Physiological expression of lens α-, β-, and γ-crystallins in murine and human corneas.
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Abstract:
How corneal transparency is formed/maintained remains largely unclear. A group of enzymes which are referred to as enzymatic crystallins were proposed to contribute to corneal transparency in various animals. This study investigated whether the three classical lens crystallins, namely α-, β-, and γ-crystallins, exist in mouse and human corneas.Mice, human tissues, and cultured corneal cells were studied. The expression of lens crystallins in corneas or in cultured corneal cells were detected at the mRNA level by quantitative reverse transcription-PCR (QRT-PCR) and at the protein level by immunohistochemistry or western blotting. To check the effect of exogenous factor on expression of lens crystallins, cultured corneal cells were challenged with lipopolysaccharide or hydrogen peroxide and the expression of lens crystallins was monitored.QRT-PCR revealed that the relative expression level of lens crystallins in C57BL/6 corneas were higher than in Balb/c corneas. Immunohistochemistry study showed that expression of αA-crystallin started from the embryonic stage, lasted untill old age, and was largely restricted to the epithelium or endothelium of the corneas. β- and γ-crystallins also were found in murine corneal epithelium. Upon treatment with lipopolysaccharide or hydrogen peroxide of cultured corneal epithelial cells, lens crystallins expression was significantly increased as detected by QRT-PCR or western blot assay. Further, both fetal corneal epithelial cultures and limbal stem cell cultures from adult human tissues were positive for lens crystallin immunofluorescence or immunohistochemistry staining.Lens crystallins are expressed in mammalian corneas and cultured corneal cells. The expression levels depended on the animal strains or cell status. The physiologic and pathological significance of lens crystallins in corneas deserves more investigation.Keywords:
Crystallin
Corneal Endothelium
Immunofluorescence
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It is unknown why human corneal endothelium exhibits limited capacity to divide while the endothelia of other species, such as rabbit, divide in vivo at wounding and in culture. A potentially valuable source of information concerning why human endothelium has such a limited proliferative capacity lies in elucidating any differences in the molecular events governing the cell cycle of these two species. A recent study of the relative expression of cell cycle-associated proteins in donor corneas suggests that human corneal endothelial cells in vivo have not exited the cell cycle but are arrested in G1-phase. The purpose of the current study was to identify differences in cell cycle protein expression in human and rabbit endothelium that would explain the difference in their relative proliferative capacities. Specifically, the authors first ascertained the relative proliferative status of rabbit corneal endothelial cells in vivo. The expression and intracellular distribution of G1-phase regulatory proteins was then determined in both species, and the results were compared.Corneas from New Zealand white rabbits (weight range, 2 to 3 kg) and from human donors (age range, 6 months to 67 years) were fresh frozen, cryostat sectioned, and prepared for indirect immunofluorescence microscopy using an established protocol. The following monoclonal antibodies were localized in rabbit corneal endothelium only: cyclins D, E, A, and B1; protein kinase p34cdc2; and Ki67, a marker of actively cycling cells. Localization patterns for the following G1-phase regulatory proteins were compared in both human and rabbit corneal endothelia: the tumor suppressors, pRb, p53, and p16INK4, and the transcription factor, E2F. Reverse transcription-polymerase chain reaction studies were conducted to detect mRNA for Ki67 in human and rabbit corneal cells.Cyclins D, E, and A were localized in the cytoplasm of rabbit corneal endothelium, whereas cyclins B1 and p34cdc2 were detected in the nucleus. No Ki67 protein or mRNA expression was detected in the endothelium of either species. In human and rabbit endothelia, p53 and p16INK4 were localized to the cytoplasm, whereas pRb was detected in the nucleus. E2F exhibited a nuclear and a cytoplasmic localization in each species.The corneal endothelium of rabbits stained positively for cyclins D, E, and A and did not stain for Ki67, suggesting that, as in humans, rabbit corneal endothelium in vivo is arrested in G1-phase upstream from Ki67 synthesis. Cyclin E was located in the cytoplasm of rabbit cells, whereas it was found in the nucleus in human endothelium. The apparent difference in cellular distribution of cyclin E in these two species may be significant because this cyclin is active during the G1-/S-phase transition. It is possible that in situ human and rabbit corneal endothelial cells are arrested at different points within G1-phase and/or that the difference in relative proliferative capacity exhibited by the corneal endothelium in these two species may be caused by differences in their relative ability to overcome G1-phase arrest.
Corneal Endothelium
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Chloride channel
Amniotic epithelial cells
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BACKGROUND:To investigate associations of senescence marker protein-30 and senescence-associated β-galactosidase expression with lens epithelial cells apoptosis among Chinese age-related cataract patients. MATERIAL AND METHODS:A total of 145 age-related cataract patients (69 cases with nuclear cataract in 91 eyes and 76 cases of cortical cataract with 102 eyes) were enrolled in our study. An annular tear of the central part of anterior lens capsules was performed for each patient. Immunohistochemical staining and real-time PCR were used to detect the protein and mRNA expression levels, and TUNEL was used to assess lens epithelial cells apoptosis. Comparisons of protein expression levels and lens epithelial cells apoptosis were made between the 2 groups. RESULTS:The results showed a higher protein expression level of senescence marker protein-30 in surrounding parts of the anterior lens capsule compared with the central part of the anterior lens capsule; however, the positive rate of senescence-associated β-galactosidase was remarkably higher in the central part than in the surrounding part. Compared with cortical cataract patients, nuclear cataract patients had elevated senescence marker protein-30 protein and mRNA expression levels, but had a decreased positive rate of senescence-associated β-galactosidase. TUNEL results showed that the lens epithelial cell apoptosis rate was higher in the central part of the anterior lens capsule than in the surrounding part in both groups. Within either central or surrounding area of anterior lens capsule, cortical cataract patients exhibited a significantly higher lens epithelial cell apoptosis rate in contrast with nuclear cataract patients. CONCLUSIONS:Our study results suggest that senescence marker protein-30 and senescence-associated β-galactosidase expressions in both nuclear cataract and cortical cataract patients were associated with lens epithelial cells apoptosis.
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To elucidate the role of perlecan (Hspg2), a large multidomain heparan sulfate proteoglycan expressed in the basement membrane, in the structure of the corneal epithelium.A previously developed perlecan-deficient (Hspg2⁻/⁻-Tg) mouse model was used. Histologic analysis of their corneas was performed by light and transmission electron microscopy. The localization of perlecan in the corneas of wild-type (WT) mice and Hspg2⁻/⁻-Tg mice was examined by immunohistochemistry. The effects of perlecan deficiency on corneal epithelial structure was analyzed with respect to the expression of corneal epithelial proliferation and differentiation markers, such as Ki67, cytokeratin12 (K12), connexin43 (Cx43), Notch1, and Pax6 by immunohistochemistry and real-time polymerase chain reaction (PCR).The Hspg2⁻/⁻-Tg mice had microphthalmos and a thinner corneal epithelium compared with that of the WT mice. Perlecan was localized in the corneal epithelial basement membrane in the WT mice, but not in the Hspg2⁻/⁻-Tg mice. The Hspg2⁻/⁻-Tg corneal epithelium exhibited thinner wing cell layers and a decreased number of Ki67-positive cells, but no dead cells, compared with the WT corneal epithelium. Immunohistochemistry and real-time PCR analysis revealed a significantly decreased expression of corneal epithelial differentiation markers such as K12, Cx43, Notch1, and Pax6 in Hspg2⁻/⁻-Tg mice, compared with those of the WT mice.The findings of this study highlight a strong correlation between the presence of perlecan in the basement membrane and the structure of corneal epithelium and that the perlecan-deficient mutation impairs corneal epithelial structure.
Perlecan
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Northern blot
Sclera
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PURPOSE. Previous studies have shown that transketolase is preferentially expressed in the corneal epithelium and comprises up to 10% of the soluble protein of the mature mouse cornea. The aim of this study is to evaluate the expression and distribution of TKT in the different ocular tissues. METHODS. We have used in situ hybridization and immunohistochemistry to localize TKT mRNA and protein in the developing and adult mouse eye. RESULTS. TKT were found to be widely distributed throughout the adult mouse eye. Among the ocular tissues examined, the corneal epithelium exhibited the highest levels of TKT mRNA and protein. Within the epithelial layer, TKT mRNA and protein were differentially distributed with the highest expression occurring in basal cells and the lowest in apical cells, suggesting that TKT expression in the corneal epithelium may be differentiation-related. Enriched expression of TKT was also found in the cornea endothelium, lens epithelium, ciliary body, and iris. Low basal levels of expression were observed in the limbus and conjunctiva. In contrast to the adult eye, TKT expression in the one-day-old mouse eye was homogeneous at low, but detectable levels, suggesting that TKT expression is developmentally regulated in the cornea as well as in the other ocular tissues. In the healing corneal epithelium, TKT expression in the single cell layer of the leading edge was completely suppressed until the cells began to stratify, at which point TKT expression increased markedly. CONCLUSIONS. The results presented here suggest that TKT is differentially expressed and developmentally regulated in the various tissues that comprise the eye.
Basal (medicine)
Transketolase
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Crystallin
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Background Peroxisome proliferator-activated receptor gamma (PPARγ) is one of nuclear transcription factors.It plays potential anti-inflammation,anti-fibrogenesis,anti-angiogenesis and neuroprotection roles in human.So the study of its physiological and pathological function in human and animals is still a focus.To understand the distribution of PPARγ in ocular tissues is important for the target treatment of eye diseases.Objective Current study was to investigate the expression of PPARγ in different parts of eye in rodent.Methods Cornea,lens,ciliary,retina and optical nerve were isolated from 6 SPF C57BL/6J mice and 1 SD rat.Western blot assay was used to detect the expressions of PPARγprotein in cornea,lens and retina.Immunohistochemistry was used to locate the distribution of PPARγ protein in cornea,lens,ciliary,retina and optical nerve.Also,the co-expression of PPARγ with glutamine synthetase (GS) (a Muller cell specific marker) and glial fibrillary acidic protein (GFAP)(an astrocyte specific marker) in retina and optic nerve was detected by immunofluorescent double staining.Results Western blot assay showed that PPARγ was expressed in the cornea,lens and retina of the mice.Immunohistochemistry revealed that PPARγ mainly located at corneal epithelium with the strongest staining in the basal cells,but only weak staining was seen in corneal endothelial cells and stroma cells.PPARγ was strongly expressed in epithelial cells and shallow cortex layer of mouse lens.In mouse retina,PPARγ was extensively and richly expressed in retinal ganglion cell layer,inner and outer plexiform layers and inner nuclear layer.In addition,PPARγ was also expressed in the non-pigmented epithelial cells in ciliary body.The co-locations of PPARγexpression with GS in retinal tissue and PPARγ expression with GFAP in optical nerve tissue were found in the mice.Conclusions PPARγis proved to distribute extensively in different ocular tissues.These results offer basis for the target treatment of relevant eye diseases.
Key words:
Peroxisome proliferater activated receptor gamma; Eye; Rodent
Inner nuclear layer
Ganglion cell layer
Inner plexiform layer
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