The molecular basis of corneal transparency

Abstract

The cornea 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. The stroma 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 and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sulfate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-beta would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.

Figure 1

Transmission electron micrographs of cornea structure. The cornea is limited by an outer epithelium, and an inner endothelium (low magnification inset in A). (A) The stroma makes up more than 90% of the corneal thickness and contains keratocytes (K) orientated parallel to the corneal surface and found between the stromal lamellae. (B) Enlargement of area in rectangle in A. The lamellae are composed of small diameter collagen fibrils with regular packing. Adjacent layer are at approximately right angles to one another forming an orthogonal lattice.

Figure 2

Stromal fibril structure. The wild type mouse cornea has small diameter collagen with regular packing consistent with corneal transparency. Type V collagen nucleates initial fibril assembly. Reduction in the concentration of type V collagen by half in the heteterozygous Col5a1 mouse results in half the nucleation sites. Therefore, fewer collagen fibrils with larger diameters are found in the stroma of this mouse model. Decorin is the major class I leucine-rich proteoglycan in the cornea. A targeted deletion of decorin and blocking the functional compensation by closely related biglycan in the compound decorin/biglycan mouse model results in a sever disruption in the regulation of fibril assembly. A target deletion of lumican a class II leucine-rich proteoglycan alters regulation of fibril assembly primarily in the posterior stroma.

Figure 3

Corneal stromal development. Neural crest derived keratoblasts invade the space between the developing corneal epithelium and endothelium, proliferate, and produce an extensive hyaluronan (HA) rich ECM. The keratoblasts then differentiate into keratocytes and replace the HA rich ECM with a collagen fibril/proteoglycan rich ECM that is transparent.

Figure 4

Corneal post-natal development. Light microscopy (Left column) and transmission electron microscopy (right column) of the developing mouse cornea at post-natal (P) day 4, 10 and 90. Four days after birth (P4) the cornea is cellular and hydrated with less regularly packed collagen fibrils (P4). The stroma reaches it maximum thickness around P10 and then begins to compact. The mature cornea (P90) has thinned and the fibrils are regularly packed.

Figure 5

Healing in a corneal stromal incisional wound. The corneal epithelium loses it hemidesmosome attachment to the basement membrane and migrates over the wound site. The quiescent keratocytes are activated to proliferate and produce α smooth muscle actin but synthesize only low levels of ECM. The resulting hypercellular myofibroblasts accumulate in regions under the epithelium. The hypercellular myofibroblasts then become either wound fibroblasts that produce a normal collagen fibril containing ECM that restores transparency or become myofibroblasts that produce a light-scattering collagen fibril containing ECM that is opaque.