Microarray Analysis of the TGF-ß Induced Phenotypic Transition in Cultured Human Corneal Stromal Cells

Abstract

Abstract: : Purpose: TGF-ß is a major factor in corneal wound healing: blockade of TGF-ß inhibits corneal opacification, edema and angiogenesis. We characterized the effects of TGF-ß1 on gene expression in cultured human corneal stromal cells (HCSCs). Methods: HCSCs (derived from three different donor eyes) were treated for 48 hours with either FGF-1 and heparin to maintain a fibroblast phenotype, or with TGF-ß1 to convert to myofibroblast phenotype. Total RNA was extracted, processed and analyzed using either Affymetrix HU95Av2 or HU133A chips. Results: relative to FGF treated cells, over 220 unique gene transcripts increased in TGF-ß1 treated cells, while over 180 decreased. TGF-ß1 treatment is known to increase elaboration of extracellular matrix (ECM), cause cytoskeletal changes (including increased expression of α-smooth-muscle actin), elicit immunosuppression, and alter rates of growth and proliferation. In six pairwise comparisons, TGF-ß1 caused the following [mean fold] consistent changes: where previously reported in the literature these are in boldface. Collagen ECM: collagens Iα1 [10] and Iα2 [3.4], IIIα1 [7.1], IVα1 [36] and IVα2 [10], Vα1 [17] and Vα2 [3.7]; also the collagen synthetic enzymes lysyl oxidase [5.2] and proline hydroxylase [3.8]. Non-collagen ECM: biglycan [9.1], lumican [12.1], versican [4.0], perlecan [2.9], leprecan [3.0] and thrombospondin [4.3]. Cytoskeleton: actin α2 [5.7], actinin α1 [3.0], filamin A [2.5], tropomyosin 1(α) [5.7], transgelin [5.5], calponin [2.2] and caldesmon [4.7]. Immunosuppression: decreased expression of IL-1ß [-32], IL-8 [-39], and the chemokines CXCL1 [-14], CXCL2 [-36], CXCL3 [-62] CXCL5 [-44] and CXCL6 [-9.6]; also downregulation of the synthetic pathway for PGE₂ (PLA₂ [-4.1] / COX₂ [-4.6] / PGE synthase [-8.5]). Cell signaling: down-regulation of the TGF-ß signaling protein Smad3 [-7.0] and up-regulation of the signaling GTPase RhoB [2.8] both will act to decrease TGF-ß signaling. RhoE [-3.2] which acts to disperse actin stress fibers is down-regulated presumably to preserve the myofibroblast phenotype. Finally, the largest increase (mean, 122 fold) in all six comparisons was for an angiopoietin-like protein (CDT6) originally cloned from human cornea. CDT6 is potently profibrotic and anti-angiogenic. Conclusions: Comparative microarray analyses accurately reflect known TGF-ß-elicited phenotypic changes in HCSC. We have identified numerous additional changes which will enable us to more fully characterize this phenotypic transition, providing novel therapeutic opportunities in corneal wound healing.

Keywords: cornea: stroma and keratocytes • gene microarray • wound healing