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.