Abstract
Trabecular meshwork (TM) and Schlemm’s canal (SC) are the main structures within the conventional outflow pathway, and TM cells and SC endothelial (SCE) cells are essential for controlling intraocular pressure. To examine the interaction between TM cells and SCE cells, we investigated whether exosomes contribute to intercellular communication. Additionally, TM cells in glaucoma acquire mesenchymal characteristics in response to transforming growth factor (TGF)-β2 and extracellular matrix proteins such as collagen type 1 (Col-1); these changes result in increased resistance of aqueous outflow. In this study, we stimulated TM cells with TGF-β2 and Col-1 and characterized the exosomal miRNAs (exomiRs) released in response to each stimulus. Isolated exosomes were rich in miRNAs, with downregulated miR-23a-5p and upregulated miR-3942-5p and miR-7515 levels following Col-1 or TGF-β2 stimulation. Next, a miRNA-mRNA network under TGF-β2 stimulation was constructed. There were no connections among the 3 miRNAs and predicted genes under Col-1 stimulation. GO and KEGG analyses revealed that the identified miRNAs were associated with various signaling pathways, including the inflammatory response. Interestingly, SCE cells treated with miR-7515 mimic showed increased VEGFA, VEGFR2, PECAM, and Tie2 expression. Ultrastructures typical of exosomes and positive staining for exosomal markers were observed in human TM cells. Our data showed that TM cells may communicate with SCE cells via exomiRs and that miR-7515 may be important for SCE cell reprogramming.
Highlights
Trabecular meshwork (TM) and Schlemm’s canal (SC) are the main structures within the conventional outflow pathway, and TM cells and SC endothelial (SCE) cells are essential for controlling intraocular pressure
TM cells were isolated from the eyes of cynomolgus monkeys (Macaca fascicularis) and were stimulated with 5 ng/ml transforming growth factor (TGF)-β2 or collagen type 1 (Col-1) to induce a mesenchymal phenotype[13], and exosomes were isolated from the cell supernatants using EXO-QUIC reagent
This study shows that TM cells secrete exosomes containing various miRNAs in response to TGF-β2 and Col-1 and that exosomal miRNAs (exomiRs) are associated with the expression of lymphatic and angiogenic genes in SCE cells
Summary
Trabecular meshwork (TM) and Schlemm’s canal (SC) are the main structures within the conventional outflow pathway, and TM cells and SC endothelial (SCE) cells are essential for controlling intraocular pressure. TM cells in glaucoma acquire mesenchymal characteristics in response to transforming growth factor (TGF)-β2 and extracellular matrix proteins such as collagen type 1 (Col-1); these changes result in increased resistance of aqueous outflow. We stimulated TM cells with TGF-β2 and Col-1 and characterized the exosomal miRNAs (exomiRs) released in response to each stimulus. Abbreviations TM Trabecular meshwork SC Schlemm’s canal microRNA MiRNA exomiR Exosomal microRNA AH Aqueous humor EV Extracellular vesicle. In glaucoma, TM cells transform into myofibroblastic cells, which lose normal TM cell characteristics and overproduce extracellular matrix (ECM) components; as a result, the resistance of AH flow and intraocular pressure (IOP) increase[8]. We hypothesized that the kind of exomiRs present depends on the status of TM cells, normal or glaucomatous/ mesenchymal, and examined which miRNA is a key regulator of SCE cells
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