Changes in gene expression in response to mechanical strain in human scleral fibroblasts
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.
References (50)
- et al.
Jun-B is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2
J. Biol. Chem.
(1998) - et al.
Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction
Biochim. Biophys. Acta
(1992) - et al.
BMP 7 null mutation in mice: development defects in skeleton, kidney, and eye
Exp. Cell Res.
(1997) - et al.
Identification and functional characterization of a Smad binding element (SBE) in the Jun B promoter that acts as a transforming growth factor-β, activin and bone morphogenetic protein-inducible enhancer
J. Biol. Chem.
(1998) - et al.
Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework: analysis of global gene expression in Escherichia coli K12
J. Biol. Chem.
(2001) - et al.
Involvement of different ion changes in osteoblast's and osteocytes’ searly response to mechanical strain
Bone
(1996) - et al.
Cathepsin B and L activities in isolated osteoclasts
Biochem. Biophys. Res. Commun.
(1991) - et al.
Muscarinic antagonist effects on experimental chick myopia
Exp. Eye Res.
(1991) - et al.
The mechanisms and regulation of procathepsin L secretion from osteoclasts in bone resorption
FEBS Lett.
(1994) - et al.
A study of biochemical and biomechanical qualities of normal and myopic eye sclera in humans of different age groups
Metab. Pediatr. Syst. Ophthalmol.
(1983)
Localization of tenascin in human skin wounds—an immunohistochemical study
Int. J. Legal Med.
Cloned, stably expressed parathyroid hormone (PTH)/PTH-related peptide receptors activate multiple messenger signals and biological responses in LLC-PK1 kidney cells
Endocrinology
The treatment of myopia with atropine and bifocals: a long-term prospective study
Ophthalmology
Expression of bone morphogenic protein (BMP) 2 and BMP4 and receptors in the cornea (ARVO Abstract)
Invest. Ophthalmol. Vis. Sci.
Regulation of osteoclast differentiation by fibroblast growth factor 2: stimulation of receptor activator of nuclear factor κB ligand/osteoclast differentiation factor expression in osteoblasts and inhibition of macrophage colony-stimulating factor function in osteoclast precursors
J. Bone Miner. Res.
Tenascin-C expression by fibroblasts is elevated in stressed collagen gels
J. Cell Biol.
Cytotactin binding: inhibition of stimulated profileration and intracellular alkalinization in fibroblasts
Proc. Nat. Acad. Sci USA
Scleral changes in pathological myopia
Trans. Am. Acad. Ophthalmol. Otolaryngol.
An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family
Cell
A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye
Genes Dev.
Tenascin: a modulator of cell growth
Eur. J. Biochem.
FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Muller and RPE cells
Invest. Ophthalmol. Vis. Sci.
Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells
J. Bone Miner. Res.
Structure and sequence of human FGF8
Genomics
A G protein-linked receptor for parathyroid hormone and parathyroid hormone-related peptide
Science.
Cited by (70)
IOP and glaucoma damage: The essential role of optic nerve head and retinal mechanosensors
2024, Progress in Retinal and Eye ResearchCharacterization of arterial flow mediated dilation via a physics-based model
2020, Journal of the Mechanical Behavior of Biomedical MaterialsCitation Excerpt :At arterial bifurcations, for example, the flow exhibits a disturbed pattern resulting in a disturbed WSS spatiotemporal distribution, thereby triggering vascular remodeling that potentially leads to atherosclerosis (Cheng et al., 2006). Many diseases have been associated with defective mechano-sensing such as deafness, arteriosclerosis, cardiomyopathy, osteoporosis, glaucoma, kidney disease, premature ageing, cancer, and several others (Cheng et al., 2006; Vollrath et al., 2007; Garcia-Cardena et al., 2001; LiYi-Shuan et al., 2005; Palmer, 2005; Heydemann and Mcnally, 2007; Lammerding et al., 2004; Chang and Potter, 2005; Klein-Nulend et al., 2003; Tan, 2006; Johnstone, 2004; Cui et al., 2004; Nauli et al., 2003; Delmas, 2004; Capell et al., 2007; Verstraeten et al., 2008; Paszek et al., 2005; Huang and Ingber, 2005; Wolf et al., 2007; Suresh, 2007; Sarntinoranont et al., 2003; Huang and Ingber, 1999; Liang and Cheng, 2008; Liang et al., 2008; Dong et al., 2005). The loss of the glycocalyx was associated with alterations in the sublingual microvascular permeability and glycocalyx thickness in lacunar stroke patients with white matter lesions (Martens et al., 2013).
Mechanical strain affects collagen metabolism-related gene expression in scleral fibroblasts
2020, Biomedicine and PharmacotherapyCitation Excerpt :Previous studies have shown that mechanical strain affects the biological behavior of scleral fibroblasts [8,15]. In addition, there is a close relationship between mechanical strain and scleral remodeling [16,17]. In the present study, the appropriate mechanical strain improved collagen synthesis and changed the MMP-2/TIMP-2 balance to reduce MMP-2 function, and it also improved collagen deposition.
Scleral structure and biomechanics
2020, Progress in Retinal and Eye ResearchThe pilocarpine-induced ciliary body contraction affects the elastic modulus and collagen of cornea and sclera in early development
2018, Biomedicine and Pharmacotherapy