Collagen XII Is a Regulator of Corneal Stroma Structure and Function

Abstract

The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics. METHODS. The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XIIdeficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy. RESULTS. Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/corneas. The difference between these two groups was significant (P < 0.001, t-test). CONCLUSIONS. Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function

“…27 In the corneal stroma, collagen XII affects fibrillogenesis and keratocyte organization and regulates stromal stiffness. 16 Collagen XII null stromas are characterized by decreased interfibrillar space and increased tissue stiffness. 16 However, the effects of collagen XIV in stromal structure and function contrast with collagen XII.…”

“…Reverse transcription and quantitative realtime PCR analysis were performed as previously described. 16 The following primer sequences were used: Col14a1, 5 0 -ACCTGTGAGTGTCCCTGGTC-3 0 (forward) and 5 0 -AGGCCAGTCAGAGCATCACT-3 0 (reverse); and Actb, 5 0 -AGATGACCCAGATCATGTTTGAGA-3 0 (forward) and 5 0 -CACAGCCTGGATGGCTACGT-3 0 (reverse). Each sample was run in duplicate PCR.…”

“…Cornea samples from WT and Col14a1 À/À mice, 30 days old, were analyzed using transmission electron microscopy (TEM), as previously described. 16 Briefly, at least three corneas per group were dissected and fixed in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1 mol/L sodium cacodylate, pH 7.4, with 8.0 mmol/L CaCl 2 , post-fixed in 1% osmium tetroxide. The corneas were dehydrated in graded ethanol series, followed by propylene oxide.…”

“…The measurement protocol has been described in detail in a recent publication. 16 Briefly, elasticity was determined via indentation of corneas using a silicon nitride cantilever with a 2.5-mm silica bead affixed to the apex (k Z 0.12 N/m; Novascan Technologies, Inc., Ames, IA). Measurements were repeated 10 times in at least three different locations around the cornea.…”

“…and XIV, in corneal structure and function. 11,16 The purpose of this study was to establish the functional role(s) of collagen XIV in the development and function of the corneal stroma.…”