Mesenchymal Stem Cell-Derived Exosomes Protect Trabecular Meshwork from Oxidative Stress

doi:10.21203/rs.3.rs-234332/v1

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Mesenchymal Stem Cell-Derived Exosomes Protect Trabecular Meshwork from Oxidative Stress

https://doi.org/10.21203/rs.3.rs-234332/v1

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published 21 Jul, 2021

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This study aims to investigate potential effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on trabecular meshwork cells under oxidative stress and predict candidate genes associated with this process. Trabecular meshwork cells were pretreated with BMSC-derived exosomes, and exposed to 0.1mM H₂O₂. Survival rate of trabecular meshwork cells was measured with CCK-8 assay. Production of iROS was measured using a flow cytometer. RT-PCR and ELISA were used to detect mRNA and protein levels of inflammatory cytokines and matrix metalloproteinases (MMPs). RNA sequencing for trabecular meshwork cells from Exo- and control groups was performed on BGISEQ500 + bgiseq-500 platform. Phenotypically, pretreatment of BMSC-derived exosomes improves survival rate of trabecular meshwork cells exposed to H₂O₂, reduces production of iROS, and inhibits expression of inflammatory cytokines, whereas increases expression of MMPs. There are 23 DEmiRNAs, 307 DElncRNAs, and 367 DEmRNAs differentially expressed between Exo- and control groups. Exosomes derived from BMSCs may protect trabecular meshwork cells from oxidative stress. Candidate genes responsible for beneficial effects ,such as DIO2 and HMOX1, such as DIO2 and HMOX1, are predicted.

Ophthalmology

Translational Medicine

exosome

mesenchymal stem cell

trabecular meshwork

oxidative stress

Glaucoma is a disease characterized by atrophy and depression of optic disc, visual field defect, and visual impairment. About 80 million people are estimated to be affected with glaucoma worldwide by 2020 ¹. Based on status of iridocorneal angle, glaucoma can be classified into open-angle, closed-angle and developmental types, which are further divided into primary and secondary subtypes ². Notably, a major risk factor for primary open-angle glaucoma (POAG) is high intraocular pressure ³. POAG is usually triggered by oxidative stress damage and abnormal accumulation of metabolites in trabecular meshwork cells, causing loss of cellular function. As a result, it is of great significance to focus on early recovery of trabecular meshwork structure and function in POAG with elevated intraocular pressure.

Mesenchymal stem cells (MSCs) are self-replicating multipotent stromal cells isolated from mesenchymal tissues such as bone marrow ⁴, umbilical cord blood ⁵, adipose ⁶ and dental pulp ⁷. MSCs are proposed to play an important role in recovery and protection of tissues ^8–10, including nerve, bone, and cornea. Moreover, several studies reported potential ability of MSCs to recover or protect biological functions of trabecular meshwork. For instance, Roubeix et al. ¹¹ found that soluble factors secreted by MSCs might inhibit dysfunction of trabecular meshwork. In addition, Manuguerra et al. ¹² discovered that pro-recovery effects of MSCs on trabecular meshwork in a model of open-angle glaucoma are mediated in a paracrine manner rather than direct differentiation and repopulation of MSCs. However, mysterious “factors” secreted by MSCs are yet to be deciphered. Nevertheless, a study ⁸ on protective effect of MSCs on injured retinas might have alluded exosome as a “factor” in question.

Exosomes, first reported in 1981 ¹³, comprise proteins, lipids, DNA, mRNA and microRNA¹⁴ surrounded by a phospholipid bi-layer secreted into extracellular space. It ranges from 30 to 150 nm in size ^15–17. Exosomal mRNAs and microRNAs may induce translation of new proteins after being transported to receipient cells via fusion of exosomes and cell membrane ¹⁸. MSCs have capability to synthesize and secrete broad-spectrum exosomes¹⁹. Exosomes, as an important secreted constituent, might mediate and amplify restorative and protective functions of MSCs ^20–22. Therefore, this study speculates that potential “factors” protecting trabecular meshwork cells in glaucoma models are exosomes secreted by MSCs. Exosomes secreted by bone marrow MSCs (BMSCs) contain over 150 different miRNA molecules ²³ that can be delivered to target cells. Therefore, BMSC-derived exosomes may become critical tools for therapy of degenerative diseases.

Characterization of exosomes isolated from human BMSCs

Exosomes from hBMSCs were isolated using ultracentrifugation. Transmission electron microscopy revealed exosomes with a round shape and central depression (Fig. 1a). Using ZetaView, exosomes were evaluated to be a diameter of 130 nm in size (Fig. 1b). Based on Western blotting, exosomes expressed classic markers including HSP70 and CD63 (Fig. 1c).

Exosomes isolated from hBMSCs are uptaken by human trabecular meshwork cells (TMCs)

The uptake of hBMSC-derived exosomes by hTMCs was observed at different time points i.e., 6, 12, and 24 hours after incubation, respectively (Fig. 2). After hTMCs incubation with labeled hBMSC-derived exosomes, green fluorescent particles surrounding the nucleus were increased over time.

hBMSC-derived exosomes improve viability of hTMCs exposed to H₂O₂

After 24 hours of pretreatment with hBMSC-derived exosomes, hTMCs were exposed to H₂O₂ (0.1mM) for certain periods i.e., 6, 12, and 24 hours, respectively. Notably, H₂O₂ (0.1mM) significantly reduced survival rate of hTMC in a time-dependent manner. Meanwhile, pretreatment with hBMSC-derived exosomes improved viability of hTMCs exposed to 0.1 mM H₂O₂ (Fig. 3), and optimal protective efficiency achieved at 6 hours.

Effects of hBMSC-derived exosomes on production of iROS in H₂O₂-exposed hTMCs

After pretreatment with hBMSC-derived exosomes for 24 hours, hTMCs were exposed to H₂O₂ (0.1mM) for 6 hours. Exposure to H₂O₂ significantly induced a high amount of iROS production in hTMCs, whereas pretreatment with hBMSC-derived exosomes decreased production of iROS at 6 hours, implying hBMSC-derived exosomes protected hTMCs against oxidative stress by reducing iROS (Fig. 4).

Effects of hBMSC-derived exosomes on expression of proinflammatory factors in H₂O₂-exposed hTMCs

After pretreatment with hBMSC-derived exosomes for 24 hours, hTMCs were exposed to H₂O₂ (0.1mM) for 6 hours. Expression levels of proinflammatory factors, including IL-1α, IL-1β, IL-6, and IL-8, were analyzed by quantitative real-time (qRT-) PCR and ELISA. High levels of IL-1α, IL-1β, IL-6, and IL-8 in hTMCs exposed to H₂O₂ were observed. Pretreatment with hBMSC-derived exosomes down-regulated IL-1α, IL-1β, IL-6, and IL-8 in hTMCs exposed to H₂O₂ (Fig. 5).

Effects of hBMSC-derived exosomes on expression of MMPs in H₂O₂-exposed hTMCs

Human TMCs were treated as described above. Expression levels of MMP-2 and MMP-3 were analyzed by qRT-PCR and ELISA. After exposure to H₂O₂, expression levels of MMP-2 and MMP-3 were unaffected. By contrast, pretreatment with hBMSC-derived exosome up-regulated MMP-2 and MMP-3 in hTMCs exposed to H₂O₂ (Fig. 6).

Effects of hBMSC-derives exoosmes on gene expression in H₂O₂-exposed hTMCs

Through expression profile sequencing, 66,891 genes were included. Totally, 804 genes were differentially expressed (DE) through DEGseq calculation, including 23 DEmiRNA, 307 DElncRNA, and 367 DEmRNA. Interestingly, expression of DEmiRNA in Exo group (hTMCs pretreated with hBMSC-derived exosomes and followed by exopure to H₂O₂) vs. control group (hTMCs pretreated by PBS and followed by exposure to H₂O₂) was idenfied (Fig. 7a). Moreover, 4,500 DEmiRNA target genes were identified through database queries, including 42 DE target genes by calculation (Fig. 7b).

Using Pearson correlation coefficient models, co-expression of DElncRNA and DEmRNA was predicted. There were 3,634 relationship pairs, including 3,375 pairs of positive correlation and 259 pairs of negative correlation. DElncRNA-DEmRNA interactional network (power-law distribution R² = 0.664) was constructed based on characteristics of a biological process. Notably, 9 subnets were identified using MCODE. Additionally, 21 lncRNA, 26 mRNA, and 263 pairs of interactional relations were identified in the highest score (11.435) subnet. Their levels were positively correlated and up-regulated in Exo group (Fig. 7c). Moreover, using DAVID, functional enrichment of DEmRNA was explored, and two KEGG pathways and a GO term were annotated (Table 1).

Table 1

Results of lncRNA KEGG pathway and GO term annotation
Term ID	Description	P-value	Count
hsa05150	Staphylococcus aureus infection	0.07583	2
hsa01230	Biosynthesis of amino acids	0.09993	2
GO:0008219	cell death	0.04324	2

Detailed mechanisms underlying dysfunctional trabecular meshwork in POAG are not completely understood. However, reports ^24,25 have revealed that oxidative stress triggers trabecular meshwork dysfunction in vitro and in vivo. TMCs under oxidative stress produce excessive iROS via mitochondria. Increased iROS production indicates oxidative stress and thus up-regulates inflammatory markers in TMCs, including IL-1, IL-6, and IL-8. Additionally, a polymorphism in IL-1α correlated with increased IL-1 gene expression and elevated risk for POAG ²⁶. As a result, consitutively high production of iROS and inflammatory factors are crucial features of TMC dysfunction in POAG ^27,28. H₂O₂-exposed hTMCs are widely adopted in glaucoma model in vitro. In this study, survival rate of trabecular meshwork cells exposed to H₂O₂ was reduced, iROS was continuously produced, and IL-1α, IL-1β, IL-6, and IL-8 were up-regulated compared to control group. Besides, we successfully constructed classical glaucoma model in vitro.

Previous studies ^27,28 were confined to antioxidant effect on trabecular meshwork cells exposed to H₂O₂. Either sufficiency or deficiency in antioxidant supplement is not always associated with eye pathology ²⁹. Whereas, exosomes, as a subcellular structure containing RNAs, might affect gene expression and protect trabecular meshwork cells from oxidative stress. MSC-derived exosomes have exhibited remarkable therapeutic effects for degenerative eye diseases. For instance, MSC-derived exosomes accelerated recovery of corneal epithelium ³⁰. In addition, protective effects of MSC-derived exosomes on retinal ganglion cells were identified in glaucoma models ³¹. However, only a few studies had explored potential effects of MSC-derived exosomes on trabecular meshwork, a key component of aqueous drainage channels. Herein, we focused on hTMCs, which were pretreated with hBMSC-derived exosomes, then exposed to hydrogen peroxide. Interestingly, hTMCs pretreated with hBMSC-derived exosome demonstrated enhnanced survival rate, lower iROS production, and lower expression of IL-1α, IL-1β, IL-6, and IL-8 compared to those only exposed to hydrogen peroxide. These findings imply that BMSC-derived exosomes potentially alleviate dysfunction of trabecular meshwork induced by oxidative stress.

Besides up-regulated proinflammatory cytokines, iROS induced by oxidative stress potentially promotes pathophysiological changes in the outflow tract by increasing intracellular oxidative damage. Matrix metalloproteinases (MMPs), a class of zinc-containing neutral proteases implicated in regulating extracellular matrix degradation as well as interacting with microenvironment, have been proposed as a vital group of enzymes to maintain outflow tract homeostasis ^32–37. In human outflow models, MMP-2 and MMP-3 could significantly increase the outflow capacity ³⁸, and regulate extracellular matrix remodeling ³⁹. Despite few previous studies on interaction between MMPs and oxidative stress, this study detected changes in MMP-2 and MMP-3 levles upon stimulation of BMSC-derived exosome in TMCs. Surprisingly, although oxidative stress might not influence MMPs levels in TMCs, MMPs were up-regulated in TMCs pretreated with hBMSC-derived exosomes. This indicates that BMSC-derived exosomes may enhance trabecular meshwork function.

To identify candidate genes responsible for alleviating oxidative stress damage in trabecular meshwork cells, gene expression was profiled in Exo and control groups. Differentially expressed genes, including 23 DEmiRNAs, 307 DElncRNAs, and 367 DEmRNAs, were identified. Many of these DEs have been reported to be related to trabecular network. For example, DIO2 might regulate phagocytosis in trabecular network and extracellular matrix remodeling, thereby maintaining homeostasis of outflow tract ⁴⁰. In addition, expression of Hmox1 was downregulated with increased production of iROS in trabecular meshwork⁴¹. In lncRNA-mRNA interactional subnet, DIO2 and HMOX1 act as hub nodes, co-expressing with 10 lncRNAs, respectively. The expression of Mir-126 in micro-vesicles was up-regulated in tears of patients diagnosed with open-angle glaucoma ⁴². However, Mir-126-5p was down-regulated in Exo group, indicating that hBMSC-derived exosomes induced decrease in production of iROS, thereby reducing risk of glaucoma. Additionally, Mir-125b-5p was down-regulated in aqueous humor of patients with POAG ⁴³. Mir-106b, which was linked to senescence, was down-regulated in trabecular meshwork under oxidative stress ⁴⁴. However, mir-125b-5P and miR106b were downregulated in Exo group compared to control group. This might be attributed to: (1) unclaried roles of mir-125b-5P and miR106b in glaucoma pathogenesis (down-regulation possibly indicating a protective effect against oxidative stress); (2) protective effect of hBMSC-derived exosomes not entirely preventing oxidative stress caused damage in trabecular meshwork cells. Meanwhile, we have discovered novel genes in trabecular meshwork under oxidative stress. For instance, expression of C5AR1 was up-regulated in retinal pigment epithelium cells under oxidative stress ⁴⁵; miR-150 regulated tissue injury triggered by high oxygen concentrations ⁴⁶.

Through gene function annotation, HMOX1 and CLUL1 promote apoptosis, so dysregulated HMOX1 and CLUL1 might cause dysfunction in trabecular meshwork. In this study, HMOX1 and CLUL1 were up-regulated in Exo group, where iROS was decreased, indicating negative correlation between HMOX1/CLUL1 expression and iROS production. Moreover, ALDOB and SDSL acting in biosynthesis of amino acids were up-regulated in this study. ALDOB-SDSL pathway might enhance antioxidant activity of trabecular meshwork cells.

Our study was limited to in vitro experiments on hTMCs. The conditions of hTMCs exposed to H₂O₂ might not entirely mirror those in POAG in vivo, however, pathological conditions may present similarity to a certain extent. Besides, hTMc are constantly exposed to aqueous humor in vivo, while H₂O₂ level in human aqueous humor can reach as high as 300 µM ²⁵. Additionally, Cyp1B1 and FOXC1 were indicated to be involved in hTMCs exoposed to H₂O₂ in vitro ^25,47. However, this finding needs to be subjected to in vivo experiments. In addition, the findings from transcriptome sequencing need to be validated by a serial of experiments. Nonetheless, this study has demonstrated that hBMSC-derived exosomes could be absorbed by hTMCs, which have exerted protective effect against oxidative stress damage, and thus enabling functional preservation of hTMCs. The transcriptomic sequencing and network analysis corroborate with several previous studies, and indicate meaningful regulatory RNAs and nodes, thus providing a basis for future research on glaucoma therapy by integraing exosomes with stem cells.

Human BMSCs culture

Human CD29⁺/CD44⁺/CD73⁺/CD90⁺/CD105⁺/CD166⁺/CD14⁻/CD31⁻/CD34⁻/CD45⁻ BMSCs were provided by Stem Cell Bank, Chinese Academy of Sciences. Cells were cultured in NutriStem MSC XF Basal Medium (Biological Industries, Israel) supplemented with 0.6% MSC XF Supplement (Biological Industries, Israel) and 1% penicillin/streptomycin. Cell cultures were maintained at 37°C in 5% CO₂ with the medium changed at intervals of 2 days. Cells were passaged with recombinant trypsin-EDTA solution (Biological Industries, Israel) at 80% confluence. For all experiments, human BMSCs (hBMSCs) were used at passage 2 to 5.

Isolation and characterization of exosomes

Fresh exosome-free medium was changed at 80% confluence of hBMSCs. After 48 hours, conditioned medium was collected and centrifuged at 300g for 10 minutes, 2000g for 10 minutes and 10000g for 30 minutes, to discard the pellets and collect the supernatant each time. Thereafter, the supernatant was subjected to centrifugation in a SW 32 Ti rotor (Beckman Coulter, USA) at 100,000g for 70 minutes. After washing twice, the pellets were wre-suspended in PBS. The exosome preparation was filtered through a 0.22µm filter and stored at -80°C until use. At passage 2 to 5, exosomes were isolated from hBMSCs.

Exosomal morphology was characterized by transmission electron microscopy. The size distribution of exosomes was measured using a ZetaView analysis system (PMX 110; Particle Metrix, Germany). Classic exosomal surface markers were examined by Western blotting analysis. Total protein from exosomes was extracted in lysis buffer, and a protein BCA assay kit was used to measure concentration. Lysates were separated on 10% SDS-polyacrylamide gel and proteins were transferred to polyvinylidene difluoride membranes. Then, membranes were incubated with 5% milk for 1 h and probed overnight at 4°C with antibodies targeting HSP70 (ab181606, 1:1000 dilution, Abcam, USA) and CD9 (ab92726, 1:2000 dilution, Abcam, USA). After washing, blots were incubated with peroxidase-conjugated antirabbit IgG horseradish and peroxidase secondary antibodies (ab205718, 1:2000 dilution, Abcam, USA) at room temperature for 1 h. The Alpha FluorChem E system (ProteinSimple, USA) was used to visualize the protein bands.

Human trabecular meshwork cells (hTMCs) culture

A human normal trabecular meshwork cell line was obtained from ScienCell Research Labs (Carlsbad, CA, USA). HTMCs were cultured in DMEM-Ham’s F12 (M&C gene technology, Beijing, China) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS). Cell cultures were maintained at 37°C in 5% CO₂ with medium being changed at intervals of 3 days and cells passaged with 0.05% trypsin/EDTA at 80% confluence. For all experiments, hTMCs were used at passage 2 to 5.

Exosome tracking

HBMSC-derived exosomes were stained with a green fluorescent dye (PKH67, Sigma-Aldrich, USA). Briefly, 200µl exosome suspension with 1.0 × 10⁸ exosomes were mixed with equal volume dilution containing 2µl dye and incubated at 37°C for 5 minutes in dark. An equal volume of 10% BSA-PBA solution was added to stop dyeing. Then, they were subjected to ultracentrifugation at 100,000g for 70 minutes. The pellets were re-suspended in 100µL PBS. HTMCs were cultured as described above. At 60% confluence, the cells were exposed to the stained exosomes for 0, 6, 12, and 24 hours respectively. Then, the hTMCs stained with 4, 6-diamidino-2-phenylindole (DAPI), were mounted on a slide and observed under a fluorescence microscope.

Oxidative stress and human BMSC-derived exosomes pretreatment

HTMCs were seeded at a density of 1×10⁶ cells per well then cultured in normal growth medium for 24 hours. After washing 3 times with PBS, the cells were cultured in a complete media (containing exosome-free serum) with hBMSC-derived exosomes (1×10⁸ particles/ml) or PBS for 24hours. After another 3-time-washing, hTMCs were treated with 0.1mM H₂O₂ for 6, 12, and 24 hours respectively. Then, the supernatant and cell lysis solution were collected for further analysis.

Cell counting Kit-8

After treatments, cell viability was quantified at 6, 12, and 24 hours using a Cell Counting Kit-8 (Sigma-Aldrich, USA). A total of 100 ml aliquots of medium was transferred to 96 well plates and absorbance at 450 nm was measured using a spectrophotometer (Bio-rad; USA) and normalized to control.

Measurement of intracellular ROS

Intracellular ROS (iROS) was determined by 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA; Sigama, USA). After the above treatments, the culture medium with H₂O₂ was discarded and hTMCs were incubated with 10µM of DCFDA for 30 minutes in PBS. This was followed by PBS wash, incubation in media for 20 minutes, and trypsinization. Lastly, cells were collected in PBS and kept on ice until analyzed by FACS Aria III flow cytometer (Becton, Dickinson and Company; USA). An average of 10,000 cells was analyzed in each experiment. All experiments were performed in triplicate.

Quantitative Real-Time PCR (RT-PCR)

Total RNA was extracted from cultured cells using Trizol reagent (TaKaRa, Japan) as per the manufacturer's protocol. After quantifying the RNA concentration, a cDNA library was constructed using a Reverse Transcriptase kit (TaKaRa, Japan) following to the manufacturer's instructions. QRT-PCR (TaKaRa, Japan) was used to measure RNA levels. Samples were normalized to internal control GAPDH. Primer sequences are listed as follows: IL-1α (forward primers 5’-AGG CTG CAT GGA TCA ATC TGT GTC-3’; reverse primers 5’-CTT CCT CTG AGT CAT TGG CGA TGG-3’), IL-1β (forward primers 5’-CTG AAA GCT CTC CAC CTC CA-3’; reverse primers 5’-TCA TCT TTC AAC ACG CAG GA-3’), IL-6 (forward primers 5’-GGT GTT GCC TGC TGC CTT CC-3’; reverse primers 5’-AGA TGC CGT CGA GGA TGT ACC G-3’), IL-8 (forward primers 5’-TCT CTT GGC AGC CTT CCT GA-3’; reverse primers 5’-TTT CTG TGT TGG CGC AGT GT-3’), MMP-2 (forward primers 5’-GCC TCT CCT GAC ATT GAC CTT GG-3’; reverse primers 5’-CAC CAC GGA TCT GAG CGA TGC-3’), MMP-3 (forward primers 5’-GCC AGG GAT TAA TGG AGA TG-3’; reverse primers 5’-ATT TCA TGA GCA GCA ACG AG-3’), and GAPDH (forward primers 5’-TCG ACA GTC AGC CGC ATC TTC TTT-3’; reverse primers 5’-ACC AAA TCC GTT GAC TCC GAC CTT-3’).

ELISA Measurement

The levels of IL-1α, IL-1β, IL-6, IL-8, MMP-2, and MMP-3 in cell culture supernatant were detected by a multi-detection microplate reader using a double-antibody sandwich ELISA kit (Boster Biological Technology, USA). All results were normalized against a standard curve.

RNA sequencing

Total RNA from Exo group (pretreated with BMSC-derived exosomes for 24 hours and exposed to 0.1mM H₂O₂ for 6 hours) and control group (pretreated wit PBS for 24hours and exposed to 0.1mM H₂O₂ for 6 hours) were sequenced on BGISEQ500 + bgiseq-500 platform. RNA sosiequencing data were filtered by SOpAPnuke ⁴⁸ to eliminate reads with adapter, unknown base N ratio greater than 5%, or ratio of bases with mass value less than 10 greater than 20%. Small RNA sequencing data removes tags based on the following criteria: less than 18nt, with 5' adapter contamination or poly A, or without 3' adapter sequence or insert. HISAT ⁴⁹ and Bowtie2 ⁵⁰ were used as reference genome comparison. RSEM ⁵¹ was used to calculate gene expression, while FPKM was used to standardize gene expression.

The differentially expressed genes were calculated by DEGseq ⁵², and P-value (after BH and ST correction) < 0.05 and |Log₂-Fold Change|≥1. The co-expression of lncRNA and mRNA was calculated using Pearson correlation coefficiency. The co-expression relation pair was selected by P value < 0.01 and |cor|>0.9. The target genes of miRNA were quested through the miRTarBase database ⁵³. The network graph was drawn using Cytoscape software, while the subnet was mined by MCODE in Cytoscape. DAVID was used for pathway enrichment analysis.

Statistical analysis

All experiments were conducted at least 3 biological replicates. Results were presented as mean ± standard deviation (SD) of three independent experiments. A P value for difference was determined by t-test using GraphPad Prism software and Microsoft Excel. A differences was considered significant when a P value were less than 0.05 (P < 0.05).

Author contributions statement

J.G., Y.L. conceived the experiments. Y.L., J.Z., XZ. W. and X.W. conducted the experiments. XZ.W. and W. L. analyzed the data. Y.L. prepared the manuscript. All authors have contributed to revising the manuscript and fnal approval.

Additional information

The authors declare that they have no competing interests.

Data availability

Supplementary information is available for this paper at https://doi.org/10.5281/zenodo.4533663.

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No competing interests reported.

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Mesenchymal Stem Cell-Derived Exosomes Protect Trabecular Meshwork from Oxidative Stress

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Characterization of exosomes isolated from human BMSCs

Exosomes isolated from hBMSCs are uptaken by human trabecular meshwork cells (TMCs)

hBMSC-derived exosomes improve viability of hTMCs exposed to H₂O₂

Effects of hBMSC-derived exosomes on production of iROS in H₂O₂-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of proinflammatory factors in H₂O₂-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of MMPs in H₂O₂-exposed hTMCs

Effects of hBMSC-derives exoosmes on gene expression in H₂O₂-exposed hTMCs

Discussion

Methods And Materials

Human BMSCs culture

Isolation and characterization of exosomes

Human trabecular meshwork cells (hTMCs) culture

Exosome tracking

Oxidative stress and human BMSC-derived exosomes pretreatment

Cell counting Kit-8

Measurement of intracellular ROS

Quantitative Real-Time PCR (RT-PCR)

ELISA Measurement

RNA sequencing

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Mesenchymal Stem Cell-Derived Exosomes Protect Trabecular Meshwork from Oxidative Stress

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Characterization of exosomes isolated from human BMSCs

Exosomes isolated from hBMSCs are uptaken by human trabecular meshwork cells (TMCs)

hBMSC-derived exosomes improve viability of hTMCs exposed to H2O2

Effects of hBMSC-derived exosomes on production of iROS in H2O2-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of proinflammatory factors in H2O2-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of MMPs in H2O2-exposed hTMCs

Effects of hBMSC-derives exoosmes on gene expression in H2O2-exposed hTMCs

Discussion

Methods And Materials

Human BMSCs culture

Isolation and characterization of exosomes

Human trabecular meshwork cells (hTMCs) culture

Exosome tracking

Oxidative stress and human BMSC-derived exosomes pretreatment

Cell counting Kit-8

Measurement of intracellular ROS

Quantitative Real-Time PCR (RT-PCR)

ELISA Measurement

RNA sequencing

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

hBMSC-derived exosomes improve viability of hTMCs exposed to H₂O₂

Effects of hBMSC-derived exosomes on production of iROS in H₂O₂-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of proinflammatory factors in H₂O₂-exposed hTMCs

Effects of hBMSC-derived exosomes on expression of MMPs in H₂O₂-exposed hTMCs

Effects of hBMSC-derives exoosmes on gene expression in H₂O₂-exposed hTMCs