Transforming growth factor‐β‐regulated gene transcription and protein expression in human GFAP‐negative lamina cribrosa cells

Abstract

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy, which is a major cause of worldwide visual impairment and blindness. Pathological hallmarks of the glaucomatous optic nerve head (ONH) include retinal ganglion cell axon loss and extracellular matrix (ECM) remodeling of the lamina cribrosa layer. Transforming growth factor-beta (TGF-beta) is an important pro-fibrotic modulator of ECM metabolism, whose levels are elevated in human POAG lamina cribrosa tissue compared with non-glaucomatous controls. We hypothesize that in POAG, lamina cribrosa (LC) glial cells respond to elevated TGF-beta, producing a remodeled ONH ECM. Using Affymetrix microarrays, we report the first study examining the effect of TGF-beta1 on global gene expression profiles in glial fibrillary acidic acid (GFAP)-negative LC glial cells in vitro. Prominent among the differentially expressed genes were those with established fibrogenic potential, including CTGF, collagen I, elastin, thrombospondin, decorin, biglycan, and fibromodulin. Independent TaqMan and Sybr Green quantitative PCR analysis significantly validated genes involved in regulation of cell proliferation (platelet-derived growth factor [PDGF-alpha]), angiogenesis (vascular endothelial growth factor [VEGF]), ECM accumulation and degradation (CTGF, IL-11, and ADAMT-S5), and growth factor binding (ESM-1). Bioinformatic analysis of the ESM-1 promoter identified putative Smad and Runx transcription factor binding sites, and luciferase assays confirmed that TGF-beta1 drives transcription of the ESM-1 gene. TGF-beta1 induces expression and release of ECM components in LC cells, which may be important in regulating matrix remodeling in the lamina cribrosa. In disease states such as POAG, the LC cell may represent an important pro-fibrotic cell type and an attractive target for novel therapeutic strategies.

Myofibroblasts

“…Keratocytes have a number of specific features that are usually lost during myofibroblast activation, such as production of crystalline proteins (Chen et al, 2011a;Jester et al, 2012). Other eye-specific myofibroblast precursors are cells derived from the lamina cribrosa in glaucoma (Kirwan et al, 2005;Wallace et al, 2014) and Müller cells in proliferative diabetic retinopathy (Guidry et al, 2009). EMT is another local source to possibly generate ocular myofibroblasts from human lens epithelial cells (Dawes et al, 2009;Liu et al, 1994;Lovicu et al, 2015;Nishi et al, 1996), retinal pigment epithelial cells (Chen et al, 2014;Parapuram et al, 2009), and corneal epithelial progenitor cells (Kawakita et al, 2005;Kawakita et al, 2013).…”

Global Changes in Optic Nerve Head Gene Expression after Exposure to Elevated Intraocular Pressure in a Rat Glaucoma Model

“…101 Exposure to TGF␤1 resulted in expression of genes for ECM components, cell proliferation, angiogenesis, and growth factor binding. 102 To explore a potential role for epidermal growth factor in glaucoma, rat ONH astrocyte cultures were stimulated with EGF, and the upregulation of EGF receptor and other messages were evaluated. 103 In common, we found an upregulation of leukemia inhibitory factor, versican, Cd44, TGF␤1, and endothelin converting enzyme 1 in the pressuredamaged ONH.…”

Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

“…16 Freshly purified CD8 ϩ T cells or CD16 ϩ /CD56 ϩ NK cells (5 ϫ 10 6 ) from patients with either T-LGL (n ϭ 5) or NK-LGL (n ϭ 7) leukemia or from normal controls were subjected to total RNA extraction using TRIzoL LS Reagent (Invitrogen) following the manufacturer's directions. First-strand cDNA was synthesized from 2 g of purified total RNA using random hexamers and Moloney murine leukemia virus reverse-transcription reagents (Invitrogen) in a total volume of 20 L. A total of 1 g of cDNA was applied in a 10-L PCR mix using a QuantiTect SYBR Green PCR kit (QIAGEN).…”