ReviewThe molecular basis of corneal transparency
Research highlights
► The first article to experimentally identify that a particular component of the corneal stromal ECM, keratan sulfate, was needed for corneal transparency was published in Volume I of this Journal 49 years ago with Dr. Anseth as author. ►The in vitro biochemical studies showing collagen type V and the proteoglycans lumican, decorin and keratocan were responsible for regulating the diameter and spacing of the collagen fibrils in the corneal stroma was confirmed in vivo by mutations and deletions in the genes for these products in both mice and men. ► The culture of keratocytes in serum free, chemically defined medium has implicated the growth factors IGF-I/II, FGF-2 and TGF-? as responsible for stromal development and the different phases of wound healing based on the synthesis of the different components of the ECM and ?-smooth muscle actin.
Introduction
The cornea is the major refractive element of the adult eye. It consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells (Fig. 1). There is also a population of transient bone marrow derived cells, monocytes (macrophages) and dendritic cells that reside in the cornea. The stroma comprises 90% of the thickness of the cornea. It consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25–30 nm fibrils that are regularly packed within lamellae. It was hypothesized, based on the physical properties of light, that this lattice-like structure produces minimal light scattering and therefore, transparency (Maurice, 1957, Benedek, 1971, Farrell et al., 1973).
The unique size and spacing of the collagen fibrils in the cornea stroma has suggested corneal stromal collagen may have unusual attributes and interactions associated with its assembly into small diameter, highly organized fibrils. However, there were several lines of evidence that indicated other components of the stromal ECM also were involved in reducing light scattering. There was a correlation between the appearance of metachromatic staining of the glycosaminoglycans in stromal ECM and appearance of the highly organized collagen lattice during the acquisition of corneal transparency in the developing embryo (Coulombre and Coulombre, 1958). There was also a loss of the metachromatic staining in opaque corneal wounds (Dunnington and Smelser, 1958) that coincided with a reduction in the levels of corneal keratan sulfate (Anseth, 1961), a glycosaminoglycan determined later to be a component of some proteoglycans. These observations generated considerable interest in characterizing the collagens and proteoglycans in the corneal stroma and determining their roles in the formation of a transparent ECM.
Section snippets
Collagen
All collagens are trimers, each composed of one, two or three distinct gene products. There are 28 known types of collagens that constitute a diverse family of glycoproteins (Gordon and Hahn, 2010). All collagens have a triple helical domain. The triple helical domains require proline for alpha helix formation and glycine at every third residue for packing into a triple helix. In addition, collagens contain two unique amino acids, hydroxyproline and hydroxylysine. Hydroxyproline is important in
Proteoglycans
All proteoglycans consist of a core protein with one or more covalently attached glycosaminoglycan (GAG) side chains (for review see (Iozzo, 1998)). Proteoglycans were originally named and grouped according to the type of GAG chain attached to the core protein. They have since also been grouped into families based homologous sequences of amino acids in their core protein that confer a particular activity. Most proteoglycans now fit into one of three major families: ones that either 1)
Stromal ECM Composition
The ECM of the corneal stroma consists primarily of collagen with lesser amounts of proteoglycans. The major fibril-forming collagens of the adult corneal stromal ECM are types I and V (Birk et al., 1986). The collagen fibrils of the corneal stroma are heterotypic fibrils: both of these 2 collagen types are present in each collagen fibril. Type V is involved in initiating fibril assembly (Wenstrup et al., 2004, Wenstrup et al., 2006) and regulating fibril diameter (Birk et al., 1990).
There are
Keratocytes
Keratocytes have a compact cell body with numerous cytoplasmic lamellapodia, that gives them a dendritic-like morphology, and are interconnected in a three dimensional network by these lamellapodia (Poole et al., 1993, Hahnel et al., 2000). The compact cell body minimizes the surface area of the keratocyte exposed to light and this probably serves to reduce light scattering while their processes provides for cell–cell communications. Light scattering by the keratocytes is also reduced by the
ECM production during stromal development
The formation of the cornea has been extensively studied in the chicken and the rabbit (Coulombre and Coulombre, 1958, Toole and Trelstad, 1971, Cintron et al., 1983, Funderburgh et al., 1986, Cornuet et al., 1994, Quantock and Young, 2008). The ectoderm, remaining after the formation of the lens placode, becomes the epithelium of the cornea. Neural crest cells (Table 1) migrate into the space between the corneal epithelium and the lens placode and form the endothelium of the cornea. Other
Corneal growth during post-natal development
Although the collagen fibrils in the chicken corneas are completely compacted at hatching, they are not at birth of mice (Fig. 4). Furthermore, the keratocytes in the corneal stroma continue to proliferate and produce ECM, although at a lower rate than during late embryonic development and this causes the cornea to continue to grow in diameter during the post-natal peroid. This growth was shown in rabbit corneas to occur as a result of uniform expansion of the cornea (Davison and Galbavy, 1985
ECM production during stromal wound healing
Although the keratocytes proliferate and are biosynthetically active during embryonic and post-natal development, they exhibit a relatively low level of activity in the adult cornea and are considered quiescent keratocytes (Table 1) (Jester et al., 1994, Muller et al., 1995, Zieske et al., 2001). However, the quiescent keratocytes can become active again after the cornea is injured (Fig. 5). When an incisional wound through the epithelium into stroma occurs, the keratocytes damaged during
A comparison of development and wound healing
Stromal development consists of an initial proliferation and hydration phase that is followed by a dehydration and collagen/proteoglycan synthesis phase. Stromal repair also consists of an initial proliferation phase but without hydration and this results in hypercellularity. This may be due, in part, to the ability of the endothelium of the adult cornea to pump salt and water out of the stroma to maintain a critical level hydration level. In addition, the hypercellularity in stromal repair can
Keratocyte activation
Keratocytes can be easily isolated from cornea by mincing the corneas into small fragments, digesting the tissue fragments with collagenase to release the keratocytes and collecting the keratocytes by centrifugation. Despite this trauma, the keratocytes that survive and attach to culture dishes in serum free medium retain their native, quiescent phenotype (Jester et al., 1996, Beales et al., 1999). This suggests that the physical abuse the keratocytes receive during isolation does not activate
Proliferation and ECM synthesis by keratocytes in vitro
While there are many different cell types that make the growth factors, it is the quiescent keratocyte that respond to the growth factors and make the new stromal ECM during wound healing. Because quiescent keratocytes isolated from the cornea stroma by collagenase digestion retain their quiescent phenotype in vitro when cultured in serum free media (Jester et al., 1996, Beales et al., 1999), they can be used to determine the effect that different growth factors have on the proliferation of
Spheroid formation by keratocytes in vitro
Recently, it has been shown that mouse and bovine keratocytes cultured in media containing both FGF-2 and ITS (a media supplement containing pharmacological levels of insulin) will proliferate, aggregate into densely packed clusters and, can be passed and expanded without loss of keratocan production (Yoshida et al., 2005, Funderburgh et al., 2008). Bovine keratocytes cultured in FGF-2 alone can be passed but stop proliferating after one or two passages and bovine keratocytes cultured in ITS
Stratification in vitro
Keratocytes cultured in media containing fetal bovine serum (FBS) proliferate at very high levels and, although a varying proportion of them are myofibroblasts (Masur et al., 1996), they are usually called corneal fibroblasts (Table 1). Corneal fibroblasts can be easily passed and expanded, and this has made them a popular substitute for keratocytes. Corneal fibroblasts will stratify when ascorbic acid is added to the media (Hata and Senoo, 1989, Saika, 1992, Guo et al., 2007b). Ascorbic acid
Candidate growth factors involved in the phases of wound healing
By comparing the expression of α smooth muscle actin and the production of ECM induced by the action of growth factors on keratocytes in vitro to that which occurs in vivo during wound repair, it is possible to speculate on the identity the growth factors that are responsible for each of the different repair phenotypes. A combination of two growth factors, FGF-2 and TGF-β, would be needed to induce the keratocytes to become the hypercellular myofibroblasts seen during the initial phase of wound
Fibroblasts
Based on the expression levels of keratocan and keratan sulfate, corneal fibroblasts differ from hypercellular myofibroblasts, wound fibroblasts and spheroids. Corneal fibroblasts make substantially reduced levels of keratocan and keratan sulfate (Beales et al., 1999, Funderburgh et al., 2003) while the hypercellular myofibroblasts and the wound fibroblasts seen in vivo (Sundarraj et al., 1998), the wound fibroblasts induced in vitro by IGF-I/II (Musselmann et al., 2005, Musselmann et al., 2008
Summary and Concluding Remarks
Transparency of the corneal stromal ECM is accomplished by regulation collagen fibril growth and spacing. The major collagen of the stroma is collagen type I, but collagen type V is also required to initiate assembly of this collagen into fibrils. The growth of the initial diameter of the fibril is regulated by type V/type I interactions and it is modulated and maintained by the core proteins of the proteoglycans decorin, lumican, keratocan, and biglycan that interfere with and limit the
Acknowledgements
Supported by Grants EY08104 (JRH) and EY05129 (DEB) from the National Institutes of Health. We thank Sheila Adams and Dr Simone Smith for help in prepartion of the figures.
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