Abstract
To establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.
Highlights
The only evidence-based therapy for the treatment of glaucomatous optic neuropathy (GON) is decreasing the intraocular pressure (IOP) to suitable levels by the administration of an anti-glaucoma medication, laser treatment or s urgery[1,2,3,4]
Effects of DEX and transforming growth factor β2 (TGFβ2) on the morphology, barrier function and permeability of the 2 and 14 (2D) cultured human TM (HTM) monolayers were studied by scanning electronic microscopy (SEM), transendothelial electron resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability, respectively
To elucidate the underlying mechanism causing these effects by DEX and TGFβ2, the mRNA expression of major extracellular matrix (ECM) comprised of HTM including COL1, 4 and 6, FN and α-smooth muscle actin (α-SMA) were evaluated
Summary
The only evidence-based therapy for the treatment of glaucomatous optic neuropathy (GON) is decreasing the intraocular pressure (IOP) to suitable levels by the administration of an anti-glaucoma medication, laser treatment or s urgery[1,2,3,4]. The molecular mechanisms responsible for the pathological changes in glaucomatous TM as well as the efficacy of several anti-glaucoma medications have been studied using these m odels[11] Most of these studies have used conventional 2D cell cultures, the HTM is composed of multiple sheets[12]. In our earlier pilot study using this methodology, we successfully obtained 3D HTM spheroids, and found that the TGFβ2-induced the formation of significantly smaller and stiffer 3D HTM s pheroids[16] Such TGFβ2-induced effects were substantially reduced by the presence of Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors. Based on these findings, we concluded that 3D cultures using HTM and TGFβ2 may be a physiologically relevant model for POAG. To establish a physiologically relevant in vitro models for POAG and SG by 3D cultures using HTM, we examined the effects of TGFβ2 or DEX on the size, morphology and physical properties of the 3D spheroids and the expression of ECM in these spheroids were compared with each other
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