Abstract
Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)–lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX–LPA signaling axis.
Highlights
In the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, ENPP2 is known as an autotaxin (ATX), a type of secreted glycoprotein [1,2,3]
To pursue this issue in more detail, we examined the effects of ATX and lysophosphatidic acid (LPA) on the size, morphology and physical properties of the 3D spheroids and the expression of several genes including extracellular matrix (ECM) molecules, their modulators including matrix metalloproteinase (MMP), tissue inhibitors metalloproteinase (TIMP), lysyl oxidase (LOX), and hypoxia inducible factor (HIF), and several endoplasmic reticulum (ER) t
To further examine the LPA-induced effects toward human TM, the barrier function of 2D cultured human trabecular meshwork (HTM) cell monolayers was evaluated by transendothelial electron resistance (TEER)
Summary
In the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, ENPP2 is known as an autotaxin (ATX), a type of secreted glycoprotein [1,2,3]. As a possible mechanism responsible for causing these ATX and LPA induced effects, the results of a conventional two-dimensional (2D) cell culture study using human trabecular meshwork (HTM) cells suggested that the ATX/LPA/LPA receptor signaling axis related to actin stress fibers, focal adhesions, and myosin light chain phosphorylation may be involved [10,11,17]. Our group recently reported on the development of a unique 3D cell drop culture system, and in our previous studies using this methodology [19,20,21,22], we were able to successfully produce spheroids, and that such TGFβ2-induced effects were substantially reduced in the presence of Rho-associated coiled-coil containing protein kinase (ROCK) inhibitors [23,24] These collective findings suggest that our 3D HTM spheroid system has the potential to serve as a physiologically relevant model that replicates human TM. This suggests that this model could be used in studies of the effects of the ATX/LPA/LPA receptor signaling axis on our
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