Extracellular vesicles miRNA-126: a potential therapeutic role in Type I diabetic rat model

Abstract

AimType 1 diabetes mellitus (TIDM) is a metabolic disorder of an unknown etiology caused mainly by autoimmune or viral destruction of pancreatic β-cells. So far, the main therapy is insulin administration with no curable treatment. The aim of the current study was to evaluate the therapeutic effect of extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BM-MSCs) and transfected with miRNA126 in treatment of TIDM in rats. Moreover, to compare transfected EVs effect versus their parent BM-MSCs and untransfected EVs.MethodsThe induction of diabetes in rats was done by intraperitoneal (i.p) injection of streptozotocin (STZ). Rats were divided randomly into groups that received either BM-MSCs or EVs or miRNA-126 mimic transfected EVs or miRNA-126 inhibitor transfected EVs.ResultsmiRNA-126 mimic transfected EVs, BM-MSCs, EVs treated groups resulted in lowering of blood glucose level and increase in plasma insulin level significantly compared to diabetic untreated group with significantly higher miRNA126 level in pancreatic tissue of miRNA126 mimic-EVs treated group compared to diabetic group. Results were superior in miRNA-126 treated group compared to BM-MSCs treated.ConclusionmiRNA-126 transfected EVs were found to be superior to BM-MSCs in the treatment of STZ induced type-1 DM. MSCsderived EVs may represent a novel cell-free treatment strategy with a potential implication of miRNA-126.

Keywords

Introduction

Type 1 DM (T1DM) is considered one of the most common metabolic disorders in childhood. Different treatment modalities were developed for the treatment of T1DM; however, due to patient inconvenience and reported complications, more studies are needed to eradicate the disease.¹
Bone marrow mesenchymal stem cells (BM-MSCs) are non-hematopoietic stem cells that can produce and secrete a wide spectrum of cytokines, chemokines, and growth factors in a paracrine fashion that affect the action of nearby cells.²
Extracellular vesicles (EVs) derived from MSCs are considered one of the promising paracrine mechanisms of stem cells that could effectively mimic the beneficial effects of MSCs. EVs mainly contribute to the action of stem cells by transferring proteins, lipids, and RNA species between cells. Many studies have assessed the potential therapeutic role of MSC-derived EVs in T1DM due to their immunoregulatory actions.³
MicroRNAs (miRNAs) are considered non-coding small-molecule RNAs that control gene expression by attaching to complementary target sequences of the 3′ untranslated regions (3′-UTR) of mRNAs.⁴ miRNAs may have cross-talks with mRNAs in pancreatic tissues of T1DM.⁵
miRNA-126 is profusely found in endothelial cells and performs an important role in regulating vascular integrity and angiogenesis. Hence, several studies have investigated its potential therapeutic role in decreasing the consequences of diabetic vascular complications in various types of DM.⁶ However, no study to date has investigated the potential paracrine role of miR-126 on pancreatic tissue regeneration and insulin production.
The current study aimed to assess the potential therapeutic role of EVs secreted from bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of T1DM as compared to BM-MSCs alone. Moreover, to study miR-126 as a potential molecular mechanism by which EVs communicate with pancreatic tissue. As far as we know, this is the first study to assess the use of BM-MSC-derived EVs in the treatment of DM with the implication of miRNA-126.

Materials and Methods

Laboratory AnimalsThe present study included sixty adult male Wistar albino rats (150–180 g, 7–8 weeks). Rats were housed under standard conditions with a 14:10 h light-dark cycle at 25 ± 2 °C, with free access to food and water, at the institutional Animal House of the Physiology Department. Experiments were done in compliance with the approved guidelines set by the institutional Research Ethics Committee and the National Institutes of Health guide for the care and use of laboratory animals.Experimental DesignBone Marrow IsolationTwo male Wistar albino rats (25–35 g, 3 weeks) were sacrificed for bone marrow extraction. The femur and tibia were dissected, disinfected, and flushed with PBS. The proximal and distal ends of the tibiae and femurs were cut.⁷Preparation of Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs)The cells obtained from the tibia and femur bones were layered on Ficoll-Paque (Gibco-Invitrogen). The cells were collected, washed, and seeded in T flasks. The cells were characterized by morphology, adherence, and flow cytometry assessment using CD44, CD90, and CD45 surface markers.EVs Isolation and Characterization ProtocolEVs were isolated from MSCs supernatant. After filtration, ultracentrifugation was done at 120,000 × g for 70 minutes at 4 °C (Thermo Fisher Micro Ultracentrifuge MX 120+, Medical Research Institute – Alexandria University). Pellets were discarded, re-suspended in PBS, and stored at –80 °C. Transmission Electron Microscope (TEM) with an accelerating voltage of 120 kV (JEM-1400 series 120 kV Transmission Electron Microscope, USA) was used for EVs characterization.MiRNA-126 AnalysisThe extraction of total RNA from BM-MSCs and their EVs was done under completely sterile and RNAse-free conditions in accordance with the manufacturer’s instructions of the miRNeasy Mini Kit (Qiagen, Hilden, Germany). The evaluation of the concentration of the extracted RNA samples was done using NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). Quantitative RT-PCR analysis for miRNA-126 was performed twice using TaqMan microRNA assays and TaqMan Universal PCR Master Mix II (Applied Biosystems, USA).⁸ StepOne real-time PCR system (Applied Biosystems, USA) was used for amplification. StepOneTM Software v2.3 was used for data analysis.⁹BM-MSCs Transfection ProtocolOne day before transfection, BM-MSCs were cultured at 2–8 × 10⁴ cells per well in a 24-well plate. On the day of transfection, 37.5 ng miRNA-inhibitor/mimic was mixed with 2 μL of Lipofectamine 2000 transfection reagent (Invitrogen Cat No. 11668-030). After incubation, the complexes were added to the cells and re-incubated for 72 h. qRT-PCR analysis was done as previously described.
The induction of T1DM was done by a single intraperitoneal (I.P.) injection of streptozotocin (STZ) at a dose of 50 mg/kg.⁷ Healthy untreated rats received an equal volume of phosphate buffer saline (PBS) intraperitoneally and served as controls (Group I, control group) (n = 10). Diabetic rats were randomly divided into five groups, each containing 10 rats.
- Group II: untreated diabetic group
- Group III: diabetic rats treated with a single I.P. injection of BM-MSCs at a dose of 10⁶ cells per rat¹⁰
- Group IV: diabetic rats received a single I.P. injection of BM-MSC-derived extracellular vesicles at a dose of 1000 μg/ml in a frozen pellet
- Group V: diabetic rats received a single I.P. injection of BM-MSC-derived extracellular vesicles expressing miR-126 mimic at a dose of 1000 μg/ml in a frozen pellet¹⁰
- Group VI: diabetic rats received a single I.P. injection of BM-MSC-derived extracellular vesicles expressing miR-126 inhibitor at a dose of 1000 μg/ml in a frozen pellet
Animal sacrifice was done by cervical dislocation at the end of the study. Blood samples were taken from the aorta in EDTA tubes (10 μL of 0.1 M EDTA for 200–400 μL of blood); plasma was separated for the assessment of insulin level and stored at –80 °C.Biochemical AnalysisPlasma Insulin LevelPlasma insulin level was determined by ELISA according to the manufacturer’s instructions (Insulin ELISA kit, Thermo Fisher, USA). Once rats’ plasma insulin level became below 9 µIU/ml, they were considered diabetic.Blood Glucose Level (Fasting/Random)Blood glucose level was measured weekly using samples from venous blood with Glucocheck strips.Histopathological Examination of the PancreasAfter sacrifice, the pancreatic tissues were harvested, fixed, and processed into paraffin blocks. Four to five micron sections were cut using a manual rotatory microtome. Hematoxylin and eosin (H&E)-stained sections were then assessed for histopathologic changes.¹¹Ethical ApprovalThis study was approved by the Ethics Committee of Alexandria Faculty of Medicine (Date: 27.06.2021, IRB Decision No: 00012098 – FWA No: 00018699).Statistical AnalysisAll statistical analyses were done using SPSS software version 20.0 (Armonk, NY: IBM Corp). Mean and standard deviation were used to summarize the data. Analysis of variance (ANOVA) test was done for comparison between groups, with post hoc test (Tukey’s) for pairwise comparison. Post hoc test (adjusted Bonferroni) for pairwise comparisons was done to compare between two durations. Statistical significance was considered at p≤0.05.

Results

Characterization of Bone Marrow Mesenchymal Stem Cells (BM-MSCs)MSCs were characterized by their fibroblast spindle-shape using inverted contrast phase microscope (Olympus CKX41SF, Japan) (Fig. 1A–D). Flow cytometric analysis of cultured BM-MSCs showed the positive expression of the mesenchymal surface markers CD44 and CD90 as well as negative expression of CD45 (Fig. 1E–F).Characterization of Extracellular VesiclesTEM was used to visualize the isolated EVs, showing spherical structures with lipid bilayer of different sizes ranging from 50.92 to 67.03 nm, indicating that most of the extracellular vesicles were exosomes (Fig. 1G).Quantification of miRNA-126 Level in BM-MSCs and Their EVs miRNA-126 Expression in BM-MSCs and Their Derived EVs Was Compared to Untransfected BM-MSCs and BM-MSCs Transfected With miRNA-126 Inhibitor (In Vitro)Cells transfected with miRNA-126 mimic showed 17-fold higher miRNA-126 expression than BM-MSCs. Additionally, miRNA-126 dropped by 4-fold following transfection with miRNA-126 inhibitor (Fig. 2A).Evaluation of Therapeutic Effect of BM-MSCs and Their EVsPlasma Insulin LevelThere was a significant increase in the level of plasma insulin in BM-MSCs, EVs, and EVs–miRNA-126 mimic treated groups compared to the untreated diabetic group and EVs–miRNA-126 inhibitor group (p≤0.001) (Fig. 2B).Blood Glucose LevelThere was a significant decrease in blood glucose levels in the BM-MSCs, EVs, and miRNA-126 mimic treated groups compared to untreated diabetic and miRNA-126 inhibitor treated groups (p≤0.001) (Fig. 2C).Histopathological AssessmentPancreatic tissues of rats treated with miRNA-126 mimic, BM-MSCs, and EVs stained with hematoxylin and eosin (H&E) showed improvement compared to untreated diabetic and miRNA-126 inhibitor groups. The diabetic group showed degenerative changes seen as mononuclear infiltration (red arrow) and diffuse cytoplasmic vacuolation of islet cells (yellow arrow) (×400) (Fig. 3B).
BM-MSCs treated group showed improvement in endocrine cell histology where vacuolation was only focally seen (yellow arrow) with residual mononuclear cells (red arrow) (×400) (Fig. 3C). EVs treated group showed improvement in endocrine cell histology where vacuolation was only focally seen (yellow arrow) (Fig. 3D). EVs with mimic showed an increased number of islet cells.

Discussion

T1DM treatment is considered a challenge mainly due to the early onset of the disease and its unknown exact etiology. Many different treatment modalities were used for the treatment of T1DM.¹ BM-MSCs have been recognized as a hopeful agent for the treatment of T1DM because of their ability to differentiate into islet-like cells. MSCs regenerative ability can be attained through certain paracrine factors released from them, which include extracellular vesicles (EVs). EVs derived from MSCs have immunomodulatory effects through induction of T-regulatory cells and suppression of autoreactive immune cells.¹² miRNAs play an important role in many aspects of diabetes as they modulate the expression of many genes associated with the pathophysiological process. In the current work, EVs were used as cargo for miRNA-126 mimic and inhibitor to prevent their digestion by different nucleases and avoid their modification.¹³
In the current work, T1DM was induced by STZ injection. Many previous studies have used STZ for induction of T1DM because of its cytotoxic effect in β-cells through inflammation activation, production of excessive amounts of reactive oxygen species, and cell apoptosis.^14-15
In the present study, T1DM was confirmed by high blood glucose >300 mg/dl, decreased plasma insulin level, and histopathological pancreatic changes. These results were consistent with previous studies that reported the confirmation of T1DM was done by fasting blood glucose level >300 mg/dl and histopathological pancreatic changes.^15-16-17
The results of the present work revealed that cultured BM-MSCs were characterized by their morphology and surface markers (Fig. 1A–F), which was consistent with a previous study by Kobolak et al.¹⁸ EVs were also characterized by their morphology (Fig. 1G), which agreed with a previous study that denoted the size of EVs derived from BM-MSCs ranged around 100 nm in diameter with typical spherical shape.⁷
The study results documented that BM-MSCs treated group showed significant lowering in blood glucose levels and elevation in plasma insulin levels, as well as improvement in histopathological pancreatic tissue findings. This may be due to their immunomodulatory effects and homing capabilities.^4,12 These findings are in agreement with previous studies that revealed the decline in blood glucose level and elevation in plasma insulin level after injection of BM-MSCs in T1DM rats.^7,19
Regarding the group that received EVs, they showed a decrease in blood glucose level, increase in plasma insulin level, and improvement in histological findings in pancreatic tissue. However, EVs results were superior to those of BM-MSCs, which may be due to the immunomodulatory ability of EVs through inhibition of reactive T cells and reduction of inflammatory cytokine levels.²⁰
Rani et al.²¹ stated that exosomes can be used as a novel substitute for whole-cell therapies. Exosomes may provide a better safety profile when compared to their parent cells. The previously mentioned study could provide an explanation for the mechanism of improvement that occurred in our present work in the EVs treated group.²¹
The findings of the present study demonstrated that miRNA-126 mimic treatment improved fasting blood glucose, plasma insulin levels, and pancreatic tissue histopathology, denoting possible therapeutic effects. Another previous study reported the anti-inflammatory role of miRNA-126, as intravitreal injection of miRNA-126 mimic improved diabetic retinopathy by enhancing retinal tissue histopathology and reducing inflammation by inhibiting specific inflammatory pathways.²²
Another study suggested that the increase in expression of miRNA-126 may play an anti-inflammatory and anti-apoptotic role in cardiomyopathy. This could explain the mechanism of miRNA-126 in reversing cytotoxic effects of STZ on islet cells.²³
Zhang and colleagues²⁴ revealed the angiogenic role of miRNA-126, reporting that diabetic wounds treated with negative pressure wound therapy (NPWT) showed large increases in miR-126, which controls VEGF-induced angiogenesis.²⁴ Thus, these findings may provide a possible explanation for the results in our present study in terms of improvement of β-islet cell mass after treatment with miRNA-126 through angiogenesis.

Conclusion

The current study highlights miRNA-126 mimic- transfected EVs as a promising, safe, cell-free treatment strategy for T1DM. In addition, molecular action of EVs could be mediated by miRNA-126 in T1DM rat model. However, the exact molecular pathways and mechanisms of miRNA-126 should be further investigated.

References

1. Kahanovitz L, Sluss PM, Russell SJ. Type 1 diabetes: a clinical perspective. Point Care. 2017;16(1):37-40. doi:10.1097/poc.0000000000000125
   Article · PubMed · Google Scholar
2. Moravej A, Karimi MH, Geramizadeh B, et al. Mesenchymal stem cells upregulate the expression of PD-L1 but not VDR in dendritic cells. Immunol Invest. 2017;46(1):80-96. doi:10.1080/08820139.2016.1225757
   Article · PubMed · Google Scholar
3. Garcia-Contreras M, Brooks RW, Boccuzzi L, Robbins PD, Ricordi C. Exosomes as biomarkers and therapeutic tools for type 1 diabetes mellitus. Eur Rev Med Pharmacol Sci. 2017;21(12):2940-2956.
   PubMed · Google Scholar
4. Negi S, Rutman AK, Paraskevas S. Extracellular vesicles in type 1 diabetes: messengers and regulators. Curr Diab Rep. 2019;19(9):69. doi:10.1007/s11892-019-1193-7
   Article · PubMed · Google Scholar
5. Hashimoto N, Tanaka T. Role of miRNAs in the pathogenesis and susceptibility of diabetes mellitus. J Hum Genet. 2017;62(2):141-150. doi:10.1038/jhg.2016.150
   Article · PubMed · Google Scholar
6. Pishavar E, Behravan J. miR-126 as a therapeutic agent for diabetes mellitus. Curr Pharm Des. 2017;23(22):3309-3314. doi:10.2174/1381612823666170424120121
   Article · PubMed · Google Scholar
7. Sabry D, Marzouk S, Zakaria R, Ibrahim HA, Samir M. Correction to: the effect of exosomes derived from mesenchymal stem cells in the treatment of induced type 1 diabetes mellitus in rats. Biotechnol Lett. 2020;42(12):2761-2762. doi:10.1007/s10529-020-02975-1
   Article · PubMed · Google Scholar
8. Xiao GY, Cheng CC, Chiang YS, Cheng WT, Liu IH, Wu SC. Exosomal miR-10a derived from amniotic fluid stem cells preserves ovarian follicles after chemotherapy. Sci Rep. 2016;6:23120. doi:10.1038/srep23120
   Article · PubMed · Google Scholar
9. Lin Q, Di YP. Determination and quantification of bacterial virulent gene expression using quantitative real-time PCR. Methods Mol Biol. 2020;2102:177-193. doi:10.1007/978-1-0716-0223-2_9
   Article · PubMed · Google Scholar
10. Braun RK, Chetty C, Balasubramaniam V, et al. Intraperitoneal injection of MSC-derived exosomes prevent experimental bronchopulmonary dysplasia. Biochem Biophys Res Commun. 2018;503(4):2653-2658. doi:10.1016/j.bbrc.2018.08.019
    Article · PubMed · Google Scholar
11. Alturkistani HA, Tashkandi FM, Mohammedsaleh ZM. Histological stains: a literature review and case study. Glob J Health Sci. 2015;8(3):72-79. doi:10.5539/gjhs.v8n3p72
    Article · PubMed · Google Scholar
12. Dabrowska S, Andrzejewska A, Janowski M, Lukomska B. Immunomodulatory and regenerative effects of mesenchymal stem cells and extracellular vesicles: therapeutic outlook for inflammatory and degenerative diseases. Front Immunol. 2020;11:591065.
    PubMed · Google Scholar
13. Martellucci S, Orefice NS, Angelucci A, Luce A, Caraglia M, Zappavigna S. Extracellular vesicles: new endogenous shuttles for miRNAs in cancer diagnosis and therapy? Int J Mol Sci. 2020;21(18):6486. doi:10.3390/ijms21186486
    Article · PubMed · Google Scholar
14. Chandirasegaran G, Elanchezhiyan C, Ghosh K, Sethupathy S. Berberine chloride ameliorates oxidative stress, inflammation, and apoptosis in the pancreas of streptozotocin-induced diabetic rats. Biomed Pharmacother. 2017;95:175-185. doi:10.1016/j.biopha.2017.08.040
    Article · PubMed · Google Scholar
15. Mostafa-Hedeab G, Hassan ME, Sabry D, Ali RI. Anti-diabetic therapeutic efficacy of mesenchymal stem cells-derived exosomes. Int J Pharm. 2020;16(6):437-446.
    PubMed · Google Scholar
16. Nurdiana S, Goh YM, Ahmad H, et al. Changes in pancreatic histology, insulin secretion, and oxidative status in diabetic rats following treatment with Ficus deltoidea and vitexin. BMC Complement Altern Med. 2017;17(1):290. doi:10.1186/s12906-017-1762-8
    Article · PubMed · Google Scholar
17. Yousef HN, Sakr SM, Sabry SA. Mesenchymal stem cells ameliorate hyperglycemia in type I diabetic developing male rats. Stem Cells Int. 2022;2022:7556278. doi:10.1155/2022/7556278
    Article · PubMed · Google Scholar
18. Kobolak J, Dinnyes A, Memic A, Khademhosseini A, Mobasheri A. Mesenchymal stem cells: identification, phenotypic characterization, biological properties, and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods. 2016;99:62-68. doi:10.1016/j.ymeth.2015.09.016
    Article · PubMed · Google Scholar
19. Hussien NI, Ebrahim N, Mohammed OM, Sabry D. Combination of obestatin and bone marrow mesenchymal stem cells prevents aggravation of endocrine pancreatic damage in type II diabetic rats. Int J Stem Cells. 2017;10(2):129-143. doi:10.15283/ijsc17035
    Article · PubMed · Google Scholar
20. Chen W, Huang Y, Han J, et al. Immunomodulatory effects of mesenchymal stromal cells-derived exosome. Immunol Res. 2016;64(4):831-840. doi:10.1007/s12026-016-8798-6
    Article · PubMed · Google Scholar
21. Rani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther. 2015;23(5):812-823. doi:10.1038/mt.2015.44
    Article · PubMed · Google Scholar
22. Zheng Y, Liu Y, Wang L, et al. MicroRNA-126 suppresses the proliferation and migration of endothelial cells in experimental diabetic retinopathy by targeting polo-like kinase 4. Int J Mol Med. 2021;47(1):151-160. doi:10.3892/ijmm.2020.4775
    Article · PubMed · Google Scholar
23. Suresh Babu S, Thandavarayan RA, Joladarashi D, et al. MicroRNA-126 overexpression rescues diabetes-induced impairment in efferocytosis of apoptotic cardiomyocytes. Sci Rep. 2016;6:36207. doi:10.1038/srep36207
    Article · PubMed · Google Scholar
24. Zhang D, Li Z, Wang Z, Zeng F, Xiao W, Yu A. MicroRNA-126: a promising biomarker for angiogenesis of diabetic wounds treated with negative pressure wound therapy. Diabetes Metab Syndr Obes. 2019;12:1685-1696. doi:10.2147/dmso.s199705
    Article · PubMed · Google Scholar

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