Dataset Information

Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

ABSTRACT: To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

SUBMITTER: Watanabe M

PROVIDER: S-EPMC9219943 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

Watanabe Megumi M Sato Tatsuya T Tsugeno Yuri Y Umetsu Araya A Suzuki Soma S Furuhashi Masato M Ida Yosuke Y Hikage Fumihito F Ohguro Hiroshi H

Biomedicines 20220606 6

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokine ...[more]

PMID: 35740359

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Project description:Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ?3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit ?, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ?3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

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.

Dataset Information

Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

Publications

Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

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