Cloning of alpha 1(IV) and alpha 2(IV) collagen cDNAs from rabbit corneal endothelial cell RNA.

To understand the role of type IV collagen in embryogenesis, regeneration, and tissue repair of the cornea at the molecular level, the authors isolated clones coding for rabbit alpha 1(IV) and alpha 2(IV) chains from a cDNA library constructed with rabbit corneal endothelial cell RNA. The isolated alpha 2(IV) cDNA clones encode a part of a 5' untranslated region, the signal peptide, the 7S domain, part of the triple-helical domain, and the entire carboxyl-terminal nontriple-helical (NC1) domain. By cross hybridization, using one of the alpha 2(IV) cDNA inserts, a cDNA clone encoding the alpha 1(IV) chain was isolated from a lambda gt10 library primed with oligo(dT). The clone covered a short portion of the triple-helical domain, the entire NC1 domain, and a short 3' untranslated region. The nucleotide-sequence analysis of these clones provides, for the first time to the authors' knowledge, the primary structure of the carboxyl-terminal portion of both rabbit alpha 1(IV) and alpha 2(IV) collagen chains. Between the alpha 1(IV)NC1 and the alpha 2(IV)NC1, 61% and 66% sequence similarities were observed at the amino acid and nucleotide levels, respectively. The locations of all the 12 cysteinyl residues were conserved in the two NC1 sequences.     

Characterization of corneal keratocyte morphology and mechanical activity within 3-D collagen matrices

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, α-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P ≤ 0.001). Time-lapse imaging of keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P < 0.01), causing increased compaction and alignment of collagen at the ends of the pseudopodia (P < 0.001). At high cell density, both keratocytes and fibroblasts appeared to form a 3-D network connected via gap junctions. Overall, this experimental model provides a unique platform for quantitative investigation of the morphological, cytoskeletal and contractile behavior of corneal keratocytes (i.e. their mechanical phenotype) in a 3-D microenvironment.     

The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures

PURPOSE: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the Mr 62,000 form of MMP-2 in keratoconic corneas. METHODS: Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-beta. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay. RESULTS: The addition of PDGF or TGF-beta to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP- 1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the Mr 62,000 species of MMP-2. CONCLUSIONS: PDGF may be involved in the production of the Mr 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.     

Human amniotic membrane, like corneal epithelial basement membrane, manifests the alpha5 chain of type IV collagen

PURPOSE: To reexamine whether the alpha5 chain of type IV (alpha5[IV]) collagen, thought to be absent, is in fact present in human amniotic membrane. METHODS: Cryosections of human amniotic membrane obtained at Cesarean section were immunohistochemically examined for the presence of alpha5(IV), with or without inclusion of the denaturing step. Amniotic membrane was digested with collagenase to release the noncollagenous NC1 domain from the alpha-chain. The NC1 domain of alpha5(IV) was then assayed on Western blot analysis. Identical experiments were performed with human corneas and conjunctivae obtained from an American eye bank. RESULTS: The basement membrane of denatured samples of amniotic membrane and cornea stained positive for alpha5(IV). Without the denaturing step, only corneal samples were positive. With or without denaturing, conjunctival epithelium did not stain. Western blot analysis detected NC1 domains of alpha5(IV) in amniotic membrane and corneal samples. CONCLUSIONS: The basement membrane of amniotic membrane resembles that of corneal epithelium but not conjunctiva. Amniotic membrane may be an excellent substrate for corneal epithelial cells.     

Aldehyde dehydrogenase (ALDH) 3A1 expression by the human keratocyte and its repair phenotypes

Transparency is essential for normal corneal function. Recent studies suggest that corneal cells express high levels of so-called corneal crystallins, such as aldehyde dehydrogenase (ALDH) and transketolase (TKT) that contribute to maintaining cellular transparency. Stromal injury leads to the appearance of repair phenotype keratocytes, the corneal fibroblast and myofibroblast. Previous studies on keratocytes from species such as bovine and rabbit indicate that the transformation from the normal to repair phenotype is accompanied by a loss of corneal crystallin expression, which may be associated with loss of cellular transparency. Here we investigated if a similar loss occurs with human keratocyte repair phenotypes. Human corneal epithelial cells were collected by scraping and keratocytes were isolated by collagenase digestion from cadaveric corneas. The cells were either processed immediately (freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. RT-PCR, western blotting and immunolabeling were used to detect mRNA and protein expression of ALDH isozymes and TKT. ALDH enzyme activity was also quantitated and immunolabeling was performed to determine the expression of ALDH3A1 in human corneal tissue sections from normal and diseased corneas. Human corneal keratocytes isolated from three donors expressed ALDH1A1 and ALDH3A1 mRNA, and one donor also expressed ALDH2 and TKT. Corneal epithelial cells expressed ALDH1A1, ALDH2, ALDH3A1 and TKT. Compared to normal keratocytes, corneal fibroblast expression of ALDH3A1 mRNA was reduced by 27% (n=5). ALDH3A1 protein expression as detected by western blotting was markedly reduced in passage zero fibroblasts and undetectable in higher passages (n=3). TKT protein expression was reduced in fibroblasts compared to keratocytes (n=2). ALDH3A1 enzyme activity was not detectable in corneal fibroblasts (n=6) but was readily detected in corneal epithelial cells (0.29+/-0.1U/mg protein, n=4) and keratocytes (0.05+/-0.009U/mg protein, n=7). ALDH3A1 expression was also reduced in corneal fibroblasts and myofibroblasts as determined by immunolabeling of the cells in culture (n=3) and in diseased corneal tissues in situ (n=2). We conclude that expression of the crystallin ALDH3A1 is decreased in repair phenotype human keratocytes, compared to normal human keratocytes. Extrapolating from studies of bovine and rabbit, the reduced expression of ALDH3A1 may contribute to the loss of corneal transparency experienced by human patients after injury and refractive surgeries.     

Postnatal corneal transparency, keratocyte cell cycle exit and expression of ALDH1A1

PURPOSE: Recent studies have shown that rabbit corneal keratocytes abundantly express two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1A1 (ALDH1A1), in vivo and that these proteins may contribute to corneal transparency at the cellular level. The purpose of this study was to determine the relationship between the expression of these proteins and the development of postnatal corneal transparency. METHODS: Rabbits 1 day to 42 days of postnatal age were evaluated by in vivo confocal microscopy (CM) to measure corneal epithelial thickness, stromal thickness, and corneal haze. Selected corneas were then processed for immunocytochemistry and Western and Northern blot analyses, to determine stromal cell density, cell cycle entry, and expression of ALDH1A1 and TKT. RESULTS: Quantitative measurement of corneal haze showed that the postnatal cornea was hazy after birth and became transparent during the first weeks after eyelid opening. Development of transparency was associated with decreased cytoplasmic light-scattering from postnatal corneal stromal cells, with the appearance of nuclear light-scattering after eyelid opening. Four days after birth, stromal cell density decreased rapidly, and the cells became quiescent, showing decreased staining by Ki67, a cell cycle marker. Whereas expression of TKT showed a gradual increase after birth, ALDH1A1 showed a marked increase after eyelid opening, and the combined expression significantly correlated with the reduction in light-scattering by postnatal stromal cells. CONCLUSIONS: The data suggest that development of postnatal corneal transparency is associated with decreased keratocyte density and quiescence and the expression of TKT/ALDH1A1.     

Avian corneal nerves: co-distribution with collagen type IV and acquisition of substance P immunoreactivity

Within the avian cornea collagen type IV is preferentially and characteristically localized to the epithelial and endothelial basement membranes. In the present paper, we demonstrate that collagen type IV also is present within the corneal stroma coincident with the development and distribution of corneal nerves indicating that intra-stromal fibers are associated with Schwann cells or an equivalent cell type. We also demonstrate intra-stromal fibers of collagen type IV orthogonal to the epithelial basement membrane. These novel structures are most prominent on the tenth day of development and become progressively less distinct until they are no longer detectable on the eighteenth day of development. Substance P immunoreactivity is prominently expressed by nerves innervating the epithelium. The first substance P immunoreactive nerves are detected on the twelfth day of development, concomitant with the initiation of epithelial innervation and not the extension of nerves through the stroma. Such nerve fibers become more numerous with progressive development and demonstrate extensive association with both basal and superficial epithelial cells. Thus, the avian cornea is richly supplied with substance P primary afferents. The expression of substance P immunoreactivity correlates directly with the initiation of innervation of the corneal epithelium.     

Type IV collagen and corneal epithelial adhesion and migration. Effects of type IV collagen fragments and synthetic peptides on rabbit corneal epithelial cell adhesion and migration in vitro

Type IV collagen, a 500-kilodalton (alpha 1)2(alpha 2)1 heterotrimer with noncollagenous domains (NC1) is the major molecule in most basement membranes in the body. In addition to its structural role as scaffolding, type IV collagen is involved in promoting adhesion and migration of various cell types in vitro, including rabbit corneal epithelial cells. This study assessed the effect of purified proteolytic fragments of type IV collagen and selected synthetic peptides derived from the alpha 1 and alpha 2 chains that are related to the adhesion and directed migration of dissociated primary cultured rabbit epithelial cells. Two homologous peptides (HEP-1 and HEP-2) derived from alpha 1 and alpha 2 NC1 regions were found to promote epithelial cell adhesion. A peptide (HEP-3) derived from an interruption of the triple helix of type IV collagen was effective in promoting corneal epithelial cell migration in both chemotaxis and haptotaxis assays. The helical fragment of type IV collagen promoted both directed migration and ample adhesion, indicating that there may be at least another moiety in the helical region responsible for cell adhesion. The results with these peptides revealed to some extent how corneal epithelial cells react at the molecular level with type IV collagen. They could serve as the basis for therapeutic agents to modify corneal epithelial behavior in situations of perturbed wound healing.     

Lattice corneal dystrophy associated with familial systemic amyloidosis (Meretoja's syndrome)

A 79-year-old white man of Irish descent presented with lattice corneal dystrophy, blepharochalasis, and peripheral seventh cranial nerve palsies. Family studies revealed that his 23-year-old daughter had early lattice cornea dystrophy. The corneal button removed by penetrating keratoplasty exhibited characteristic amyloid accumulation by light and electron microscopy. Biopsy of the patient's normal appearing conjunctiva and skin of the lower lid revealed amyloid. Biopsy of the daughter's conjunctiva was negative for amyloid, but her lid skin had characteristic amyloid deposits by light and electron microscopy. Immunoperoxidase strains were negative for AA and AP and serum prealbumin and SAA proteins were normal. Meretoja's syndrome has rarely been described outside a small geographic region in Finland. The clinical and histopathologic findings of this entity are discussed and contrasted to isolated "lattice corneal dystrophy."     

紫外线照射大鼠角膜胶原ⅠmRNA的表达

目的：观察紫外线照射大鼠角膜胶原ⅠmRNA的表达变化，探讨紫外线辐射对角膜的损伤机制。方法：用20mW、260～365nm的紫外线照射大鼠角膜，用RT-PCR方法检测照射后不同时间点角膜胶原ⅠmRNA的表达变化。结果：角膜经过紫外线辐射后30分钟，角膜胶原ⅠmRNA的表达量稍有上升，2小时下降最多，6小时回升超过正常，在24小时、48小时及7天、14天中角膜胶原ⅠmRNA的表达量呈持续缓慢上升。结论：紫外线辐射可影响大鼠角膜胶原ⅠmRNA的表达。     

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.     

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.     

Progress of corneal collagen cross-linking combined with refractive surgery

As a photochemical reaction that can stiffen the cornea, corneal collagen cross-linking (CXL) is the only promising method of preventing the progress of keratectasia, such as keratoconus and secondary ectasia following refractive surgery. The aim of CXL is to stabilize the underlying condition, with a small chance of visual improvement. Combining CXL with refractive surgery targeting both stabilization and reshaping of the corneal tissue for visual function improvement is a good treatment option. This review aims to provide a comprehensive and unbiased summary of the published research regarding combined CXL and refractive surgery, including measures and results, to help elucidate the future direction of CXL.     

Two mutations in the TGFBI (BIGH3) gene associated with lattice corneal dystrophy in an extensively studied family

PURPOSE: To determine the genetic basis for lattice corneal dystrophy (LCD) in an extensively studied family. METHODS: Ten affected family members were examined clinically, and three individuals were studied with in vivo confocal microscopy and optical coherence tomography (OCT). Corneal tissues from eight affected family members were examined histopathologically. The status of the transforming growth factor beta-induced gene (TGFBI) gene was determined in each consenting family member (six affected, seven nonaffected) by amplifying, sequencing, and analyzing exons 4 and 12 of TGFBI for mutations. All exons from the entire coding region of TGFBI of one affected person were analyzed for mutations. RESULTS: Slit lamp biomicroscopy disclosed the clinical features of LCD in both eyes of affected individuals. In vivo confocal microscopy confirmed the presence of deposits as bright lesions within the corneal stroma. OCT revealed increased reflectivity within the corneal stroma. The corneal stroma in persons undergoing penetrating keratoplasty contained amyloid. Affected members of the family were found to have two heterozygous single-nucleotide mutations in exon 12 of the TGFBI gene (C1637A and C1652A) leading to predicted amino acid substitutions in the encoded TGFbeta-induced protein (A546D and P551Q). Mutations were not detected in exon 4. In addition, an inconsequential single-nucleotide polymorphism T1620C (F540F) was found in some affected and nonaffected family members. CONCLUSIONS: Two mutations in the TGFBI gene (A546D and P551Q) cosegregated with LCD in an extensively studied family that lacked the R124C mutation that frequently accompanies this form of corneal amyloidosis.