(E-177) Decorin Coating of Collagen Surfaces Inhibits TGF-β1 Mediated Myofibroblast Differentiation in Corneal Keratocytes

Introduction: Within the native corneal stroma, proteoglycans such as decorin play an important role in the development and maintenance of the extracellular matrix (ECM). During wound healing, transforming growth factor β 1 (TGF-β1), along with other growth factors, is released into the stroma where it activates quiescent corneal keratocytes into highly contractile and ECM-secreting myofibroblasts which can disrupt the ECM and result in corneal scarring and blindness¹. Previous studies have found that decorin exhibits high affinities for many molecules including type I collagen, TGF-β1, and various cell surface receptors². By binding to TGF-β1 or cell receptors related to TGF-β1 signaling, decorin has shown promise in reducing myofibroblast differentiation as a potential therapeutic against fibrosis. However, the details of how decorin accomplishes this reduction in the context of the highly organized corneal stroma ECM are still not known. A better understanding of these details would aid in the development of more effective strategies for utilizing decorin as a therapeutic for corneal wound healing. In this study we investigated the effects of a decorin coating on the ability of various type I collagen surfaces (i.e., collagen monomers, random fibrils, or aligned fibrils) to inhibit corneal keratocyte myofibroblast differentiation in the presence of TGF-β1.

Materials and Methods: Random and aligned collagen fibril micropatterns were fabricated as described previously by either polymerizing a drop of collagen in a polydimethylsiloxane (PDMS) well or perfusing a solution of type I collagen through PDMS microchannels onto glass cover slips coated with a thin layer of PDMS³. Monomeric collagen substrates were also prepared to compare cell response to the decorin coating in the absence of fibrillar topography. Substrates were then blocked with 0.1% Bovine Serum Albumin (BSA), coated with a decorin solution (10 μg/mL), and briefly washed with Phosphate Buffered Saline (PBS, 1X). Normal Rabbit Keratocytes (NRK) were then seeded onto these substrates in basal media with or without TGF-β1 (5 ng/mL). NRK were then fixed, permeabilized, and blocked with 1% BSA before fluorescent labeling of actin (Phalloidin), nucleus (DAPI), and α-smooth muscle actin (α-SMA primary antibodies followed by Alexa-Fluor conjugated secondary antibodies) as a marker of myofibroblast differentiation.

Results: NRK were cultured for 120 hours in basal media with or without TGF-β1 on type I collagen monomer, random fibril, or aligned fibril substrates with or without a decorin coating. Fluorescent imaging of Alexa-Fluor labeled decorin confirmed its attachment to the collagen substrates. Exposure to TGF-β1 resulted in α-SMA expression in NRK on all collagen surface types, with an increase in expression on collagen monomer versus all fibrillar substrates (Figure 1). The addition of the decorin coating to TGF-β1 containing cultures yielded a roughly 50%, 45%, and 20% decrease in α-SMA expression on monomer, random fibril, and aligned fibril substrates, respectively. NRK cultured in basal media maintained their quiescent phenotypes and did not express α-SMA irrespective of the presence of a decorin coating.

Discussion: Decreased α-SMA expression in cells cultured on decorin coated collagen substrates suggests that the decorin bound to the collagen can inhibit TGF-β1 signaling and myofibroblast differentiation. This effect may be a result of decorin sequestering growth factor to the collagen surface or by detaching from the collagen to inhibit TGF-β1 in solution before it can bind to cell receptors. This effect was preserved in both monomer and fibrillar collagen substrates, suggesting that decorin can bind to both assembled and non-assembled collagen molecules. However, the decrease in α-SMA expression on fibrillar versus monomer substrates suggests a potential inhibitory effect of collagen fibrils on myofibroblast differentiation. This effect however, seemed to be erased with the addition of the decorin coating, possibly due to changes in the amount of decorin that can bind to each type of collagen surface.

Conclusions: Taken together, these data suggest that (i) decorin can bind to collagen in both monomer or fibrillar form, and that (ii) as a coating, decorin can inhibit TGF-β1 signaling and myofibroblast differentiation in corneal keratocytes.


Acknowledgements: This work was supported by the National Institutes of Health Grant R01 EY030190.

References: [1] Jester et al. TGFβ induced myofibroblast differentiation of rabbit keratocytes requires synergistic TGFβ, PDGF and integrin signaling. Exp. Eye Res. 75, 645–657 (2002). [2] Sylvester & Ratner. Collagen affinity coating for surface binding of decorin and other biomolecules: Surface characterization. Biointerphases 12, 02C419 (2017). [3] Lam et al. A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior. Biomed. Microdevices 21, (2019).