TGFβ Induced Myofibroblast Differentiation of Rabbit Keratocytes Requires Synergistic TGFβ, PDGF and Integrin Signaling

Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas, Dallas, TX 75390-9057, USA.
Experimental Eye Research (Impact Factor: 2.71). 01/2003; 75(6):645-57. DOI: 10.1006/exer.2002.2066
Source: PubMed


There is a growing consensus that corneal myofibroblasts are derived from adjacent stromal keratocytes which undergo an orderly phenotypic transition from quiescent keratocyte to activated fibroblast to myofibroblast. Both in vivo and in vitro studies have shown this transition to be dependent, in part, on transforming growth factor beta (TGFbeta). In many fibroblastic cells autocrine production of platelet derived growth factor (PDGF) is known to mediate the growth up-regulation by TGFbeta. In this study, blocking antibodies to PDGF significantly reduced by 80% (P<0.025) the TGFbeta1 stimulated cell cycle entry of serum-free cultured rabbit corneal keratocytes. AntiPDGF treatment also markedly reduced the TGFbeta1-induced intracellular actin filament re-organization, fibronectin fibril assembly, and focal contact formation as well as reducing by 80% the expression of alpha-smooth muscle (alpha-SM) specific isoform of actin characteristic of myofibroblast differentiation. Although PDGF treatment of quiescent keratocytes produced an activated, fibroblastic cell type, PDGF stimulated keratocytes exhibited the same temporal, myofibroblastic differentiation response to TGFbeta1 as did quiescent keratocytes. Furthermore, blocking TGFbeta1 induction of myofibroblast differentiation with the Arg-Gly-Asp containing peptide, GRGDdSP, for 3 days followed by allowing progression of myofibroblast differentiation by removing GRGDdSP did not change the temporal response or tyrosine phosphorylation cascade (2-72 hr) leading to myofibroblast differentiation. Nor did PDGF treatment of keratocytes reverse the RGD blockade of TGFbeta1 induced myofibroblast differentiation. Overall these cumulative findings indicate that myofibroblast differentiation in the rabbit corneal keratocyte requires synergistic growth factor/integrin signaling involving TGFbeta, PDGF, and the fibronectin receptor. Additionally, the similar TGFbeta1 temporal response of PDGF-stimulated compared to nai;ve keratocytes suggests that myofibroblast differentiation does not require transition through a fibroblast phenotype.

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Available from: Walter Matthew Petroll, Dec 01, 2014
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    • "TGF-b isoforms play distinct functions in wound healing with TGF-b1/2 having predominantly pro-scarring roles and TGF-b3 having mainly anti-scarring effects (Finnson et al., 2013; Karamichos et al., 2014; Matsuba et al., 2011). PDGF is another cytokine that has been shown to be an important modulator of myofibroblast development in the cornea (Jester et al., 2002; Kaur et al., 2009b; Singh et al., 2011, 2014a; Stramer and Fini, 2004). IL-1 produced by stromal cells has been shown to trigger apoptosis of myofibroblasts when TGF-b is withdrawn from the cells (Barbosa et al., 2010b; Kaur et al., 2009a). "
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    ABSTRACT: The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or "haze". Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes in corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and corneal fibroblasts produce critical EBM components, such as nidogen-1, nidogen-2 and perlecan, that are essential for complete regeneration of a normal EBM once laminin secreted by epithelial cells self-polymerizes into a nascent EBM. Mature myofibroblasts that become established in the anterior stroma are a barrier to keratocyte/corneal fibroblast contributions to the nascent EBM. These myofibroblasts, and the opacity they produce, often persist for months or years after the injury. Transparency is subsequently restored when the EBM is completely regenerated, myofibroblasts are deprived of TGFβ and undergo apoptosis, and the keratocytes re-occupy the anterior stroma and reabsorb disordered extracellular matrix. The aim of this review is to highlight factors involved in the generation of stromal haze and its subsequent removal.
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    • "A similar increase in stress fiber formation and myofibroblast transformation of corneal keratocytes is observed within compressed 3-D collagen matrices; further demonstrating that increased substrate stiffness enhances TGFb-induced myofibroblast transformation of corneal keratocytes. Both connective tissue growth factor (CTGF) and PDGF have been shown to participate in TGFb-induced myofibroblast differentiation through an autocrine feedback loop, which would be amplified at higher cell density (Garrett et al., 2004; Jester et al., 2002). While stress fibers were observed in all cells within compressed ECM irrespective of cell density, the percentage of cells with a-SM-actin incorporated into stress fibers was greater at higher cell density (~60% versus ~20%), suggesting that both mechanical stiffness and autocrine signaling promote myofibroblast transformation in vitro (Lakshman and Petroll, 2012). "
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    ABSTRACT: The generation of cellular forces and the application of these physical forces to the ECM play a central role in mediating matrix patterning and remodeling during fundamental processes such as developmental morphogenesis and wound healing. In addition to growth factors and other biochemical factors that can modulate the keratocyte mechanical phenotype, another key player in the regulation of cell-induced ECM patterning is the mechanical state of the ECM itself. In this review we provide an overview of the biochemical and biophysical factors regulating the mechanical interactions between corneal keratocytes and the stromal ECM at the cellular level. We first provide an overview of how Rho GTPases regulate the sub-cellular pattern of force generation by corneal keratocytes, and the impact these forces have on the surrounding ECM. We next review how feedback from local matrix structural and mechanical properties can modulate keratocyte phenotype and mechanical activity. Throughout this review, we provide examples of how these biophysical interactions may contribute to clinical outcomes, with a focus on corneal wound healing. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Preview · Article · Apr 2015 · Experimental Eye Research
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    • "TGFβ signaling also plays multiple roles in the anterior segment of the eye: TGFβ1 plays a predominant role in the differentiation of keratocytes to myofibroblasts [12], [73], TGFβ2 is immunosuppressive in normal human and rabbit aqueous humor [74], [75] and is a key cytokine that can influence corneal wound healing [76]. In this study, no alteration to TGFß2 was observed with separate knockdown of either YAP or TAZ. "
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