identified a novel therapeutic strategy to improve cardiac function by regulating the expression of miR-21 with PB-sEVs as an miR-21 or anti-miR-21 delivery vehicle and confirmed the miR-21-associated nitrogen metabolic disorders in MI.

Anamorsin attenuates cupric chloride-induced dopaminergic neuronal cell death.

Neurodegenerative diseases are associated with elevated levels of metal elements, which are well-known inducers of reactive oxygen species (ROS) in cells. Because dopaminergic neurons in the substantia nigra are vulnerable to ROS, dysregulation of metals and the resulting accumulation of ROS could be a cause of dopaminergic neurodegeneration. In this study, we showed that overexpression of anamorsin protected MN9D dopaminergic neuronal cells from cupric chloride-induced death. This cytoprotection was achieved by specifically decreasing ROS levels. As determined by mini two-dimensional electrophoretic assay, an acidic shift of anamorsin occurred during drug-induced death, which seemed to be mediated by oxidative modification of three of its CXXC motifs. Consequently, drug-induced dissociation of ASK1 from Trx1 and subsequent phosphorylation of JNK and p38 MAPK were inhibited in MN9D cells overexpressing anamorsin. Taken together, our results indicate that anamorsin exerts a neuroprotective effect by reducing intracellular ROS levels and subsequently attenuating activated stress-activated MAP kinases pathways.

Site-specific phosphorylation of Fbxw7 by Cdk5/p25 and its resulting decreased stability are linked to glutamate-induced excitotoxicity.

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine protein kinase that regulates brain development and neurodegeneration. Cdk5 is activated by p25 that is generated from calpain-dependent cleavage of p35. The generation of p25 is responsible for the aberrant hyper-activation of Cdk5, which causes neurodegeneration. Using in vitro assays, we discovered that F-box/WD repeat-containing protein 7 (Fbxw7) is a new substrate of Cdk5. Additionally, Cdk5-dependent phosphorylation of Fbxw7 was detected in the presence of p25, and two amino acid residues (S349 and S372) were determined to be major phosphorylation sites. This phosphorylation was eventually linked to decreased stability of Fbxw7. Using a culture model of cortical neurons challenged with glutamate, we confirmed that decreased stability of Fbxw7 was indeed Cdk5-dependent. Furthermore, diminished levels of Fbxw7 led to increased levels of transcription factor AP-1 (c-Jun), a known substrate of Fbxw7. Given that previous reports demonstrate that c-Jun plays a role in accelerating neuronal apoptosis in these pathological models, our data support the concepts of a molecular cascade in which Cdk5-mediated phosphorylation of Fbxw7 negatively regulates Fbxw7 expression, thereby contributing to neuronal cell death following glutamate-mediated excitotoxicity.

Calcium chloride enhances the delivery of exosomes.

Exosomes might have an unimproved potential to serve as effective delivery vehicles. However, when exosomes are developed for therapeutic applications, a method to enhance their delivery is important. This study aimed to evaluate wheather calcium chloride (CaCl2) or other chloride compounds could enhance exosome delivery to various cells without causing toxicity. Exosomes were purified from human serum by using the ExoQuick exosome precipitation kit. Isolated exosomes were mixed with CaCl2 at concentrations ranging from 100 µM to 1 mM, and then washed using Amicon filter for treating the cells. The delivery efficiency of exosomes and the viability of the cells [HEK 293 (human kidney cells) and H9C2 (rat cardiomyocytes)] were evaluated. Cellular uptake of exosomes was observed using a confocal microscope based on PKH26 labeling of exosomes. CaCl2 increased the delivery of exosomes in a dose- and treatment time-dependent manner. In HEK 293 cells, a CaCl2 concentration of 400 µM and exposure time of 12 h increased the delivery of exosomes by >20 times compared with controls. In H9C2 cells, a CaCl2 concentration of 400 µM and exposure time of >24 h increased the delivery of exosomes by >400 times compared with controls. The viability of both cell lines was maintained up to a CaCl2 concentration of 1 mM. However, cobalt chloride, cupric chloride, and magnesium chloride did not change the delivery of exosomes in both cell lines. These results suggest that the use of CaCl2 treatment might be a useful method for enhancing the delivery of exosomes.

[Corrigendum] Human peripheral blood­derived exosomes for microRNA delivery.

Exosomes serve important functions in cell­to­cell communication and biological functions by serving as a delivery cargo shuttle for various molecules. The application of an improved delivery method for microRNAs (miRNAs/miRs) may enhance their potential as a therapeutic tool in cardiac diseases. Thus, the present study investigated whether human peripheral blood­derived exosomes may be used as a delivery cargo system for miRNAs, and whether the delivery of miR­21 using a human peripheral blood derived­exosome may influence the degree of remodeling following myocardial infarction (MI). In H9C2 and HL­1 cells, miR­21 expression was successfully regulated by treatment with human peripheral blood derived­exosomes loaded with an miR­21 mimic or inhibitor compared with untreated cells. In addition, the mRNA and protein expression levels of SMAD family member 7 (Smad7), phosphatase and tensin homolog (PTEN) and matrix metalloproteinase 2 (MMP2), which are involved in cardiac fibrosis, were associated with the uptake of miR­21 mimic­ or inhibitor­loaded exosomes. Similarly, the in vivo mRNA and protein expression of Smad7, PTEN and MMP2 were altered following treatment with miR­21 mimic­ or inhibitor­loaded exosomes. Furthermore, miR­21 mimic­loaded exosomes enhanced fibrosis, whereas miR­21 inhibitor­loaded exosomes reduced fibrosis in a mouse MI model. These results suggested that miRNA­loaded human peripheral blood derived­exosomes may be used as a therapeutic tool for cardiac diseases. [the original article was published in International Journal of Molecular Medicine 43: 2319­2328, 2019; DOI:10.3892/ijmm.2019.4150].