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Hannah Youngblood, Hongfang Yu, Patricia V. Schoenlein, Hongyan Xu, Yutao Liu; Response of trabecular meshwork cells to estradiol under TGFβ2- and stretch-induced stress. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2724 – A0088.
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The only modifiable risk factor for primary open-angle glaucoma, elevated intraocular pressure (IOP), is regulated by trabecular meshwork (TM)-related aqueous humor outflow. Estrogen signaling may play a role in lowering IOP. We sought to determine if 17β-estradiol (E2) treatment of primary human TM (HTM) cells could reverse the transcriptional profile induced by ocular hypertension (OHT)-inducing TGFβ2.
Primary HTM cells from 10 donors were cultured for >24 hours in estradiol-free medium before treating in the following groups: medium only, DMSO, 50nM E2, 10ng/mL TGFβ2, and 50nM E2 + 10ng/mL TGFβ2. For each cell line, treatments were performed simultaneously on cells cultured statically on plastic 6-well plates and cells undergoing cyclical stretch (1 cycle/sec) on a Flexcell Tension System for 24 hours. qRT-PCR was performed to assay the expression of 17 selected TGFβ2-responsive genes. The ΔΔCt method followed by one-way ANOVA was used to calculate expression changes in response to treatments normalized to ACTB expression. Using relative fold changes, a linear mixed effect model was used to test for the overall effects of stretch and sex on gene expression, using treatment, stretch, and sex as covariates.
Of the 17 TGFβ2-responsive genes profiled, TGFβ2 treatment significantly altered the expression of 4 genes in static and 8 genes in stretched HTM cells. Conversely, E2 treatment significantly altered the expression of 11 genes in static and BMP1 in stretched HTM cells. In several cases, genes that were significantly downregulated in response to TGFβ2 were significantly upregulated in response to E2 (e.g., SMAD2 and SMAD3 in static cells and BMP1 in stretched cells). Furthermore, in cells treated with both E2 and TGFβ2, E2 lessened or reversed the effects of TGFβ2 for several genes (e.g., NFATC1 and SMAD2 in static cells and BMP1, FST, GREM1, LAMC1, NFATC1, SMAD2, and SMAD3 in stretched cells). In addition to treatment response, nine genes were significantly impacted by stretch while two genes were significantly impacted by the sex of the cell donor.
TGFβ2 had more significant effects under stretch, suggesting that stretch may exacerbate TGFβ2 effects. In many cases, E2 was able to mitigate or reverse the transcriptional effects of TGFβ2. Therefore, E2 may ameliorate the effects of OHT-inducing TGFβ2 to reduce IOP, especially under conditions of mechanical stress.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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