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Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

ABSTRACT: 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. 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. 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. The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX-LPA signaling axis.

SUBMITTER: Watanabe M

PROVIDER: S-EPMC8584821 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

Watanabe Megumi M Furuhashi Masato M Tsugeno Yuri Y Ida Yosuke Y Hikage Fumihito F Ohguro Hiroshi H

International journal of molecular sciences 20211107 21

<h4>Purpose</h4>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.<h4>Methods</h4>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 (E ...[more]

PMID: 34769470

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Project description:It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.

Project description:BackgroundElevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.MethodsThe level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions.ResultsTGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition.ConclusionsTrans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Dataset Information

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

Publications

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

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