Differences in the TGF-β1-induced profibrotic response of anterior and posterior corneal keratocytes in vitro.

Keratocytes are the main cellular component of the stroma. They display a dendritic morphology and have gap junctions at the end of their processes that allow for rapid intercellular communication.1 By generating and maintaining the structure of the collagen lamellae and the stromal extracellular matrix, keratocytes play an important role in maintaining corneal clarity.2 In healthy corneas, keratocytes are believed to regulate stromal balance by synthesizing new collagens and proteoglycans and secreting enzymes to degrade old or unhealthy stroma.3 In response to wounding (e.g., during penetrating keratoplasty, laser refractive surgery, and accidental corneal insult), keratocytes become activated and can transform into repair phenotypes that include corneal fibroblasts and myofibroblasts. Expression of the cell surface protein Thy-1 indicates keratocyte transformation into corneal fibroblasts.4 Myofibroblasts are further characterized by their expression of α-smooth muscle actin (α-SMA), which provides them with contractile ability. Using electron microscopy and fluorescent probes for microfilaments, such corneal fibroblasts can be identified adjacent to wounds.5 Wound contraction correlates with intracellular F-actin expression in activated cells and with the synthesis and extracellular deposition of fibronectin and collagen type I. These intracellular and extracellular components are aligned and linked by α5β1, an integrin expressed by activated cells. The contraction of actin filaments causes a tightening of the interconnected network of cells and extracellular matrix.6 The expression of α-SMA, a biochemical marker for myofibroblast transformation, is exclusively localized within wounds and associates temporally with their contraction.7 TGF-β induces α-SMA expression and myofibroblast transformation in cultured corneal keratocytes, and antibodies to TGF-β reduce corneal fibrosis and fibronectin deposition in vivo.8–10 Increasing data suggest that not all fibroblasts behave in the same manner. There is heterogeneity in fibroblast phenotypic response to TGF-β, depending on the tissue of origin.11 These phenotypic differences may be the result of different signal transduction cascades that are elicited in response to TGF-β stimulation in different tissue types.11 However, phenotypic heterogeneity has also been observed in cells derived from the same tissue, and differences in the response to TGF-β have been correlated with certain cell surface markers. For example, orbital fibroblasts that are Thy-1 positive differentiate into myofibroblasts, whereas Thy-1–negative orbital fibroblasts differentiate into lipofibroblasts.12 There can also be heterogeneity in the proliferation rates of fibroblasts, depending on whether the cells are derived from quiescent or fibrotic tissue from the same source.13 Heterogeneity in the phenotypic response of fibroblasts to different growth factors has also been identified.14 Generally, though differences tend to be more remarkable when comparing cells derived from different tissues, they can also occur for cells derived from the same tissue. Although there is a growing body of knowledge regarding how keratocytes respond to injury, no systematic investigation of corneal keratocyte heterogeneity has been conducted based on corneal location (i.e., anterior vs. posterior, central vs peripheral cornea). Yet, normal, healthy corneas show clear differences between the anterior and posterior stroma in vivo. The anterior stroma has a greater density of keratocytes than the posterior stroma.15 Keratocytes in the anterior stroma are smaller than those in the posterior stroma.15 The anterior stroma also has smaller diameter collagen fibrils than the posterior stroma,16 and there are differences in glucose concentrations and proteoglycan ratios between the anterior and posterior stroma, with the anterior stroma having a lower concentration of glucose and a higher ratio of dermatan sulfate to keratan sulfate.17,18 The difference in the proteoglycan distribution likely affects stromal hydration; the anterior stroma has a lower water content than the posterior stroma.17 It may also impact optical properties across the stroma. For example, the anterior stroma has been found to have a higher refractive index than the posterior stroma,19 and differential corneal swelling20 in the stroma has been shown to affect corneal light scatter21 and transparency.22 Although structural differences between anterior and posterior stroma have been well characterized in situ, there is a dearth of information on how anterior and posterior keratocytes respond to injury and wounding stimuli. This knowledge has pertinent clinical applications as corneal surgeons increasingly turn toward lamellar and endothelial keratoplasties. These procedures entail differential surgical manipulation and wounding of the anterior and posterior corneal stroma. Given that corneal keratocyte activation and transformation into scar-forming myofibroblasts ultimately impacts postoperative ocular optics and visual performance, understanding differences between the anterior and posterior keratocytic response to wounding stimuli may be critical to patient outcomes. In this study, we asked whether feline cultured anterior and posterior primary keratocytes differ in their entry into the cell cycle, activation, and transformation into myofibroblasts in response to TGF-β1 stimulation and whether these differences translate into differences in wound closure in vitro. The feline cornea was selected for the present set of experiments because its curvature, size, thickness, and histologic and optical properties are more similar to those of humans than other mammals.23 For this reason, cats have been used extensively to study incisional and laser-induced corneal wound healing and its optical and biomechanical correlates.6,10,24–28 Feline and human corneas behave similarly after injury; thus, the cat cornea is considered a good model for understanding penetrating and posterior lamellar keratoplasties in humans.29–31