Expression of the keratan sulfate proteoglycans lumican, keratocan and osteoglycin/mimecan during chick corneal development

The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence.The expression of each of the three corneal KSPGs in the developing and mature chick cornea was investigated by competitive PCR and immuno-biochemical analysis of corneal extracts. Competitive PCR was used to determine the message levels for chick lumican, keratocan and osteoglycin in embryonic day 9, 12, 15, 18 and adult corneas. Results showed that lumican mRNA fluctuated during development but remained at a relatively high level while keratocan and osteoglycin message levels declined steadily from day 9 to adult. Additionally, lumican mRNA was present at higher levels, during all stages of corneal development, than keratocan and at much higher levels than osteoglycin. Antibodies shown to be specific for each KSPG were used to characterize proteoglycans isolated from embryonic and adult chick corneas. KSPGs from embryonic corneas eluted 1-2 fractions earlier on Q-Sepharose than KSPG from adult corneas. Additionally, Western blot analysis showed that embryonic KSPGs were more keratanase-resistant, endo-beta-galactosidase sensitive than adult KSPGs. The results of this study indicate an alteration in sulfation or the fine structure of the glycosaminoglycan chains occurs during corneal maturation for the 3 KSPGs.     

Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation

PURPOSE: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation. METHODS: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents. RESULTS: Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels. CONCLUSIONS: The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.     

Identification of novel mutations in the carbohydrate sulfotransferase gene (CHST6) causing macular corneal dystrophy

PURPOSE: Macular corneal dystrophy (MCD) is a rare corneal dystrophy that is characterized by abnormal deposits in the corneal stroma, keratocytes, Descemet's membrane, and endothelium, accompanied by progressive clouding. It has been classified into three immunophenotypes--MCD types I, IA, and II--according to the serum level of sulfated keratan sulfate (KS) and immunoreactivity of the corneal tissue. Recently, mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of MCD. Mutation screening of the CHST6 gene has been undertaken to identify the underlying mutations in five unrelated British families with MCD. METHODS: DNA was extracted from venous blood obtained from all participants, and the coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the presence of KS in serum from the probands of MCD-affected families participating in the study. RESULTS: Six novel missense mutations--four homozygous and two compound heterozygous--were identified in the CHST6 gene. The ELISA showed that the disease in all patients participating in the study was of MCD type I, including the subtype IA. CONCLUSIONS: These novel mutations are thought to result in loss of corneal sulfotransferase function, which would account for the MCD phenotype.     

Stimulation of collagen synthesis by insulin and proteoglycan accumulation by ascorbate in bovine keratocytes in vitro

PURPOSE: Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma. The keratan sulfate proteoglycans (KSPGs) lumican and keratocan are also present, and they interact with collagen and modulate its assembly into fibrils. In this study, ascorbate was added to a defined medium containing insulin, and its effects on the synthesis of collagen and KSPGs by keratocytes were determined. METHODS: Collagenase-isolated keratocytes were cultured with or without insulin with or without ascorbate. Collagen and glycosaminoglycan synthesis was determined by collagenase digestion of incorporated 3H-glycine and by chondroitinase ABC or endo-beta-galactosidase digestion of incorporated 35SO4. KSPGs were detected by Western blot. Collagen stability was determined by pepsin digestion. Ethyl-3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation. RESULTS: Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans. The accumulation of PGDS, a KSPG that does not interact with collagen, was not affected by ascorbate. Only the collagen synthesized in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation. CONCLUSIONS: The results of this study indicate that the accumulation of lumican and keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.     

Novel CHST6 nonsense and missense mutations responsible for macular corneal dystrophy

PURPOSE: To identify the underlying mutations in two unrelated British families with macular corneal dystrophy (MCD) by screening the carbohydrate sulfotransferase (CHST6) gene. DESIGN: Case reports and results of DNA analysis. METHODS: Two subjects from two British families with MCD were studied. The genetic status of CHST6 was determined for all members of these MCD families. In addition, sulfated keratan sulfate (KS) assay from the probands was also undertaken. CHST6 gene was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by sequencing and restriction digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess KS presence in serum. RESULTS: Four compound heterozygous mutations were identified, three of which are novel. The ELISA showed that the probands were of MCD type I. CONCLUSIONS: These novel mutations are expected to result in loss of CHST6 function, which would account for the MCD phenotype.     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

Immunohistochemical expression and distribution of proteoglycans and collagens in sclerocornea

To immunolocalize corneal keratan sulfate (KS) and its core protein lumican, aggrecan, type I and type III collagens in sclerocornea specimens and compare their expression and distribution to age-matched healthy corneas and scleras. Sclerocornea specimens (n = 3) and age-matched normal corneoscleral rim specimens (n = 3) were studied by light microscopy and histochemically. KS, lumican, aggrecan, type I and type III collagens were immunolocalized in the specimens using indirect immunofluorescence. The fluorescence intensity in each specimen was scored from 0 to 4, with 0 representing no fluorescence and 4 representing intense fluorescence. The sclerocornea specimens showed histologic features typical of sclerocornea. KS and lumican immunolabeling in the corneal stroma in sclerocornea was decreased, whereas aggrecan immunolabeling was increased compared to that seen in normal cornea and normal sclera. KS and lumican staining was more intense in the posterior part of sclerocornea specimens, whereas aggrecan staining was distributed throughout the stroma. The staining intensity and distribution of type I collagen in sclerocornea was similar to that seen in normal cornea. Type III collagen was faint to absent in both normal cornea and sclerocornea but strong labeling was noted in normal sclera. The immunophenotype of sclerocornea is similar to that of normal cornea but with reduced labeling intensity of KS and lumican and increased labeling intensity of aggrecan. This change could potentially contribute to the abnormal fibril assembly in sclerocornea.     

Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes

PURPOSE. To characterize the major proteoglycans produced and secreted by collagenase-isolated bovine keratocytes in culture. METHODS. Freshly isolated keratocytes from mature bovine corneas were cultured in serum-free Dulbecco's modified Eagle's medium/ F12. Secreted proteoglycans were radiolabeled with protein labeling mix ((35)S-Express; Dupont NEN Life Science Products, Boston, MA) and digested with chondroitinase ABC, keratanase, and endo-beta-galactosidase to remove glycosaminoglycan chains, and core proteins were analyzed by autoradiography and Western blot analysis. An unidentified keratan sulfate proteoglycan (KSPG) was purified by gel filtration (Superose 6; Amersham Pharmacia, Piscataway, NJ) and anion-exchange chromatography (Resource Q; Amersham Pharmacia) and subjected to amino acid sequencing. RESULTS. Keratanase digestion of proteoglycans produced approximately 50 kDa core proteins that immunoreacted with antisera to lumican, keratocan, and osteoglycin-mimecan. Chondroitinase ABC digestion produced a approximately 55-kDa core protein that immunoreacted with antisera to decorin. A 28-kDa band generated by keratanase or endo-beta-galactosidase digestion did not react with these antibodies. Chromatographic purification and amino acid sequencing revealed that the protein was prostaglandin D synthase (PGDS). Identity was confirmed by Western blot analysis using antisera to recombinant PGDS. PGDS isolated from corneal extracts was not keratanase sensitive but was susceptible to endo-beta-galactosidase, suggesting that it contains unsulfated polylactosamine chains in native tissue and is therefore present as a glycoprotein. CONCLUSIONS. These results indicate that bovine keratocytes, when cultured under serum-free conditions, produce the four known leucine-rich proteoglycans decorin, keratocan, lumican, and osteoglycin/mimecan and maintain a phenotype that is comparable to that of in situ keratocytes. Additionally, these cells produce PGDS, a known retinoid transporter, as a KSPG.     

Macular dystrophy of the cornea. A systemic disorder of keratan sulfate metabolism

The serum of most patients with type 1 macular corneal dystrophy (MCD), the most prevalent subtype, lacks detectable antigenic keratan sulfate (KS), and it has been postulated that such individuals may lack antigenic KS in their cartilage as well. To test this hypothesis, we studied the cornea, serum, and nasal cartilage from an MCD patient using light and electron microscopy, immunohistochemistry, and a quantitative enzyme-linked immunosorbent assay (ELISA) which uses a monoclonal antibody against a sulfated epitope on the KS chain to measure KS content. Histologically, corneal deposits seen were characteristic of MCD. No abnormal deposits were noted in the cartilage. The lack of immunoreactivity in corneal sections with antibodies against sulfated epitope on KS and the absence of this epitope in serum showed that the patient had type 1 MCD. The cartilage specimen showed no immunoreactivity in the chondrocytes or extracellular matrix. Quantitative analysis by ELISA demonstrated that the antigenic KS content of the cornea and cartilage was at least 800 times lower than that in normal controls. This provided direct evidence that the abnormality in the sulfation of keratan in type 1 MCD involves the cornea and cartilage.     

An x-ray diffraction study of corneal structure in mimecan-deficient mice

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.     

Macular corneal dystrophy: A review

Macular corneal dystrophy is a corneal stromal dystrophy which leads to progressive vision loss. Macular corneal dystrophy is an autosomal recessive condition in which there is abnormality of proteoglycan synthesis. Mutations in the carbohydrate sulfotransferase gene prevent normal sulfation of corneal keratan. Different immunophenotypes have been described depending on the presence of keratan sulfate in cornea and/or serum. The deposition of abnormal proteoglycans leads to loss of corneal transparency and decreased vision. Imaging techniques such as in vivo confocal microscopy and anterior segment ocular coherence tomography have helped enhance our understanding of the corneal ultrastructural changes in this condition. These imaging modalities provide additional information without the need for a tissue biopsy or excision. Traditionally, full-thickness penetrating keratoplasty to replace the opacified cornea has remained the standard of treatment to improve vision. However, newer surgical interventions such as deep anterior lamellar keratoplasty and phototherapeutic keratectomy have also been shown to play a role in the treatment. Disease recurrence remains a challenge and the reason for poor visual prognosis. Newer techniques such as gene-targeting therapies and enzyme replacement therapies are being studied for a potential permanent solution in macular corneal dystrophy. Recent research is directed toward development of genetically modified products to integrate into host corneal DNA and block the mutant genes and hence overcome the underlying pathophysiology. Enzyme replacement therapy is another intervention with potential to treat macular corneal dystrophy. Animal studies show clearance of accumulated keratan sulfate from the body tissues in the treatment of systemic mucopolysaccharidosis by long-term enzyme replacement therapy. Future research should be directed toward elucidation of the relationship between the mutated carbohydrate sulfotransferase gene, the mechanism of deposit formation, and the development of pharmaceutical agents based on gene therapy.     

Immunohistochemical classification of primary and recurrent macular corneal dystrophy in Germany: subclassification of immunophenotype I A using a novel keratan sulfate antibody.

Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by abnormal deposition of glycosaminoglycans in corneal stroma, keratocytes, Descemet's membrane and corneal endothelium. According to the presence and distribution of sulfated keratan sulfate (KS)-epitopes in serum and cornea (using mAb 5-D-4), MCD can be classified into three immunophenotypes: type I, I A and II. The purpose of this study is to evaluate the immunophenotype of primary and recurrent MCD and to analyze the reactions of a novel KS-antibody in MCD corneas, which recognizes an epitope localized in the binding region of KS-chains to the core protein (mAb 3D12/H7). Indirect immunohistochemistry for KS (mAbs 3D12/H7 and 5-D-4) was performed on 44 corneas of 37 patients with MCD including two recurrences. Immunogold labeling was used to localize KS ultrastructurally within keratocytes. The serum concentration of KS (cKS) was determined in a serum antigen-inhibition assay. Immunohistochemically, no reaction was observed using mAb 5-D-4 in 18 corneas of 16 patients (43% of 37 patients; immunophenotype I). Positive reactions within single keratocytes but not in the stroma, were seen in 22 corneas of 17 patients (46% of 37 patients; immunophenotype I A) and positive reactions in keratocytes and extracellular stroma were found in four corneas of four patients (11% of 37 patients: immunophenotype II). For analysis of cKS a total of seven samples was available. Whereas in the samples of the five patients with immunophenotypes I and I A cKS was below the limit of detection, in the two sera from patients with immunophenotype II, cKS was normal (cKS = 1243 and 1380 nmol l(-1)). The two recurrences demonstrated immunophenotype II. Using mAb 3D12/H7, MCD immunophenotype I A can be further subclassified in type I A 1 (lacking reaction with mAb 3D12/H7 in keratocytes; 77%) and type I A 2 (positive reaction with mAb 3D12/H7 within keratocytes; 23%). MCD immunophenotype I A can not only be found in Saudi Arabia, but is as common as immunophenotype I in German patients. The only recurrences of MCD necessitating regrafting occurred in two patients with immunophenotype II possibly suggesting a higher risk for recurrence in this immunophenotype. The mAb 3D12/H7 allows a further subclassification of immunophenotype I A into type I A1 and 2. This points to a broader spectrum of MCD immunophenotypes and indirectly to a broader corneal proteoglycan pathology in MCD.     

Lumican suppresses cell proliferation and aids Fas-Fas ligand mediated apoptosis: implications in the cornea

Lumican, an extracellular matrix (ECM) keratan sulfate proteoglycan, binds fibrillar collagen and limits collagen fibril diameter in the cornea, skin and tendon. Lumican-deficient mice (Lum(-/-)) develop abnormally thick collagen fibrils, translucent corneas and fragilities of the skin and the tendon. In addition to modulating interstitial ECM structure, here we hypothesized that lumican regulates proliferation and apoptosis of cells residing in the interstitium. Corneal and embryonic fibroblasts from the Lum(-/-) mouse show increased growth in culture. Lum(-/-) mouse embryonic fibroblasts (MEF), compared to their wild type counterparts, display increased rates of proliferation and decreased apoptosis. Ectopic expression of lumican in Lum(-/-) MEF or exogenous recombinant lumican in the culture medium reduces proliferation to rates seen in the Lum(+/+) MEF. We further investigated the implications of lumican's proliferation and apoptosis regulatory role in the cornea where lumican is a major component of the stromal matrix. Stromal keratocytes undergo proliferation and apoptosis during corneal maturation and in the healing of injured cornea. The Lum(-/-) mouse shows increased proliferation and decreased apoptosis of stromal keratocytes during postnatal corneal maturation at the 10-day age. Apoptosis is also significantly down regulated in Lum(-/-) vis-à-vis Lum(+/+) mice during stromal wound healing in the adult 6-week age. Lumican appears to regulate these cellular functions by modulating specific cell growth and apoptosis mediators. Thus, Lum(-/-) MEF have decreased p21(WAF1/CIP1), a universal inhibitor of cyclin-dependent kinases and a consequent increase in cyclins A, D1 and E. Furthermore, the tumor suppressor p53, an upstream regulator of p21 is down regulated in the MEF and the cornea of Lum(-/-) mice. The evidence suggests regulation of p21 by lumican in a p53-dependent way. The MEF and the cornea of Lum(-/-) mice also show a dramatic decrease in Fas (CD95). The Lum(-/-) MEF fail to induce Fas upon treatment with Fas ligand. Fas-Fas ligand interaction is an initiating event in apoptosis and its disruption in lumican-deficiency may partly explain the observed decrease in apoptosis. Fas-Fas ligand mediated apoptosis is critical for maintaining the immune privileged status of the cornea, which implies a new and exciting role for lumican in the cornea.     

Immunochemical localization of keratan sulfate proteoglycans in cornea, sclera, and limbus using a keratanase-generated neoepitope monoclonal antibody

PURPOSE: To evaluate the use of neoepitope monoclonal antibody BKS-1, which recognizes keratanase-generated keratan sulfate (KS) stubs on keratan sulfate proteoglycans in human cornea, limbus, and sclera. METHODS: BKS-1 specifically recognizes a keratanase-generated neoepitope [N-acetyl-glucosamine-6-sulfate (GlcNAc-6-S)] at the nonreducing terminal of corneal and skeletal KS glycosaminoglycan chains. It was produced by using keratanase-digested KS peptides from bovine cartilage aggrecan as the immunizing antigen. BKS-1 was used in conjunction with 5D4 to analyze the KS distribution in human cornea, limbus, and sclera using Western blotting, immunohistochemistry, and electron microscopy. RESULTS: 5D4 Western blot analysis displayed a diffuse staining pattern, and it was difficult to distinguish differences among cornea, sclera, and limbus. However, BKS-1 showed differences in KS levels, with higher levels in the cornea and lower levels in the limbus and sclera. Ultrastructural studies showed that the monoclonal antibody (mAb) BKS-1 neoepitope was not observed in the epithelium or basement membrane; however, 5D4 was present in these layers. Large quantities of both antibodies were present in Bowman's layer, stroma, and Descemet's membrane, but the quantity of 5D4 was significantly higher (P < 0.001) than the quantity of BKS-1 in all these layers of the cornea. CONCLUSIONS: mAb 5D4 recognizes oversulfated structures within KS chains, whereas BKS-1 recognizes a single neoepitope on KS after keratanase digestion of monosulfated KS disaccharides. With the use of BKS-1, the authors identified a more clearly defined pattern for KS distribution in the cornea than was seen with 5D4. The presence of a large quantity of BKS-1 immunostaining in the cornea suggests that KS-substituted proteoglycans are more prevalent in the cornea than in the limbus or sclera.     

重视角膜基质细胞的作用

位于角膜基质胶原纤维板层之间的角膜基质细胞处于静息状态,可以分泌胶原以及硫酸角质素,对角膜透明性的维持发挥着重要作用。当角膜受到损伤时,角膜基质细胞可发生凋亡,向成纤维细胞以及肌成纤维细胞等修复细胞表型转化,进而促进细胞再生,诱导角膜纤维瘢痕形成。此外,大量角膜基质细胞还具有干细胞特性。目前已知角膜基质细胞是多种机体功能的积极参与者,应重视角膜基质细胞的作用。     

Mutations in the CHST6 gene in patients with macular corneal dystrophy: immunohistochemical evidence of heterogeneity

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive disorder leading to severe visual impairment. The carbohydrate sulfotransferase 6 (CHST6) gene, which encodes the corneal N-acetylglucosamine 6-O-sulfotransferase on 16q22 has been identified as a causative gene for MCD. The purpose of this study was to identify mutations in CHST6 in Japanese patients with MCD and evaluate them by means of immunohistochemistry. METHODS: CHST6 was screened in 7 patients and 45 healthy control subjects. Genomic DNA was isolated, and the open reading frame (ORF) of CHST6 was amplified by polymerase chain reaction (PCR). PCR products were analyzed by direct sequencing and restriction enzyme digestion. Immunohistochemistry with a monoclonal anti-keratan sulfate (KS) antibody was performed on corneas of four patients from three families. RESULTS: Three novel mutations (P204Q, R205L, and R177H) and two previously reported mutations (R211W and A217T) were identified in the ORF of CHST6. P204Q, R205L, and R211W were found to be homozygous and R177H and A217T compound heterozygous with R211W on another allele. Immunohistochemistry revealed that R205L homozygous cornea had negative reactivity against the anti-KS antibody, representing type I MCD, and that R211W homozygous and R211W/A217T compound heterozygous corneas had negative or very weak reactivity in the stroma with antibody positive deposits, which were distinct from any previously reported types. CONCLUSIONS: Two mutations (homozygoous R211W and compound heterozygous R211W/A217T) should be subclassified immunohistochemically into new phenotypes of MCD. This heterogeneity could provide further insights into the pathogenesis of MCD.     

Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.

PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.     

Truncating mutations in the carbohydrate sulfotransferase 6 gene (CHST6) result in macular corneal dystrophy

PURPOSE: Identification of mutations in the CHST6 gene in 15 patients from 11 unrelated families affected with recessive macular corneal dystrophy (MCD). METHODS: Genomic DNA was extracted from peripheral blood leukocytes of the affected patients and their healthy family members, and the mutational status of the CHST6 gene was determined for each patient by a PCR-sequencing approach. Serum concentrations of antigenic keratan sulfate for each proband were determined by ELISA. RESULTS: ELISA indicated that all affected patients, except one, were of MCD type I or IA. Fourteen distinct mutations were identified within the CHST6 coding region: 2 nonsense, 2 frameshift, and 10 missense. Of these, 12 were novel, and a nonsense mutation in the homozygous state is reported for the first time. CONCLUSIONS: These molecular results in French patients with MCD combined with those reported in previous studies indicated CHST6 mutational heterogeneity. The characterization herein of nonsense mutations is in keeping with the fact that MCD results from loss of function of the CHST6 protein product.     

A Case of Korean Patient with Macular Corneal Dystrophy Associated with Novel Mutation in the CHST6 Gene

To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.     

Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. 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.