Regulation of corneal stroma extracellular matrix assembly

Abstract

The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus.

Keywords: collagens; corneal stroma; extracellular matrix; fibrillogenesis; small leucine-rich proteoglycans; stromal organization.

Figure 1. The ultrastructure of the cornea stroma

Three layers specifically define the cornea: the outer epithelium, the stroma, and the inner endothelium. All three layers can be seen; the stroma comprises more than 90% of the corneal thickness (A, inset). (A) The stroma contains keratocytes (K) positioned parallel to the corneal surface, between the stromal lamellae. (B). The lamellae are composed of small diameter collagen fibrils with regular packing; adjacent layers are at approximately right angles to one another (B, is an enlarged area of the rectangle in A). P30 mouse cornea; A, inset light micrograph; A and B transmission electron micrographs. Figure modified from (Hassell and Birk, 2010).

Figure 2. Keratoblast micro-domains during stromal embryonic development

(A,B) Transmission electron microscopy of keratoblasts from 14 d (stage 40) chicken embryos cut perpendicular to the cornea surface. (A) A keratoblast process contains an extracellular micro-domain, termed channels with several collagen fibrils in longitudinal section (white arrows). The fibrils in the in the process of being deposited, are perpendicular to the orientation of the fibrils (F) on either side of the cell process. (B) A corneal keratoblast that contains 4 small extracellular micro-domains. Three of them are similar in size and contain 7–10 collagen fibrils (white arrows) while the fourth one contains 19 collagen fibrils. Two of the smaller micro-domains are joining as indicated by the membranous connection (black arrow). (C) High voltage electron microscopy of the corneal keratoblasts from 14 d (stage 40) chicken embryo corneas cut parallel to the corneal surface (0.5 μm thick section). An extracellular micro-domain (indicated by white arrows) is seen running through most of this section, indicating how extensive this first level of extracellular micro-domains can be; running from deep with the keratoblast, opening at the cell surface and continuing into the extracellular environment. The inset shows a transverse section of the micro-domain within the keratoblast (black arrow) that contains eight collagen fibrils. (Tv: Transport Vesicle) Figure modified from Birk and Trelstad, 1984.

Figure 3. The keratoblast surface compartments and collagen fibrils deposited have an orthogonal organization

A high voltage electron microscopy of a 0.5 μm-thick-section from a 14 d (stage 40) chicken embryo cut parallel to the cornea surface. The corneal keratoblast and its processes demonstrate a roughly orthogonal organization. Bundles of the collagen fibrils, fibers are seen within cell surface foldings. Cell processes are seen separating fibers. A fusion of these compartments and a retraction of cell surface forms large surface associated compartments as seen at the black thin arrows. The compartment is seen along two major axes at approximately right angles to one another (white vs. black thick arrows). It is within these keratoblast-associated compartments that collagen fibril, fiber, and lamellar (L) formation occur. The inset shows a section cut perpendicular to a similar region within which fibers coalesce to form large bundles and lamellae (L) in a large compartment. (Tv: Transport vescicle; G: Golgi; N: Nucleus; Nu: Nucleolus; Fil: Filopodia.) Figure modified from Birk and Trelstad, 1984.

Figure 4. Corneal stroma collagen fibril assembly model

Collagen fibrillogenesis is a multiple-step process that is tightly regulated by the interaction of many molecules. (A) Initially, fibrils nucleate at the cell surface, due to interaction of collagen I and collage V, to form a heterotypic fibril. (B) Then fibril-associated collagens with interrupted triple helices (FACITs) interact with protofibrils to regulate fibril packing, lamellar assembly, and organization into the stroma. (C) Moreover, small leucine-rich proteoglycans (SLRPs) bind to the protofibrils’ surface to regulate linear and lateral fibril growth as protofibrils mature to collagen fibrils, thus resulting in a “corneal block,” wherein fibril diameters are limited and organization occurs in such a way to allow for proper refraction of light and transparency. Figure adapted from (Chen and Birk, 2013).

Figure 5. Heterotypic collagen fibrils

Collagen fibrils are heterotypic. (A) They are co-assemblages of quantitatively major fibril-forming collagens like collagen I, and regulatory fibril-forming collagens like collagen V or XI. Regulatory fibril-forming collagens have a partially processed N-terminal propeptide, retaining a non-collagenous domain that must be in/on the gap region/fibril surface. The heterotypic interaction is involved in nucleation during fibril assembly; typically these interactions promote regular packing of the lamellae, as in wild-type mice (B, D). However, an absence of collagen V, as in Col5a1^Δst/Δst cornea stroma-specific conditional null mice, causes disorganized lamellae packing associated with very large fibrils and abnormal structures (C, E). Panel A has been adapted from (Birk and Bruckner, 2011); panels B–E have been adapted from (Sun et al., 2011).

Figure 6. SLRP roles in regulating fibril assembly

Small leucine-rich repeat proteoglycans (SLRPs) regulate extracellular matrix (ECM) assembly, particularly linear and lateral fibril growth, by binding to collagen fibril surface (A). SLRPs also interact with ECM components besides collagens; these include cytokines and cell surface receptors. (B) SLRPs affect fibril diameter and spacing in the corneal stroma. (C, D) When SLRPs are absent, as in null mice or in gene mutations in human patients, the fibril structure, organization and spacing are impacted. Panel A has been adapted from (Chen and Birk, 2013).

Figure 7. FACIT roles in regulating fibril assembly

Fibril-Associated Collagens with Interrupted Triple Helices (FACIT) have 2–3 COL domains and 3–4 NC domains with a large N-terminal NC domain that projects into the inter-fibrillar space. The FACIT collagens all associate with the surface of collagen fibrils. Figure adapted from (Birk and Bruckner, 2011) and (Linsenmayer et al., 1998).