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1 From the Departments of Medicine and Genetics and 2 Ophthalmology, Case Western Reserve University and University Hospitals of Cleveland, Ohio; the 3 Department of Ophthalmology, University of Texas, Southwestern Medical Center at Dallas; the 4 Department of Biochemistry and Molecular Biology, University of South Florida, Tampa; and 5 Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
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Abstract |
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METHODS. Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically.
RESULTS. Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice.
CONCLUSIONS. The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.
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Introduction |
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Lumican is a member of the leucine-rich proteoglycan (LRP) family.10 It is a major keratan sulfate proteoglycan of the corneal stroma as well as other collagenous extracellular matrices (skin, cardiac valves, cartilage, and bone).11 Other LRP members include decorin, fibromodulin, biglycan, keratocan, osteoglycin, and epiphycan.12 Decorin, a chondroitin sulfate (CS) proteoglycan widely expressed during mouse embryonic development, is also a major component of the corneal stroma.13 Previous studies have shown that the core proteins of lumican, decorin, and other LRPs from tendons can delay spontaneous collagen fibril formation and inhibit the lateral growth of fibrils in fibrillogenesis assays in vitro.14 15 16 Also, the abnormal lateral growth of isolated corneal fibrils stripped of their surface-associated macromolecules is prevented by the corneal proteoglycans.17 Recent gene-targeting studies of LRPs suggest a similar role for these proteoglycans in vivo. Thus, absence of lumican in our lumtm1sc/lumtm1sc mouse model of corneal dystrophy affected collagen architecture of the cornea and skin with consequent corneal opacity and reduced dermal biomechanical tensile strength. In addition to lumican, gene-targeted null mutations in decorin and fibromodulin also led to abnormal collagen fibril architecture in skin and tendons.18 19 However, to date only the lumican-deficient mice have demonstrated a corneal phenotype.
The purpose of the present study was to assess corneal opacification in the lumtm1sc/lumtm1sc mice and define its source in the corneal stroma by in vivo confocal microscopy. Parallel analyses of collagen fibril structure, fibril packing, and organization in the lumican-deficient and wild-type control mice and lumican expression in the mature normal cornea indicate that lumican serves a key role in the establishment and maintenance of corneal transparency.
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Methods |
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In Vivo Confocal Microscopy
In vivo examinations were performed using a scanning confocal
microscope with a z-axis resolution of 9 µm (Tandem
Scanning, Reston, VA). Before examination, mice were anesthetized with
an intramuscular injection of ketamine HCl (50 mg/ml), xylazine HCl
(20mg/ml), and acepromazine (10 mg/ml) in sterile water. A drop of
2.5% hydroxypropyl methylcellulose was then applied to the objective
tip as an immersion fluid, because the objective was brought into
contact with the mouse eye. With the gain, kilovolts, and black level
on the video camera kept in automatic mode, initially high-quality
images were obtained from all corneal sublayers. These were then
switched to manual settings that were kept constant throughout the
study, to allow for direct comparison of scans obtained at different
time points. Digital image acquisition and CMTF have been described
earlier.20
21
Conventional Transmission Electron Microscopy
Corneas of
lumtm1sc/lumtm1sc
and wild-type controls of similar age
(lum+/lum+)
were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M
sodium cacodylate, (pH 7.4) with 8.0 mM CaCl2 for
2 hours on ice. The corneas were dissected and postfixed with 1%
osmium tetroxide for 1 hour. After dehydration in a graded ethanol
series followed by propylene oxide, the corneas were infiltrated and
embedded in a mixture of Polybed 812, nadic methyl anhydride,
dodecenylsuccinic anhydride, and 2,4,6-tris(dimethylaminomethyl)phenol
(DMP-30; Polysciences, Warrington, PA). Thick sections (1 µm)
were cut and stained with methylene blue-azur blue for examination and
selection of specific regions for further analysis. Thin sections (100
nm) were prepared using an ultramicrotome (UCT; Reichert Jung, Vienna,
Austria) and a diamond knife and stained with 2% aqueous uranyl
acetate followed by 1% phosphotungstic acid (pH 3.2). The sections
were examined and photographed at 80 kV using a transmission electron
microscope (model 7000; Hitachi, Tokyo, Japan). The microscope was
calibrated using a line grating.
Fibril Diameter Measurements
The corneal stroma was divided into three regions for analysis:
anterior, mid and posterior stroma. The anterior stroma was defined as
the 10 µm subjacent to the epithelium, and the posterior stroma was
the 10 µm adjacent to Descemet’s layer. When specifically stated,
regions of the posterior stroma were chosen for analysis based on the
presence of structurally abnormal fibrils. In all other experiments
calibrated micrographs from each region were chosen randomly in a
masked manner. Micrographs of appropriate regions were taken at
x48,700. The micrographs were digitized, and diameters were measured
by computer (RM Biometrics-Bioquant Image Analysis System; Nashville,
TN).
Immunofluorescence Microscopy
Corneas from 3-month-old and more than 7-month-old wild-type and
lumican-deficient mice were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS; pH 7.3) for 30 minutes on ice. The
tissues were cryoprotected with 2 M sucrose-PBS, and frozen in optimal
cutting temperature compound (OCT; Tissue Tek, Miles Laboratories,
Naperville, IL). Sections (6 µm) were cut and mounted on
poly-L-lysine–coated slides. Sections were treated with
sodium borohydride (50 mg/100 ml PBS) for 15 minutes at room
temperature, and nonspecific binding sites were blocked by incubation
in 5% bovine serum albumin (BSA) in PBS overnight at 4°C. Sections
were then incubated with a polyclonal anti-mouse lumican antiserum
(1:250) obtained from Ake Oldberg (University of Lund, Sweden). The
lumican antiserum was generated against a bacterially produced lumican
fusion protein and characterized as lumican specific.19
The sections were then incubated with a secondary goat anti-rabbit
dichlorotriazynyl amino fluorescein–conjugated antibody (1:150;
Jackson ImmunoResearch, West Grove, PA). Negative control samples were
incubated identically, except the primary antibody was excluded. To
visualize nuclei, the slides were mounted in glycerol solution with 1
mg/ml Hoechst stain. Images were captured using a digital camera
(Optronics, Goletta, CA), with set integration times and identical
conditions to facilitate comparisons between samples. However, for
optimal presentation of the data, images presented had exposure times
as indicated in the figure legend.
KS and CS Content
Glycosaminoglycan (GAG) content per whole eye was determined by
modifying a previously published method that uses the ability of
proteoglycans to bind the dimethylmethylene blue (DMB)
dye.22
Whole eyes were extracted in 0.05 M sodium acetate
containing 4 M guanidium-HCl (GuHCl). The soluble supernatant
containing proteoglycans was dialyzed against 0.1 M Tris (pH 7.4)
containing phenylmethylsulfonyl fluoride (500 mM),
N-ethylmaleimide (100 mM), and EDTA (100 mM) and incubated
with DMB reagent prepared as described. The change in absorbance was
determined in an enzyme-linked immunosorbent assay reader (model II
ELISA reader; Dynatech, Alexandria, VA) as a ratio of 550:610 nm.
To estimate the amount of KS and CS in the total GAG content, the
extracts were digested with either keratanase or chondroitinase ABC
(Seikagaku, America, Rockville, MD) to specifically remove KS or CS,
and DMB-reactive GAGs were estimated as described. KS or CS in
micrograms per eye was calculated as follows. Commercially available KS
and CS (Seikagaku America) at various concentrations (0–10 µg/well)
was mixed with DMB, and absorbance at 550:610 nm was determined to
generate a standard curve to estimate proteoglycan-DMB binding.
Absorbance at 550:610 nm of whole-eye extracts reacted with DMB, before
and after enzymatic removal of GAGs, was compared with the standard
curve to estimate micrograms of proteoglycan per eye.
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Results |
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Abnormal Collagen Fibril Architecture Localizes to Posterior Stroma
in lumtm1sc/lumtm1sc Mice
Corneas from adult mice (>7 months old) were analyzed by
transmission electron microscopy. The stroma was divided into three
regions for analysis: anterior, mid, and posterior. In
lumican-deficient and wild-type corneas, the fibrils in the anterior
stroma had regular packing and cylindrical collagen fibrils. In
contrast, in the posterior stroma of the lumican-deficient mice, fibril
diameters were larger. In addition, there were fibrils laterally
associated with one another and fibrils with irregular contours (Fig. 2)
. The midstromal region was unremarkable in fibril structure, except
for the rare appearance of abnormal laterally fused fibrils typical of
the posterior stroma. The midstroma closer to the posterior stroma had
a higher incidence of these abnormal fibrils (data not shown). Packing
of the collagen fibrils in the lumican-null mutants was fairly
unperturbed in the anterior stroma but was affected severely in the
posterior stroma, with frequent fibril-poor spaces or
lakes.23
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Abnormal Fibril Packing and Lamellar Organization in Posterior
Stromas of lumtm1sc/lumtm1scMice
In addition to the defects in fibril structure, the posterior
stroma of the lumican-deficient corneas harbored alterations in
interfibrillar spacing associated with abnormal fibril packing and a
dramatic disruption in lamellar organization of fibrils. These features
become apparent when corneal sections were viewed under low
magnification from the anterior to the posterior stroma (Fig. 5)
. Relative to lamellae in wild-type controls (bold arrows in Fig. 5A
),
lamellae in the posterior stroma of the mutant corneas (Fig. 5B)
were
poorly organized and difficult to differentiate from adjacent lamellae.
In particular, fibrils lost their regular packing adjacent to the
Descemet’s layer (asterisks, Figs. 5B
5C
). In the anterior stroma,
lamellar disorganization was less apparent, and approximately 10 to 15
µm away from Descemet’s layer approaching the anterior stroma,
lamellar architecture was relatively normal.
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Discussion |
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The lumican-deficient mouse model links abnormal collagen architecture with loss of corneal transparency, providing the first genetic mouse model for corneal opacity.9 Mutant mice were assessed qualitatively for clouding by slit lamp biomicroscopic examination of the corneas. Loss of corneal transparency could be detected as early as 5 weeks. In the present study, in vivo CMTF was used to obtain a quantitative assessment of corneal opacity. Backscattered light can be used as an indication of the extent of corneal opacity. Regional differences in the quantity of backscattered light can provide additional information on the status of corneal transparency across the entire thickness of the cornea. This technique has been used on a rabbit model for wound repair after photorefractive keratectomy.24 Three-dimensional reconstruction of the CMTF scans of the mouse cornea in our study showed a distinct highly reflective zone in the posterior stroma. Profiles of the scanned images confirmed a more than threefold increase in backscattered light in the lumican-deficient mice.
The CMTF data provided the expected indication of loss of transparency long before the loss was detected by slit lamp examination.9 However, it was not expected that the intensity of backscattered light in the younger mice would be as high as it was in the older mutants. Given the extreme sensitivity of CMTF scanning, the backscattering in the lumican-deficient mice examined in the current study may already be at saturation levels. Earlier examinations of the same animal over time, from postnatal to adult stages, will provide further insight on the progressive nature of corneal opacity.
The analysis of corneal opacification clearly indicated that the defect resides in the posterior stroma. Therefore, we carefully examined the collagen architecture in the mutant mice, from the anterior stroma containing newly synthesized collagen fibrils to the posterior stroma containing mature collagen fibrils. The architectural alterations coincided with the functional defect, in that the anterior stroma was normal in fibril diameter and packing, but in the posterior stroma, fibril diameter had increased by 6%. The posterior stroma also contained a population of large-diameter fibrils, many with irregular contours indicative of abnormal lateral growth of fibrils.25
A recent study also reported irregular large fibrils in the posterior stroma in lumican-deficient mice.26 Abnormal lateral growth requires lateral associations of collagen fibrils that would disrupt fibril packing and organization. Both packing and general lamellar organization of the fibrils were dramatically disrupted in the posterior stroma of the lumican-deficient mice. These structural defects in the lumican-null mice agree with our immunohistologic results that demonstrate a much stronger presence of lumican in the posterior stroma of wild-type normal corneas. An almost total restriction of the fibril- and matrix-disruptions to the posterior stroma of the mutant mice implies a unique role for lumican in this region. Lumican-collagen interactions in the posterior stroma may be of particular significance in limiting interfibrillar associations between mature fibrils. Perhaps other KS-LRPs usurp this role in other regions of the stroma. Certainly, the heterogeneity of the defect also suggests other regulators of stroma matrix assembly that remain to be elucidated. The increase in fibril diameter and thinning of cornea that we have reported in lumtm1sc/lumtm1sc mice occurs in several types of mucopolysaccharidosis3 25 27 and keratoconus,28 respectively. However, the massive matrix disorganization of the posterior stroma in lumican-deficient mice was not seen in the human dystrophies.
The structural and functional differences in the anterior versus the posterior stroma of the lumican-null mutants should be reviewed in the context of the differences between the anterior and posterior stroma noted previously.29 30 31 32 In agreement with our finding that the posterior stroma is rich in lumican is the observation that this region of the stroma also has a higher concentration of KS than the anterior stroma. Our results for KS estimation in whole eyes detected a lowering of total ocular KS levels in the lumican-null mutants. We speculate a similar reduction of KS from the posterior stroma in the mutants may contribute to the anomalies seen in these mutants. It was pointed out earlier that low oxygen content in the posterior stroma allowed efficient synthesis of KSPG and not CS proteoglycan (CSPG), which is oxygen-dependent.33 Yet, even the very thin mouse cornea, less than 20% the thickness of rabbit and bovine corneas, is rich in KSPG in the posterior stroma, indicating an intrinsic need for KSPG in this region. Studies on the bovine cornea show increased hydration in the posterior stroma, and KSPG is reported to have higher water retention capability. Thus, it is likely that lumican in the posterior stroma is required for a KS-rich environment to maintain a highly hydrated state.
In addition to lumican, keratocan and mimecan are two major KSPGs of the cornea.34 35 Yet, these are not sufficient to complement the contributions of lumican to maintenance of stromal architecture. It is possible that their contribution to the KS pool in the posterior stroma is not high enough to compensate for a reduction in KS associated with the absence of lumican. Ocular phenotypes of the keratocan- and mimican-deficient mice will provide further clues about their role in the cornea. Keratocan, specifically expressed throughout the cornea during development and in adult mice, may adequately maintain anterior stromal architecture as well as stromal physiological properties.36 Mice deficient in keratocan may have corneal developmental anomalies, anterior stromal collagen defects, and changes in corneal physiology. The heterogeneity in the posterior stroma of the lumican-deficient corneas implies that keratocan and/or mimican also have a secondary role in this region.
The molecular mechanism underlying the considerable thinning of the stroma in the lumican-null mice remains to be elucidated. It may be due to a smaller pool of stromal keratocytes in the stroma, implying that lumican may play a role in the early stages of cell migration into the developing stroma. Alternatively, absence of lumican in the null mice may have an adverse effect on the biosynthesis of other stromal components. In the 4- to 5-month age group we also noted a small increase in the epithelial thickness in the lumican-null mice. Recently, the corneal epithelium has been shown to express lumican after wounding, indicating a biologic role for lumican in the epithelium as well.26 Thus, it is possible that the observed thickening of the epithelium in our older lumican-null mice may be a compensatory wound-healing response to lumican-deficiency and the thinner structurally impaired stroma.
The present study showed lumican to be present in high quantities in the posterior stroma. The collagen structural and organizational anomalies observed in its absence in the posterior stroma of lumican-null mutants establishes a key role for lumican in maintaining the unique properties of the posterior stroma.
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Footnotes |
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Submitted for publication February 23, 2000; revised May 18, 2000; accepted June 16, 2000.
Commercial relationships policy: N.
Corresponding author: Shukti Chakravarti, Departments of Medicine, Johns Hopkins University, School of Medicine, Ross 954, 720 Rutland Avenue, Baltimore, MD 21205. sxc76{at}po.cwru.edu
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K. J. Jepsen, F. Wu, J. H. Peragallo, J. Paul, L. Roberts, Y. Ezura, A. Oldberg, D. E. Birk, and S. Chakravarti A Syndrome of Joint Laxity and Impaired Tendon Integrity in Lumican- and Fibromodulin-deficient Mice J. Biol. Chem., September 13, 2002; 277(38): 35532 - 35540. [Abstract] [Full Text] [PDF]
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 L. Ameye and M. F. Young Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis, osteoarthritis, Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases Glycobiology, September 1, 2002; 12(9): 107R - 116R. [Abstract] [Full Text] [PDF]
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V. B. Mahajan, C. Wei, and P. J. McDonnell III Microarray Analysis of Corneal Fibroblast Gene Expression after Interleukin-1 Treatment Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
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B. A. Austin, C. Coulon, C.-Y. Liu, W. W.-Y. Kao, and J. A. Rada Altered Collagen Fibril Formation in the Sclera of Lumican-Deficient Mice Invest. Ophthalmol. Vis. Sci., June 1, 2002; 43(6): 1695 - 1701. [Abstract] [Full Text] [PDF]
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A. H. Plaas, L. A. West, E. J. A. Thonar, Z. A. Karcioglu, C. J. Smith, G. K. Klintworth, and V. C. Hascall Altered Fine Structures of Corneal and Skeletal Keratan Sulfate and Chondroitin/Dermatan Sulfate in Macular Corneal Dystrophy J. Biol. Chem., October 19, 2001; 276(43): 39788 - 39796. [Abstract] [Full Text] [PDF]
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A. J. Quantock, K. M. Meek, and S. Chakravarti An X-ray Diffraction Investigation of Corneal Structure in Lumican-Deficient Mice Invest. Ophthalmol. Vis. Sci., July 1, 2001; 42(8): 1750 - 1756. [Abstract] [Full Text] [PDF]
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D. F. Holmes, C. J. Gilpin, C. Baldock, U. Ziese, A. J. Koster, and K. E. Kadler Corneal collagen fibril structure in three dimensions: Structural insights into fibril assembly, mechanical properties, and tissue organization PNAS, June 19, 2001; 98(13): 7307 - 7312. [Abstract] [Full Text] [PDF]
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