Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.

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Lumican Regulates Collagen Fibril Assembly: Skin Fragility and Corneal Opacity in the Absence of Lumican

It has long been recognized that collagen architecture of the corneal stroma is crucially important in the ultimate transparency of the cornea.1 Collagen fibrils in the stroma are maintained in the range of 20 to 40 nm and organized into a highly ordered, latticelike configuration. The highly ordered architecture of the corneal stroma is affected by multiple factors. Recent studies of several types of hereditary corneal dystrophies elucidated abnormal collagen fibril architecture of the corneal stroma. For example, corneal opacification is a clinical feature of Scheie’s syndrome or mucopolysaccharidosis (MPS) type I, a lysosomal storage disorder with an iduronidase A deficiency.2 In addition to featuring granular deposits, transmission electron microscopy of MPS I–affected corneas revealed the presence of thicker collagen fibrils and localized disorganization of the matrix.3,4 Macular corneal dystrophy, with deficiencies in keratan sulfate (KS) biosynthesis, also causes clouding of the cornea and similar disruptions in stromal fibril structure and organization.5–8 In both cases, altered proteoglycan synthesis and composition are to be expected. Recently, a mouse model for corneal dystrophy was developed by targeted disruption of the lumican gene (lumtm1sc/lumtm1sc).9 The mutant mice had cloudy corneas and stromal collagen fibrils with increased diameter and altered structure.

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.14–16 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.

Corneal Opacity in Lumican-Null Mice: Defects in Collagen Fibril Structure and Packing in the Posterior Stroma

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene.

Lumican and fibromodulin are collagen-binding leucine-rich proteoglycans widely distributed in interstitial connective tissues. The phenotypes of lumican-null (Lum(-/-)), Fibromodulin-null (Fmod(-/-)) and compound double-null (Lum(-/-)Fmod(-/-)) mice identify a broad range of tissues where these two proteoglycans have overlapping and unique roles in modulating the extracellular matrix and cellular behavior. The lumican-deficient mice have reduced corneal transparency and skin fragility. The Lum(-/-)Fmod(-/-) mice are smaller than their wildtype littermates, display gait abnormality, joint laxity and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis and extreme tendon weakness are the likely cause for joint-laxity. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum(+/+)Fmod(-/-) mice, with further loss in stiffness in a lumican gene dose-dependent way. At the level of ultrastructure, the Lum(-/-) cornea, skin and tendon show irregular collagen fibril contours and increased fibril diameter. The Fmod(-/-) tendon contains irregular contoured collagen fibrils, with increased frequency of small diameter fibrils. The tendons of Lum(-/-)Fmod(-/-) have an abnormally high frequency of small and large diameter fibrils indicating a de-regulation of collagen fibril formation and maturation. In tissues like the tendon, where both proteoglycans are present, fibromodulin may be required early in collagen fibrillogenesis to stabilize small-diameter fibril-intermediates and lumican may be needed at a later stage, primarily to limit lateral growth of fibrils

Functions of lumican and fibromodulin: Lessons from knockout mice

We performed anterior keratectomies on six monkey eyes, four by excimer laser large-area ablation at 193 nm and two by mechanical keratectomy. Immunofluorescence was used to study the wound healing response histopathologically. The distribution of fibrinogen, fibronectin, laminin, collagen types III, IV, and VI, and keratan sulfate was determined at postoperative intervals of 24 hours, 6 days, and 1 month. At 24 hours, fibrinogen and fibronectin coated the ablated surface, but corneal epithelial cells had not yet migrated over the wound. By 6 days and persisting at 1 month, an epithelial ingrowth of seven to 10 layers, mild stromal hypercellularity, and new collagen formation were present in the repair region. At 1 month, fibrinogen, fibronectin, laminin, and type III collagen were strongly detected in the repair region. Type VI collagen was present in both normal and healed corneal stroma at all intervals, and type IV collagen was present in Descemet's membrane only. Sulfated keratan sulfate was absent from the newly synthesized collagen stroma at all intervals. Slit-lamp photographs demonstrated corneal haze in the ablation zone in all cases at 24 hours, persisting for 1 month. The fluorescence patterns produced by excimer laser ablation and mechanical keratectomy were qualitatively identical.

Immunofluorescence study of corneal wound healing after excimer laser anterior keratectomy in the monkey eye.

An ELISA assay using a monoclonal antibody (ET-4-A-4) that recognizes a sulfated carbohydrate epitope in both keratan sulfate type I (corneal) and type II (skeletal) was employed to quantify keratan sulfate in serum and corneal tissue from patients with macular corneal dystrophy (MCD). This assay disclosed significant quantities of keratan sulfate in the serum in 45 healthy individuals (251 +/- 78 ng/ml), and in 66 patients with various corneal diseases (273 +/- 101 ng/ml). In contrast keratan sulfate was not detected (less than 2 ng/ml) in the serum of 16 patients with histopathologically confirmed MCD. Keratan sulfate was also detected in extracts of normal corneas and corneal tissue with a variety of pathologic conditions, but was virtually absent in corneal tissue from five patients with MCD. In corneas with MCD the chondroitin sulfate/keratan sulfate ratio was considerably higher than that of all normal and pathologic corneas studied. Since keratan sulfate in the serum appears to be derived predominantly from the normal turnover of cartilage these studies strongly suggest that the defect in keratan sulfate synthesis in MCD is not restricted to corneal cells and that MCD is one manifestation of a systemic disorder of keratan sulfate. The cartilage changes, however, do not have clinical significance. Moreover, since keratan sulfate can be detected in the blood of newborns it should be possible to diagnose MCD prior to corneal opacification.

R. Dennis

Papers

Macular corneal dystrophy. Lack of keratan sulfate in serum and cornea.