Fig. 3: Glaucoma phenotypes are induced by ALK5/VEGFC-mediated SC endothelial dysfunction in the presence of TM.
a, ELISA-based examination of VEGFC expression in TM cells with or without DEX and SB431542 (n = 9 wells). b, Ocular fluid outflow on-chip models using human TM and SC cells. To validate the roles of VEGFR3 and TGF-β receptor signaling in DEX-induced glaucoma, the average flow rate after dexamethasone treatment (DEX, 1 μM) with or without TGF-β receptor inhibitor (SB431542, 10 μM) and VEGFR3 kinase inhibitor (MAZ51, 20 μM) was obtained (n = 12, 19, 20 and 19 chips, respectively). c, IOP measured in the steroid-induced mouse glaucoma model with or without SB431542 (10 mg kg−1 day−1, i.p. injection) or MAZ51 (10 mg kg−1 day−1, i.p. injection) treatments (n = 5 mice). d, Prox1 and VE-Cad staining of mouse SC endothelium in the steroid-induced glaucoma model with or without SB431542 or MAZ51 treatments. e, Junction widths of mouse SC endothelium in the steroid-induced glaucoma model with or without SB431542 or MAZ51 treatments (n = 7, 11, 10 and 13 FOVs in five mice). f, VEGFC expression in TM cells with ALK4, ALK5 or ALK7 knockdown under DEX or DMSO treatment (n = 9, 9, 9, 9, 9, 9, 3 and 6 wells, respectively). g, Average flow rate of fluid outflow in TM + SC coculture models using wild-type SC cells and silenced TM cells with ALK4, ALK5, or ALK7 siRNAs under DEX or DMSO treatment (n = 14, 14, 21, 18, 18, 14, 18 and 17 chips, respectively). Experiments described in a,f were performed using commercially obtained TM cells (ScienCell); experiments described in b,g were performed using both commercial TM cells (ScienCell) and primary TM cells isolated from donor eyes (Stamer laboratory). Scale bars, 25 μm (d). All data are presented as mean ± s.d. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (two-sided) in a–c,e; using unpaired two-sided Student’s t-tests in f,g. Exact P values are shown in a–c,e–g.
