Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas.

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.

Regulation of a Rho-associated kinase expression during the corneal epithelial cell cycle.

PURPOSE: It has been recognized that an increased expression of the Rho-associated kinase (ROCK-I), a downstream target of Rho (a Ras-related small guanosine triphosphatase [GTPase]), is associated with limbal-to-corneal epithelial transition. The purpose of the present study was to determine whether the expression of ROCK-I is regulated during the cell cycle of corneal epithelial cells. METHODS: Rabbit corneal epithelial cells in culture were subjected to different culture conditions to enrich them in the G0, G1, and S phases of the cell cycle. Indirect immunofluorescence staining and western blot techniques were used for analyzing the changes in the relative intracellular concentrations of ROCK-I. Northern blot analysis of the isolated cellular RNA was performed to estimate the relative concentrations of ROCK-I mRNA. RESULTS: Serum deprivation did not cause all the corneal epithelial cells in culture to be arrested in the G0 phase of the cell cycle. However, the cells could be arrested in G0 by treating them with culture medium supplemented with transforming growth factor (TGF)-beta1. The relative concentration of ROCK-I in the G0-arrested cells was higher than in the corresponding control untreated cultures. G0-arrested cells were induced to enter G1, followed by the S phase of the cell cycle, by refeeding them with the medium devoid of TGF-beta1. The total intracellular concentration of ROCK-I significantly decreased during the G1 phase of the cell cycle and increased again during the S phase. The decrease in intracellular ROCK-I during the G1 phase was confirmed by arresting the cells in G1 with isoleucine deprivation and thymidine-mimosine treatments. ROCK-I mRNA levels were also found to be decreased during the G1 phase of the cell cycle. CONCLUSIONS: The levels of ROCK-I in the corneal epithelial cells were significantly lower in the G1 phase than those in the S and G0 phases of the cell cycle. Therefore, a Rho signaling pathway(s) involving ROCK-I may be regulated during the corneal epithelial cell cycle. The downregulation of ROCK-I during the G1 phase, at least in part, is due to the decreased levels of its mRNA. Based on these findings, ROCK-I may have a role in the progression of the cell cycle in the corneal epithelial cells as they migrate centripetally from the limbal to the corneal surface.

Upregulation of DAF (CD55) on orbital fibroblasts by cytokines. Differential effects of TNF-beta and TNF-alpha.

PURPOSE: Decay accelerating factor (DAF) and membrane cofactor protein (MCP) are membrane complement regulators that protect self cells from deposition of autologous C3b on their surfaces. CD59, a third downstream regulator of the cascade, prevents the assembly on self cells of autologous membrane-attack complexes. All three proteins are highly expressed on corneal and conjunctival epithelia, and are present in lower levels on multiple intraocular and adnexal cell types. The purpose of this study was to determine whether, and if so, how DAF, MCP and CD59 expression by ocular and adnexyl cells is modulated by cytokines. METHODS: Primary cultures of orbital fibroblasts and corneal epithelial cells were incubated with TNF-alpha, TNF-beta, TGF-beta1, IFN-gamma, MIF or blocking anti-MIF mABs and extracts of the cells quantitated for DAF, MCP and CD59 by two-site immunoradiometric assays. Where inductions occurred, the kinetics of the increases, the effect of combining cytokines, and the effect of protein kinase-C inhibition were studied. RESULTS: DAF expression on orbital fibroblasts was upregulated 6.3-, 3.7- and 4.2-fold by TGF-beta1, TNF-beta and IFN-gamma, respectively, but that its expression on corneal epithelial cells was minimally affected. These same (or other) cytokines did not significantly upregulate MCP or CD59. The cytokine-induced upregulation of DAF expression on orbital fibroblasts requires 24 hr for IFN-gamma or 48 hr for TGF-beta1 or TNF-beta, is dependent on new protein synthesis, and does not involve protein kinase-C activation. CONCLUSIONS: TGF-beta1-, TNF-beta- and IFN-gamma-mediated upregulation of DAF should serve to prevent complement-mediated injury to orbital fibroblasts in the course of ocular inflammation. The induction by TNF-beta rather than TNF-alpha contrasts with that on all other cell types studied.

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.

Cloning, chromosomal localization, and characterization of cDNA from a novel gene, SH3BP4, expressed by human corneal fibroblasts.

The cornea contains, as a major element, a transparent stroma produced and maintained by keratocytes (fibroblasts). Through molecular biology studies using cultured human corneal fibroblasts, a cDNA that was shown to be novel was isolated and sequenced. This novel gene product, named SH3-domain binding protein 4 (SH3BP4), contains a 5.6-kb message that is present in normal human corneal fibroblasts and all tissues examined, with higher levels in pancreas, placenta, heart, and kidney. SH3BP4 was localized by FISH analysis to human chromosome 2q37.1-q37.2 near the telomere. The deduced sequence for SH3BP4 was found to contain a 963-amino-acid open reading frame that has homology to a 479-amino-acid protein in GenBank called EH-binding protein. Although the entire sequence of EH-binding protein aligns with SH3BP4, the alignment is not complete or contiguous. Therefore, SH3BP4 has an additional 73 amino acids at the N-terminus and an additional 411 amino acids near the C-terminus that are not present in EH-binding protein. Consensus sequence domains identified in SH3BP4 include a SH3 domain, three N-P-F motifs, a P-X-X-P motif noted for binding to SH3 domains, a bipartite nuclear targeting signal, and a tyrosine phosphorylation site. SH3BP4 homologies and consensus sequence sites indicate that it may be involved in a newly identified cascade of proteins involved in endocytosis, intracellular sorting, and the cell cycle.

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.

A Rho-associated protein kinase: differentially distributed in limbal and corneal epithelia.

PURPOSE: The authors have developed monoclonal antibodies (mAbs) to characterize the sequential biochemical changes in corneal epithelial cells after they differentiate from stem cells, located in the limbus, and migrate centripetally to follow the pathway of terminal differentiation. The purpose of this study was to identify a protein (recognized by mAb HE1/11F) with increased expression associated with the transition of the limbal epithelium to corneal epithelium. METHODS: The distribution and identification of the protein(s) were performed using an indirect immunohistochemical staining technique and a western blot analysis, respectively. A rabbit corneal epithelial cDNA library, constructed in the Uni-Zap XR vector, was screened with mAb HE1/11F to select cDNA clones expressing polypeptide(s) recognized by this mAb. Additional overlapping cDNA clones were obtained from a primer extension cDNA library to determine the sequence of the complete open reading frame encoding the protein recognized by mAb HE1/11F. RESULTS: Rabbit corneal epithelium exhibited strong immunostaining with mAb HE1/11F, however, the limbal epithelial cells stained weakly. HE1/11F recognized 160-kDa (HEBM1) and 100-kDa (HEBM2) polypeptides in the corneal epithelial extracts. The amino acid sequence of the protein deduced from the nucleotide sequence of the cDNA exhibited a close homology to that of a RhoA (Ras-related small GTPase)-associated serine-threonine kinase (ROCK-I or Rho-associated coiled-coil kinase). A 160-kDa RhoA-binding polypeptide with a molecular mass similar to that of HEBM1 and ROCK-I was detected in the corneal epithelial extracts. These findings strongly suggested that HEBM1 was rabbit ROCK-I. The identity of HEBM1 was further confirmed from the reactivity of mAb HE1/11F with ROCK-I immunoprecipitated from rabbit corneal epithelial extracts using anti-ROCK-I antibodies. CONCLUSIONS: The increased expression of a protein identified as ROCK-I from cDNA analyses is associated with rabbit corneal epithelial differentiation and transition from the limbal to corneal surface. Therefore, a RhoA signaling pathway is likely to be associated with corneal epithelial differentiation (maturation). A close homology among the cDNA sequences of rabbit, mouse, rat, and human ROCK-I indicates that this RhoA-associated kinase is a well-conserved protein.

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.

Immunohistochemical analysis of unsutured and sutured corneal wound healing.

In the unsutured partial thickness penetrating wounds of the cornea, the epithelium migrates over the wounded stromal surface prior to the onset of stromal regeneration. To determine the possible affects of the epithelial ingrowth on the organization of the stromal scar tissues, the healing of unsutured and sutured wounds was compared immunohistochemically. Immunostaining patterns for fibronectin, types III, VI and VII collagen, keratan sulfate proteoglycan (KSPG), and intermediate filament-associated protein (IFAP 130) in fibroblasts, were analyzed in unsutured and adjacent sutured keratotomy wounds in monkeys, at 2-9 weeks after surgery. At 2-4 weeks, fibronectin, type III and type VI collagen showed a lamellar interweaving pattern across unsutured wounds that was absent in sutured wounds. Type VII collagen was detected along the entire depth of regenerated stroma in unsutured wounds, but not in sutured wounds indicating that the epithelium had formerly been present in the regenerated stroma in unsutured wounds. Fibroblasts in both types of wounds expressed IFAP 130, but staining was more pronounced in sutured wounds. At 5-9 weeks, cellular re-activation, as judged from the expression for IFAP 130, was concomitant with a loss of lamellar interweaving with fibronectin, type III and type VI collagen across unsutured wounds, and proceeded in a posterior to anterior direction. In contrast, in sutured wounds, lamellar interweaving was established in anterior to posterior direction. At all postoperative times, unsutured and sutured wounds showed minimal staining for KSPG in the anterior scar.(ABSTRACT TRUNCATED AT 250 WORDS)

Scar tissue orientation in unsutured and sutured corneal wound healing.

AIMS: This study aimed to evaluate stromal wound healing morphology in short term unsutured compared with sutured corneal wounds, to define regional variation in healing within radial keratotomy wounds. METHODS: Stromal scar tissue orientation (fibroblast and collagen fibre orientation) was analysed in unsutured and adjacent sutured keratotomy wounds in monkeys, 2 to 9 weeks after surgery, using light and transmission electron microscopy. RESULTS: At 2 to 4 weeks, scar tissue orientation was transverse to the wound edge in unsutured wounds, but sagittal in sutured wounds. At 5 to 9 weeks, a reorientation of scar tissue sagittal to the wound was seen in the unsutured wounds, proceeding from the posterior to anterior wound regions. In sutured wounds, a scar tissue reorientation transverse to the wound was seen, proceeding from the anterior wound region in a posterior direction. CONCLUSIONS: Within the same cornea, sutured and unsutured wounds showed opposite patterns of healing. Sutured wounds initially healed more slowly, but obtained pseudolamellar continuity over time. In contrast, healing of unsutured wounds was characterised by an early approximation towards lamellar repair that was followed by an ineffective reorganisation of the scar. This latter pattern of healing, that may be associated with a variable weakening of the wound, may relate to the clinical findings of unpredictability and/or progression of refractive effect following radial keratotomy.

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.

Primary structure of human lumican (keratan sulfate proteoglycan) and localization of the gene (LUM) to chromosome 12q21.3-q22.

A human corneal fibroblast cDNA library was screened with a bovine lumican cDNA probe to obtain three clones. Sequencing of the longest clone (1.75 kb) yielded an open reading frame of 1014 bp coding for a 338-amino-acid core protein. Amino acid sequencing of a tryptic peptide resulted in a 9-amino-acid match with the derived primary structure, confirming the identity of these clones. Human lumican displays all of the features of small interstitial proteoglycans: N- and C-terminal domains with highly conserved cysteines and a central domain containing nine repeats of slight variations of the leucine motif LXXLXLXXNXL. Like bovine lumican, the human core protein contains four possible N-glycosylation sites in the central domains, all or some of which are substituted with keratan sulfate side chains. At the amino acid level, it is 90% identical with bovine and 72% identical with the chicken core protein. The gene (LUM) was localized to human chromosome 12 by hybridizing a cDNA probe to a Southern blot containing a human/hamster monochromosomal mapping panel DNA. Further sublocalization to 12q21.3-q22 was performed by the fluorescence in situ hybridization technique using a lumican P1 genomic clone. By immunohistochemical staining, we show lumican's presence, not only in the corneal stroma as shown previously, but also in the dermal area of the skin, indicating a wider distribution of this proteoglycan.

Characterization of immortalized rabbit corneal epithelial cells with SV40 large T antigen.

The authors developed a line of immortalized rabbit corneal epithelial cells by transfecting a primary culture of New Zealand White (NZW) rabbit corneal epithelial cells with Simian virus 40 (SV40) T antigen gene using a calcium phosphate precipitation method. The transfected cells, which survived more than 40 passages and 400 population doublings, showed some ability to form colonies in soft agar, and had doubling times and plating efficiencies not significantly different from those of untransfected cells. The epithelial nature of the transfected cells was confirmed by immunofluorescence staining with the antibody to 64 kD keratin (AE5), a specific marker for corneal type epithelial cells. Immunofluorescence microscopic examination revealed that the transfected cells expressed major basement membrane components of epithelial cells, including collagen types IV and VII and laminin. Electron microscopic studies demonstrated the presence of microvilli and intercellular in cultured transfected cells. These results indicate that the transfected cells retain some of the normal phenotypic characteristics of corneal epithelial cells and may therefore be used to study the corneal epithelium in vitro.

Basement membrane synthesis by human corneal epithelial cells in vitro.

PURPOSE: Collagen gels may prove to be potential carriers for transplantation of cultured corneal epithelial cells. The purpose of this study was to evaluate the suitability of collagen gels in comparison with corneal stromal blocks as the substrate to support the growth of human corneal epithelial cells in culture and the synthesis and deposition of the basement membrane components by these cells. METHODS: Corneal epithelial sheets, freed from the culture dishes using Dispase II (Boehringer Mannheim, Indianapolis, IN), were cultured on corneal stromal blocks. Deposition of laminin, type IV collagen, type VII collagen, and perlecan (heparan sulfate proteoglycan) were evaluated immunohistochemically after 4 days, 7 days, 2 weeks, and 3 weeks. Human limbal explant cultures were established on collagen gels prepared from bovine type I collagen with or without addition of cultured human corneal fibroblasts. After 1, 2, 3, and 4 weeks, the deposition of the basement membrane components was evaluated immunohistochemically. RESULTS: Corneal epithelial cells, cultured on corneal stromal blocks as well as on collagen gels with or without fibroblasts, deposited laminin, type IV collagen, perlecan, and type VII collagen at the interface of the cells and the substrates. However, different substrates differentially influenced the temporal pattern of the deposition of various basement membrane components. On the stromal blocks, deposition of laminin, type IV collagen, and perlecan by the epithelial cells was evident at 1 week. Type VII collagen was detected at 2 weeks. On the collagen gels with fibroblasts, deposition of laminin, type IV collagen and perlecan was detectable at 1 week. In the epithelial cultures on the collagen gels without fibroblasts, only perlecan was detectable at 1 week. At 2 weeks, all of the basement membrane components, including type VII collagen were detectable on the collagen gels, either with or without fibroblasts. CONCLUSION: Human corneal epithelium cultured on collagen gels or on corneal stromal blocks can synthesize and deposit basement membrane components, including laminin, type IV collagen, type VII collagen, and perlecan within 2 weeks in culture. Therefore, collagen gels may serve as potential carriers for human corneal epithelial transplantation.

Regulation of fibronectin and laminin synthesis by retinal capillary endothelial cells and pericytes in vitro.

Retinal capillaries are composed of endothelial cells resting on a basement membrane, in which are embedded pericytes. In diabetes mellitus, the basement membrane becomes thickened, and there is a loss of pericytes. The relative contributions of endothelial cells and pericytes to the synthesis of extracellular matrix proteins which are components of the basement membrane are not well-characterized. To determine how a selective loss of pericytes might affect the composition of retinal capillary basement membranes, we used primary cultures of bovine retinal capillary endothelial cells and pericytes to determine the forms and quantify the amounts of laminin and fibronectin synthesized and secreted by these cell types as well as to determine how high glucose concentrations alter these parameters. Results of ELISAs showed that pericyte cell/matrix layers contained nearly ten times more fibronectin than endothelial cells (288 +/- 24 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.001), but the amounts of laminin were similar. D-glucose (40 mM) tripled the amount of fibronectin incorporated into the endothelial cell/matrix layer (102 +/- 4 vs. 34 +/- 5 ng micrograms-1 DNA, P < 0.05), but had a lesser effect on pericytes. The non-metabolizable analogue L-glucose, also increased the amount of fibronectin incorporated in both pericyte and endothelial cell/matrix layers. The effects of D- and L-glucose on fibronectin secreted into the medium by both cell types were similar to the effects on incorporation of fibronectin into cell/matrix layers. Glucose had no effect on laminin synthesis. [35S]methionine radiolabeling and immunoprecipitation showed that pericytes and endothelial cells synthesize different forms of fibronectin. Both pericytes and endothelial cells synthesized an A and two B chains of laminin which were of similar apparent size, but the two cell types post-translationally modified the subunits differently. We conclude that pericytes and endothelial cells may contribute different forms and amounts of fibronectin and laminin to the retinal capillary basement membrane, so the preferential loss of pericytes in diabetes could result in basement membrane abnormalities which might lead to endothelial cell dysfunction.

Transforming growth factor-beta stimulates collagen and fibronectin synthesis by human corneal stromal fibroblasts in vitro.

The effects of transforming growth factor-beta (TGF beta) and epidermal growth factor (EGF) on the synthesis of collagen and fibronectin, and on the proliferation of human corneal stromal fibroblasts in vitro, were evaluated. Human corneal stromal fibroblasts in culture were incubated for 48 hours with TGF beta or EGF in the absence of serum. Collagen and fibronectin in the culture media were measured by a collagenase-digestion assay and a competitive ELISA, respectively. The effects of the growth factors on proliferation were assessed by 3H-thymidine incorporation. Collagen synthesis was dose-dependently stimulated by TGF beta; at a concentration of 1 ng/ml of TGF beta, a 120% increase in collagen synthesis was seen over that of controls (p < 0.01). EGF, at a concentration of 10 ng/ml, induced a 40% increase in collagen synthesis over that of controls (p < 0.01). The maximum stimulation by TGF beta was greater than that by EGF (p < 0.05). Fibronectin synthesis was stimulated by TGF beta and EGF in a dose-dependent manner; 230% (p < 0.001) and 210% (p < 0.01) increases in fibronectin synthesis were caused by 10 ng/ml TGF beta and EGF, respectively. TGF beta and EGF dose-dependently stimulated 3H-thymidine incorporation. The maximum increases in 3H-thymidine incorporation reached 180% (p < 0.001) and 190% (p < 0.001) over that in controls, at 10 ng/ml concentrations of TGF beta and EGF, respectively. In conclusion, both TGF beta and EGF are potent stimulants of collagen and fibronectin synthesis and proliferation. Therefore, these two growth factors may be effective alternatives or additional choices for the treatment of corneal ulcer.

Corneal epithelial cell attachment with endogenous laminin and fibronectin.

PURPOSE: To evaluate the role of endogenously produced laminin and fibronectin as well as the effect of exogenous laminin and fibronectin in the attachment of human corneal epithelial cells in vitro. METHODS: Primary cultured human corneal epithelial cells labeled with 3H-thymidine were seeded onto plates coated with laminin or fibronectin, or onto uncoated bacteriologic plates. Attachment of cells was measured in the presence or absence of antisera against laminin or fibronectin, by counting radioactivity. RESULTS: Human corneal epithelial cells attached to plates coated with human laminin or human fibronectin in a dose-dependent manner, with 69% and 50% of cells attached to the wells coated with 40 micrograms/ml of laminin and fibronectin, respectively (P < 0.001). The percentage of attachment to uncoated bacteriologic plates increased from 1.2% at 45 min of incubation to 6.7% at 90 min, 22.2% at 3 hr, and 40.1% at 6 hr of incubation. Cycloheximide, a protein synthesis inhibitor, completely inhibited cell attachment. Rabbit antiserum against human fibronectin reduced cell attachment to the uncoated plates to 67% of the control value (P < 0.01), whereas rabbit antiserum against human laminin decreased the attachment to 52% of the control (P < 0.01). A combination of these two antisera reduced cell attachment to 46% of the control (P < 0.01). CONCLUSIONS: Endogenous laminin and fibronectin as well as exogenous laminin and fibronectin play significant roles in the attachment of human corneal epithelial cells in culture.

Trigeminal ganglion neurons affect corneal epithelial phenotype. Influence on type VII collagen expression in vitro.

PURPOSE: To examine whether trigeminal ganglion (TG) neurons in tissue culture influence expression of Type VII collagen (a major component of the anchoring fibrils involved in the attachment of the epithelium to the underlying stroma) by cultured corneal epithelial cells. METHODS: A two-chambered coculture system was used. Fetal rabbit TG neurons were cultured into a central chamber on collagen- or laminin-coated tissue culture dishes. After good neurite outgrowth (average 7 days), rabbit corneal epithelial explants were placed into the outer chamber. Once neurite-epithelial cell interaction occurred, the cultures were immunostained for Type VII collagen. Direct coculture of TG neurons onto confluent passaged rabbit corneal epithelium also was studied. RESULTS: Neurites in contact with the epithelial cells in the outer chamber formed branching complexes, but staining for Type VII collagen was negative. In cocultures of TG neurons onto confluent passaged rabbit corneal epithelium, there was extensive neurite branching on and around the epithelial cells within a week. Scattered epithelial cells, many in clusters, were found to express Type VII collagen, as determined by immunofluorescence staining. CONCLUSIONS: Based on the finding from this study that TG neurons influence production of Type VII collagen by rabbit corneal epithelium in vitro, it is likely that TG neurons influence corneal epithelial phenotypic characteristics that are critical to the maintenance of healthy epithelium in vivo.

Biosynthesis of stromal matrix proteoglycans and basement membrane components by human corneal fibroblasts.

The proteoglycans produced by intact human corneas and corneal cells in culture were compared by characterizing the biosynthetically radiolabeled proteoglycans and by using antibodies to detect their core proteins. Organ cultures of corneas primarily produce a keratan sulfate proteoglycan (KSPG) and a chondroitin and dermatan sulfate proteoglycan (decorin). Immunostaining with antibodies specific for the core proteins of KSPG and decorin showed that these proteoglycans are localized to the corneal stroma. The stroma also contained trace amounts of matrix that stained with antibodies to basement membrane heparan sulfate proteoglycan (perlecan) and laminin. Corneal fibroblasts in culture produced decorin, but the synthesis of KSPG appeared to be blocked at the level of core protein synthesis. Corneal fibroblasts in culture, however, produced perlecan in greater amounts than they did in organ cultures, and they synthesized both perlecan and laminin in greater amounts than did corneal epithelial cells in culture. These results indicate that the synthesis of proteoglycans by human corneal fibroblasts in culture is altered, resulting in increased production of basement membrane-associated proteoglycans and decreased synthesis of corneal stroma-associated proteoglycans.

Expression of vimentin by rabbit corneal epithelial cells during wound repair.

Intermediate filaments of epithelial cells generally consist of specific combinations of keratins. However, cultured epithelial cells from certain tissues and some epithelial tumors have been shown also to express vimentin. In the present study, the expression of vimentin by epithelial cells in healing corneal wounds (partial thickness penetrating wounds) and in tissue culture was analyzed. Both immunohistochemical and immunotransblot analyses indicated that although vimentin was not detected in the normal rabbit corneal epithelium in vivo, cultured rabbit corneal epithelial cells co-express keratins and vimentin. At 1 day post-wounding, vimentin was not detectable in the epithelial cells that had covered the denuded stroma. However, at 2 days postwounding, the epithelium at the base of the epithelial plug immunoreacted with both anti-vimentin and antikeratin monoclonal antibodies. Immunotransblot analyses of the extracts of the epithelial plugs confirmed the presence of vimentin (Mr = 58k). The 58k band was not detected in the extract of normal rabbit corneal epithelium. At day/5, vimentin was no longer detectable in the epithelium. This study demonstrated that corneal epithelial cells transiently co-express vimentin and keratins in vivo during wound healing and in tissue culture. The time-course of the transient expression of vimentin suggests that the vimentin expression in the epithelial cells during healing is not linked to cell proliferation or to the centripetal migration of the epithelium during early stages (first 24 h) of healing, but may be linked to cell-matrix interactions or the migration of basal cells in the upward direction at the following stage of healing.