Keratocan Research Articles

PAX6, FABP5 and keratocytes‐characteristic marker expression in human limbal fibroblasts and keratocytes of congenital aniridia and healthy subjects, in vitro

Aims/Purpose: To investigate PAX6, FABP5 and keratocyte‐characteristic marker expression in human limbal fibroblasts and keratocytes (LFCs and LKCs) of congenital aniridia (AN) and healthy corneas, in vitro.Methods: Primary human limbal fibroblasts (LFCs/ AN‐LFCs) were isolated from aniridia (n = 8) and healthy corneas (n = 8) and were cultured in DMEM/F12 with 5% fetal calf serum. Limbal keratocytes (LKCs/ AN‐LKCs) were cultured in low‐glucose serum‐free cell culture medium (LGSF‐medium). Stem cell marker PAX6 and ABCG2, retinoic acid marker FABP5 and keratocyte‐characteristic marker collagen I (COL1A1), collagen III (COL3A1), collagen V (COL5A1), lumican (LUM), keratocan (KER), α‐smooth muscle actin (ACTA2), CD34, aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) expression analysis has been performed by qPCR and Western blot.Results: PAX6 mRNA expression was significantly lower in AN‐LFCs and AN‐LKCs than in LFCs and LKCs (p = 0.04; p = 0.014). FABP5 mRNA expression was significantly higher in LKC and AN‐LKCs than in LFCs and AN‐LFCs (p = 0.036; p = 0.012) COL5A1 mRNA expression was significantly lower (p = 0.007), KER and ALDH3A1 mRNA expression was significantly higher (p = 0.013; p = 0.007) in AN‐LFCs than in LFCs. COL1A1 and COL5A1 mRNA expression was significantly higher in AN‐LKCs than in LKCs (p = 0.009; p = 0.001). Protein expression did not differ significantly between LFCs/ AN‐LFCs or LKCs/ AN‐LKCs for any of the analysed markers (p ≥ 0.05).Conclusions: Our study confirmed altered gene expression in aniridia limbal fibroblasts and keratocytes compared to healthy controls. These may play an important role in development and progression of aniridia associated keratopathy.

Early biological responses in ocular tissue after SMILE and LASIK surgery

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

IC3D second edition: an update

AbstractThe International Committee for Classification of Corneal Dystrophies (IC3D) was created by the Cornea Society in Chicago in 2005 to revise the corneal dystrophy nomenclature. The initial group consisted of 15 corneal specialists and 2 ophthalmic pathologists from North America, Europe and Asia, chaired by Dr. Jayne Weiss. The first IC3D edition was published in 2008. Already 6 years later, sufficient new data had become available to begin revision of the IC3D by 13 original and 2 new corneal specialists and 1 original ophthalmic pathologist. The second edition was published in 2015. Like the first one it presents each dystrophy as a template with key characteristics and typical clinical images, but is now supplemented with typical anterior segment OCT and histopathologic images as well. The category of ‘Bowman layer dystrophies’ was replaced by ‘Epithelial–stromal TGFBI dystrophies’ and ‘Descemet Membrane and Endothelial Dystrophies’ was simplified to ‘Endothelial Dystrophies’ because Bowman and Descemet are acellular and secondarily affected. Congenital Hereditary Endothelial Dystrophy 1 (CHED1) was merged with posterior polymorphous dystrophy 1 (PPMD1). Posterior amorphous corneal dystrophy (PACD) was found to be caused by a joint deletion of keratocan (KERA), lumican (LUM), decorin (DCN), and epiphycan (EPYC) genes. After publication of the second IC3D, epithelial recurrent erosion dystrophy (ERED) has been linked with COL17A1 mutations across the world, and the so called ‘variant Thiel‐Behnke dystrophy’, that was dubious, was found to be identical with ERED. The corneal dystrophy field continues to develop, making a third edition likely in the future.

Effects of low-dose of TGF-β1 on maintaining bovine corneal stromal cell growth and retarding extra cellular matrix fibrosis in a three-dimensional culture model

Background Transforming growth factor-β1 (TGF-β1) plays an important role in corneal wound healing.The effects of TGF-β1 on the synthesis of extra cellular matrix (ECM) vary upon different concentrations.Previous studies focused on the effects of high concentration of TGF-β1 on keratocytes under the two-dimensional culture condition, and the effect of low concentration of TGF-β1 on the synthesis of ECM in keratocytes remains unclear. Objective This study was to investigate the growth of Pellet, a three-dimensional model of corneal stroma cells in vitro, and its ECM synthesis under a low concentration of TGF-β1. Methods Bovine corneal stromal cells were isolated from fresh bovine eyeballs by two-step digestion by collagenase and cultured using DMEM/F12 medium with 10% fetal bovine serum (FBS). Pellets derived fresh bovine keratocytes with culture medium containing 0.25 ng/ml TGF-β1+ 5% FBS and 0.50 ng/ml TGF-β1+ 5% FBS were established, respectively.The morphology of Pellets was observed under the natural light at 48 hours, 1 week, 2 weeks and 3 weeks after culture.In 3 weeks after culture, the cell structures was observed by hematoxylin-eosin staining, and Calcein-AM/propidium (Calcein-AM/PI) staining was used to assay the cell viability.Real-time fluorescence quantitative PCR and immunofluorescence technology were applied to analyze the expressions of α-smooth muscle actin (α-SMA), fibronectin (FN), type Ⅰ collagen (ColⅠ) and type Ⅲ collagen (Col Ⅲ) mRNA and proteins.RT-PCR was employed to detect the expressions of lumican (LUM) mRNA and keratocan (KERA) mRNA in the cells. Results Cells in Pellet clustered throughout the culture duration.Hematoxylin-eosin staining showed the mass red-dyed collagen fibers in both 0.25 ng/ml TGF-β1+ 5% FBS group and 0.50 ng/ml TGF-β1+ 5% FBS group, and most cells possessed complete structures.The death rate of the cells was (33.60±1.65)% in the 0.25 ng/ml TGF-β1+ 5% FBS group and (30.90±0.78)% in the 0.50 ng/ml TGF-β1+ 5% FBS group, showing an insignificant difference between them (t=0.144, P=0.887). The expressions of α-SMA, FN and Col Ⅲ proteins in 0.25 ng/ml TGF-β1+ 5% FBS group were lower than those in the 0.50 ng/ml TGF-β1+ 5% FBS group (tα-SMA=4.622, P=0.010; tFN =2.973, P=0.040; t Col Ⅲ=7.845, P<0.001), but the expression of Col Ⅰ in 0.25 ng/ml TGF-β1+ 5% FBS group was higher than that in 0.50 ng/ml TGF-β1+ 5% FBS group (tColⅠ=4.022, P=0.016). The ratio of Col Ⅲ/ColⅠ in 0.25 ng/ml TGF-β1+ 5% FBS group was lower than that in the 0.50 ng/ml TGF-β1+ 5% FBS group in both mRNA and protein level (tmRNA =-3.039, P=0.038; tprotein=3.215, P=0.032). The expression of LUM mRNA and KERA mRNA were detected in Pellet at different time points.The expression of LUM mRNA in 0.25 ng/ml TGF-β1+ 5% FBS group increased over time.While in 0.50 ng/ml TGF-β1+ 5% FBS group, the expression of LUM mRNA peaked at 1 week but declined at 2 weeks.The expression of KERA mRNA in two groups were all peaked at 1 week but declined at 2 weeks. Conclusions Low-dose TGF-β1 in Pellet can maintain the normal growth of keratocytes and synthesize ECM.The expression of ECM tends to the normal condition after reducing the concentration of TGF-β1, implying a scarless expression. Key words: Corneal keratocyte; Transforming growth factor-β1; Three-dimensionl model; Bovine

A Novel KERA Mutation in a Case of Autosomal Recessive Cornea Plana With Primary Angle-Closure Glaucoma.

Keratocan is a cornea-specific keratan sulfate proteoglycan found predominantly in the adult vertebrate eye. In human beings, mutations in keratocan (KERA) are associated with autosomal recessive cornea plana (CNA2), which is characterized by a flattened forward convex curvature of the cornea. Here, we report a novel mutation in a case of autosomal recessive bilateral cornea plana presenting with primary angle-closure glaucoma in a 41-year-old woman from Eastern India. The KERA gene of the patient and her sons was directly sequenced. Mutational analysis of the KERA revealed 2 novel mutations. The first mutation was a 3 base-pair deletion (c.371_373delTCT), leading to the loss of a highly conserved amino acid (p.Phe125del). The second mutation was a base substitution resulting in a silent mutation (c.69G>A). One of her 2 sons carried the homozygous substitution (c.69G>A), whereas the other son was heterozygous (c.69G>R). The mutation that we report here leads to the deletion of a conserved amino acid (p.Phe125del) from the third LRR motif of the keratocan protein, which might lead to an abnormal tertiary structure of the protein, thereby leading to the disease.

Corneal strength change and keratocan expression of corneas during the healing of penetrating injury in rabbits

Background Corneal walleye formation is a result of excessive corneal wound healing. Controlling the corneal trauma or excessive healing reaction after operation of penetrating corneal trauma is a focus in the study of corneal wound healing. Objective This study was to investigate the changes of corneal strength and the expression of keratocan, a marker of corneal stroma, after corneal penetrating injury operation, and to determine the optimal removal time of corneal suture. Methods Full-thickness incisions of 5 mm along the vertical diameter were done at the central cornea on 80 eyes of 80 six-month-old New Zealand rabbits in this study. Then the incisions were interruptedly sutured with 10-0 nylon thread to establish the corneal wound healing models. Each 4 rabbits were sacrificed in 1 week, 2, 3, 4, 5, 6, 7, 8 weeks after operation, respectively, and the central corneal stripes were prepared with the size of 7 mm×5 mm. The mean maximal strength of the corneal bands was measured by uniaxial tensile test with electroforce3220-AT biomachanics machine. Then, each 6 whole corneas were obtained at the above time points, and the dynamic changes of keratocan mRNA expression in the specimens were detected by reverse trancription PCR (RT-PCR). The care and use of the animals followed the rules of ARVO. Results The mean maximal strength of corneal stripes was 0, (1.007±0.041), (1.991±0.034), (2.512±0.030), (3.630±0.049) and (4.935±0.004) MPa in 1 week, 2, 3, 4, 5, 6 weeks after modeling, and the corneal strength values from 3 weeks through 6 weeks were significantly enhanced in comparison with the value at the adjacent before timepoint (q=6.35, 7.54, 8.21, 5.86, all at P<0.01). The relative expression levels (absorbance) of keratocan mRNA in the corneas were 0.869±0.015, 0.779±0.065, 0.621±0.027, 0.460±0.018, 0.393±0.057 and 0.255±0.045 in 1 week, 2, 3, 4, 5, 6 weeks after operation, and each value was lower than that of the adjacent before timepoint (q=5.24, 5.61, 7.49, 4.75, 5.47, all at P<0.01). The intensity of corneal stripes and the expression levels of keratocan mRNA in the corneas were stable in 7 weeks and 8 weeks after operation. Conclusions The dynamic change of corneal strength during the repair of penetrating corneal injury is associated with the down-regulation of keratocan in cornea. Rabbit cornea reaches a maximal strength capacity in 6 weeks after penetrating injury, therefore, it is the optimal time to remove suture. Key words: Ocular trauma; Cornea/physiopathology; Biomechanics; Corneal stroma/cytology; Fibrosis; Reverse transcriptase PCR

Differentiation of human embryonic stem cells into cells with corneal keratocyte phenotype.

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.

Evaluation of Proteoglycan Gene Polymorphisms as Risk Factors in the Genetic Susceptibility to High Myopia

PURPOSE. To investigate the relationship between high myopia and single nucleotide polymorphisms (SNPs) in six proteoglycan genes: aggrecan (ACAN), fibromodulin (FMOD), decorin (DCN), lumican (LUM), keratocan (KERA), and epiphycan (EPYC). These genes were selected for study because they are involved in induced myopia in animals and/or are within the human MYP3 locus identified by linkage analysis of families with high myopia. METHODS. Two groups of Chinese subjects were studied: group 1 (300 cases and 300 controls) and group 2 (356 cases and 354 controls). Cases were high myopes with spherical equivalent (SE) ≤ -8.00 D, and controls had SE between +1.0 and -1.0 D. From these candidate genes, 60 tagging SNPs were selected. First, 12 DNA pools were each constructed from 50 samples of the same phenotype from group 1 subjects and were tested for association with the SNPs. Second, putatively positive SNPs were confirmed by individual genotyping of group 1 subjects. Finally, positive results were replicated in group 2 subjects. RESULTS. Of the 58 SNPs successfully screened by DNA pooling, 8 ACAN SNPs passed the threshold of P ≤ 0.10 (nested ANOVA) and were then genotyped in the individual samples. Haplotypes rs3784757 and rs1516794 showed significant association with high myopia. However, the positive result could not be replicated in the second subject group. CONCLUSIONS. These six proteoglycan genes were not associated with high myopia in these Chinese subjects and hence are unlikely to be important in the genetic predisposition to high myopia.

Inhibitory effect of curcumin on corneal keratocytes fibrosis

Background The injury or surgery of cornea cause the proliferation of corneal stromal cells and scar formation.Recent research showed that cureumin can obviously reduce the degree of fibrosis of tissue.But if curcumm play inhibitory effect on corneal keratocytes fibrosis is rarely reported.Objecttve This studv was to investigate the effect of curcumin on the transformation of corneal keratocytes into fibroblasts in vitro and further explore the antifibrotic effect of curcumin on corneal keratocytes.Methods The murine corneal keratocytes from 150 BALB/c mice were isolated and primary culture in DMEM culture medium containing 10% fetal bovine serum and then divided into blank control group(inducer group,CG),low-dose group(CG+7.5 mg/L curcumin),mediumdose group(CG+10.0 mg/L curcumin),high-dose group(CG+12.5 mg/L curcumin),non-inducer group.Seven days following intervention,the expression of cell markers such as keratocan,aldehyde dehydrogenase(ALDH),decorin and fibronectin-1 in keratocytes were analyzed by RT-PCR.The effect of curcumin on cultured murine corneal keratocytes proliferation was evaluated by MTS technique.The expression of fibronectin-1 in murine cornea was investigated by immunofluorescence assay.Results The primarily cultured keratocytes showed tlIe fusiform-like shape with the abundant cytoplasm and big nuclei.In the presence of curcumin,the mRNA levels of keratocan and ALDH were down-regulated and those of CD90 and decorin were up-regulated,showing the significantly differences with the increase of dose(P<0.05),but the expression pf fibronectin-i was not obviously changed with the alteration of dose of curcumin. MTS showed that the inhibitory rates of curcumin on keratocytes in 10.0 mg/L and 2. 5 mg/L groups were enhanced in comparison with 7.5 mg/L group, showing statistically significant difference among three groups( F = 956.00, P<0.05). The expression of fibronectin-1 was found in the corneal keratocytes with the red fluorescence in stroma. Conclusion Curcumin can inhibit the fibrosis of corneal keratoeytes in a dose-dependent manner. These results offer a preliminary theoretical basis for the application of curcumin in controlling corneal scar formation during wound healing. Key words: Curcumin; Keratocyte; Fibroblast; Fibrosis

Reconstruction of rabbit corneal stroma with skin fibroblasts

To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. It was an experimental study. Skin fibroblast cells were isolated from newborn rabbits, cultured and expanded in vitro. Cells were labeled with green fluorescence protein (GFP) gene by retro-viral infection. Fibroblasts at passage 3 were seeded on polyglycolic acid (PGA) non-woven fibers to form a cell-scaffold construct. Constructs were then implanted into the adult rabbit corneal stroma layer after being cultured in vitro for 1 week. Engineered stroma were observed continuously and harvested after 8 weeks of transplantation for gross, histological evaluation and Keratocan examination. PGA alone was used as control. The engineered tissue in the cornea became transparent gradually over a period of 8 weeks. Histological analysis showed that engineered stromal lamellar was relatively regular and the orientation of fibers was parallel to the surface of cornea, which is similar to normal cornea. The implanted cells were confirmed by GFP expression under fluorescent microscope, which also express Keratocan. By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between engineered stroma (33.08 + or - 2.47) nm and normal stroma (t = 1.80, P = 0.0771). Skin fibroblast cells could be used as seed cells for reconstruction of the corneal stroma.

Immunolocalisation and expression of keratocan in tendon

Short Communication Immunolocalisation and expression of keratocan in tendon S. G. Rees Ph.D.y*, A. D. Waggett Ph.D.z, B. C. Kerr Ph.D.z, J. Probert B.Sc.z, E. C. Gealy Ph.D.z, C. M. Dent M.D.x, B. Caterson Ph.D.z and C. E. Hughes Ph.D.z ySchool of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, UK zConnective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, UK xSchool of Medicine, Department of Orthopaedic and Trauma Surgery, Cardiff University, Llandough Hospital, Penlan Road, Penarth, Vale of Glamorgan, CF64 2XX, UK

Autosomal Dominant Cornea Plana is not Associated with Pathogenic Mutations in DCN, DSPG3, FOXC1, KERA, LUM, or PITX2

Purpose: To determine the genetic basis of autosomal dominant cornea plana (CNA1) through the performance of a genome-wide linkage analysis and screening of the decorin (DCN), dermatan sulfate proteoglycan 3 (DSPG3), forkhead box C1 (FOXC1), keratocan (KERA), lumican (LUM,) and paired-like homeodomain transcription factor 2 (PITX2) genes in members of an affected multigenerational family. Methods: Cycloplegic refraction, slit lamp biomicroscopy, corneal pachymetry, and corneal topography were performed to determine each patient's affected status. DNA was obtained from affected and unaffected subjects for the performance of a genome-wide linkage analysis as well as PCR amplification and sequencing of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2. Results: Five affected and three unaffected individuals were examined and provided a peripheral blood sample for DNA isolation. All affected individuals demonstrated an average corneal dioptric power less than 39 D, as well as one or more of the following anomalies: high hyperopia, strabismus, microcornea, posterior embryotoxon, iridocorneal adhesions, iris atrophy, and pupillary irregularities. A genome-wide linkage analysis did not indicate or exclude linkage to the region on chromosome 12 to which CNA1 has been previously mapped, and did not provide a single or multipoint LOD score greater than 2.0 for any of the 400 microsatellite markers. Screening of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2 revealed 12 previously described single nucleotide polymorphisms, 2 previously described duplications, and 1 previously described insertion. None of the mutations previously associated with autosomal recessive cornea plana (CNA2) were identified. Seven novel sequence variants were described, including 5 single nucleotide substitutions, 1 insertion and 1 deletion. None of the identified sequence variants demonstrated complete segregation with the affected phenotype in the pedigree. Conclusion: Although missense and nonsense mutations in KERA are associated with CNA2, we did not identify any of the previously described mutations or novel mutations that segregated with the disease phenotype in a family with CNA1. In addition, no pathogenic sequence variations were found in DCN, DSPG3, LUM, PITX2 and FOXC1, which have also been implicated in corneal and anterior segment dysgenesis.

Mutations in KERA, encoding keratocan, cause cornea plana.

Specialized collagens and small leucine-rich proteoglycans (SLRPs) interact to produce the transparent corneal structure. In cornea plana, the forward convex curvature is flattened, leading to a decrease in refraction. A more severe, recessively inherited form (CNA2; MIM 217300) and a milder, dominantly inherited form (CNA1; MIM 121400) exist. CNA2 is a rare disorder with a worldwide distribution, but a high prevalence in the Finnish population. The gene mutated in CNA2 was assigned by linkage analysis to 12q (refs 4, 5), where there is a cluster of several SLRP genes. We cloned two additional SLRP genes highly expressed in cornea: KERA (encoding keratocan) in 12q and OGN (encoding osteoglycin) in 9q. Here we report mutations in KERA in 47 CNA2 patients: 46 Finnish patients are homozygous for a founder missense mutation, leading to the substitution of a highly conserved amino acid; and one American patient is homozygous for a mutation leading to a premature stop codon that truncates the KERA protein. Our data establish that mutations in KERA cause CNA2. CNA1 patients had no mutations in these proteoglycan genes.