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 to 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 upregulated and correlated with nitric oxide synthase 3 (NOS3) levels, which in turn influences cardiac function. The downregulation 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 upregulation of miR-21 expression by PB-sEVs loaded with miR-21 reversed these effects. Mechanistically, miR-21 targeted and downregulated 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.

pathophysiological consequences of MI [15,16]. As abnormal expression of miRNAs is a key 23 phenomenon in MI pathogenesis, there is a growing interest in attempts to regulate miRNAs 24 expression to normal levels [17]. However, poor stability and inefficient delivery in vivo 25 restrict the clinical applications of miRNAs [18]. Thus, efficient delivery approaches are 26 highly desired. 27 Among miRNAs, miRNA-21 (miR-21) is known to play a crucial role in several 28 physiological and pathological MI processes by targeting various signaling pathways [19][20][21]. Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191077 5 Taking the advantages of the effects of miR-21 on MI, we propose to deliver miR-21 into 1 cardiac tissue through PB-sEVs to promote the recovery of cardiac function. In the present 2 study, we evaluated whether the regulation of miR-21 expression using PB-sEVs may 3 provide an effective strategy for MI treatment and investigated the mechanisms affected by Health System; approval no. YUMC 4-2011-0872), and signed informed consent was 5 obtained from all subjects. Human peripheral blood was obtained from relatively healthy 6 subjects without serious cardiac disease at Yonsei University Health System (Seoul, Korea). 7 The clinical profiles of patients are listed in Supplementary Table S1. All patients did not had 8 tachycardia at least 1 month before blood sampling.    For transmission electron microscopy (TEM), sEVs were adsorbed to a Formvar-12 carbon-coated electron microscope grid (Leica Microsystems, Inc., Buffalo Grove, IL, USA) 13 for ~1 min. Then, the samples were stained with 2% uranyl acetate and observed using a 14 JEM-1011 electron microscope (JEOL Ltd., Tokyo, Japan). at 600 x g for 5 min to remove debris. The supernatants were added to 96-well plates and the 5 reagents were added and incubated at room temperature. The absorbance was measured at    was observed using a confocal microscope (Zeiss LSM 710; Carl Zeiss, Jena, Germany).

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To assess cardiac function, echocardiography was performed using a Vevo 2100 11 system (VisualSonics, Toronto, Ontario, Canada), and left ventricle ejection fraction (LVEF) 12 and left ventricle fractional shortening (LVFS) were measured. 13 Tissues, including heart, liver, spleen, kidney and lung, were fixed in 4% The data are represented as mean ± standard deviation (SD), and statistical 27 significance between the experimental groups was determined using either the unpaired, two-   2 We investigated the effects of MI on miR-21 expression and found that miR-21 3 levels were significantly upregulated in MI mice, compared to those in non-MI mice (p < 4 0.01) ( Figure 1A). Consistent with the changes in miR-21 expression in mice, miR-21 5 levels were also upregulated in cultured H9C2 cells exposed to hypoxia. The expression of were also gradually increased in both AC16 and HL-1 cells exposed to hypoxia. In addition, 28 29 We examined the effects of anti-miR-21-loaded PB-sEVs on the recovery of infarcted hearts ( Figure 3A). We first evaluated the uptake and distribution of sEVs in vivo.

Effect of anti-miR-21-loaded PB-sEVs in mouse infarcted heart
1 PKH26 fluorescence was concentrated in the hearts 24 h after intramyocardial injection of 2 PKH26-labeled PB-sEVs, observed using an IVIS ® Spectrum in vivo imaging system 3 ( Figure 3B). In addition, we performed immunofluorescent staining for cardiomyocyte-4 specific (cTnI + ) antigens. PKH26-labeled PB-sEVs co-localized with cTnI + cells at 24 h 5 after injection ( Figure 3C). Therefore, these results suggested that PB-sEVs effectively 6 distributed into cardiomyocytes in the hearts. We did not observe any inflammation or 7 changes in morphology of the liver and other organs after PB-sEVs injection 8 (Supplementary Figure S4A). The results of quantitative RT-PCR analysis confirmed that 9 miRNA-loaded PB-sEVs effectively regulated miR-21 levels in the hearts after injection, 10 indicating that PB-sEVs successfully delivered of miRNAs to mouse heart ( Figure 3D).

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Taken together, these results support PB-sEVs as a suitable approach for the effective 12 delivery of therapeutic miRNAs in vivo. 13 After validating the potential utility of the PB-sEVs platform in vivo, we evaluated   30 To elucidate the molecular mechanisms underlying the MI pathogenesis driven by  Western blot analysis also demonstrated the downregulation of NOS3 expression after 29 hypoxia; the treatment with anti-miR-21 reversed this effect on NOS3 expression ( Figure 5B).  Figure S5).

Identification of STRN as a direct target of miR-21
2 Furthermore, we found that NO production was reduced after exposure to hypoxia for 24 h 3 compared with normoxic controls. Also, miR-21 overexpression could impair NO production, 4 while anti-miR-21 enhanced NO production, respectively. Taken together, miR-21 attenuated 5 the expression of NOS3, which in turn repressing NO production ( Figure 5C). Consistently, 6 the in vivo results revealed that STRN expression levels were significantly downregulated in 7 MI mice compared with non-MI mice, and further decreased in the mice treated with miR-21-8 loaded PB-sEVs. In contrast, the mice treated with anti-miR-21-loaded PB-sEVs 9 significantly increased STRN expression. Moreover, it was observed that the downregulation 10 of NOS3 expression after MI, which was significantly altered following treatment with miR-11 21-loaded PB-sEVs or anti-miR-21-loaded PB-sEVs ( Figure 5D). In addition, 12 immunofluorescence analysis showed that the loose distribution of NOS3 was reversed after 13 treatment with anti-miR-21-loaded PB-sEVs specific for cardiomyocytes ( Figure 5E).
14 Overall, miR-21 could lead to a significant decrease in NOS3 levels in response to 15 decreases in STRN expression both in vitro and in vivo.

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The major highlights of the present study are as follows: miR-21 expression was 2 significantly upregulated in hypoxic conditions and correlated with the expression of NOS3, 3 thereby conferring cardiac dysfunctions in response to MI. Second, the regulation of miR-21 4 expression using PB-sEVs had an impact on the pathophysiology of MI in mouse model. 5 Finally, miR-21 targeted and suppressed the mRNA and protein expression of STRN, which 6 may regulate NOS3 expression.  and has been used safely and routinely for blood transfusions, isolating sEVs from human 20 peripheral blood itself may allow for effective yield system. We have previously reported 21 that PB-sEVs may serve as a miRNAs delivery system to overcome these limitations [23].

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Here, we provided further evidence that PB-sEVs may act as carriers and efficient delivery   Competing interests 13 The authors declare that there are no competing interests associated with the 14 manuscript.