Developmentally regulated appearance of spliced variants of type XII collagen in the cornea.

PURPOSE: To determine whether temporal and spatial changes in the distribution of the long and short alternatively spliced variants of type XII collagen are associated with any specific morphogenetic events in pre- and postnatal development of the cornea and surrounding tissues. METHODS: The distribution of alternatively spliced variants of type XII collagen in fetal and newborn rabbit tissues was analyzed immunohistochemically using monoclonal antibodies that recognize either only the long form or both the short and the long forms of type XII collagen. RESULTS: During early fetal development of the cornea in rabbit (days 14 -17), the short form of type XII collagen was detected in the corneal stroma, the sclera, and the stroma in the rudimentary eyelid folds, whereas the long form was present only in the sclera. The long form was first evident in the cornea at day 24 but only in the posterior stroma. At later stages of prenatal development, the distribution of the long variant gradually extended toward the anterior stroma and in the newborn rabbit, the long variant was distributed throughout the entire stroma. However, in the eyelid, although the short form was present along the entire subepidermal regions both during fetal and neonatal development, the long form was transiently expressed between days 21 and 24 and was restricted to the subepidermal regions at the junction of the opposing eyelids. The long form of type XII collagen was first detectable in the basal epithelial cells and in its basement membrane (BM) at day 12 after birth, just before the opening of the eyelids. It continued to be present in the corneal BM zone in the adult rabbit but was not present in the limbal or conjunctival BM zone. CONCLUSIONS: The expression and distribution of the alternatively spliced forms of type XII collagen are developmentally and differentially regulated in the cornea, the sclera, and the eyelid. Although the short form is expressed in the stromal matrices of the cornea and surrounding tissues from early stages of corneal development, the appearance and distribution of the long variant form of type XII collagen coincide with the pattern of stromal condensation. Its first appearance in the corneal epithelial BM precedes the eyelid opening by 1 to 2 days, possibly suggesting that it may be involved in the tighter anchoring of the corneal epithelium to the underlying tissue or in promoting stromal condensation to assist in the separation of the corneal epithelium from the juxtaposed palpebral conjunctival epithelium of the eyelid.

Proteoglycan distribution during healing of corneal stromal wounds in chick.

Proteoglycan distribution during corneal stromal healing in growing corneas of young chicks were histologically and immunohistochemically analysed. Single linear incisions to produce partial-thickness wounds were made in the corneas of 5 day old chicks. The corneas were harvested at different times after wounding and processed for either histochemical analyses using periodic acid-Schiff's reaction (PAS) or for indirect immunofluorescence analyses of lumican, keratocan, keratan sulfate, perlecan and laminin. Linear corneal stromal incisions were completely covered by migrated stratified epithelium by day 2 post wounding and resulted in a gaping wound with a thinner stroma. New stromal scar tissue formed between the epithelium and the original stroma that resulted in partial restoration of stromal thickness. During the first two to three weeks of healing, the stromal tissue filling the depression formed from the gaping wound, was hypercellular and PAS positive, indicating significantly higher levels of glycoprotein content but no new Bowman's membrane was formed. By four weeks, the scar tissue occupied a 2-3 mm wide region. Immunofluorescence analyses indicated that other major differences in the healing and normally growing stroma were the increased synthesis and deposition of perlecan and laminin. No differences were evident in the immunofluorescence for keratocan or keratan sulfate in the scar tissue, but the scar tissue did contain markedly decreased levels of lumican. Thus, the regulation of proteoglycan and glycoprotein synthesis is altered in the keratocytes that are recruited to the wounded regions in the growing corneal stroma of post-hatched young chicks. While synthesis and deposition of adhesive molecules including laminin and perlecan are elevated, the synthesis of one of the keratan sulfate proteoglycans, lumican, is reduced in the scar tissue as compared to the normally growing stroma.

Type XII collagen contributes to diversities in human corneal and limbal extracellular matrices.

PURPOSE: To characterize diversities in the extracelhtlar matrices (ECMs) of the corneal and the surrounding limbal epithelium and stroma. METHODS: Immunohistochemical analyses were employed for screening monoclonal antibodies (mAbs) developed against ECM components of the human corneal epithelial basement membrane (BM) zone. In the current study, mAb BM8 was used as the monospecific probe to characterize its antigen (AgBM8) immunochemically, and to immunoselect a complementary DNA (cDNA) clone encoding AgBM8. Direct biochemical and cDNA sequence analyses were performed for the further characterization of AgBM8. An indirect colloidal gold-conjugated antibody technique was employed for immunoelectron microscopic analysis to study the distribution of AgBM8 in the corneal ECMs. RESULTS: The protein AgBM8, isolated from rabbit corneal stromal and epithelial tissues, was identified as the long-splice variant form of type XII collagen based on its size (approximately 340 kDa disulfide-linked subunits), the presence of collagenous domain(s) and a noncollagenous domain of approximately 300 kDa in its subunit structure, and its internal amino acid sequences. The identity of AgBM8 was further confirmed from the amino acid sequence (517 amino acids) deduced from the sequence of a cDNA immunoselected with mAb BM8. Immunofluorescence analyses indicated that the long form of type XII collagen is present in the ECMs of corneal stroma and in the sclera, as well as in the corneal epithelial BM zone but is absent in the limbal and conjunctival epithelial BM zones. It was not detectable in the subepithelial loose connective tissues in the limbus and in the bulbar conjunctiva. Immunoelectron microscopic analyses indicated that the long variant form of type XII collagen is present in corneal epithelial BM, Bowman's membrane, and the interfibrillar matrix of the corneal stroma. In the stroma, colloidal gold was distributed along the collagen fibrils with a periodicity of 150 to 200 nm. CONCLUSIONS: The long variant form of human type XII collagen, a member of the fibril-associated collagens with interrupted triple helices, referred to as FACITs, contributes to the differences in the BM zones of the cornea and limbus. Although many of the dense connective tissues in adult animals contain the short variant form of type XII collagen, human corneal stroma, the BM zone, and the sclera contain the long variant form as the predominant form of type XII collagen. In the corneal stroma, type XII collagen may be organized along the collagen fibrils in a uniform head-to-tail pattern.

Perlecan is a component of cartilage matrix and promotes chondrocyte attachment.

Aggrecan, a chondroitin/keratan sulfate-containing proteoglycan, is a major component of cartilaginous tissues. Immunolocalization studies, using antibodies directed to perlecan, a heparan sulfate proteoglycan first detected in basement membranes, and laminin (another major component of basement membranes), indicate that perlecan and laminin are also present in the matrices of hyaline cartilage in the nasal septum, the articular surface of the bone and the growth plate of the developing bone. Consequently, we used antibodies to both aggrecan and perlecan to characterize their synthesis and secretion by primary cultures of chondrocytes derived from the rat chondrosarcoma. Chondrocytes were pulsed for 20 minutes with [35S]methionine and then chased for up to six hours. The radiolabeled perlecan and aggrecan were immunoprecipitated and analyzed by SDS-PAGE. The results show that chondrocytes synthesize precursor proteins to both proteoglycans, but that only the aggrecan precursor protein is secreted as a proteoglycan. Perlecan was also secreted but with less posttranslational modifications than aggrecan. Northern blot analyses of the RNAs from immortalized rat chondrocytes indicated that the major mRNA encoding for perlecan was approximately 13 kb in length, similar in size to that expressed by other cell types, which synthesize 400 kDa core protein perlecan. Analyses of the proteoglycan fractions from the extracts of bovine articular surface indicated that perlecan in this tissue contains both chondroitin and heparan sulfate side-chains. Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro. These studies indicated that perlecan, once thought to be a unique component of the basement membranes, is more widely distributed and is an important component of the cartilage matrix, where it may provide for cell adhesion to the matrix.