Collagen XII Is a Regulator of Corneal Stroma Structure and Function.

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.

Abnormal corneal endothelial maturation in collagen XII and XIV null mice.

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.

Proteomic signatures of the desmoplastic invasion front reveal collagen type XII as a marker of myofibroblastic differentiation during colorectal cancer metastasis.

Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.

Association of type XII collagen with regions of increased stability and keratocyte density in the cornea.

The anterior avian cornea possesses several distinct cellular and extracellular regions including the epithelial basal lamina, Bowman's layer and the interfacial matrix that separates Bowman's layer from the stroma. These unique regions differ biochemically, physically and morphologically but all contain type XII collagen. Previously, the collagen fibrils of several of these interfacial regions were shown to be stable to thermal and enzymatic denaturation. We reasoned that type XII collagen, a fibril-associated collagen, would be a good candidate to confer such stabilizing properties. The studies described herein were performed to localize type XII collagen and to assess its role in the interfacial matrices (IM). Using antibodies that react with both the short and long type XII collagen isoforms and that react specifically with the long isoform, we demonstrate that it is the short isoform that is present in Bowman's layer and the associated interfacial matrix lying between Bowman's and the stroma proper. In situ hybridization analyses demonstrate that both the epithelial and endothelial cells synthesize type XII collagen. In vitro cell culture analyses, however, demonstrate that in addition to epithelial cell synthesis, the stromal fibroblasts are capable of synthesizing type XII collagen as well. Immunofluorescence analyses performed at elevated temperature demonstrate that type XII collagen is thermally stable in Bowman's layer, but not in the anterior interfacial matrix or Descemet's layer. In addition, we observed that the distribution of type XII collagen during the development of the anterior extracellular matrices correlates precisely with an elevated density of keratocytes populating the interfacial matrix just deep to Bowman's layer. We show that this cellular density is developmentally regulated and does not arise from a localized increase in cell proliferation. These data demonstrate that Bowman's layer and the anterior interfacial matrix have unique biochemical and morphologic properties. Type XII collagen is thermally stable in Bowman's layer and, as a surface component of type I collagen fibrils, may contribute to the stability of the fibrils in this region. Neither type XII nor type I collagen is stable in the adjacent interfacial matrix, suggesting that differences in the type I-XII collagen fibril organization may exist between Bowman's layer and IM.