Purchase this article with an account.
Jingwen Cai, Kristin Marie Perkumas, William D Stamer, Yutao Liu; The role of TGFβ2 on mechano-responsive genes of bovine angular aqueous plexus cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5134. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
TGFβ2 plays a role in the aqueous humor (AH) outflow, but little is known about its specific role in Schlemm’s canal (SC) endothelial cells. SC cells are under constant mechanical strain generated by the pressure gradient across the outflow tissues. The glaucomatous SC cells are stiffer, likely contributing to elevated intraocular pressure (IOP). To study the effect of TGFβ2 on mechanical response of SC cells, we aimed to establish an in vitro model using bovine angular aqueous plexus (AAP) cells to study the function of SC endothelial cells.
Three strains of human primary SC endothelial cells were mechanically stretched 15%, 1 cycle/second for 24 hours using Flexcell FX-5000 Tension system, followed by isolation of total RNA. Expression of 9 previously identified glaucoma-related genes were examined for their mechano-response using droplet digital PCR assays. To establish an in vitro model of SC cells, we dissected and pooled bovine ocular tissues containing trabecular meshwork (TM) and AAP cells from 6 fresh bovine eyes, followed by collagenase I digestion. Cells were washed, filtered, and cultured in a 2% gelatin-coated T25 flask. After 10 days to reach confluence, cells were treated with 4ug/mL puromycin for 2 days to eliminate TM cells. Remaining AAP cells repopulated the dish and characterized by positive immunostaining of von Willerbrand factor (vWF) and VE-cadherin. Validated cell strains were treated with human TGFβ2 for 24 and 48 hours at 5 or 10 ng/mL. Then we profiled the expression of mechano-responsive genes using real-time PCR to investigate their response to TGFβ2 treatment in AAP cells.
Using human SC cells, we identified 9 genes with stretch-induced expression (CLDN23, DCN, EZR, TGFBR3, CAV2, GAS7, CYP1B1, ABCA1, and FMOD). IPA-based pathway analysis revealed their common connection to TGFβ pathway. Upon TGFβ2 treatment, AAP cells displayed reduced expression of DCN, ABCA1, CYP1B1, and elevated expression of TGFBR1, GAS7, and FMOD.
We have established an in vitro model using bovine AAP cells to study the function of SC cells. TGFβ2 may affect the mechano-response of AAP cells and may relate to IOP regulation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only