Human corneal fibrosis: an in vitro model.

One of the critical objectives associated with corneal biology is the understanding of how an organized, dense collagen matrix develops from a population of cells. To be able to even approach this question, we must develop techniques that allow us to understand the underlying mechanisms of collagen production and organization. In the human cornea, scarring is an inevitable result after trauma, infection, or refractive surgery and can produce blinding complications. Currently, treatment options are limited and consist primarily of corneal transplantation. Pharmacologic intervention is available to slow corneal wound repair; however, it can lead to ulceration rather than prevent scarring. Except for the epithelium, the human cornea exhibits little-to-no regenerative capacity. A mature human corneal stroma is a relatively acellular extracellular matrix (ECM) comprised primarily of hydrated type I/V collagen fibrils (15% wet weight) of uniform diameter (∼30–35 nm), glycosaminoglycans (GAGs), keratan and dermatan sulfates, various proteoglycan (PG) core proteins, and miscellaneous other protein constituents, including type VI collagen.1,2 Stromal function is critically dependent on its nanoscale structure. The major structural collagen of the stroma (type I/V heterotypic collagen—20% type V) is arranged in approximately 250 to 400 lamellae of parallel, non–cross-linked fibrils.3 The fibrils of adjacent lamellae are nearly perpendicular to each other. On corneal injury, the keratocytes are stimulated to proliferate (termed fibroblasts) and migrate to the wound site.4 In some types of wounds, the fibroblasts differentiate further into myofibroblasts and exhibit filaments consisting of smooth muscle actin (SMA). Fibroblasts and myofibroblasts synthesize and secrete a variety of ECM components, including type I and III collagens and fibronectin. When keratocytes are isolated and placed into culture in the presence of serum or growth factors, they become proliferative and then become increasingly quiescent as they reach confluence. Several groups found that the addition of ascorbic acid (vitamin C) increases the proliferative rate of cultured fibroblasts and a loss of contact inhibition.4–9 In addition, it was reported that vitamin C (VitC) stimulates the synthesis and secretion of ECM components.5,6,10,11 VitC acts as a cofactor for the enzymes responsible for hydroxylation of the lysine and proline residues on procollagen. These hydroxylations are essential for the stabilization of the interaction of the α chains in the triple helical regions of collagen. This stabilization may allow stratification of cells and accumulation of matrix. Subsequently, it was found that more stable forms of VitC—l-ascorbic acid 2-phosphate and 2-O-α-d-glucopyranosyl-l-ascorbic acid—have a far more potent effect on deposition of ECM materials.5,7,8,12–14 VitC also has been found to stimulate the stratification of several fibroblast types, including dermal5 and corneal.7 Little is known about the growth factors associated with the developmental process; however, the growth factor most commonly associated with corneal fibrosis and fibrosis in general is TGF-β1.15–22 TGF-β1 has been demonstrated to stimulate the overproduction and deposition of ECM components, such as type I and III collagen,15–22 as well as, extradomain A fibronectin (EDA-Fn).23,24 The objective of the present study was to determine whether the stimulation of collagen synthesis by TGF-β1 increases the level of stratification and whether the deposited ECM express fibrotic markers similar to those in corneal scars.