Determining How Biophysical Cues Regulate the Myofibroblast Differentiation of Corneal Keratocytes

Following traumatic injury or refractive surgery, corneal wound healing can initiate a fibrotic response which can lead to a decrease in ocular function. This fibrosis is due to, in part, the differentiation of quiescent corneal keratocytes into mechanically active myofibroblasts. Signaling downstream of transforming growth factor beta 1 (TGF-β1) has been shown to be a key regulator of this transformation. Myofibroblast differentiation is characterized by an increase is contractility and secretion of unorganized extracellular matrix (ECM) proteins, which can disrupt the organization of the stroma’s highly-aligned ECM structure and lead to corneal hazing. Past works have shown that ECM stiffness can modulate keratocyte behaviors; however, the biophysical cues that guide these changes are still unclear. Here, to better understand how ECM stiffness can modulate keratocyte behavior in response to TGF- β1, we fabricated soft (1 kPa) or stiff (10 kPa) polyacrylamide gels, functionalized with unpolymerized collagen I, to mimic normal or fibrotic corneal tissue. Harvested rabbit corneal keratocytes (NRKs) were then plated on these substrata or collagen-coated glass coverslips in the presence or absence of exogenous TGF-β1 with or without pharmacological inhibitors for either contractility (blebbistatin) or focal adhesion assembly (PF- 573228). After 5 days of culture, cells were fixed and stained for molecular markers of myofibroblast differentiation (α-SMA), contractility (pMLC), focal adhesion formation (vinculin), or TGF-β1 signaling (pSmad3). In other experiments, polystyrene microspheres were embedded within the gels to preform traction force microscopy (TFM). Results from these in vitro experiments show that when cultured in serum-free media regardless of substratum stiffness or the addition of inhibitors, NRKs exhibited morphologies characteristic of quiescent keratocytes, exerted low contractile forces with negligible levels myofibroblast differentiation, and focal adhesions were observed to be small, few and localized at the tips of cellular extensions. When NRKs were cultured in the presence of TGF-β1 without inhibitors, NRKs on stiff PA gels or collagen-coated glass coverslips displayed phenotypes typical of a myofibroblast differentiation: broad morphologies, increased numbers α-SMA-positive cells, increased contractility corresponding with large, abundant focal adhesions. On softer substrata, cells exhibited a more quiescent phenotype displaying long dendritic processes, less myofibroblast differentiation, decreased contractility and small focal adhesions localized at the tips of cellular extensions. The use of pharmacological inhibitors targeted the phosphorylation of non-muscle myosin light chain or focal adhesion kinase (FAK) significantly reduced stiffness-dependent differences in contractility and morphology. Treatment with FAK inhibitor also resulted in a significant decrease in myofibroblast differentiation and striking changes in subcellular patterning and size of focal adhesions. In other experiments, lamellar constructs were made by sandwiching FBS- or TGF-β1- treated NRKs between functionalized soft or stiff PA gels. In lamellar constructs of varying stiffness, FBS-induced fibroblasts remained viable and exerted relatively equal peak contractile stresses on both top and bottom substrates of varying stiffness. pSMAD3 nuclearization was observed in nearly all TGF-β1-treated NRKs cultured in the lamellar constructs regardless of ECM stiffness. Together, these data provide insight into the potential role of ECM stiffness on the myofibroblast differentiation of corneal keratocytes in response to TGF-β1 during wound healing.