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Growth Differentiation Factor-15-Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

ABSTRACT: Purpose:To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-?-related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP. Methods:Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), ?-smooth muscle actin (?SMA), SMAD signaling, and gene expression were determined by immunoblot, immunofluorescence, mass spectrometry, cDNA microarray, and real-time quantitative PCR (RT-qPCR) analyses. Results:Growth differentiation factor-15, a common constituent of ECM derived from the human TM cells, was confirmed to be distributed throughout the conventional aqueous humor outflow pathway of the human eye. Growth differentiation factor-15 protein levels were significantly increased in human TM cells in response to TGF-?2, dexamethasone, endothelin-1, lysophosphatidic acid, TNF-?, IL-1? treatment, and by cyclic mechanical stretch. Stimulation of human TM cells with rGDF-15 caused a significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, SMAD signaling, gene expression, and the levels of ?SMA and ECM proteins. Conclusions:The results of this study, including a robust induction of GDF-15 expression by several external factors known to elevate IOP, and rGDF-15-induced increase in contractility, cell adhesion, and the levels of ECM proteins and ?SMA in TM cells, collectively suggest a potential role for GDF-15 in homeostasis and dysregulation of AH outflow and IOP in normal and glaucomatous eyes, respectively.

SUBMITTER: Muralidharan AR

PROVIDER: S-EPMC5152563 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Growth Differentiation Factor-15-Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

Muralidharan Arumugam Ramachandran AR Maddala Rupalatha R Skiba Nikolai P NP Rao Ponugoti Vasantha PV

Investigative ophthalmology & visual science 20161201 15

<h4>Purpose</h4>To determine the role and regulation of growth differentiation factor-15 (GDF-15), a TGF-β-related cytokine in human trabecular meshwork (TM) cells in the context of aqueous humor (AH) outflow and IOP.<h4>Methods</h4>Regulation of expression by external cues, and the distribution and secretion of GDF-15 by human TM primary cell cultures, and the effects of recombinant (r) GDF-15 on TM cell contractile characteristics, actin cytoskeleton, cell adhesion, extracellular matrix (ECM), ...[more]

PMID: 27918822

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Project description:PurposeBiophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models (in vitro or ex vivo) to study this phenomena.MethodsPrimary trabecular meshwork cells were isolated from segmental flow regions, and cells were cultured for 4 weeks in the presence or absence or dexamethasone to obtain cell derived matrices (CDM). The biomechanical attributes of the CDM, composition of the matrisome, and incidence of crosslinks were determined by atomic force microscopy and mass spectrometry.ResultsData demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. However, no correlations were observed between elastic moduli and presence or absence of mature and immature crosslinks in the CDMs.ConclusionRegardless of a direct correlation between matrix stiffness and crosslinks, we observed distinct differences in the composition and mechanics of the matrices deposited by segmental flow cells. These results suggest distinct differences in cellular identify and likely a basis for mechanical memory post isolation and culture. Nevertheless, we conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

Dataset Information

Growth Differentiation Factor-15-Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

Publications

Growth Differentiation Factor-15-Induced Contractile Activity and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

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