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Purpose: 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 XII-deficient 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.

Purpose: Maturation of the endothelium and the adjacent matrix was characterized in wild-type (WT) mice. The influence of FACIT collagen XII and XIV deficiency on the morphology, maturation, and function of the corneal endothelium was examined., Methods: Analysis of the endothelium and Descemet's membrane (DM) was performed using transmission electron microscopy at postnatal day (P)4, P14, and P30 in WT, Col12a1(-/-), Col14a1(-/-), and Col12a1(-/-)/Col14a1(-/-) mice. Endothelial junctions were analyzed using ZO-1. The presence of endothelial-stromal communications was evaluated with phalloidin staining as well as electron microscopy. Finally, corneal thickness was assessed., Results: A thin DM, clefts between endothelial cells and DM, and large "vacuole-like" structures were present in the endothelial cells of WT mice at P4 but not noted at P30. The endothelia of Col12a1(-/-), Col14a1(-/-), and compound Col12a1(-/-)/Col14a1(-/-) in the P30 cornea maintained the vacuole-like structures seen at P4. A mature endothelial junction pattern was delayed in the null corneas. Expression of ZO-1 in WT endothelia at P14 was diffuse and localized to the basolateral and apical cell membrane. At P30, staining was localized to intercellular junctions. ZO-1 reactivity was patchy in Col12a1(-/-), Col14a1(-/-), and compound Col12a1(-/-)/Col14a1(-/-) corneas at P14 and P30. Stromal thickness was increased in P30 null corneas. Endothelial cell processes were demonstrated penetrating the DM and into the underlying stroma, throughout the entire endothelial layer in the P4 cornea., Conclusions: Collagen XII and XIV null mice demonstrate delayed endothelial maturation. The structural alterations suggest functional changes in endothelial function resulting in increased corneal thickness. Endothelial-stromal interactions suggest a pathway for signal transduction.