Corneal epithelium in keratoconus underexpresses active NRF2 and a subset of oxidative stress-related genes.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Transepithelial Corneal Cross-linking With Supplemental Oxygen in Keratoconus: 1-Year Clinical Results.

PURPOSE: To evaluate the efficacy and safety of transepithelial corneal cross-linking (CXL) with supplemental oxygen. METHODS: This was a prospective, non-comparative, pilot cohort study conducted at the National Reference Center for Keratoconus (Toulouse, France) on patients with progressive keratoconus. Transepithelial, pulsed, accelerated CXL was performed in an oxygen-rich atmosphere. Oxygen goggles were applied to the eyes to maintain a high level of oxygen during treatment. The main efficacy outcome was the mean change from baseline in maximum keratometry (Kmax) and the secondary outcomes were the mean changes in flat keratometry (K1), steep keratometry (K2), mean keratometry (Km), corrected distance visual acuity (CDVA), uncorrected distance visual acuity (UDVA), and demarcation line depth. The safety outcomes were the incidence of adverse events, the mean change in pachymetry, and endothelial cell count. RESULTS: Thirty-four eyes of 34 patients were included. At 12 months postoperatively, the Kmax decreased by 1.56 ± 1.71 diopters (D) (P < .0001) and CDVA improved by 0.093 ± 0.193 logMAR (P < .02). The K2 and Km decreased by 0.51 ± 1.03 D (P < .02) and 0.40 ± 0.78 D (P < .01), respectively. There was no change in K1 and UDVA. The most frequent adverse event was corneal haze (64.78%). There were neither cases of infectious keratitis or loss of more than two lines in CDVA nor changes in pachymetry or endothelial cell count. CONCLUSIONS: Transepithelial CXL performed in an oxygen-rich atmosphere results in improved Kmax and CDVA with good safety. These promising findings suggest that this procedure could be safe and capable of halting the progression of keratoconus. [J Refract Surg. 2021;37(1):42-48.].

Customized Topography-Guided Corneal Collagen Cross-linking for Keratoconus.

PURPOSE: To compare the efficacy and safety of topography-guided corneal collagen cross-linking (TG-CXL) to conventional corneal CXL (C-CXL) in progressive keratoconus. METHODS: In this prospective, nonrandomized clinical trial, 60 eyes of 60 patients were scheduled to receive either TG-CXL (30 eyes with deepithelialization focused on the cone, riboflavin application for 10 minutes, and 30 mW/cm2 pulsed ultraviolet-A irradiance pattern according to topography) or C-CXL (30 eyes treated in accordance with the Dresden protocol). Patients were observed for 1 year postoperatively. Maximum keratometry (Kmax), mean keratometry in the inferior part of the cornea (I index), corrected distance visual acuity (CDVA), demarcation line observed in optical coherence tomography, and nerves and cell densities analyzed by confocal microscopy were compared preoperatively and at 1 year postoperatively. RESULTS: The difference was significant for both Kmax (P < .01) and I index (P < .01) between the two groups. CDVA improved significantly in the TG-CXL (0.2162 ± 0.2495 logMAR, P < .05) versus the C-CXL (0.2648 ± 0.2574 logMAR, P = .104) group. A stromal demarcation line was observed in both treatment groups, with similar depth at the top of the cone (P = .391), but it was shallower at the surrounding area in the TG-CXL group (P < .0001). Stromal evaluation by confocal microscopy showed less damage and faster healing in the surrounding area than on the cone area in the TG-CXL group. CONCLUSIONS: At 1 year postoperatively, TG-CXL seems to be as safe as C-CXL with stronger flattening in Kmax and I index and better improvement in CDVA. TG-CXL induces a biological gradient between the cone and the surrounding area that facilitates nerve and cell recovery. [J Refract Surg. 2017;33(5):290-297.].

Collagens and proteoglycans of the cornea: importance in transparency and visual disorders.

The cornea represents the external part of the eye and consists of an epithelium, a stroma and an endothelium. Due to its curvature and transparency this structure makes up approximately 70% of the total refractive power of the eye. This function is partly made possible by the particular organization of the collagen extracellular matrix contained in the corneal stroma that allows a constant refractive power. The maintenance of such an organization involves other molecules such as type V collagen, FACITs (fibril-associated collagens with interrupted triple helices) and SLRPs (small leucine-rich proteoglycans). These components play crucial roles in the preservation of the correct organization and function of the cornea since their absence or modification leads to abnormalities such as corneal opacities. Thus, the aim of this review is to describe the different corneal collagens and proteoglycans by highlighting their importance in corneal transparency as well as their implication in corneal visual disorders.

Iontophoresis Transcorneal Delivery Technique for Transepithelial Corneal Collagen Crosslinking With Riboflavin in a Rabbit Model.

PURPOSE: We compared an iontophoresis riboflavin delivery technique for transepithelial corneal collagen crosslinking (I-CXL) with a conventional CXL (C-CXL). METHODS: We designed three experimental sets using 152 New Zealand rabbits to study riboflavin application by iontophoresis using charged riboflavin solution (Ricrolin+) with a 1-mA current for 5 minutes. The first set was to compare riboflavin concentration measured by HPLC in corneas after iontophoresis or conventional riboflavin application. The second set was to analyze autofluorescence and stromal collagen modification immediately and 14 days after I-CXL or C-CXL, by using nonlinear two-photon microscopy (TP) and second harmonic generation (SHG). In the third set, physical modifications after I-CXL and C-CXL were evaluated by stress-strain measurements and by studying corneal resistance against collagenase digestion. RESULTS: Based on HPLC analysis, we found that iontophoresis allowed riboflavin diffusion with 2-fold less riboflavin concentration than conventional application (936.2 ± 312.5 and 1708 ± 908.3 ng/mL, respectively, P < 0.05). Corneal TP and SHG imaging revealed that I-CXL and C-CXL resulted in a comparable increased anterior and median stromal autofluorescence and collagen packing. The stress at 10% strain showed a similar stiffness of corneas treated by I-CXL or C-CXL (631.9 ± 241.5 and 680.3 ± 216.4 kPa, respectively, P = 0.908). Moreover, we observed an increased resistance against corneal collagenase digestion after I-CXL and C-CXL (61.90% ± 5.28% and 72.21% ± 4.32% of remaining surface, respectively, P = 0.154). CONCLUSIONS: This experimental study suggests that I-CXL is a promising alternative methodology for riboflavin delivery in crosslinking treatments, preserving the epithelium.

Upregulation of bone morphogenetic protein-1/mammalian tolloid and procollagen C-proteinase enhancer-1 in corneal scarring.

PURPOSE: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. METHODS: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. RESULTS: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. CONCLUSIONS: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis.

BACKGROUND: Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. METHODS AND RESULTS: A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the proband's mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. CONCLUSIONS: We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases.

Development of ex vivo organ culture models to mimic human corneal scarring.

PURPOSE: To develop ex vivo organ culture models of human corneal scarring suitable for pharmacological testing and the study of the molecular mechanisms leading to corneal haze after laser surgery or wounding. METHODS: Corneas from human donors were cultured ex vivo for 30 days, either at the air-liquid interface (AL) or immersed (IM) in the culture medium. Histological features and immunofluorescence for fibronectin, tenascin C, thrombospondin-1, and α-smooth muscle actin were graded from 0 to 3 for control corneas and for corneas wounded with an excimer laser. The effects of adding 10 ng/ml transforming growth factor-ß1 (TGF-ß1) to the culture medium and of prior complete removal of the epithelium and limbus, thus preventing reepithelialization, were also analyzed on wounded corneas. Collagen III expression was detected with real-time PCR. RESULTS: Wounding alone was sufficient to induce keratocyte activation and stromal disorganization, but it was only in the presence of added TGF-ß1 that intense staining for fibronectin and tenascin C was found in the AL and IM models (as well as thrombospondin-1 in the AL model) and that α-smooth muscle actin became detectable. The scar-like appearance of the corneas was exacerbated when TGF-ß1 was added and reepithelialization was prevented, resulting in the majority of corneas becoming opaque and marked upregulation of collagen III. CONCLUSIONS: THE MAIN FEATURES OF CORNEAL SCARRING WERE REPRODUCED IN THESE TWO COMPLEMENTARY MODELS: the AL model preserved differentiation of the epithelium and permits the topical application of active molecules, while the IM model ensures better perfusion by soluble compounds.

NC1 long and NC3 short splice variants of type XII collagen are overexpressed during corneal scarring.

PURPOSE: To investigate type XII collagen expression in corneal scars in vivo. METHODS: Type XII collagen protein expression was evaluated by immunohistochemistry in human corneal scars and in a mouse model of corneal scarring at several time points (from day 7 to day 210) after full-thickness excision. Alternative splice variants of the NC3 and NC1 domains of type XII collagen were investigated in the mouse wound-healing model using RT-PCR. RESULTS: Type XII collagen was overexpressed in human corneal scars in areas that were also positive for alpha-smooth muscle actin staining. In a mouse model of corneal wound injury we found that at 14 and 21 days postexcision, type XII collagen was largely concentrated in the subepithelial region of the cornea, especially in and near the wound bed. By 28 days postexcision, expression of type XII collagen decreased but remained higher than that in controls. NC3 short form is the main form expressed in the cornea during the wound-healing process. After injury, the NC1 long splice variant mRNA was the most highly overexpressed variant in the cornea, especially in the epithelium (×2.7, 3.72, and 5.57 at days 7, 14, and 21, respectively, P < 0.01 to 0.001 compared with uninjured samples). Corneal scars from a 7-month-old mouse revealed an overexpression of type XII collagen in the wound area similar to what we observed in human corneal scars. CONCLUSIONS: Type XII collagen is overexpressed in permanent human and mouse corneal scars and could represent a new target to treat corneal scarring.

Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology.

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.

Comparative transcriptome and network biology analyses demonstrate antiproliferative and hyperapoptotic phenotypes in human keratoconus corneas.

PURPOSE: To decipher the biological pathways involved in keratoconus pathophysiology by determining the patterns of differential gene expression between keratoconus and control corneas. METHODS: RNA was extracted from surgically removed corneas of 10 keratoconus patients and from normal corneas of 10 control patients who had undergone enucleation of an eye for ocular melanoma. Several hundred thousand RNA transcripts were assessed using exon microarrays. Statistical comparison and identification of differentially regulated and differentially spliced RNA transcripts was performed by comparing keratoconus cases and controls. In addition, relevant biological pathways were identified by information extraction using network biology. RESULTS: Eighty-seven genes showed significant differences in expression levels. Among these, 69 were downregulated in keratoconus patients, particularly partners of the transcription factor AP-1. The 18 overexpressed genes include mucins, keratins, and genes involved in fibroblast proliferation. In addition, 36 genes were shown to be differentially spliced, including 9 among those that were differentially expressed. Network biology and analysis using Gene Ontology descriptors suggest that many members of both groups belong to pathways of apoptosis and regulation of the balance between cellular differentiation and proliferation. CONCLUSIONS: This work constitutes the first genome-wide transcriptome analysis of keratoconus patient corneas that include all currently known genes and exons. Differential expression suggests that mechanisms of cell loss resulting from antiproliferative and hyperapoptotic phenotypes may be responsible for the pathogenesis of keratoconus. Array information, experimental design, raw intensities, and processed log(2) ratios were deposited at the European Bioinformatic Institute's ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/). The accession number is E-MEXP-2777.

An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins.

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.