Elsevier

Experimental Eye Research

Volume 78, Issue 2, February 2004, Pages 275-284
Experimental Eye Research

Changes in gene expression in response to mechanical strain in human scleral fibroblasts

https://doi.org/10.1016/j.exer.2003.10.007Get rights and content

Abstract

Scleral fibroblasts are involved in scleral remodeling during axial elongation in myopia. Mechanical load is a potent stimulator of gene expression. This study seeks to identify changes in gene expression of scleral fibroblasts in response to mechanical load and speculate on possible mechanisms of scleral remodeling in the development of myopia. Human scleral fibroblasts (HSFs) were mechanically stretched for 30 min and 24 hr. A gene microarray analysis was used to measure changes in gene expression. A total of 237 genes revealed differential and significant changes in expression (P<0·01) after 30 min of stretching. Of these, 28 unexpressed genes began to be expressed (turned on), while 31 expressed genes were no longer expressed (turned off). After 24 hr, 308 genes showed reproducible changes in expression (P<0·01), while 29 genes were turned on and 17 genes were turned off. After 30 min, 25 genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell growth/differentiation factors, extracellular matrix (ECM) proteins, lipid metabolism, protein metabolism, transcription factors, binding proteins and water channels. After 24 hr, 21 genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell growth/differentiation factors, lipid metabolism, ECM proteins, transcription factors, and carbohydrate metabolism. RT-PCR and Southern blotting confirmed the changes in expression of selected genes. In this study we identified a large number of early and late mechanical response genes in HSFs. These changes in gene expression will provide potential candidate genes that might be involved in scleral remodeling during axial elongation in myopia.

Introduction

Axial elongation in the development of myopia is facilitated by the sclera. High myopia in humans and animals is associated with a thinner sclera, particularly at the posterior pole of the eye (Curtin and Teng, 1958). Changes in the biochemical composition of myopic sclera include decreased glycosaminoglycan (GAG) and decreased collagen content (Avetisov et al., 1983). Scleral thinning in myopic humans was previously believed to be due to passive stretching of the tissue to cover the enlarged globe (Young, 1977). Studies of experimentally-induced myopia in both avian and mammalian models demonstrate that axial elongation in myopia is induced by active remodeling of the scleral ECM rather than by passive stretching of the scleral shell. The active remodeling reduces the scleral ECM content, including GAG and collagen, reducing scleral resistance to expansion in response to the normal range of intraocular pressures and resulting in axial elongation. Altered mechanical properties (such as increased creep rate) of the sclera in myopia (Phillips et al., 2000), likely promote scleral expansion, increase matrix remodeling and axial elongation.

Tissue remodeling involves multiple ECM proteins and degradative enzymes, such as the matrix metalloproteinases (MMPs) that degrade ECM proteins. The levels of many proteins are significantly influenced at the mRNA level. Analysis of mRNA expression levels of a large number of genes may help elucidate the molecular mechanisms responsible for the scleral changes described above. However, classical methods, such as reverse-transcriptase-polymerase chain reaction (RT-PCR), Northern blotting and nuclease protection assays, have limitations. Although differential display of amplified subsets of RNA allows a broad search for expression differences, the results are generally not quantitative and false positive findings are common (Locklin et al., 2001). Because cellular processes are controlled by a large number of genes that are expressed at different times and levels, the ability to simultaneously monitor a large number of genes is desirable. cDNA arrays have the potential to simultaneously quantify the expression of many genes in parallel.

Studies of both avian and mammalian models of myopia suggest that scleral remodeling processes contribute to axial elongation; but little is known about gene expression by human scleral fibroblasts and changes in expression that may occur during scleral remodeling. In this experiment, we used a gene expression microarray containing 12 000 genes to study the effects of mechanical stretch on early and late gene expression and the types of genes expressed in the HSFs.

Section snippets

Reagents

Tissue culture media, fetal bovine serum (FBS), trypsin–EDTA, Trizol Reagent and penicillin-streptomycin-fungizone were purchased from Gibco BRL (Carlsbad, CA, USA) and 6-well culture plates coated with laminin and constructed with flexible, silicone bottoms were obtained from Flexcell International Corporation (Hillsborough, NC, USA). Reverse transcriptase was purchased from Qiagen (Valencia, CA, USA). PCR primers were synthesized by Sigma-Genosys (The Woodlands, TX). Taq DNA polymerase was

Results

The gene expression of the cells that were mechanically stretched for 30 min and 24 hr, as well as that of the control cells, was analysed using the gene chip. Only those genes having consistent and significant changes (P<0·01) in expression compared with their control in the three independent experiments were considered to be significant. In response to 30 min of stretch, 237 genes showed significant changes in expression. Twenty-eight genes that were not expressed in the resting state became

Discussion

Changes in scleral biomechanical properties can induce scleral remodeling and axial elongation in the development of myopia. Scleral fibroblasts are the key cell in the scleral remodeling process. The changes in gene expression induced by mechanical load are not completely understood. Our previous studies have confirmed that scleral fibroblasts can produce MMPs, which are involved in scleral remodeling, in response to mechanical forces (Wei et al., 2001). The actin cytoskeletal network, some

Acknowledgements

Supported in part by NIH grant EY03040 and by an unrestricted grant from Research to Prevent Blindness, New York, NY, USA. The authors have no proprietary interest in any of the materials discussed in this article.

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