An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment

Abstract

Due to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes. Methods: We harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators. To compare the cellular responses to candidate regulators of these mechanoresponsive genes, AAP and human SC cells (n = 3) were treated with 5 or 10 ng/mL transforming growth factor beta-2 (TGFβ2) for 24 or 48 hours, followed with expression profiling using real-time PCR or ddPCR. Results: We found that the isolated AAP cells displayed uniform cobblestone-like morphology and positive expression of two endothelial markers. In stretched SC cells, nine glaucoma-related genes were upregulated, and IPA implicated TGFβ as a potential upstream regulator. The effects of TGFβ2 treatment were similar for both AAP and SC cells in a dose-and time-dependent manner, activating TGFBR1 and SMAD2, inhibiting BMP4, and altering expression of three glaucoma-related genes (DCN, EZR, and CYP1B1). Conclusions: Bovine AAP cells may serve as an alternative cellular model of human SC cells. Translational Relevance: These AAP cells may be used to study the functional pathways related to the outflow facility.

A ROCK inhibitor suppresses the transforming growth factor-beta-2-induced endothelial–mesenchymal transition in Schlemm’s canal endothelial cells

“…SC cells from glaucoma patients show increased expression of the fibrosis markers α-SMA, fibronectin, and collagen, and increased cell proliferation 19 , 20 . These changes are similar to those induced by TGF-β2 stimulation 17 , 18 . Our findings imply induction of EndMT by TGF-β2 in SC cells.…”

“…TGF-β2 increases aqueous humor outflow resistance, which is thought to be related to epithelial–mesenchymal transition (EMT) induction of TM cells by TGF-β2, such as increased expression of ECM (fibronectin, collagen) and alpha smooth muscle actin (α-SMA) 12 – 16 . Moreover, TGF-β2 induces the endothelial mesenchymal transition (EndMT) in SC cells 17 , 18 . In addition, SC cells from glaucoma patients have higher expression of α-SMA, fibronectin, and collagen, and higher proliferative potential than those from non-glaucoma eyes 19 , 20 .…”

Fundamental Biomaterial Considerations in the Development of a 3D Model Representative of Primary Open Angle Glaucoma

“…Type I collagen coated polyacrylamide (PA) gels of varying stiffness (1–75 kPa) have been largely used in both TM and eSC culturing [ 25 , 125 , 126 ], illustrating the impact of substratum stiffness on TM/eSC functionality, modulating several characteristics such as actin stress fibre formation, focal adhesion size, cytoskeletal contractility and expression of several genes implicated in POAG [ 25 , 55 , 125 , 126 ]. When TM or eSC cells were subjected to static or cyclic biaxial strain when cultured on type I collagen [ 127 , 128 , 129 , 130 , 131 , 132 , 133 ] or laminin coated [ 134 ] PDMS, it was observed that the rate or magnitude of strain at differing time intervals significantly altered genes involved in cellular stress and ECM remodelling [ 127 , 128 , 131 ]. As TM cells experience pulsatile mechanical stress, stretch–strain and static models are intended to mimic these changes; however, they may not be wholly accurate [ 29 , 135 ].…”