Keratocan-deficient mice display alterations in corneal structure

Keratocan (Kera) is a cornea-specific keratan sulfate proteoglycan (KSPG) in the adult vertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP) gene family and is one of the major components of extracellular KSPG in the vertebrate corneal stroma. The Kera gene is expressed in ocular surface tissues including cornea and eyelids during morphogenesis. Corneal KSPGs play a pivotal role in matrix assembly, which is accountable for corneal transparency. In humans, mutations of the KERA gene are associated with cornea plana (CNA2) that manifests decreases in vision acuity due to the flattened forward convex curvature of cornea. To investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Northern and Western blotting and immunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. The expression levels of other SLRP members including lumican, decorin, and fibromodulin were not altered in the Kera-/- cornea as compared with that of the wild-type littermates. Mice lacking keratocan have normal corneal transparency at the age of 12 months. However, they have a thinner corneal stroma and a narrower cornea-iris angle of the anterior segment in comparison to the wild-type littermates. As demonstrated by transmission electron microscopy, Kera-/- mice have larger stromal fibril diameters and less organized packing of collagen fibrils in stroma than those of wild type. Taken together, our results showed that ablation of the Kera gene resulted in subtle structural alterations of collagenous matrix and did not perturb the expression of other SLRPs in cornea. Keratocan thus plays a unique role in maintaining the appropriate corneal shape to ensure normal vision.     

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

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.     

Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.     

Keratocan and lumican regulate neutrophil infiltration and corneal clarity in lipopolysaccharide-induced keratitis by direct interaction with CXCL1

Keratocan and lumican are keratan-sulfate proteoglycans (KSPG), which have a critical role in maintaining corneal clarity. To determine whether these KSPGs have a role in corneal inflammation, we examined Kera(-/-) and Lum(-/-) mice in a model of lipopolysaccharide (LPS)-induced keratitis in which wild-type mice develop increased corneal thickness and haze due to neutrophil infiltration to the corneal stroma. Corneal thickness increases caused by LPS mice were significantly lower in Kera(-/-) and Lum(-/-) than wild-type mice. Further, LPS-injected Lum(-/-) mice had elevated corneal haze levels compared with that of Kera(-/-) and wild-type. At 24 h post-injection, total enhanced green fluorescent protein-positive bone marrow-derived inflammatory cells in chimeric mice was significantly lower in Kera(-/-) mice and Lum(-/-) mice compared with wild-type mice. Neutrophil infiltration was inhibited in Kera(-/-) and Lum(-/-) mice at 6 and 24 h post-stimulation, with Lum(-/-) corneas having the most profound defect in neutrophil migration. Reconstitution of keratocan and lumican expression in corneas of Kera(-/-) and Lum(-/-) mice using adeno-keratocan and adeno-lumican viral vectors, respectively, resulted in normal neutrophil infiltration in response to LPS. Immunoprecipitation/Western blot analysis showed that lumican and keratocan core proteins bind the CXC chemokine KC during a corneal inflammatory response, indicating that corneal KSPGs mediate neutrophil recruitment to the cornea by regulating chemokine gradient formation. Together, these data support a significant role for lumican and keratocan in a corneal inflammatory response with respect to edema, corneal clarity, and cellular infiltration.     

Role of lumican in the corneal epithelium during wound healing

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.     

TGFbeta2 in corneal morphogenesis during mouse embryonic development.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.     

TGFβ2 in Corneal Morphogenesis during Mouse Embryonic Development

To examine the roles of TGFβ isoforms on corneal morphogenesis, the eyes of mice that lack TGFβs were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb^−/− mice, only Tgfb2^−/− mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2^−/− mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFβ2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2^−/− mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2^−/− mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.     

An X-ray scattering investigation of corneal structure in keratocan-deficient mice.

The transparency of the cornea has been closely linked with the characteristic size and arrangement of its constituent collagen fibrils. This arrangement, in turn, is thought to depend on interactions with intervening matrix proteoglycans. The purpose of this investigation was to examine fibrillar collagen organisation in the corneas of mice homozygous for a null mutation in keratocan, a keratan sulfate-containing proteoglycan. Low-angle synchrotron X-ray scattering techniques were used. We found that keratocan-deficient mice had corneal collagen fibrils with significantly larger diameters than those in wild-type littermates. Furthermore, there was an increase in the centre-to-centre spacing of the collagen fibrils that was accompanied by a decrease in nearest-neighbour fibrillar order. We hypothesise that a lack of keratocan might lower the number of keratan sulfate proteoglycans that associate with collagen, leading to alterations in their diameters and spatial arrangements. Alternatively, it might change the osmotic balance between the inside and outside of fibrils, causing them to swell and move further apart.     

Corneal cell proteins and ocular surface pathology

The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structure, the basement membrane, composed of a specific subset of collagen type IV and laminin isoforms in addition to ubiquitous extracellular matrix molecules. Matrix metalloprotein-ases have been identified in normal corneal tissue and cells and may play a role in the development of ulcerative corneal diseases. Changes in extracellular matrix molecule localization and synthesis have been noted in other types of corneal diseases as well, including bullous keratopathy and keratoconus.     

Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues

Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.     

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn^−/0/Lum^−/− mouse model and single Lum^−/− or Bgn^−/0 mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn^−/0/Lum^−/− corneas demonstrated gross opacity that was not seen in the Bgn^−/0 or wild type corneas and greater than that in the Lum^−/− mice. The Bgn^−/0/Lum^−/− corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum^−/− and no opacity in Bgn^−/0 or wild type corneas. In the Bgn^−/0/Lum^−/− corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn^−/0 or wild type corneas and was restricted to the posterior stroma in Lum^−/− mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.     

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)(+) BM cells into keratocan null (Kera(-/-)) and lumican null (Lum(-/-)) mice. EGFP(+) BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP(+) BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP(+) due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera(-/-) mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera(-/-) mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera(-/-) mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.     

Functions of lumican and fibromodulin: lessons from knockout mice

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 Published in 2003.     

Fibromodulin and lumican bind to the same region on collagen type I fibrils

Fibromodulin and lumican are closely related members of the extracellular matrix leucine-rich repeat glycoprotein/proteoglycan family. Similar to decorin, another member of this protein family, they bind to fibrillar collagens and function in the assembly of the collagen network in connective tissues. We have studied the binding of recombinant fibromodulin, lumican and decorin, expressed in mammalian cells, to collagen type I. Using a collagen fibril formation/sedimentation assay we show that fibromodulin inhibits the binding of lumican, and vice versa. Fibromodulin and lumican do not affect the binding of decorin to collagen, nor does decorin inhibit the binding of fibromodulin or lumican. Binding competition experiments and Scatchard plot analysis indicate that fibromodulin binds to collagen type I with higher affinity than lumican.     

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Tenomodulin is necessary for tenocyte proliferation and tendon maturation

Tenomodulin (Tnmd) is a member of a new family of type II transmembrane glycoproteins. It is predominantly expressed in tendons, ligaments, and eyes, whereas the only other family member, chondromodulin I (ChM-I), is highly expressed in cartilage and at lower levels in the eye and thymus. The C-terminal extracellular domains of both proteins were shown to modulate endothelial-cell proliferation and tube formation in vitro and in vivo. We analyzed Tnmd function in vivo and provide evidence that Tnmd is processed in vivo and that the proteolytically cleaved C-terminal domain can be found in tendon extracts. Loss of Tnmd expression in gene targeted mice abated tenocyte proliferation and led to a reduced tenocyte density. The deposited amounts of extracellular matrix proteins, including collagen types I, II, III, and VI and decorin, lumican, aggrecan, and matrilin-2, were not affected, but the calibers of collagen fibrils varied significantly and exhibited increased maximal diameters. Tnmd-deficient mice did not have changes in tendon vessel density, and mice lacking both Tnmd and ChM-I had normal retinal vascularization and neovascularization after oxygen-induced retinopathy. These results suggest that Tnmd is a regulator of tenocyte proliferation and is involved in collagen fibril maturation but do not confirm an in vivo involvement of Tnmd in angiogenesis.