Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.     

Molecular genetics of Meesmann's corneal dystrophy: ancestral and novel mutations in keratin 12 (K12) and complete sequence of the human KRT12 gene

Recently, we identified the first mutations in corneal keratins K3 and K12 in families with Meesmann's corneal dystrophy (MCD). Here, we sequenced all regions of the human K12 gene, to enable mutation detection for all exons using genomic DNA as a template. The human K12 genomic sequence spans 5919 bp and consists of eight exons. A microsatellite dinucleotide repeat was identified within intron 3, which was highly polymorphic and which we developed for use in genotype analysis. In addition, two mutations in the helix initiation motif of K12 were found in families with MCD. A novel mutation was detected in an American kindred, 410T-->C, which predicts the amino acid substitution M129T. In a German family, mutation 428G-->C was identified, predicting amino acid change R135T. The latter mutation was identical to that which we identified in the original kindred described by Meesmann. Using the intragenic microsatellite polymorphism in K12 and additional flanking markers, we were able to show that this family shares a common haplotype with the original Meesmann kindred. These results strongly imply that R135T represents an ancestral mutation in the German population. Both mutations occur in the highly conserved helix initiation motif of the K12 polypeptide. A total of eight mutations have now been reported in the K12 gene.     

Corneal wound healing in an osteopontin-deficient mouse

PURPOSE: To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS: Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS: In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor beta1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS: OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing.     

Corneal wound healing in tenascin knockout mouse

PURPOSE: Tenascin (TN) is a large hexameric extracellular matrix glycoprotein that is expressed in developing organs and tumors. It has also been reported that TN is expressed in the embryonic cornea and during corneal wound healing. However, the role of TN in the cornea is not fully known. In this study, the role of TN in corneal wound healing was examined using the TN knockout (KO) mouse. METHODS: Two different injuries (a linear perforation wound and two 10-0 nylon suture wounds) were made separately on the corneas of both TNKO and congenic wild-type mice. The corneal wound healing was compared histologically, and the expression of TN and fibronectin (FN) on the injured cornea was examined immunohistochemically and by immunoblot analysis. RESULTS: Based on histologic analysis, there was no significant difference in the wound healing process between wild-type and TNKO mice in the linear incision experiment. However, the corneal stromata of TNKO mice were compressed prominently and devoid of migrating keratocytes in suture injury, which induced a more significant amount of TN than perforation wounds. Although FN expression on the sutured corneas of TNKO mice was upregulated during suture injury, the amount of FN protein was smaller than that of wild-type mice at the same time points after injury. CONCLUSIONS: In suture wounds, TN appears to enhance the amount of FN expression, and a lack of TN may impair stromal cell migration. TN plays a significant role in corneal wound healing, especially for wounds with mechanical stress.     

A novel mutation in the helix termination motif of keratin K12 in a US family with Meesmann corneal dystrophy

PURPOSE: Meesmann corneal dystrophy is an autosomal dominant disorder characterized by fragility of the anterior corneal epithelium. We have previously demonstrated that this disease can be caused by mutations in the genes encoding keratins K3 or K12, the major intermediate filament proteins expressed in corneal epithelial cells. Here, we have carried out mutation analysis in a United States kindred presenting with typical features of Meesmann corneal dystrophy. METHODS: Exons 1 and 6 of the K12 gene (KRT12) were polymerase chain reaction amplified from the proband's and control DNA and subjected to direct automated sequencing. RESULTS: A heterozygous missense mutation 1300A-->G was detected in exon 6 of KRT12, predicting amino acid substitution 1426V in the helix termination motif of the K12 polypeptide. The mutation was confirmed in the proband and excluded from 50 normal individuals by restriction enzyme analysis of polymerase chain reaction products. CONCLUSION: We report a novel mutation in a critical molecular overlap region of K12 in a United States family with Meesmann corneal dystrophy. The results confirm that mutations in the corneal keratins (K3 or K12) can underlie Meesmann corneal dystrophy.     

简化K液保存角膜的动物实验

用未除去小分子的纯化硫酸软骨素(Chondroitin Sulfate,CS)配成简化K 液,(SimplifiedK-Solution,SK 液),保存兔角膜。对保存后的角膜和用此角膜作角膜移植手术后的角膜植片进行电镜和角膜厚度观察。以MK 液作对照。结果表明,术后短期内SK 液组植片的水肿消退较MK 液组者快,内皮细胞密度也较高。说明SK 液保存的角膜在移植手术后其功能恢复较MK 液者快。     

Corneal parameters of six- to 12-year-old Chinese children

Purpose: The aim was to determine the corneal parameters in low to moderate myopic Chinese children and to investigate the differences in these corneal parameters between male and female subjects. Methods: Refractive errors and corneal parameters were retrieved from subjects who had participated in studies on myopia and astigmatism in 2008 and 2009. Corneal parameters including simulated K (Sim K) and asphericity (Q) at 9.0 mm chord and horizontal visible iris diameter (HVID) were determined for 217 children (112 males and 105 females aged from six to 12 years) using the Medmont E300 topographer. Results: The Q of the corneas was ‐0.44 ± 0.12 along the flat meridian and ‐0.22 (‐0.70 to 0.63) along the steep meridian. The HVID and Sim K of male subjects were larger and flatter than female subjects. The mean HVID of male and female subjects was 11.3 ± 0.3 mm and 11.1 ± 0.30 mm, respectively. The mean Sim K value in male and female subjects was 7.91 ± 0.24 mm and 7.79 ± 0.21 mm, respectively, along the flat meridian, and was 7.65 ± 0.26 mm and 7.53 ± 0.24 mm, respectively, along the steep meridian. Conclusions: All corneas were prolate elliptical in shape along the flat meridian and the peripheral flattening rate was found to be greater along the flat meridian. A greater flattening rate was observed in the corneas of Chinese children when compared to that of Caucasian children. The Q between male and female subjects was not significantly different. Male subjects tended to have larger HVID and flatter corneas than female subjects.     

Novel mutations in the helix termination motif of keratin 3 and keratin 12 in 2 Taiwanese families with Meesmann corneal dystrophy

PURPOSE: To analyze mutations of the keratin 3 gene (KRT3) and keratin 12 gene (KRT12) in 2 Taiwanese families with Meesmann corneal dystrophy (MCD). METHODS: Diagnosis of MCD was confirmed by slit-lamp examination of the cornea in 4 members of family 1 and 6 members of family 2. All exons and flanking intron boundaries of KRT3 and KRT12 were amplified by polymerase chain reaction (PCR), and products were subjected to direct sequencing. Restriction fragment length polymorphism analysis (RFLP) with created mismatch primers, Bst XI and Nsp I, was used to confirm the presence of the mutations in affected individuals in family 1 and family 2, respectively. RESULTS: A novel heterozygous missense mutation (1508G-->C), predicting the substitution of a proline for an arginine (R503P) was detected in the helix termination motif of the keratin 3 polypeptide in family 1. Another novel heterozygous missense mutation (1286A-->G), predicting the substitution of a cysteine for a tyrosine at codon 429 (Y429C) was detected in the helix termination motif of the keratin 12 polypeptide in family 2. These 2 mutations were excluded from 50 normal controls by RFLP analysis, indicating that they were not common polymorphisms. CONCLUSIONS: A novel missense mutation (R503P) in KRT3 and another novel missense mutation (Y429C) in KRT12 lead to MCD in 2 unrelated Taiwanese families. The mutant codons in our study are all located in the highly conserved alpha-helix-termination motif, which is essential for keratin filament assembly. Mutation at this area may account for the disruption of keratin filament assembly, leading to MCD.     

Corneal disorders in KKAy mouse: a type 2 diabetes model

PURPOSE: To observe the clinical and histopathological changes occurring in corneas of KKAy mice, a model of type 2 diabetes, and to elucidate the possible mechanisms involved in these changes. METHODS: Corneal epithelial cell proliferation was analyzed in KKAy and age-matched non-diabetic C57BL/6J control mice using (3)H-thymidine autoradiography. Clinical examination and histopathological analysis were also conducted on both types of mice. RESULTS: KKAy mice showed a significant elevation in blood glucose concentration and body weight compared to age-matched control mice. Fragile corneal epithelial cell attachment and subepithelial opacities were observed in the central area of the cornea of 10-week-old KKAy mice. Corneal epithelial cell proliferation decreased significantly in the 16-week-old KKAy mice. Histological study in the older KKAy mice groups revealed the presence of subepithelial deposits, widening of the intracellular spaces between corneal epithelial cells with poor adherence to the basement membrane (BM) and thickening of the BM itself. At the central area of the cornea, remnants of cell components with deposits and lacuna formation were observed, perhaps secondary to the continuous presence of poor adhesion and detachment of epithelial cells in the area. In the 50-week and older KKAy mice, thinning and atrophy of the corneal epithelial cell layer became more prominent at the central cornea with increases in deposition of materials, blood vessel invasion and activation of keratocytes. The deposits were stained black by von Kossa's method, indicating the presence of tissue calcium. Type IV collagen immunoreactivity was observed not only in the corneal and conjunctival BM but also between the stroma, particularly around the central cornea and in the walls of invading vessels. Laminin staining was intense at the BM around the central cornea, and in the walls of invading vessels along the stroma. Pyrraline, which is one of the major components of advanced glycation end products, was also present in the stroma, and around blood vessels. All these corneal changes were not observed with aging in the age-matched C57BL/6J mice. CONCLUSIONS: Our findings provide evidence of the existence of corneal disorders in KKAy mice. These observations may provide useful information for the explanation of the mechanisms involved in corneal disorders in non-insulin-dependent diabetes mellitus patients.     

A novel mutation in the cornea-specific keratin 12 gene in Meesmann corneal dystrophy

PURPOSE: To report a novel mutation in the keratin 12 gene (KRT12) found in a Japanese family in association with Meesmann corneal dystrophy (MECD). METHODS: After informed consent was obtained, genomic DNA was extracted from the leukocytes of the peripheral blood of the proband, her affected father, normal mother, and 50 normal unrelated volunteers. Exons 1-8 of the KRT12 gene were amplified by polymerase chain reaction and directly sequenced. RESULTS: A novel heterozygous T to G transversion at the second nucleotide position of codon 433 (CTG > CGG), resulting in the replacement of leucine by arginine at codon 433 of the KRT12 gene (L433R), was detected in the proband and her affected father but not in her normal mother or the 50 controls. CONCLUSIONS: The novel L433R mutation of the KRT12 gene found in two members of this Japanese family caused MECD.     

A novel keratin 12 mutation in a German kindred with Meesmann's corneal dystrophy

AIM: To study a kindred with Meesmann's corneal dystrophy (MCD) to determine if a mutation within the cornea specific K3 or K12 genes is responsible for the disease phenotype. METHODS: Slit lamp examination of the cornea in four members of the kindred was carried out to confirm the diagnosis of MCD. The region encoding the helix initiation motif (HIM) of the K12 polypeptide was polymerase chain reaction (PCR) amplified from genomic DNA derived from affected individuals in the kindred. PCR products generated were subjected to direct automated sequencing. Restriction enzyme analysis employing Ban I was used to confirm the presence of the mutation in affected individuals of the family. RESULTS: Sequencing of the K12 gene in an affected individual from the family revealed a novel heterozygous missense mutation (413A-->C), predicting the substitution of a proline for a glutamine at codon 130 (Q130P) in the HIM of the K12 protein. The mutation was excluded from 50 normal, unaffected individuals by restriction enyzme analysis and was therefore unlikely to be a common polymorphism. CONCLUSION: A novel missense mutation in the K12 gene leads to MCD in a German kindred. Missense mutations have now been identified within the region encoding the helix initiation motif of the K12 protein in eight of 11 MCD kindreds analysed at the molecular level.     

Corneal epithelial proliferation and thickness in a mouse model of dry eye

Although several studies have previously focused on the conjunctival epithelial response to surface dryness, little is known about the effect of a dry environment on corneal epithelium, which is the most clinically significant tissue affected in dry eye. The aim of this study was to quantitatively evaluate the effect of desiccating stress on the number of proliferating corneal epithelial cells and corneal epithelial thickness in mice placed in a controlled-environment chamber (CEC) that induces dry eye. Corneal epithelial cell proliferation and thickness were studied in 8- to 12-week-old female BALB/c mice placed in the CEC (temperature: 22.3 ± 0.7 °C; relative humidity: 22.5 ± 4.5%; airflow: 15 L/min) for 7 days and compared to a control group of mice with no dry eye. Actively proliferating cells were identified by immunofluorescence using a FITC-conjugated antibody against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle. To detect the spatial distribution of proliferative cells, Ki-67⁺ cells were counted in three areas of the epithelium: center, periphery, and limbus. Corneal epithelial thickness was evaluated in the central cornea after staining with hematoxylin-eosin. Results from each experimental group were compared using the Mann-Whitney test. The number of Ki-67⁺ cells observed in the corneal epithelium of mice exposed to the CEC was significantly higher in each area (center: 32.1 ± 1.1; periphery: 94.2 ± 5.3; limbus: 4.0 ± 1.5) than in the control group (center: 13.2 ± 1.0, p = 0.02; periphery: 42.9 ± 2.3, p = 0.02; limbus: 0.0, p = 0.01). In mice subjected to desiccating stress, a significant number of Ki-67⁺ positive cells were detected in the basal and suprabasal cell layers (central area 46%; periphery 30.8%: limbus 0%), whereas in the control group the cells were exclusively distributed through the basal cell layer. Ki-67⁺ cells were not found in the corneal stroma or endothelium in any group. The corneal epithelium was found to be significantly thicker in dry eye mice (54.94 ± 6.09 μm) as compared to the controls (43.9 ± 6.23 μm, p < 0.0001) by a mean of 25%. These results demonstrate that desiccating stress increases corneal epithelial turnover and thickness, similar to what is observed in other chronic inflammatory states of other epithelialized surfaces. The CEC can facilitate the study of the regulation of epithelial cell function and turnover at the molecular and cellular levels under desiccating stress conditions.     

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (相似文献   

Localization of keratin in the cells of the cornea in aphakia and normal mouse embryos

Abnormal lens morphogenesis in the aphakia mutant in the mouse often results in a club-shaped elongated 'lens' that remains attached to the surface epithelium by a persistent connecting stalk, which is partially solid and partially cystic. Usually, the cells are continuous with the surface epithelium of the cornea and also with the cuboidal cells lining the corneal inner surface. Immunofluorescence with keratin antiserum not only gave positive reactions with the corneal epithelial cells, but also with many cells of the lens stalk, including its cysts, and with islands of cells on the inside of the cornea. These keratin-containing 'endothelial' cells may be the product of metaplasia of the endothelium into epithelium-like cells. Alternatively, they may also be the result of abnormal migration of epithelial cells into the eye or of abnormal differentiation of neural crest cells.     

Heterozygous Ala137Pro mutation in keratin 12 gene found in Japanese with Meesmann's corneal dystrophy

PURPOSE: To report the molecular genetic analysis of a Japanese pedigree with Meesmann's corneal dystrophy (MCD). METHODS: Sequencing of the keratin 3 and keratin 12 genes was performed in 2 patients who were siblings and in an unaffected individual in the same family. The patients had the typical corneal microcysts and recurrent erosions with mild photophobia. RESULTS: A novel mutation resulting in the substitution of alanine to proline in codon 137 of the keratin 12 gene (Ala137Pro) was found in the 2 patients, but not in the unaffected member of the family and the 50 controls. CONCLUSIONS: This novel mutation (Ala137Pro) of the keratin 12 gene found in a Japanese family had caused MCD.     

Severe Meesmann's epithelial corneal dystrophy phenotype due to a missense mutation in the helix-initiation motif of keratin 12

Purpose

To describe a severe phenotype of Meesmann''s epithelial corneal dystrophy (MECD) and to determine the underlying molecular cause.

Methods

We identified a 30-member family affected by MECD and examined 11 of the 14 affected individuals. Excised corneal tissue from one affected individual was examined histologically. We used PCR and direct sequencing to identify mutation of the coding regions of the KRT3 and KRT12 genes.

Results

Cases had an unusually severe phenotype with large numbers of intraepithelial cysts present from infancy and they developed subepithelial fibrosis in the second to third decade. In some individuals, the cornea became superficially vascularized, a change accompanied by the loss of clinically obvious epithelial cysts. Visual loss from amblyopia or corneal opacity was common and four individuals were visually impaired (≤6/24 bilaterally) and one was blind (<6/60 bilaterally). In all affected family members, there was a heterozygous missense mutation c. 395T>C (p. L132P) in exon 1 of the KRT12 gene, which codes for the helix-initiation motif of the K12 polypeptide. This sequence change was not found in unaffected family members or in 100 unaffected controls.

Conclusions

The Leu132Pro missense mutation is within the helix-initiation motif of the keratin and is predicted to result in a significant structural change of the K12 protein. The clinical effects are markedly more severe than the phenotype usually associated with the Arg135Thr mutation within this motif, most frequently seen in European patients with MECD.