Corneal Crystallins Research Articles

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence.In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed.In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls.Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by altered DNA damage response in human corneal epithelial cells

Aldehyde dehydrogenase 3A1 is constitutively expressed in a taxon-specific manner in the cornea, where, due to its high abundance, it has been characterized as a corneal crystallin. ALDH3A1 has been proposed to be a multifaceted protein that protects cellular homeostasis through several modes of action. The present study examines the mechanisms by which ALDH3A1 exerts its cytoprotective role under conditions of oxidative stress. To this end, we have utilized an isogenic HCE-2 (human corneal epithelium) cell line pair differing in the expression of ALDH3A1. Single cell gel electrophoresis assay and H2DCFDA analysis revealed that the expression of ALDH3A1 protected HCE-2 cells from H2O2-, tert-butyl peroxide- and etoposide-induced oxidative and genotoxic effects. Furthermore, comparative qPCR analysis revealed that a panel of cell cycle (Cyclins B1, B2, D, E), apoptosis (p53, BAX, BCL-2, BCL-XL) and DNA damage response (DNA-PK, NBS1) genes were up-regulated in the ALDH3A1 expressing HCE-2 cells. Moreover, the expression profile of a variety of DNA damage signaling (DDS)-related genes, was investigated (under normal and oxidative stress conditions) by utilizing the RT2 profiler™ PCR array in both isogenic HCE-2 cell lines. Our results demonstrated that several genes associated with ATM/ATR signaling, cell cycle regulation, apoptosis and DNA damage repair were differentially expressed under all conditions tested. In conclusion, this study suggests that ALDH3A1 significantly contributes to the antioxidant defense of corneal homeostasis by maintaining DNA integrity possibly through altering the expression of specific DDS-related genes. Further studies will shed light on the precise role(s) of this multifunctional protein.

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.

Corneal haze phenotype in Aldh3a1-null mice: In vivo confocal microscopy and tissue imaging mass spectrometry

ALDH3A1 is a corneal crystallin that protects ocular tissues from ultraviolet radiation through catalytic and non-catalytic functions. In addition, ALDH3A1 plays a functional role in corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation. We have previously shown that Aldh3a1 knockout mice in a C57B6/129sV mixed genetic background develop lens cataracts. In the current study, we evaluated the corneal phenotype of Aldh3a1 knockout mice bred into a C57B/6J congenic background (KO). In vivo confocal microscopy examination of KO and wild-type (WT) corneas revealed KO mice to exhibit corneal haze, manifesting marked light scattering from corneal stroma. This corneal phenotype was further characterized by Imaging Mass Spectrometry (IMS) with spatial resolution that revealed a trilayer structure based on differential lipid localization. In these preliminary studies, no differences were observed in lipid profiles from KO relative to WT mice; however, changes in protein profiles of acyl-CoA binding protein (m/z 9966) and histone H4.4 (m/z 11308) were found to be increased in the corneal epithelial layer of KO mice. This is the first study to use IMS to characterize endogenous proteins and lipids in corneal tissue and to molecularly explore the corneal haze phenotype. Taken together, the current study presents the first genetic animal model of cellular-induced corneal haze due to the loss of a corneal crystallin, and strongly supports the notion that ALDH3A1 is critical to cellular transparency. Finally, IMS represents a valuable new approach to reveal molecular changes underlying corneal disease.

Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro.

Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.

ALDH3A1 Plays a Functional Role in Maintenance of Corneal Epithelial Homeostasis

Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1-/-/3a1-/- double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.

Application of retinoic acid improves form and function of tissue engineered corneal construct

ABSTRACTRetinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.

Mechanisms for PDGF, a Serum Cytokine, Stimulating Loss of Corneal Keratocyte Crystallins

As corneal stromal cells (keratocytes) become activated before transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. The authors previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. The goal of this study was to further define the molecular mechanisms for PDGF-stimulated loss of crystallins to elucidate the pathway for keratocyte activation. Freshly isolated rabbit corneal keratocytes were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components, PDGF receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquitinated TKT levels were assessed by immunoprecipitation, and TKT messenger RNA (mRNA) levels were quantified by quantitative reverse transcription-polymerase chain reaction. PDGF treatment at the same time as inhibition of PDGF receptor, Akt, JNK, and ubiquitin-proteasome pathway prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels. The results suggest that PDGF-stimulated TKT loss is mediated through cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to the degradation of existing TKT protein but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes after surgical procedures in the cornea.

Efficient E. coli Expression Strategies for Production of Soluble Human Crystallin ALDH3A1

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli’s molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli’s molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin.

Ocular aldehyde dehydrogenases: Protection against ultraviolet damage and maintenance of transparency for vision

Aldehyde dehydrogenase (ALDH) enzymes catalyze the NAD(P)(+)-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some members of the ALDH superfamily of enzymes are abundantly expressed in the mammalian cornea and lens in a taxon-specific manner. Considered to be corneal and lens crystallins, they confer protective and transparent properties upon these ocular tissues. ALDH3A1 is highly expressed in the cornea of most mammals, with the exception of rabbit that expresses exclusively ALDH1A1 in the cornea. ALDH1A1 is present in both the cornea and lens of several animal species. As a result of their catalytic and non-catalytic functions, ALDH3A1 and ALDH1A1 proteins protect inner ocular tissues from ultraviolet radiation and reactive oxygen-induced damage. In addition, these corneal crystallins contribute to cellular transparency in corneal stromal keratocytes, supporting a structural role of these ALDH proteins. A putative regulatory function of ALDH3A1 on corneal cell proliferation has also been proposed. Finally, the three retinaldehyde dehydrogenases cooperatively mediate retinoic acid signaling during the eye development.

Aldehyde dehydrogenase inhibitors: a comprehensive review of the pharmacology, mechanism of action, substrate specificity, and clinical application.

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of enzymes that play a key role in the metabolism of aldehydes of both endogenous and exogenous derivation. The human ALDH superfamily comprises 19 isozymes that possess important physiological and toxicological functions. The ALDH1A subfamily plays a pivotal role in embryogenesis and development by mediating retinoic acid signaling. ALDH2, as a key enzyme that oxidizes acetaldehyde, is crucial for alcohol metabolism. ALDH1A1 and ALDH3A1 are lens and corneal crystallins, which are essential elements of the cellular defense mechanism against ultraviolet radiation-induced damage in ocular tissues. Many ALDH isozymes are important in oxidizing reactive aldehydes derived from lipid peroxidation and thereby help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes have been reported in various human cancers and are associated with cancer relapse. As a direct consequence of their significant physiological and toxicological roles, inhibitors of the ALDH enzymes have been developed to treat human diseases. This review summarizes known ALDH inhibitors, their mechanisms of action, isozyme selectivity, potency, and clinical uses. The purpose of this review is to 1) establish the current status of pharmacological inhibition of the ALDHs, 2) provide a rationale for the continued development of ALDH isozyme-selective inhibitors, and 3) identify the challenges and potential therapeutic rewards associated with the creation of such agents.

αA-crystallin in the pathogenesis and intervention of experimental murine corneal neovascularization

This study was to determine the potential roles of lens crystallins in the pathogenesis of corneal neovascularization (CorNV) and implications in therapy of CorNV-related diseases. Suture- or chemical burn-induced CorNV in different strains of mice were used. Changes of gene expression patterns were analyzed by microarray, and the results of interesting genes were confirmed by real-time quantitative PCR and Western blot. Mice deficient in αA-crystallin gene were used to evaluate the role of αA-crystallin in the development of CorNV. In some animals, exogenous αA-crystallin proteins were injected around time of CorNV induction. CorNV was assessed by slit-lamp, flat-mounts and histology. In BALB/C mice, the expression of α-, β-, and γ-crystallins were up-regulated at day 5 and returned to baseline level at day 10 of suture-induced CorNV, but remained up-regulated from day 6 to day 14 of chemical burn-induced CorNV. In chemical burn-induced CorNV in C57BL/6J mice, however, they were down-regulated at day 6. Corneal crystallins were down-regulated in both CorNV models at all time points in both BALB/c and C57BL/6J mice. Comparison of CorNV development in αA-crystallin-deficient mice and that in wild-type mice revealed no significant difference. Subconjunctival injection of αA-crystallin significantly attenuated suture-induced CorNV, and the inhibitory activity might be implemented by the increased expression of soluble VEGFR-1. In conclusion, the expression patterns of lens crystallins were time- and strain-dependent but different from that of corneal crystallins in mouse CorNV models. Exogenous αA-crystallin protein attenuated CorNV, potentially by increasing the expression of soluble VEGFR-1.

Myofibroblast Differentiation Modulates Keratocyte Crystallin Protein Expression, Concentration, and Cellular Light Scattering

The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering. Serum-free cultured rabbit corneal keratocytes and TGFβ (5 ng/mL) induced myofibroblasts were harvested and counted and protein/RNA extracted. Expression of myofibroblast and keratocyte markers was determined by real-time PCR and Western blot analysis. The cell volume of calcein AM-loaded keratocytes and myofibroblasts was determined by using nonlinear optical microscopy. Cellular light scattering of transformed myofibroblasts expressing human keratocyte crystallins was measured by reflectance confocal microscopy. Differentiated myofibroblasts showed a significant decrease in RNA levels for the keratocyte markers ALDH1A1, lumican, and keratocan and a significant increase in the myofibroblast marker α-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P < 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P < 0.002) compared to keratocytes. Western blot analysis showed that the level of ALDH1A1 protein per cell was similar between myofibroblasts and keratocytes, but was substantially reduced as a percentage of total water-soluble protein. Light scattering measurements showed that induced expression of corneal crystallins significantly decreased light scattering. These data suggest that myofibroblast differentiation leads to a marked increase in cell volume and dilution of corneal crystallins associated with an increase in cellular light scattering.

Differential response of lens crystallins and corneal crystallins in degenerative corneas

Corneal degenerations, occurring either spontaneously or as a complication to other diseases, cause vision problems by endangering corneal transparency. Our past cornea research projects involving mice revealed that some recruited mice presented corneal problems similar to human corneal degeneration. The present study examines the histology of diseased mice corneas, including ultrastructure. Genome-wide microarray and proteomic methods were utilized to screen for molecular changes in the diseased corneas. It was found that abnormalities affected mainly anterior layers of the corneas. The most often observed histological abnormalities included neoplasm or detachment of the epithelial layer, erosion or breakage of Bowman membranes, blood vessel formation, and bleeding in the stroma. Microarray assay showed that among the 46 up-regulated probes in diseased corneas, 13 were for lens crystallins. However, all corneal crystallins genes remained unchanged. αA-crystallin was among the proteins that showed the greatest increase in diseased corneas, as detected by gel electrophoresis. We propose that lens crystallins, rather than corneal crystallins, are involved in the pathological process of corneal degeneration. Further study along these lines would provide insight into the mechanism of corneal transparency.

Adhesion, phenotypic expression, and biosynthetic capacity of corneal keratocytes on surfaces coated with hyaluronic acid of different molecular weights

In ophthalmology, hyaluronic acid (HA) is an important extracellular matrix (ECM) component and is appropriate for use in generating a microenvironment for cell cultivation. The aim of this work was to evaluate the rabbit corneal keratocyte (RCK) growth in response to HA coatings under serum-free conditions. After modification with HA of varying molecular weights (MWs: 35–1500kDa), the surfaces were characterized by atomic force microscopy and contact angle measurements, and were used for cell culture studies. Our data indicated that the substrates coated with higher negatively charged HA become rougher and are more hydrophilic, resulting in the decrease of cell adhesion and cell–matrix interaction. This early cellular event was likely responsible for the determination of keratocyte configuration. Additionally, for the growth of RCKs on dry HA coatings with surface roughness of 1.1–1.7nm, a strong cell–cell interaction was observed, which may facilitate the formation of multicellular spheroid aggregates and maintenance of mitotically quiescent state. At each culture time point from 1 to 5days, a better biosynthetic capacity associated with a higher prevalence of elevated ECM production was found for the cells in a spherical configuration. Irrespective of polysaccharide MW of surface coatings, the RCKs presented good viability without hypoxia-induced death. As compared with a monolayer of adherent keratocytes on tissue culture polystyrene plates and low MW HA-modified samples, the cell spheroids (76–110μm in diameter) showed significantly higher expressions of keratocan and lumican and lower expressions of biglycan, similar to those of keratocytes in vivo. Moreover, the expression levels of corneal crystallin aldehyde dehydrogenase (7–9-fold increase) and nestin (10–16-fold increase) were greater in larger-sized spheroids, indicating higher ability to maintain cellular transparency and self-renewal potential. It is concluded that the cultured RCKs on surfaces coated with HA of different MWs can sense ECM cues, and the multicellular spheroids may potentially be used for corneal stromal tissue engineering applications.

Genetic basis of corneal diseases and the role of keratocytes in corneal transparency – a review

AbstractThe current paper reviews the genetic basis of diseases affecting the cornea, including corneal dystrophies and systemic diseases with corneal manifestations (e.g. the mucopolysaccharidoses), discusses the mechanisms by which corneal injury and disease can affect corneal transparency and discusses stem cell therapy as a potential therapy for corneal injury and disease. Several diseases of the cornea have been described, many of which are inheritable. The identification of the genetic mutations responsible for these diseases has led to a better understanding of the mechanisms underlying their corneal manifestations. Corneal disease and injury can affect corneal transparency. Stromal keratocytes play an important role in cellular and extracellular transparency of the cornea. They synthesize type I and V collagen and keratan sulfate proteoglycans that together regulate collagen fibril size and spacing important for extracellular matrix transparency. There are strong indications that cellular transparency of keratocytes relies on the presence of corneal crystallins, which provide a uniform density of proteins in the cytoplasm that increase the spatial order necessary for transparency. After injury, activated keratocytes show heterogeneity of cytoplasmic proteins, decreased spatial ordering and increased light scattering. Stem cell transplantation is regarded as a potential new therapy that can replace keratocytes and restore corneal transparency and normal structure after injury or corneal disease.

Corneal aldehyde dehydrogenases: Multiple functions and novel nuclear localization

Aldehyde dehydrogenases (ALDHs) represent a superfamily of NAD(P) +-dependent enzymes which catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some ALDHs have been identified as corneal crystallins and thereby contribute to the protective and refractive properties of the cornea. ALDH3A1 is highly expressed in the cornea of most mammals with the exception of rabbit that abundantly expresses ALDH1A1 in the cornea instead of ALDH3A1. In this study, we examined the gene expression of other ALDHs and found high messenger levels of ALDH1B1, ALDH2 and ALDH7A1 in mouse cornea and lens. Substantial evidence supports a protective role for ALDH3A1 and ALDH1A1 against ultraviolet radiation (UVR)-induced oxidative damage to ocular tissues. The mechanism by which this protection occurs includes UVR filtering, detoxification of reactive aldehydes generated by UVR exposure and antioxidant activity. We recently have identified ALDH3A1 as a nuclear protein in corneal epithelium. Herein, we show that ALDH3A1 is also found in the nucleus of rabbit keratocytes. The nuclear presence of ALDH3A1 may be involved in cell cycle regulation.

Collagen vitrigel membranes for the in vitro reconstruction of separate corneal epithelial, stromal, and endothelial cell layers

The goal of this study was to evaluate the potential suitability of collagen Vitrigel (CV) membrane as a substrate for the separate reconstruction of the three main cellular layers of the cornea. Limbal explants, keratocytes, and endothelial cells were cultured on transparent membranes made of type I collagen. The resulting cell sheets were evaluated using RT-PCR, in addition to light and electron microscopy. Tensile testing was also performed to examine the mechanical properties of CV. Limbal explant cultures resulted in partially stratified epithelial sheets with upregulation of the putative stem cell marker p63. Keratocytes cultured in serum on CV exhibited stellate morphology along with a marked increase in expression of corneal crystallin ALDH and keratocan, (a keratan sulphate proteoglycan: KSPG), compared to identical cultures on tissue culture plastic. Endothelial cells formed dense monolayers with uniform cell size, tight intercellular junctions, and expression of voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2, and sodium bicarbonate transporter NBC1. Epithelial and endothelial cells exhibited adhesive structures (desmosomes and hemidesmosomes) and evidence of apical specialization (microplicae), while endothelial cells also produced a Descemet's membrane-like basal lamina. CV was found to possess ultimate tensile strengths of 6.8 +/- 1.5 MPa when hydrated and 28.6 +/- 7.0 MPa when dry. Taken together, these results indicate that CV holds promise as a substrate for corneal reconstruction.

Identification of Candidate Klf4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of Klf4 in the Mouse Cornea

Krüppel-like factor 4 (Klf4) plays a crucial role in the development and maintenance of the mouse cornea. In the current study, wild-type (WT) and Klf4-conditional null (Klf4CN) corneal gene expression patterns were examined, to gain understanding of the molecular basis of the Klf4CN corneal phenotype. Expression of more than 22,000 genes in 10 WT and Klf4CN corneas was compared by microarrays, analyzed using BRB ArrayTools (National Cancer Institute, Bethesda, MD) and validated by Q-RT-PCR. Transient cotransfections were used to test whether Klf4 activates the aquaporin-3, Aldh3a1, and TKT promoters. Scatterplot analysis identified 740 and 529 genes up- and downregulated by more than twofold, respectively, in the Klf4CN corneas. Cell cycle activators were upregulated, whereas the inhibitors were downregulated, consistent with the increased Klf4CN corneal epithelial cell proliferation. Desmosomal components were downregulated, consistent with the Klf4CN corneal epithelial fragility. Downregulation of aquaporin-3, detected by microarray, was confirmed by immunoblot and immunohistochemistry. Aquaporin-3 promoter activity was stimulated 7- to 10-fold by cotransfection with pCI-Klf4. The corneal crystallins Aldh3A1 and TKT were downregulated in the Klf4CN cornea, and their respective promoter activities were upregulated 16- and 9-fold by pCI-Klf4 in cotransfections. The expression of epidermal keratinocyte differentiation markers was affected in the Klf4CN cornea. Although the cornea-specific keratin-12 was downregulated, most other keratins were upregulated, suggesting hyperkeratosis. Functionally diverse candidate Klf4 target genes were identified, revealing the molecular basis of the diverse aspects of the Klf4CN corneal phenotype. These results establish Klf4 as an important node in the genetic network of transcription factors regulating the corneal homeostasis.

Gene Expression of the Mouse Corneal CrystallinAldh3a1: Activation by Pax6, Oct1, and p300

Aldehyde dehydrogenase 3a1 (Aldh3a1) represents approximately 50% of the water-soluble protein of the mouse corneal epithelial cells and thus, by analogy with the abundant lens crystallins, is considered a corneal crystallin. This study was conducted to examine the developmental pattern and transcriptional activation of Aldh3a1 gene expression in the mouse cornea. Aldh3a1 mRNA and protein were analyzed by quantitative (Q)-PCR and Western immunoblot analysis. Functional promoter analysis was examined by cotransfecting plasmids containing variable portions of the Aldh3a1 promoter fused to the luciferase reporter gene into COS-7 cells with selected transcription factors. Transcription factor binding sites were identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP). In situ hybridization and immunohistochemistry were used to assess expression of Aldh3a1, Pax6, and Oct1 in the cornea. Aldh3a1 expression is temporally regulated in the cornea beginning at birth and increasing 100-fold by 6 weeks of age. Pax6, Oct1, and p300 synergistically activate the Aldh3a1 promoter approximately 116-fold. One Pax6 and two Oct1 binding sites were identified in vitro and in vivo in the Aldh3a1 promoter fragment analyzed. Pax6 and Oct1 are both present in the nuclei of corneal epithelial cells of the 6-week-old mouse. Finally, a reduction of Aldh3a1 correlated with reduced Pax6 in the corneas of heterozygous Small eye Pax6(+/-) mice. Pax6, Oct1, and p300 activate gene expression of the corneal crystallin Aldh3a1 in the mouse. These transcription factors are also implicated in the high expression of crystallin genes in the lens, consistent with the "refracton hypothesis" unifying many aspects of the lens and cornea.