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(Investigative Ophthalmology and Visual Science. 2007;48:4989-4999.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0654
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Compositional Differences between Infant and Adult Human Corneal Basement Membranes

Andrea Kabosova,1 Dimitri T. Azar,2 Gregory A. Bannikov,3 Kevin P. Campbell,4 Madeleine Durbeej,5 Reza F. Ghohestani,6 Jonathan C. R. Jones,7 M. Cristina Kenney,8 Manuel Koch,9 Yoshifumi Ninomiya,10 Bruce L. Patton,11 Mats Paulsson,9 Yoshikazu Sado,12 E. Helene Sage,13 Takako Sasaki,11 Lydia M. Sorokin,14 Marie-France Steiner-Champliaud,15 Tung-Tien Sun,16 Nirmala SundarRaj,17 Rupert Timpl,18,19 Ismo Virtanen,20 and Alexander V. Ljubimov1,21

1From the Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, and the 21David Geffen School of Medicine at UCLA, Los Angeles, California; the 2Department of Ophthalmology and Visual Science, University of Illinois at Chicago, Chicago, Illinois; the 3Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Ohio State University, Columbus, Ohio; the 4University of Iowa College of Medicine and Howard Hughes Medical Institute, Iowa City, Iowa; the 5Division for Cell and Matrix Biology, Department of Experimental Medical Science, University of Lund, Lund, Sweden; the 6Division of Dermatology and Cutaneous Surgery, University of Texas Health Sciences Center at San Antonio, San Antonio, Texas; the 7Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois; the 8Department of Ophthalmology, University of California Irvine Medical Center, Orange, California; the 9Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany; the 10Okayama University Medical School, Okayama, Japan; the 11Oregon Health and Science University School of Medicine, Portland, Oregon; the 12Shigei Medical Research Institute, Okayama, Japan; the 13Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington; the 14Institute for Physiological Chemistry and Pathobiochemistry, Münster University, Münster, Germany; the 15Department of Dermatology, University Hospital of Geneva, Geneva, Switzerland; the 16Department of Cell Biology, New York University Medical School, New York, New York; the 17Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; the 18Max-Planck-Institut für Biochemie, Martinsried, Germany; and the 20Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Adult human corneal epithelial basement membrane (EBM) and Descemet’s membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.

METHODS. Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.

RESULTS. Type IV collagen composition of infant corneal central EBM over Bowman’s layer changed from {alpha}1-{alpha}2 to {alpha}3-{alpha}4 chains after 3 years of life; in the adult, {alpha}1-{alpha}2 chains were retained only in the limbal BM. Laminin {alpha}2 and ß2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, ß2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin {gamma}3 chain. In the infant DM, type IV collagen {alpha}1-{alpha}6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.

CONCLUSIONS. The distribution of laminin {gamma}3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.

Basement membranes (BMs) are a specialized form of extracellular matrix (ECM) that separate parenchymal from stromal cells and are important for cell adhesion, migration, differentiation, and signal transduction.1 2 3 The cornea contains a complex ECM that undergoes extensive remodeling during embryonic and postnatal development. It comprises the epithelial basement membrane (EBM), an adjacent collagenous Bowman’s layer, a stromal ECM composed of orthogonal collagen lamellae and several distinct proteoglycans, and an endothelial BM, or Descemet’s membrane (DM). The corneal ECM and BMs contribute to the transparency and refractive properties of the cornea and play major roles in various corneal cell functions.

Many ECMs including BMs undergo considerable changes in development. Both corneal BMs have been shown to increase in thickness during the transition from fetal to adult stages,4 5 6 especially, the DM that acquires a posterior nonbanded region after birth.5 6 7 8 These changes have been revealed by light and electron microscopy, but the molecular alterations of BM components responsible for corneal BM maturation remained largely unknown.

In recent years, the complexity of corneal BMs has been appreciated, largely because of the availability of specific antibodies against most of the components and their isoforms. Studies from our group and others have shown that adult human continuous corneal EBM differs in composition in its various regions.9 10 11 12 13 14 15 16 It appears to have regional horizontal heterogeneity among the central part, limbus, and conjunctiva with respect to the distribution of type IV collagen and laminin isoforms, as well as thrombospondin-1 and types XII and XV collagen.10 14 16 17 The DM was also shown to be vertically heterogeneous with respect to type IV collagen and laminin, as well as fibronectin.10 It remained unclear whether these regional differences in corneal BM structure appeared early in development or were acquired later in life. To answer this question, we compared infant and adult human corneas immunohistochemically, with attention to BM components. Both corneal BMs displayed distinct signs of postnatal maturation with shifts in the expression of major components in specific regions of the EBM and at both stromal and endothelial faces of the DM.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue
Ten infant corneas obtained at autopsy from 5 individuals (aged 12 days to 3 years) and 30 healthy adult human corneas obtained at autopsy from 27 individuals (aged 13–75 years) were received from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 30 hours of death. The NDRI has a human subject protection protocol that is approved and enforced by the managerial committee operating under National Institutes of Health oversight. All such tissues are thus qualified as exempt from Institutional Review Board review (exemption 4). Immediately on arrival, the corneas were bisected, placed in OCT compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at –80°C. Nonfixed 5-µm cryostat sections collected on glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA) were subjected to indirect or double-indirect immunofluorescence analysis, as described previously.10 Photographs were taken by microscope (BX40; Olympus USA, Melville, NY). Routine specificity controls10 were negative. Samples with only very low levels of background staining were analyzed. In some experiments, to unmask type IV collagen epitopes, sections were air dried for 5 minutes, after which they were denatured in 6 M urea at pH 3.5 for 1 hour at 4°C, washed, and ultimately fixed with 1% formalin for 5 minutes at room temperature before staining.10 To unmask laminin epitopes, sections (with or without fixation in acetone at –20°C for 10 minutes) were pretreated with 0.05% SDS solution in PBS for 5 to 10 minutes at room temperature.18 Monoclonal antibodies were used as undiluted hybridoma supernates or at 10 to 20 µg/mL as purified IgGs, and polyclonal antibodies were used at 20 to 30 µg/mL. At least two independent experiments were performed for each marker, with identical results. All infant corneas and nearly all adult corneas were stained for all markers. The staining patterns were mostly reproducible within each group of corneas. Some variations in staining intensity were observed only in the central EBM for some laminin chains.

Antibodies
Monoclonal and polyclonal antibodies against type IV collagen {alpha}1-{alpha}6 chains; laminin chains {alpha}1-{alpha}5, ß1-ß3, and {gamma}1-{gamma}2; nidogen-1 and -2; BM-40/SPARC/osteonectin; perlecan core protein domain IV; fibronectin 8th type III repeat and ED-A domain (cellular fibronectin); types VII, VIII, XII, XVII, and XVIII collagen; netrin-4 (ß-netrin); various tenascin-C isoforms; fibrillin-1; thrombospondin-1; vitronectin; matrilin-2 and -4; {alpha}- and ß-dystroglycan; integrin subunits {alpha}6 and ß4; {alpha}-enolase, and cornea-specific keratin 3 have been described (see Table 1 for references). Antibodies against laminin {gamma}3 chain (Steiner-Champliaud et al., unpublished data, September 2001) will be described in detail elsewhere. Laminin isoform nomenclature follows recent recommendations.52 Cross-species adsorbed fluorescein- and rhodamine-conjugated secondary antibodies were from Chemicon International (Temecula, CA).


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  		TABLE 1. Antibodies Used in This Study

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The compositions of normal human adult corneal epithelial BM and DM have been previously examined in immunohistochemistry studies.10 11 12 14 17 19 53 54 55 56 57 58 59 60 Briefly, EBM was found to be positive throughout the central cornea and limbus (and also conjunctiva) for laminin-332 and -511 (possibly, also for laminin-111 and -311), nidogen-1, perlecan, fibronectin (total and cellular), and types IV ({alpha}5-{alpha}6 chains) and VII collagen. The central part of the corneal EBM with the underlying Bowman’s layer in addition was positive for types IV ({alpha}3-{alpha}4 chains) and XII (long-form) collagen, thrombospondin-1, and vitronectin (not corroborated here possibly due to tissue fixation differences). In contrast, limbal and conjunctival EBM was also positive for types IV ({alpha}1-{alpha}2 chains) and XV collagen, laminin chains {alpha}2 and ß2 (compatible with laminin-211, -121, -221, and -521), tenascin-C, fibrillin-1, and BM-40/SPARC, but lacked type IV collagen {alpha}3-{alpha}4 chains or the long form of type XII collagen.

In DM, the stromal face was found to be positive for fibronectin, vitronectin, and types IV ({alpha}1-{alpha}2 chains) and VIII collagen. The endothelial face stained for types IV ({alpha}3-{alpha}6 chains) and XII collagen, laminin-511, nidogen-1, thrombospondin-1, and perlecan.

In this report, the staining patterns of many of the studied components in both EBM and DM were found to be different between infant and adult corneas. It should be noted that the observed differences in the BM composition of infant compared with adult corneas could still be seen in 3-year-old corneas. In contrast, 13-year-old corneas already had the adult distribution of all studied markers.

Epithelial Basement Membrane
Results are summarized in Table 2 . Both infant and adult human corneal central EBM were positive for chains of laminin-311 ({alpha}3ß1{gamma}1), -332 ({alpha}3ß3{gamma}2), -411 ({alpha}4ß1{gamma}1), and -511 ({alpha}5ß1{gamma}1); nidogen-1 and -2; perlecan; types IV ({alpha}5-{alpha}6 chains), VII, XII (both forms), XVII, and XVIII collagen; thrombospondin-1; matrilin-2 (Fig. 1 , right column) and -4; and the hemidesmosomal component {alpha}6ß4 integrin (data not shown). Weak staining was also seen for SPARC/BM-40, laminin {gamma}3 chain (Fig. 1 , left column), and the laminin receptors, {alpha}- and ß-dystroglycans (data not shown). Staining for laminin {alpha}1 chain (component of laminin-111) was weak and inconsistent and could be revealed by only one of three antibodies. Therefore, its presence in the corneal EBM cannot be documented with certainty; some data indicate that it might be expressed only in the embryonic EBM.19 61 Distinct EBM staining for laminin {alpha}4 chain was revealed by two of five antibodies. These antibodies (377 and 1101+) did not stain muscle tissue from a laminin {alpha}4 chain null mouse confirming lack of cross-reactivity with other chains. Epithelial BM staining was only distinct after a mild pretreatment with SDS18 suggesting that the respective epitopes were masked. This may explain the recently documented lack of central EBM staining (with limbal EBM positivity) using another {alpha}4 antibody, FC10.62


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  		TABLE 2. Distribution of BM Components in Adult and Infant Corneal Epithelial BM

 

Figure 1
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  		FIGURE 1. New BM components in infant and adult human corneas. Antibodies against laminin {gamma}3 chain (LM {gamma}3; pAb 6296 is shown) showed weak staining of infant and adult central corneal EBM (left). Limbal BM staining was more prominent (middle); some capillary BMs were also positive. Bowman’s layer is located to the left; its end is marked by an arrow. Matrilin-2 (right) was present in both infant and adult central EBM. e, epithelium; s, stroma.

 
In the limbal EBM, the following components were found by immunostaining of both adult and infant corneas (Table 2) : chains of laminin-311, -332, -333, -411, -423, -511, and -523; nidogen-1 and -2; perlecan, types IV ({alpha}5-{alpha}6 chains), VII, XII (short form only), XVII, and XVIII collagen; SPARC/BM-40 (weak); tenascin-C; fibrillin-1; matrilin-2 and -4; and {alpha}6ß4 integrin (data not shown). Strong staining was seen for laminin {gamma}3 chain (Fig. 1 , middle column) that also continued in the conjunctival BM. The data on adult corneas agree well with results in many previous reports. In contrast to the adult corneas, infant corneas displayed some staining for fibronectin (data not shown), perlecan, and type VII collagen in Bowman’s layer (Fig. 2) . In some cases, very short, regularly spaced, delicate streaks running perpendicular to the EBM plane could be clearly seen in Bowman’s layer (Fig. 2 , top). At the infant corneal periphery only, Bowman’s layer was also positive for tenascin-C splice variants containing insertional FN-III repeats A1, D, and, to a lesser extent, B (data not shown). Adult corneas were negative in these areas.


Figure 2
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  		FIGURE 2. BM components and Bowman’s layer. Infant corneas displayed distinct staining for perlecan, total fibronectin, and collagen VII (COL VII) in Bowman’s layer (arrows). Such staining was absent in adult corneas. e, epithelium; s, stroma.

 
Contrary to the adult corneas, infant corneas were positive for type IV collagen {alpha}1-{alpha}2 chains in the central portion (Fig. 3 , left column). Conversely, the central EBM displayed very weak staining for {alpha}3-{alpha}4 chains of type IV collagen (abundant in the adult corneas), which could be revealed only after pretreatment of the sections with urea (Fig. 3 , right column). Cellular fibronectin staining was minimal or absent in the EBM, in contrast to its distinct presence in the adult corneas (data not shown). In contrast to adult corneas, infant corneas exhibited no laminin {alpha}2 chain in the limbal (and conjunctival) EBM (Fig. 4 , left column). Laminin ß2 chain appeared in corneal limbal BM between 6 months and 3 years of age (data not shown).


Figure 3
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  		FIGURE 3. Collagen type IV chains in infant and adult human corneas. Infant corneas contained the {alpha}1(IV) and {alpha}2(IV) (not shown) chains in the central part, contrary to their absence in adult corneas. However, the infant central EBM displayed very weak staining for the {alpha}3(IV) (not shown) and {alpha}4(IV) chains that were abundant in the adult central corneas. e, epithelium; s, stroma.

 

Figure 4
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  		FIGURE 4. Specific laminin chains and keratin 3 in infant and adult peripheral cornea. Infant corneas, unlike adult corneas, did not stain for laminin chains {alpha}2 (LM {alpha}2) and ß2 (not shown) in the limbal EBM. The peripheral basal epithelium did not stain for keratin 3 in infant corneas, in contrast to adult corneas. Arrows: LM {alpha}2-positive limbal vessels. e, epithelium; s, stroma.

 
Cornea-specific keratin 3 was similarly distributed in infant and adult corneas. All epithelial cells in the central part and suprabasal cells in the limbus were stained. However, the staining of peripheral corneal basal cells in infant corneas was weak or negative over larger distances toward the center, in comparison to that in adult corneas (Fig. 4 , right column). Apparently this difference was not due to the regional heterogeneity in its expression63 because all infant corneas showed similar patterns. A marker of limbal basal epithelial cells, {alpha}-enolase, was confined to the limbus in most infant corneas, similar to that of adults (data not shown).

Descemet’s Membrane
Results are summarized in Table 3 . In infant corneas, fibronectin, both total and cellular, was found at the stromal face, as in the adult corneas (data not shown). Thrombospondin-1 and type XVIII collagen also exhibited the adult staining pattern in infant corneas, at the endothelial DM face (data not shown). Other major BM components showed different patterns between infant and adult DM. Type IV collagen ({alpha}1-{alpha}6 chains), nidogen-1 and -2, laminin-411 and -511, perlecan, and netrin-4 were found on both DM faces of infant corneas ("railroad" pattern), contrary to the endothelial face location in the adult (Fig. 5) . The major DM component, type VIII collagen, was observed mostly on the endothelial face, contrary to the adult corneas where it was mostly seen on the stromal face (Fig. 6 , left column). Staining for the long form of type XII collagen was positive on the endothelial DM face in the infant but not in the adult corneas. In contrast, adult corneal stroma was stained more prominently for the long form of type XII collagen (characteristic beads-on-a-string pattern) than was infant corneal stroma (Fig. 6 , right column). Similar developmental accumulation of this protein was previously reported in the rabbit.35


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  		TABLE 3. Distribution of BM Components in Adult and Infant Descemet’s Membrane

 

Figure 5
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  		FIGURE 5. Different distribution of BM components in infant and adult DM. Type IV collagen {alpha}1 chain (COL {alpha}1(IV)), nidogen-2, the {alpha}4 chain of laminin 411 (LM {alpha}4; pAb 1101+), and netrin-4 were found on both faces of the DM (arrows) of infant corneas ("railroad" pattern). In adult corneas, these and other (see Table 3 ) BM components were found only on one DM face.

 

Figure 6
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  		FIGURE 6. Collagen types VIII and XII in infant and adult DM. Infant and adult DM (arrows) displayed inverse patterns of type VIII collagen (COL VIII). Only infant DM contained the long form of type XII collagen (COL XII long; mAb I1C8) on its endothelial face. s, stroma.

 
Some components were observed only in infant but not in adult corneas. The stromal DM face stained for laminin-332, tenascin-C, and fibrillin-1 (Fig. 7 , LM-332, TN-C, and FIB-1, respectively) in infant corneas only; adult corneas displayed no staining. Staining was seen only with antibodies to total tenascin-C and to FN-III repeat A1 (Fig. 7 , column TN-C A1). These data indicate that in infant corneal DM, only tenascin-C variants without insertional repeats or with insertional repeat A1 (see Ref. 64 ) were present. Matrilin-4 was present on both DM faces in the infant corneas but had largely disappeared in the adult DM (Fig. 7 , right column).


Figure 7
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  		FIGURE 7. Differential expression of specific BM components in infant and adult DM. Infant DM (arrows) contained laminin-332 (LM-332), tenascin-C (TEN-C, alternatively spliced repeat A1 is shown), fibrillin-1 (FIB-1), and matrilin-4, in contrast to adult DM. s, stroma.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The results showed that in the infant human corneas studied the composition of both corneal BMs, the EBM and DM, is reproducibly different from that of adult corneas. In the infant corneal EBM, the central part above the Bowman’s layer contained little type IV collagen {alpha}3-{alpha}4 chains (revealed only after urea treatment) but stained well for {alpha}1-{alpha}2 chains, opposite to the adult central EBM pattern.10 The distribution of {alpha}1-{alpha}2 type IV collagen in the infant corneas is in keeping with continuing expression of {alpha}1(IV) mRNA during postnatal life.65 Subsequently, {alpha}1-{alpha}2 type IV collagen synthesis may be developmentally inhibited at the posttranscriptional level, or the epitopes may become masked, because various monoclonal antibodies no longer detected it in the adult central corneas.9 10 53 65 66 {alpha}3-{alpha}4(IV) chains accumulate in the EBM later in postnatal life, in agreement with previous data.54 Because {alpha}5-{alpha}6(IV) chains are expressed in both infant and adult central corneal EBM, they appear to be regulated differently from {alpha}3-{alpha}4(IV) chains, and probably form trimers mostly without {alpha}3-{alpha}4(IV) chains.

In adult corneas, basal epithelial precursor (stem) cells are localized in the limbus, although in the embryonic and newborn corneas they may be also present in the central part.50 67 68 Limbal epithelial stem cells strongly adhere to placental type IV collagen,69 composed mostly of the {alpha}1-{alpha}2 chains70 typical of adult limbal BM. Limbal explants on amniotic membranes also make {alpha}1-{alpha}2 type IV collagen.71 In addition, embryonic stem cells undergo epithelial differentiation on the same {alpha}1-{alpha}2 type IV collagen and then can be used for replacement of injured limbus.72 These data suggest that limbal stem cells and their more abundant early progeny (transient amplifying cells) may contribute to a unique BM composition with respect to type IV collagen isoforms and also to some other molecules (e.g., specific laminin isoforms containing {alpha}2, ß2, and {gamma}3 chains, tenascin-C, fibrillin-1, types XII [long form] and XV collagen, and thrombospondin-1).10 14 16 35 57 64 68 73 The horizontal EBM heterogeneity between the limbus and the central part that develops during embryonic and postnatal life could reflect the need for limbal stem cells and transient amplifying cells to maintain a specific BM composition to preserve their undifferentiated state. Interactions of stem cells with the BM appear to be regulated through specific integrin receptors.74 75 Patches of some limbal BM components (agrin, SPARC/BM-40, tenascin-C, and versican) were reported to colocalize with p63/ABCG2-positive and Cx43-negative cell clusters in the limbal basal epithelium (Kruse FE et al. IOVS 2005;46:ARVO E-Abstract 2081). These BM proteins may be products and markers of putative stem cells that are a minor population of limbal basal epithelial cells. The other BM components differentially expressed in the limbus (described in the Results section) may be related to a bigger population of transient amplifying cells.

The distribution of type IV collagen in the infant central corneal EBM resembles that of the adult limbal EBM that is probably produced by the epithelial cells.55 Such EBM composition may favor the existence of epithelial stem cells and early transient amplifying cells in the maturing cornea.68 Accordingly, the corneal epithelial differentiation marker, keratin 3, is absent from the basal cells of the embryonic central cornea until birth.67 It was also less abundant in the peripheral basal cells of the infant versus adult corneas (Fig. 4) . Basal cells of the infant central corneal epithelium might therefore exist at a similar level of differentiation as most of the basal cells of the adult limbus. However, they seem to be somewhat more differentiated by {alpha}-enolase expression. During postnatal maturation, corneal epithelial basal cells begin to accumulate type IV collagen {alpha}3-{alpha}4 chains in the EBM with concomitant decrease in the production of limbal {alpha}1-{alpha}2(IV) chains. Our data suggest that the differentiation level of the corneal epithelial basal cells influences the expression pattern of type IV collagen isoforms in the EBM (see Ref. 68 ). Supporting this hypothesis are our previous results on the limbal pattern of type IV collagen and keratin 3 expression of the central epithelial cells in epithelial plugs over radial keratotomy scars.76 Cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) staining, may not be an important factor in the regulation of postnatal BM protein expression because after 6 months of life, PCNA-positive cells are already confined to the limbal area.77

Laminin {alpha}2 and ß2 chains were not seen in the infant EBM, whereas they were both found in the limbal and also conjunctival EBM in the adult corneas. These chains may be produced by stromal cells (hence, their absence from central corneas), and their presence could reflect stromal rather than epithelial maturation in the postnatal cornea, a situation previously described in the intestine for {alpha}2 chain.78 79 Together with type IV collagen chains (described earlier), laminin {alpha}2 and ß2 chains seem to be developmentally regulated in the cornea, although the exact timing and mechanisms of their appearance in the adult life are unknown. These findings may be one of the first indications of significant maturation (further differentiation) of epithelial and stromal limbal and conjunctival cells and their mutual BM during postnatal development. This system could be useful for the study of developmental regulation of specific integrin receptors for laminin. One integrin, {alpha}6ß4, was expressed in an adult pattern in infant corneas (data not shown), but others (such as {alpha}3ß1or {alpha}2ß1) might change their expression levels and/or patterns during corneal maturation.

In DM, infant corneas exhibited laminin-511, nidogen-1 and -2, type IV collagen {alpha}1-{alpha}6 chains, perlecan, and netrin-4 on both DM faces. However, in adult corneas, these components were located only on one DM face. {alpha}1-{alpha}2(IV) chains were found on the stromal face, and all others, on the endothelial face. Type VIII collagen was primarily located on the endothelial DM face in infant corneas, but was found mostly on the stromal face in the adult corneas. The mechanisms of these changes are not known.

Alterations in the distribution of DM components during postnatal development may reflect cellular differentiation and/or proliferation changes, especially in the case of type VIII collagen, a major DM component. It is made by proliferating corneal endothelial cells in culture but its production is inhibited on confluence.80 Human corneal endothelial cells largely cease to proliferate in the last trimester of fetal life and after birth.81 Therefore, this collagen’s network may not be made by endothelial cells after birth and may be gradually distanced from them as DM thickens. It can still remain there because of increased stability of its hexagonal network to degradation compared with the trimer.82 In adult life, it could also be produced by posterior stromal keratocytes, which is supported by data on knockout mice for {alpha}1(VIII) and {alpha}2(VIII) chains that display severe stromal alterations.83

DM components found on both sides in the infant corneas may be initially laid down by stromal and endothelial cells. During postnatal corneal maturation, the stromally located components (e.g., laminin-332, tenascin-C, fibrillin-1, netrin-4, and matrilin-4) may be degraded and replaced by those (e.g., fibronectin) made by the differentiated adult stromal cells. It would be interesting to verify this hypothesis by in situ hybridization on corneas at various stages of postnatal development. It is noteworthy that developmental vertical heterogeneity with respect to type IV collagen chains was observed in glomerular BM,84 85 which is also a product of more than one cell type.86

Tenascin-C and fibrillin-1 could be detected on the stromal face of the DM only in infant corneas. Both of these proteins can reappear in DM of adult corneas affected by bullous keratopathy and Fuchs’ endothelial dystrophy.11 87 These conditions are characterized by the inability of corneal endothelial cells to pump fluid efficiently out of the cornea resulting in corneal swelling. It is possible that the infant endothelium also cannot pump out fluid as efficiently as adult endothelium, leading to the accumulation of tenascin-C and fibrillin-1 in the infant DM. Previously, tenascin-C isoforms were also found in the central corneas of fetal and infant eyes, and their expression diminished with postnatal aging.88 However, in the Maseruka et al. paper,88 more isoforms were seen in infant corneas than we have observed and epithelial (rather than ECM) staining was notable. Moreover, they did not observe the DM staining described in the present study. These discrepancies are probably due to the use of cryosectioned tissues in our study versus paraffin-embedded sections in the Maseruka et al. paper.

To the best of our knowledge, we provide the first account of the distribution of laminin {gamma}3 chain, nidogen-2, matrilin-2, matrilin-4, and netrin-4 in human corneas (Figs. 1 5 7) . Staining for the laminin {gamma}3 chain was strong in limbal and conjunctival BM, similar to that of {alpha}1-{alpha}2(IV) chains and laminin {alpha}2 and ß2 chains (Table 2) . The staining in the EBM was weak and irregular, especially in adult corneas. In several tissues, this chain was found at non-BM locations.89 90 91 However, in skin, testis, retina-choroid, and kidney, {gamma}3 chain was observed in BMs including Bruch’s membrane and epidermal BM92 93 and was markedly reduced in mouse testis in the absence of laminin {alpha}2 chain.94 It is not known which laminin isoforms containing the {gamma}3 chain are present in limbal BM, but this region has all {alpha} and ß chains that were previously shown to complex with {gamma}3 to form laminin-213, -333, -423, and -523.52 90 91

Nidogen-1 and -2 are close homologs and both bind to laminin.95 In the human cornea, nidogen-2 was codistributed with nidogen-1/entactin and was a prominent component of corneal epithelial and limbal vascular BMs. Nidogen-1 and -2 were also both observed around keratocytes. Staining of a human meningioma (data not shown) revealed that nidogen-2 was present in both tumor stroma and vascular BMs, but nidogen-1 was seen only in the vessel walls. These data exclude antibody cross-reactivity supporting the presence of both nidogens in the infant and adult corneal EBM and DM.

Matrilin-2 and -4 have been found in noncorneal BMs, such as skin epithelial BM.45 46 These von Willebrand factor A-like domain-containing ECM adapter proteins interact with various BM components96 and may reinforce corneal BMs, especially the infant DM, where matrilin-4 is found in a "railroad" pattern. Netrin-4 (also known as ß-netrin), a BM protein with homology to laminin, may have a similar function in the DM.

Our results indicate that human corneal BMs undergo significant compositional changes from the infant to the adult, possibly related to the differentiative and/or proliferative processes of contributing cells. It is important to identify mechanisms responsible for these changes, for a better understanding of the pathogenesis of certain corneal diseases. BM structure alterations have been described in many common corneal disorders, such as keratoconus, Fuchs’ endothelial dystrophy, and bullous and diabetic keratopathies.7 11 56 58 62 64 97 98 99 100 101 Elucidation of the underlying abnormalities in BM gene and protein expression may provide a means to alleviate symptoms or to prevent the development of these common vision-threatening diseases.

Note Added in Proof
While this article was in press, the paper appeared (Schneiders FI, Maertens B, Böse K, et al. Binding of netrin-4 to laminin short arms regulates basement membrane assembly. J Biol Chem. 2007;282:23750–23758) showing the importance of laminin-binding netrin-4 for proper BM assembly.


   Acknowledgements
 
The authors thank Joshua R. Sanes for producing the antibodies against the laminin ß2 chain and Heinz Furthmayr for producing the antibodies to type IV collagen {alpha}1/{alpha}2 chains, which were obtained from the Developmental Studies Hybridoma Bank (DSHB), Department of Biology, University of Iowa (Iowa City, IA), under contract N01-HD-2-3144 from the National Institute of Child Health and Human Development; Luciano Zardi (Department of Experimental and Clinical Immunology, Advanced Biotechnology Center, Istituto Giannina Gaslini, Genoa, Italy); Eva Engvall (The Burnham Institute, La Jolla, CA); Marion K. Gordon (Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ); James D. Zieske (Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA); Jeffrey H. Miner (Renal Division, Washington University School of Medicine, St. Louis, MO); and Robert E. Burgeson (MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital East, Charlestown, MA) for the generous gifts of antibodies; Julia Y. Ljubimova (Department of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA) for providing sections of human meningioma procured under Cedars-Sinai IRB protocol 3637; and Annette M. Aoki and Nadia C. Zorapapel for expert assistance. The authors acknowledge the immense and lasting contribution of the late Dr. Rupert Timpl, who was a co-author of this article, not only to the present work, but also to the entire extracellular matrix and basement membrane field.


   Footnotes
 
19 Deceased October 20, 2003. Back

Presented in part at the XVth International Congress of Eye Research, Geneva, Switzerland, October 2002, and at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2006.

Supported by National Eye Institute Grants R01 EY10836 (MCK) and R01 EY13431 (AVL); the Skirball Program in Molecular Ophthalmology and a seed grant from the Department of Surgery, Cedars-Sinai Medical Center (AVL); and Deutsche Forschungsgemeinschaft Grants PA 660/10-1 and WA 1338/2-6 (MP).

Submitted for publication June 1, 2007; revised June 28, 2007; accepted August 14, 2007.

Disclosure: A. Kabosova, None; D.T. Azar, None; G.A. Bannikov, None; K.P. Campbell, None; M. Durbeej, None; R.F. Ghohestani, None; J.C.R. Jones, None; M.C. Kenney, None; M. Koch, None; Y. Ninomiya, None; M. Paulsson, None; B.L. Patton, None; Y. Sado, None; E.H. Sage, None; T. Sasaki, None; L.M. Sorokin, None; M.F. Steiner-Champliaud, None; T.T. Sun, None; N. SundarRaj, None; R. Timpl, None; I. Virtanen, None; A.V. Ljubimov, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Alexander V. Ljubimov, Ophthalmology Research Laboratories, Burns and Allen Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Davis-2025, Los Angeles, CA 90048; ljubimov{at}cshs.org.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kalluri R. Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer. 2003;3:422–433.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  2. Lonai P. Epithelial mesenchymal interactions, the ECM and limb development. J Anat. 2003;202:43–50.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  3. Nguyen NM, Senior RM. Laminin isoforms and lung development: all isoforms are not equal. Dev Biol. 2006;294:271–279.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  4. Alvarado J, Murphy C, Juster R. Age-related changes in the basement membrane of the human corneal epithelium. Invest Ophthalmol Vis Sci. 1983;24:1015–1028.[Abstract/Free Full Text]
  5. Murphy C, Alvarado J, Juster R. Prenatal and postnatal growth of the human Descemet’s membrane. Invest Ophthalmol Vis Sci. 1984;25:1402–1415.[Abstract/Free Full Text]
  6. Lesueur L, Arne JL, Mignon-Conte M, Malecaze F. Structural and ultrastructural changes in the developmental process of premature infants’ and children’s corneas. Cornea. 1994;13:331–338.[CrossRef][Medline][Order article via Infotrieve]
  7. Johnson DH, Bourne WM, Campbell RJ. The ultrastructure of Descemet’s membrane. II. Aphakic bullous keratopathy. Arch Ophthalmol. 1982;100:1948–1951.[Abstract/Free Full Text]
  8. Bourne WM. Biology of the corneal endothelium in health and disease. Eye. 2003;17:912–918.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  9. Cleutjens JPM, Havenith MG, Kasper M, Vallinga M, Bosman FT. Absence of type IV collagen in the centre of the corneal epithelial basement membrane. Histochem J. 1990;22:688–694.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  10. Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest. 1995;72:461–473.[Web of Science][Medline][Order article via Infotrieve]
  11. Ljubimov AV, Burgeson RE, Butkowski RJ, et al. Extracellular matrix alterations in human corneas with bullous keratopathy. Invest Ophthalmol Vis Sci. 1996;37:997–1007.[Abstract/Free Full Text]
  12. Tuori A, Uusitalo H, Burgeson RE, Terttunen J, Virtanen I. The immunohistochemical composition of the human corneal basement membrane. Cornea. 1996;15:286–294.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  13. Qin P, Piechocki M, Lu S, Kurpakus MA. Localization of basement membrane-associated protein isoforms during development of the ocular surface of mouse eye. Dev Dyn. 1997;209:367–376.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  14. Wessel H, Anderson S, Fite D, et al. Type XII collagen contributes to diversities in human corneal and limbal extracellular matrices. Invest Ophthalmol Vis Sci. 1997;38:2408–2422.[Abstract/Free Full Text]
  15. Fukuda K, Chikama T, Nakamura M, Nishida T. Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva. Cornea. 1999;18:73–79.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  16. Sekiyama E, Nakamura T, Cooper LJ, et al. Unique distribution of thrombospondin-1 in human ocular surface epithelium. Invest Ophthalmol Vis Sci. 2006;47:1352–1358.[Abstract/Free Full Text]
  17. Määttä M, Heljasvaara R, Sormunen R, et al. Differential expression of collagen types XVIII/endostatin and XV in normal, keratoconus, and scarred human corneas. Cornea. 2006;25:341–349.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  18. Kramerov AA, Saghizadeh M, Pan H, et al. Expression of protein kinase CK2 in astroglial cells of normal and neovascularized retina. Am J Pathol. 2006;168:1722–1736.[Abstract/Free Full Text]
  19. Ninomiya Y, Kagawa M, Iyama K, et al. Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J Cell Biol. 1995;130:1219–1229.[Abstract/Free Full Text]
  20. Virtanen I, Gullberg D, Rissanen J, et al. Laminin {alpha}1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues. Exp Cell Res. 2000;257:298–309.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  21. Durbeej M, Fecker L, Hjalt T, et al. Expression of laminin {alpha}1, {alpha}5 and ß2 chains during embryogenesis of the kidney and vasculature. Matrix Biol. 1996;15:397–413.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  22. Ettner N, Göhring W, Sasaki T, Mann K, Timpl R. The N-terminal globular domain of the laminin {alpha}1 chain binds to {alpha}1ß1 and {alpha}2ß1 integrins and to the heparan sulfate-containing domains of perlecan. FEBS Lett. 1998;430:217–221.[CrossRef][Medline][Order article via Infotrieve]
  23. Engvall E, Earwicker D, Haaparanta T, Ruoslahti E, Sanes J. Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul. 1990;1:731–740.[Web of Science][Medline][Order article via Infotrieve]
  24. Marinkovich MP, Lunstrum GP, Burgeson RE. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem. 1992;267:17900–17906.[Abstract/Free Full Text]
  25. Sorokin LM, Maley M, Moch H, et al. Laminin {alpha}4 and integrin {alpha}6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury. Exp Cell Res. 2000;256:500–514.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  26. Sasaki T, Mann K, Timpl R. Modification of the laminin {alpha}4 chain by chondroitin sulfate attachment to its N-terminal domain. FEBS Lett. 2001;505:173–178.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  27. Talts JF, Sasaki T, Miosge N, et al. Structural and functional analysis of the recombinant G domain of the laminin {alpha}4 chain and its proteolytic processing in tissues. J Biol Chem. 2000;275:35192–35199.[Abstract/Free Full Text]
  28. Miner JH, Patton BL, Lentz SI, et al. The laminin {alpha} chains: expression, developmental transitions, and chromosomal locations of {alpha}1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel {alpha}3 isoform. J Cell Biol. 1997;137:685–701.[Abstract/Free Full Text]
  29. Petäjäniemi N, Korhonen M, Kortesmaa J, et al. Localization of laminin {alpha}4-chain in developing and adult human tissues. J Histochem Cytochem. 2002;50:1113–1130.[Abstract/Free Full Text]
  30. Ljubimov AV, Afanasjeva AV, Litvinova LV, Senin VM. Basement membrane components produced by a mouse ascites teratocarcinoma TB24: analysis with monoclonal and polyclonal antibodies. Exp Cell Res. 1986;165:530–540.[CrossRef][Medline][Order article via Infotrieve]
  31. Ljubimov AV, Bartek J, Couchman JR, et al. Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies. Int J Cancer. 1992;50:562–566.[CrossRef][Medline][Order article via Infotrieve]
  32. Goldfinger LE, Stack MS, Jones JC. Processing of laminin-5 and its functional consequences: role of plasmin and tissue-type plasminogen activator. J Cell Biol. 1998;141:255–265.[Abstract/Free Full Text]
  33. Koch M, Murrell JR, Hunter DD, et al. A novel member of the netrin family, ß-netrin, shares homology with the ß chain of laminin: identification, expression, and functional characterization. J Cell Biol. 2000;151:221–234.[Abstract/Free Full Text]
  34. Karelina TV, Bannikov GA, Eisen AZ. Basement membrane zone remodeling during appendageal development in human fetal skin: the absence of type VII collagen is associated with gelatinase-A (MMP2) activity. J Invest Dermatol. 2000;114:371–375.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  35. Anderson S, SundarRaj S, Fite D, Wessel H, SundarRaj N. Developmentally regulated appearance of spliced variants of type XII collagen in the cornea. Invest Ophthalmol Vis Sci. 2000;41:55–63.[Abstract/Free Full Text]
  36. Ghohestani RF, Hudson BG, Claudy A, Uitto J. The {alpha}5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency. J Biol Chem. 2000;275:16002–16006.[Abstract/Free Full Text]
  37. Lin HC, Chang JH, Jain S, et al. Matrilysin cleavage of corneal collagen type XVIII NC1 domain and generation of a 28-kDa fragment. Invest Ophthalmol Vis Sci. 2001;42:2517–2524.[Abstract/Free Full Text]
  38. Katz A, Fish AJ, Kleppel MM, et al. Renal entactin (nidogen): isolation, characterization and tissue distribution. Kidney Int. 1991;40:643–652.[Medline][Order article via Infotrieve]
  39. Katz A, Fish AJ, Pe’er J, et al. Entactin/nidogen: synthesis by bovine corneal endothelial cells and distribution in the human cornea. Invest Ophthalmol Vis Sci. 1994;35:495–502.[Abstract/Free Full Text]
  40. Kohfeldt E, Sasaki T, Göhring W, Timpl R. Nidogen-2: a new basement membrane protein with diverse binding properties. J Mol Biol. 1998;282:99–109.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  41. Couchman JR, Ljubimov AV. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies. Matrix. 1989;9:311–321.[Medline][Order article via Infotrieve]
  42. Sage H, Vernon RB, Funk SE, Everitt EA, Angello J. SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2-dependent binding to the extracellular matrix. J Cell Biol. 1989;109:341–356.[Abstract/Free Full Text]
  43. Sweetwyne MT, Brekken RA, Workman G, et al. Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies. J Histochem Cytochem. 2004;52:723–733.[Abstract/Free Full Text]
  44. Dziadek M, Paulsson M, Aumailley M, Timpl R. Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor. Eur J Biochem. 1986;161:455–464.[Web of Science][Medline][Order article via Infotrieve]
  45. Piecha D, Hartmann K, Kobbe B, et al. Expression of matrilin-2 in human skin. J Invest Dermatol. 2002;119:38–43.[CrossRef][Medline][Order article via Infotrieve]
  46. Klatt AR, Nitsche DP, Kobbe B, et al. Molecular structure, processing, and tissue distribution of matrilin-4. J Biol Chem. 2001;276:17267–17275.[Abstract/Free Full Text]
  47. Balza E, Siri A, Ponassi M, et al. Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin. FEBS Lett. 1993;332:39–43.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  48. Siri A, Knäuper V, Veirana N, et al. Different susceptibility of small and large human tenascin-C isoforms to degradation by matrix metalloproteinases. J Biol Chem. 1995;270:8650–8654.[Abstract/Free Full Text]
  49. Durbeej M, Henry MD, Ferletta M, Campbell KP, Ekblom P. Distribution of dystroglycan in normal adult mouse tissues. J Histochem Cytochem. 1998;46:449–457.[Abstract/Free Full Text]
  50. Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986;103:49–62.[Abstract/Free Full Text]
  51. Zieske JD, Bukusoglu G, Yankauckas MA, Wasson ME, Keutmann HT. Alpha-enolase is restricted to basal cells of stratified squamous epithelium. Dev Biol. 1992;151:18–26.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  52. Aumailley M, Bruckner-Tuderman L, Carter WG, et al. A simplified laminin nomenclature. Matrix Biol. 2005;24:326–332.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  53. Kleppel MM, Santi PA, Cameron JD, Wieslander J, Michael AF. Human tissue distribution of novel basement membrane collagen. Am J Pathol. 1989;134:813–825.[Abstract]
  54. Kleppel MM, Michael AF. Expression of novel basement membrane components in the developing kidney and eye. Am J Anat. 1990;187:165–174.[CrossRef][Medline][Order article via Infotrieve]
  55. Ohji M, SundarRaj N, Hassell JR, Thoft RA. Basement membrane synthesis by human corneal epithelial cells in vitro. Invest Ophthalmol Vis Sci. 1994;35:479–485.[Abstract/Free Full Text]
  56. Ljubimov AV, Huang Z, Huang GH, et al. Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy. J Histochem Cytochem. 1998;46:1033–1041.[Abstract/Free Full Text]
  57. Ljubimov AV, Saghizadeh M, Spirin KS, et al. Increased expression of fibrillin-1 in human corneas with bullous keratopathy. Cornea. 1998;17:309–314.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  58. Tuori AJ, Virtanen I, Aine E, et al. The immunohistochemical composition of corneal basement membrane in keratoconus. Curr Eye Res. 1997;16:792–801.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  59. Hiscott P, Seitz B, Schlötzer-Schrehardt U, Naumann GOH. Immunolocalisation of thrombospondin 1 in human, bovine and rabbit cornea. Cell Tissue Res. 1997;289:307–310.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  60. Xiao J, Natarajan K, Rajala MS, et al. Vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium. Am J Ophthalmol. 2005;140:363–369.[CrossRef][Medline][Order article via Infotrieve]
  61. Byström B, Virtanen I, Rousselle P, Gullberg D, Pedrosa-Domellöf F. Distribution of laminins in the developing human eye. Invest Ophthalmol Vis Sci. 2006;47:777–785.[Abstract/Free Full Text]
  62. Byström B, Virtanen I, Rousselle P, et al. Laminins in normal, keratoconus, bullous keratopathy and scarred human corneas. Histochem Cell Biol. 2007;127:657–667.[CrossRef][Medline][Order article via Infotrieve]
  63. Wiley L, SundarRaj N, Sun TT, Thoft RA. Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation. Invest Ophthalmol Vis Sci. 1991;32:594–602.[Abstract/Free Full Text]
  64. Ljubimov AV, Saghizadeh M, Spirin KS, et al. Expression of tenascin-C splice variants in normal and bullous keratopathy human corneas. Invest Ophthalmol Vis Sci. 1998;39:1135–1142.[Abstract/Free Full Text]
  65. Ishizaki M, Westerhausen-Larson A, Kino J, Hayashi T, Kao WWY. Distribution of collagen IV in human ocular tissues. Invest Ophthalmol Vis Sci. 1993;34:2680–2689.[Abstract/Free Full Text]
  66. Kolega J, Manabe M, Sun TT. Basement membrane heterogeneity and variation in corneal epithelial differentiation. Differentiation. 1989;42:54–63.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  67. Rodrigues M, Ben-Zvi A, Krachmer J, Schermer A, Sun TT. Suprabasal expression of a 64-kilodalton keratin (no. 3) in developing human corneal epithelium. Differentiation. 1987;34:60–67.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  68. Lavker RM, Tseng SC, Sun TT. Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res. 2004;78:433–446.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  69. Li DQ, Chen Z, Song XJ, et al. Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness. Exp Eye Res. 2005;80:581–590.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  70. Than ME, Bourenkov GP, Henrich S, Mann K, Bode W. The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?. Biol Chem. 2005;386:759–766.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  71. Li W, He H, Kuo CL, et al. Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. Invest Ophthalmol Vis Sci. 2006;47:2381–2389.[Abstract/Free Full Text]
  72. Homma R, Yoshikawa H, Takeno M, et al. Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice. Invest Ophthalmol Vis Sci. 2004;45:4320–4326.[Abstract/Free Full Text]
  73. Schlötzer-Schrehardt U, Kruse FE. Identification and characterization of limbal stem cells. Exp Eye Res. 2005;81:247–264.[Web of Science][Medline][Order article via Infotrieve]
  74. Pajoohesh-Ganji A, Pal-Ghosh S, Simmens SJ, Stepp MA. Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse. Stem Cells. 2006;24:1075–1086.[Abstract/Free Full Text]
  75. Stepp MA. Corneal integrins and their functions. Exp Eye Res. 2006;83:3–15.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  76. Ljubimov AV, Alba SA, Burgeson RE, et al. Extracellular matrix changes in human corneas after radial keratotomy. Exp Eye Res. 1998;67:265–272.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  77. Yew DT, Sha O, Li WW, Lam TT, Lorke DE. Proliferation and apoptosis in the epithelium of the developing human cornea and conjunctiva. Life Sci. 2001;68:2987–3003.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  78. Teller IC, Auclair J, Herring E, Gauthier R, Ménard D, Beaulieu JF. Laminins in the developing and adult human small intestine: relation with the functional absorptive unit. Dev Dyn. 2007;236:1980–1990.[CrossRef][Medline][Order article via Infotrieve]
  79. Lefebvre O, Sorokin L, Kedinger M, Simon-Assmann P. Developmental expression and cellular origin of the laminin {alpha}2, {alpha}4, and {alpha}5 chains in the intestine. Dev Biol. 1999;210:135–150.[CrossRef][Medline][Order article via Infotrieve]
  80. Kapoor R, Sakai LY, Funk S, et al. Type VIII collagen has a restricted distribution in specialized extracellular matrices. J Cell Biol. 1988;107:721–730.[Abstract/Free Full Text]
  81. Joyce NC. Proliferative capacity of the corneal endothelium. Prog Retin Eye Res. 2003;22:359–389.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  82. Shuttleworth CA. Type VIII collagen. Int J Biochem Cell Biol. 1997;29:1145–1148.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  83. Hopfer U, Fukai N, Hopfer H, et al. Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye. FASEB J. 2005;19:1232–1244.[Abstract/Free Full Text]
  84. Desjardins M, Bendayan M. Ontogenesis of glomerular basement membrane: structural and functional properties. J Cell Biol. 1991;113:689–700.[Abstract/Free Full Text]
  85. Zhu D, Kim Y, Steffes MW, et al. Application of electron microscopic immunocytochemistry to the human kidney: distribution of type IV and type VI collagen in normal human kidney. J Histochem Cytochem. 1994;42:577–584.[Abstract]
  86. Lee LK, Pollock AS, Lovett DH. Asymmetric origins of the mature glomerular basement membrane. J Cell Physiol. 1993;157:169–177.[CrossRef][Medline][Order article via Infotrieve]
  87. Ljubimov AV, Atilano SR, Garner MH, et al. Extracellular matrix and Na+,K+-ATPase in human corneas following cataract surgery: comparison with bullous keratopathy and Fuchs’ dystrophy corneas. Cornea. 2002;21:74–80.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  88. Maseruka H, Ridgway A, Tullo A, Bonshek R. Developmental changes in patterns of expression of tenascin-C variants in the human cornea. Invest Ophthalmol Vis Sci. 2000;41:4101–4107.[Abstract/Free Full Text]
  89. Koch M, Olson PF, Albus A, et al. Characterization and expression of the laminin {gamma}3 chain: a novel, non-basement membrane-associated, laminin chain. J Cell Biol. 1999;145:605–618.[Abstract/Free Full Text]
  90. Yan HH, Cheng CY. Laminin {alpha}3 forms a complex with ß3 and {gamma}3 chains that serves as the ligand for {alpha}6ß1-integrin at the apical ectoplasmic specialization in adult rat testes. J Biol Chem. 2006;281:17286–17303.[Abstract/Free Full Text]
  91. Libby RT, Champliaud MF, Claudepierre T, et al. Laminin expression in adult and developing retinae: evidence of two novel CNS laminins. J Neurosci. 2000;20:6517–6528.[Abstract/Free Full Text]
  92. Gersdorff N, Kohfeldt E, Sasaki T, Timpl R, Miosge N. Laminin {gamma}3 chain binds to nidogen and is located in murine basement membranes. J Biol Chem. 2005;280:22146–22153.[Abstract/Free Full Text]
  93. Aisenbrey S, Zhang M, Bacher D, et al. Retinal pigment epithelial cells synthesize laminins, including laminin 5, and adhere to them through {alpha}3- and {alpha}6-containing integrins. Invest Ophthalmol Vis Sci. 2006;47:5537–5544.[Abstract/Free Full Text]
  94. Häger M, Gawlik K, Nyström A, Sasaki T, Durbeej M. Laminin {alpha}1 chain corrects male infertility caused by absence of laminin {alpha}2 chain. Am J Pathol. 2005;167:823–833.[Abstract/Free Full Text]
  95. Salmivirta K, Talts JF, Olsson M, et al. Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens. Exp Cell Res. 2002;279:188–201.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  96. Piecha D, Wiberg C, Morgelin M, et al. Matrilin-2 interacts with itself and with other extracellular matrix proteins. Biochem J. 2002;367:715–721.[CrossRef][Medline][Order article via Infotrieve]
  97. Bourne WM, Johnson DH, Campbell RJ. The ultrastructure of Descemet’s membrane. III. Fuchs’ dystrophy. Arch Ophthalmol. 1982;100:1952–1955.[Abstract/Free Full Text]
  98. Waring GO, III. Posterior collagenous layer of the cornea: ultrastructural classification of abnormal collagenous tissue posterior to Descemet’s membrane in 30 cases. Arch Ophthalmol. 1982;100:122–134.[Abstract/Free Full Text]
  99. Hsu JKW, Rubinfeld RS, Barry P, Jester JV. Anterior stromal puncture: immunohistochemical studies in human corneas. Arch Ophthalmol. 1993;111:1057–1063.[Abstract/Free Full Text]
  100. Kenney MC, Nesburn AB, Burgeson RE, Butkowski RJ, Ljubimov AV. Abnormalities of the extracellular matrix in keratoconus corneas. Cornea. 1997;16:345–351.[Web of Science][Medline][Order article via Infotrieve]
  101. Tuori A, Virtanen I, Aine E, Uusitalo H. The expression of tenascin and fibronectin in keratoconus, scarred and normal human cornea. Graefes Arch Clin Exp Ophthalmol. 1997;235:222–229.[CrossRef][Web of Science][Medline][Order article via Infotrieve]



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A. M.-H. Yeung, U. Schlotzer-Schrehardt, B. Kulkarni, N. L. Tint, A. Hopkinson, and H. S. Dua
Limbal Epithelial Crypt: A Model for Corneal Epithelial Maintenance and Novel Limbal Regional Variations
Arch Ophthalmol, May 1, 2008; 126(5): 665 - 669.
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