Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency

The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.     

Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness–the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.     

Genetics of corneal endothelial dystrophies

The corneal endothelium maintains the level of hydration in the cornea. Dysfunction of the endothelium results in excess accumulation of water in the corneal stroma, leading to swelling of the stroma and loss of transparency. There are four different corneal endothelial dystrophies that are hereditary, progressive, non-inflammatory disorders involving dysfunction of the corneal endothelium. Each of the endothelial dystrophies is genetically heterogeneous with different modes of transmission and/or different genes involved in each subtype. Genes responsible for disease have been identified for only a subset of corneal endothelial dystrophies. Knowledge of genes involved and their function in the corneal endothelium can aid understanding the pathogenesis of the disorder as well as reveal pathways that are important for normal functioning of the endothelium.     

角膜生物力学的测量及临床应用进展

摘要：角膜是重要的屈光间质,约占眼光学系统总屈光力的70％;因其特殊的生理结构,可表现出复杂的生物力学性质。随着近年来科学技术的进步,用于测量角膜生物力学的方法也在不断更新,获得的生物力学参数也更加精确。越来越多的国内外研究团队将对角膜生物力学的研究同临床相结合,发现当角膜形态发生变化时,其生物力学参数也会发生相应的改变。因此,可以通过对患者角膜生物力学的测量来判断病变的发展程度,也可以根据所测得的力学参数来进行手术设计,甚至可以初判患者的愈后情况。但在角膜生物力学方面的研究仍缺乏深度,对其与部分临床疾病的联系仍缺乏充分的认知,仍需探讨如何将角膜生物力学检查更好地服务于临床。本文将对角膜生物力学的离体、活体测量方法及其目前在圆锥角膜、青光眼、翼状胬肉、屈光不正及屈光不正的矫正等临床方面的应用研究作一综述。     

角膜内皮细胞再生研究新进展

人角膜内皮细胞的主要功能是维持角膜透明性,角膜内皮单层发育成熟形成细胞接触后,内皮细胞会停止分裂增殖,但并没有退出细胞周期。角膜内皮细胞的增殖有多种因素的参与和影响,接触抑制和G1期抑制使细胞增殖暂时停止;细胞因子TGF-β2抑制人角膜内皮细胞进入细胞周期S期,而EGF、FGF、NGF则能够促进细胞的增殖;ROCK抑制剂Y-27632能够促进角膜内皮细胞的粘连,有助于内皮细胞的损伤修复。体外培养角膜内皮前体细胞、诱导多潜能干细胞向角膜内皮细胞分化,为今后治疗角膜内皮失代偿提供了新方向。     

Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.     

Corneal cell proteins and ocular surface pathology

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

Regeneration and fibrosis of corneal tissues

In this review, the features of the regeneration of corneal tissue and its disorders leading to the development of fibrosis are considered. The data on the presence of stem (clonogenic) cell pool in the corneal tissues (epithelium, endothelium, stroma) are given; these cells can serve as a source for regeneration of the tissues at injury or various diseases. The main steps of regeneration of corneal tissues and their disorders that lead to outstripping proliferation of myofibroblasts and secretion of extracellular matrix in the wound area and eventually cause the formation of connective tissue scar and corneal opacity are considered. Particular attention is given to the successes of translational medicine in the treatment of corneal tissue fibrosis. The methods of cell therapy aimed at the restoration of stem cell pool of corneal tissues are the most promising. Gene therapy provides more opportunities; one of its main objectives is the suppression of the myofibroblast proliferation responsible for the development of fibrosis.     

Corneal Viscoelastic Properties from Finite-Element Analysis of In Vivo Air-Puff Deformation

Biomechanical properties are an excellent health marker of biological tissues, however they are challenging to be measured in-vivo. Non-invasive approaches to assess tissue biomechanics have been suggested, but there is a clear need for more accurate techniques for diagnosis, surgical guidance and treatment evaluation. Recently air-puff systems have been developed to study the dynamic tissue response, nevertheless the experimental geometrical observations lack from an analysis that addresses specifically the inherent dynamic properties. In this study a viscoelastic finite element model was built that predicts the experimental corneal deformation response to an air-puff for different conditions. A sensitivity analysis reveals significant contributions to corneal deformation of intraocular pressure and corneal thickness, besides corneal biomechanical properties. The results show the capability of dynamic imaging to reveal inherent biomechanical properties in vivo. Estimates of corneal biomechanical parameters will contribute to the basic understanding of corneal structure, shape and integrity and increase the predictability of corneal surgery.     

Corneal dystrophies

The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (stromal corneal dystrophies), or Descemet membrane and the corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage diseases (mucopolysaccharidoses, lipidoses, mucolipidoses), and several skin diseases (X-linked ichthyosis, keratosis follicularis spinolosa decalvans). The management of the corneal dystrophies varies with the specific disease. Some are treated medically or with methods that excise or ablate the abnormal corneal tissue, such as deep lamellar endothelial keratoplasty (DLEK) and phototherapeutic keratectomy (PTK). Other less debilitating or asymptomatic dystrophies do not warrant treatment. The prognosis varies from minimal effect on the vision to corneal blindness, with marked phenotypic variability.     

Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing

Connective Tissue Growth Factor (CTGF) and Transforming growth factor-β1 (TGF-β1) are key growth factors in regulating corneal scarring. Although CTGF was induced by TGF-β1 and mediated many of fibroproliferative effects of TGF-β1, the signaling pathway for CTGF production in corneal scarring remains to be clarified. In the present study, we firstly investigated the effects of c-Jun N-terminal kinase (JNK) on CTGF expression induce by TGF-β1 in Telomerase-immortalized human cornea stroma fibroblasts (THSF). Then, we created penetrating corneal wound model and determined the effect of JNK in the pathogenesis of corneal scarring. TGF-β1 activated MAPK pathways in THSF cells. JNK inhibitor significantly inhibited CTGF, fibronectin and collagen I expression induced by TGF-β1 in THSF. In corneal wound healing, the JNK inhibitor significantly inhibited CTGF expression, markedly improved the architecture of corneal stroma and reduced corneal scar formation, but did not have a measurable impact on corneal wound healing in vivo. Our results indicate that JNK mediates the expression of CTGF and corneal scarring in corneal wound healing, and might be considered as specific targets of drug therapy for corneal scarring.     

花背蟾蜍蝌蚪变态期角膜发育的研究

作者用光镜和电镜研究了花背蟾蜍蝌蚪变态期角膜的发育。在后肢发育晚期,内、外角膜在中央部位首先愈台,在完全变态期角膜完全愈合,此时角膜上皮细胞增殖,上皮基质变为Bowman’s膜,内、外角膜之间的成纤维细胞和由它分泌的胶原纤维形成角膜基质,内角膜细胞形成单层的角膜内皮,它与角膜基质间的Descemet’s膜最晚形成。     

Aquaporin deletion in mice reduces corneal water permeability and delays restoration of transparency after swelling

Two aquaporin (AQP)-type water channels are expressed in mammalian cornea, AQP1 in endothelial cells and AQP5 in epithelial cells. To test whether these aquaporins are involved in corneal fluid transport and transparency, we compared corneal thickness, water permeability, and response to experimental swelling in wild type mice and transgenic null mice lacking AQP1 and AQP5. Corneal thickness in fixed sections was remarkably reduced in AQP1 null mice and increased in AQP5 null mice. By z-scanning confocal microscopy, corneal thickness in vivo was (in microm, mean +/- S.E., n = 5 mice) 123 +/- 1 (wild type), 101 +/- 2 (AQP1 null), and 144 +/- 2 (AQP5 null). After exposure of the external corneal surface to hypotonic saline (100 mosm), the rate of corneal swelling (5.0 +/- 0.3 microm/min, wild type) was reduced by AQP5 deletion (2.7 +/- 0.1 microm/min). After exposure of the endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7.1 +/- 1.0 microm/min, wild type) was reduced by AQP1 deletion (1.6 +/- 0.4 microm/min). Base-line corneal transparency was not impaired by AQP1 or AQP5 deletion. However, the recovery of corneal transparency and thickness after hypotonic swelling (10-min exposure of corneal surface to hypotonic saline) was remarkably delayed in AQP1 null mice with approximately 75% recovery at 7 min in wild type mice compared with 5% recovery in AQP1 null mice. Our data indicate that AQP1 and AQP5 provide the principal routes for corneal water transport across the endothelial and epithelial barriers, respectively. The impaired recovery of corneal transparency in AQP1 null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium. The up-regulation of AQP1 expression and/or function in corneal endothelium may reduce corneal swelling and opacification following injury.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

A Native-Like Corneal Construct Using Donor Corneal Stroma for Tissue Engineering

Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived well after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium. Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct may have great potential for clinical use in corneal reconstruction.     

Corneal crystals

A limited number of conditions are associated with corneal crystals. This clinical challenge discusses a patient who presented with fine, refractile, crystalline deposits throughout the entire depth of corneal stroma in a limbal to limbal pattern. The differential diagnoses of corneal crystals are presented and discussed, along with the causative condition in this case.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."