Evaluation of the results of contact lens assisted corneal cross-linking treatment in keratoconus patients with thin corneas.

PURPOSE: We aimed to compare the efficacy and safety of accelerated contact lens-assisted cross-linking (CA-CXL) with Lotrafilcon B and Comfilcon A lenses in keratoconus (KC) patients with thin corneas. STUDY DESIGN: Retrospective, single-center study. MATERIALS AND METHODS: We retrospectively included 51 eyes of 39 KC patients with corneal thickness <400µm after epithelial scraping (Epi-off), who underwent accelerated CA-CXL treatment with Lotrafilcon B (n=20) and Comfilcon A (n=31). Uncorrected and corrected distance visual acuity (UDVA and CDVA), manifest refraction values, corneal topographic data and endothelial cell density were recorded at preoperative and postoperative 1st, 3rd and 6th month controls. RESULTS: CDVA in the Comfilcon A group was higher than CDVA before surgery at 6 months postoperatively (p<0.001). When the two lenses were compared, CDVA was found to be significantly higher in the Lotrafilcon B group in the preoperative, postoperative 1st month and 3rd month values, but there was no significant difference between the postoperative 6th month values (p=0.028, p=0.018, p=0.044, p=0.181, respectively). The maximum keratometry (Kmax) value at the 6th month after surgery in the Comfilcon A group was significantly lower than in the Lotrafilcon B group (p=0,009). There was no significant difference between the endothelial cell density values between the groups (p=0.623, p=0.609, p=0.794, p=0.458, respectively). There was no significant difference between the progression, regression, and stability rates of the two groups (p=0.714). CONCLUSIONS: Accelerated CA-CXL with Lotrafilcon B and Comfilcon A silicone hydrogel lenses is a safe and effective method to stop progression in patients with thin corneas.

RiTe conjugate mediated corneal collagen crosslinking, a novel therapeutic intervention for keratoconus - in vitro and in vivo study.

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5'Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.

Balanced activation of Nrf-2/ARE mediates the protective effect of sulforaphane on keratoconus in the cell mechanical microenvironment.

Keratoconus (KC) is a progressive degenerative disease that usually occurs bilaterally and is characterized by corneal thinning and apical protrusion of the cornea. Oxidative stress is an indicator of the accumulation of reactive oxygen species (ROS), and KC keratocytes exhibit increased ROS production compared with that of normal keratocytes. Therefore, oxidative stress in KC keratocytes may play a major role in the development and progression of KC. Here, we investigated the protective effect of sulforaphane (SF) antioxidants using a hydrogel-simulated model of the cell mechanical microenvironment of KC. The stiffness of the KC matrix microenvironment in vitro was 16.70 kPa and the stiffness of the normal matrix microenvironment was 34.88 kPa. Human keratocytes (HKs) were cultured for 24 h before observation or drug treatment with H2O2 in the presence or absence of SF. The levels of oxidative stress, nuclear factor E2-related factor 2 (Nrf-2) and antioxidant response element (ARE) were detected. The high-stress state of HKs in the mechanical microenvironment of KC cells compensates for the activation of the Nrf-2/ARE signaling pathway. H2O2 leads to increased oxidative stress and decreased levels of antioxidant proteins in KC. In summary, SF can reduce endogenous and exogenous oxidative stress and increase the antioxidant capacity of cells.

The candidate proteins associated with keratoconus: A meta-analysis and bioinformatic analysis.

PURPOSE: Keratoconus (KC) is a multifactorial disorder. This study aimed to conduct a systematic meta-analysis to exclusively explore the candidate proteins associated with KC pathogenesis. METHODS: Relevant literature published in the last ten years in Pubmed, Web of Science, Cochrane, and Embase databases were searched. Protein expression data were presented as the standard mean difference (SMD) and 95% confidence intervals (CI). The meta-analysis is registered on PROSPERO, registration number CRD42022332442 and was conducted in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA). GO and KEGG enrichment analysis were performed, as well as the miRNAs and chemicals targeting the candidate proteins were predicted. PPI was analyzed to screen the hub proteins, and their expression was verified by RT-qPCR. RESULTS: A total of 21 studies were included in the meta-analysis, involving 346 normal eyes and 493 KC eyes. 18 deregulated proteins with significant SMD values were subjected to further analysis. In which, 7 proteins were up-regulated in KC compared with normal controls, including IL6 (SMD 1.54, 95%CI [0.85, 2.24]), IL1B (SMD 2.07, 95%CI [0.98, 3.16]), TNF (SMD 2.1, 95%CI [0.24, 3.96]), and MMP9 (SMD 1.96, 95%CI [0.68, 3.24]). While 11 proteins were down-regulated in KC including LOX (SMD 2.54, 95%CI [-4.51, -0.57]). GO and KEGG analysis showed that the deregulated proteins were involved in inflammation, extracellular matrix (ECM) remodeling, and apoptosis. MMP9, IL6, LOX, TNF, and IL1B were regarded as hub proteins according to the PPI analysis, and their transcription changes in stromal fibroblasts of KC were consistent with the results of the meta-analysis. Moreover, 10 miRNAs and two natural polyphenols interacting with hub proteins were identified. CONCLUSION: This study obtained 18 candidate proteins and demonstrated altered cytokine profiles, ECM remodeling, and apoptosis in KC patients through meta-analysis and bioinformatic analysis. It will provide biomarkers for further understanding of KC pathogenesis, and potential therapeutic targets for the drug treatment of KC.

Revisiting rabbit models for keratoconus: A long-term study on collagenase-induced disease progression.

Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.

Comparison of refractive surgeries (SMILE, LASIK, and PRK) with and without corneal crosslinking: systematic review and meta-analysis.

Corneal crosslinking (CXL) is used for treating keratoconus and post-laser in situ keratomileusis ectasia. However, refractive surgery is not usually performed with prophylactic CXL. Therefore, we performed a meta-analysis comparing outcomes of refractive surgeries with vs without prophylactic CXL. We systematically searched databases for studies comparing refractive surgeries for myopic correction with vs without prophylactic corneal crosslinking. Review Manager 5.4.1 was used to perform statistical analysis. We included 2820 eyes from 28 studies. Compared with refractive surgery alone, surgery with prophylactic CXL resulted in decreased central corneal thickness, corrected distance visual acuity logMAR, and safety and efficacy indices. There were no significant differences in postoperative uncorrected distance visual acuity of 20/20 or better at ≥12 months and other visual outcomes among both groups. More randomized controlled trials with standard crosslinking protocols are needed to analyze the prophylactic use of crosslinking with refractive surgeries.

Collagen type XII is undetectable in keratoconus Bowman's layer.

PURPOSE: Corneal biomechanical failure is the hallmark of keratoconus (KC); however, the cause of this failure remains elusive. Collagen type XII (COL12A1), which localises to Bowman's layer (BL), is thought to function in stress-bearing areas, such as BL. Given the putative protective role of COL12A1 in biomechanical stability, this study aims to characterise COL12A1 expression in all corneal layers involved in KC. METHODS: TaqMan quantitative PCR was performed on 31 corneal epithelium samples of progressive KC and myopic control eyes. Tissue microarrays were constructed using full-thickness corneas from 61 KC cases during keratoplasty and 18 non-KC autopsy eyes and stained with an antibody specific to COL12A1. Additionally, COL12A1 was knocked out in vitro in immortalised HEK293 cells. RESULTS: COL12A1 expression was reduced at transcript levels in KC epithelium compared with controls (ratio: 0.58, p<0.03). Immunohistochemical studies demonstrated that COL12A1 protein expression in BL was undetectable, with reduced expression in KC epithelium, basement membrane and stroma. CONCLUSIONS: The apparent absence of COL12A1 in KC BL, together with the functional importance that COL12A1 is thought to have in stress bearing areas, suggests that COL12A1 may play a role in the pathogenesis of KC. Further studies are necessary to investigate the mechanisms that lead to COL12A1 dysregulation in KC.

Tear Fluid Inflammatory Proteome Analysis Highlights Similarities Between Keratoconus and Allergic Conjunctivitis.

Purpose: Keratoconus is characterized by the progressive thinning of the cornea, which leads to a cone-like appearance of the eye over time. Although conventionally defined as a noninflammatory condition, a number of recent studies have associated keratoconus (KC) with allergic conjunctivitis (AC) based on clinical parameters. This study aimed to consolidate this association by performing a proteomic analysis of tear fluid from patients with keratoconus and/or allergic conjunctivitis. Methods: Of 51 patients, 17 were diagnosed with KC, 17 were diagnosed with AC, and 17 were diagnosed with both KC and AC (combined). Nine of 34 patients with KC had a progressive form of the disease. Tear fluid samples (n = 51, one eye per patient) were collected by the Schirmer's strips. Tear proteins were extracted from the Schirmer's strips. Proteomic profiling of 384 inflammatory proteins was assessed by a multiplex proximity extension assay (Olink Explore 384 Inflammation Panel I). Results: A total of 384 inflammatory proteins were measured. Two hundred seventy-two of the 384 proteins passed stringent data cleaning and were compared among the patient groups. Compared to the 2 other groups, LGALS9 was upregulated uniquely in KC, whereas FGF19, PDGFB, HPCAL1, OSM, and FCAR were downregulated in KC. Similarly, TNFRSF4 and CCL13 were specifically upregulated in AC, whereas ectodysplasin A receptor (EDAR) was uniquely downregulated in AC. Conclusions: High-throughput proteomic profiling of tear fluid confirms the association between KC and AC on a molecular level and raise the importance of redefining KC as an inflammatory condition.

Integrated analysis of murine cornea identifies JAK/STAT signaling pathway upregulated specifically in female Vitamin A Deficient mice.

The Keratoconus (KC) is a corneal ectatic disease with unclear etiology. There are increasing studies that reported its association with a variety of inflammatory mechanisms. Vitamin A(VA) is an important nutrient related to inflammation regulation, and its deficiency may cause abnormalities of the ocular surface. However, the proportion of Vitamin A deficiency(VAD) was found surprisingly high among KC patients in our clinic practice. The aim of this study is to explore the effects of VAD on the transcriptome of corneas with the help of the VAD murine model and transcriptomics techniques. Blood samples of KC patients and non-KC controls (NC) were collected and the serum VA concentrations were measured and analyzed. A total of 52 NC and 39 KC were enrolled and the comparison of serum VA showed that the proportion of VAD in KC patients was 48.7% versus 1.9% in NC group. The further analysis of gender differences showed the proportion of VAD in female KC was 88.9% versus 36.7% in KC male patients. To explore the influence of VAD on cornea, the VAD mice fed with VAD diets were used. The RNA sequencing was employed to compare the corneal transcriptomic characteristics between the VAD female mice, NC female mice, VAD male mice and NC male mice. The transcriptome analysis revealed that the upregulated differential genes were mainly enriched in the immune response related pathways in VAD female mice versus NC female mice, especially the genes of JAK-STAT signaling pathway. The downstream molecules of JAK-STAT pathway were also significant after corneal mechanical scratching in female VAD mice. While, the differential genes between VAD male mice and NC male mice were estrogen signaling pathway instead of JAK-STAT pathway. This study indicates that VAD affects the transcriptomics of murine cornea with gender differences, which specifically affects the inflammatory status of the female murine cornea.

Role of Fibroblast Growth Factor Receptor 2 (FGFR2) in Corneal Stromal Thinning.

Purpose: To determine the role of fibroblast growth factor receptor 2 (FGFR2)-mediated signaling in keratocytes during corneal development, a keratocyte-specific FGFR2-knockout (named FGFR2cKO) mouse model was generated, and its phenotypic characteristics were determined. Methods: A FGFR2cKO mouse model was generated by the following method: FGFR2 flox mice were crossed with the inducible keratocyte specific-Cre mice (Kera-rtTA/tet-O-Cre). Both male and female FGFR2cKO- and control mice (1 to 3-months-old) were analyzed for changes in corneal topography and pachymetry maps using the optical coherence tomography (OCT) method. The comparative TUNEL assay and immunohistochemical analyses were performed using corneas of FGFR2cKO and control mice to determine apoptotic cells, and expression of collagen-1 and fibronectin. Transmission electron microscopic analysis was conducted to determine collagen structures and their diameters in corneas of FGFR2cKO and control mice. Results: OCT-analyses of corneas of FGFR2cKO mice (n = 24) showed localized central thinning and an increased corneal steepness compared to control mice (n = 23). FGFR2cKO mice further showed a decreased expression in collagen-1, decreased collagen diameters, acute corneal hydrops, an increased fibronectin expression, and an increased number of TUNEL-positive cells suggesting altered collagen structures and keratocytes' apoptosis in the corneas of FGFR2cKO mice compared to control mice. Conclusions: The FGFR2cKO mice showed several corneal phenotypes (as described above in the results) that are also exhibited by the human keratoconus corneas. The results suggested that the FGFR2cKO mouse model serves to elucidate not only the yet unknown role of FGFR2-mediated signaling in corneal physiology but also serves as a model to determine molecular mechanism of human keratoconus development.

Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells.

Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-ß1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

Genetic Correlations Among Corneal Biophysical Parameters and Anthropometric Traits.

Purpose: The genetic architecture of corneal dysfunction remains poorly understood. Epidemiological and clinical evidence suggests a relationship between corneal structural features and anthropometric measures. We used global and local genetic similarity analysis to identify genomic features that may underlie structural corneal dysfunction. Methods: We assembled genome-wide association study summary statistics for corneal features (central corneal thickness, corneal hysteresis [CH], corneal resistance factor [CRF], and the 3 mm index of keratometry) and anthropometric traits (body mass index, weight, and height) in Europeans. We calculated global genetic correlations (rg) between traits using linkage disequilibrium (LD) score regression and local genetic covariance using ρ-HESS, which partitions the genome and performs regression with LD regions. Finally, we identified genes located within regions of significant genetic covariance and analyzed patterns of tissue expression and pathway enrichment. Results: Global LD score regression revealed significant negative correlations between height and both CH (rg = -0.12; P = 2.0 × 10-7) and CRF (rg = -0.11; P = 6.9 × 10-7). Local analysis revealed 68 genomic regions exhibiting significant local genetic covariance between CRF and height, containing 2874 unique genes. Pathway analysis of genes in regions with significant local rg revealed enrichment among signaling pathways with known keratoconus associations, including cadherin and Wnt signaling, as well as enrichment of genes modulated by copper and zinc ions. Conclusions: Corneal biophysical parameters and height share a common genomic architecture, which may facilitate identification of disease-associated genes and therapies for corneal ectasias. Translational Relevance: Local genetic covariance analysis enables the identification of associated genes and therapeutic targets for corneal ectatic disease.

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment.

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Comparison of topographic outcomes between HPMC based and vitamin E TPGS based riboflavin solutions after corneal cross-linking.

PURPOSE: To compare the visual and topographic results between patients who underwent epithelium-off cross-linking using riboflavin solutions compounds hydroxypropyl methylcellulose (HPMC) 1.1% and D-alpha-tocopheryl polyethylene-glycol 1000 succinate (VE-TPGS). METHODS: In this study, 37 eyes treated with HPMC and 29 eyes treated with VE-TPGS were evaluated retrospectively. Spherical equivalent (SE), refractive cylinder, corrected distance visual acuity tests (CDVA), corneal topography indices (flat and steep meridians' keratometry (K1 and K2)), maximum keratometry (K max), central, thinnest, and apical corneal thicknesses, the front and back keratoconus vertex index (KVf, KVb), and the surface asymmetry index of the front and back surface (SIf, SIb), and endothelial cell density were compared at baseline and postoperative follow-up visits (1, 3, 6, and 12months). RESULTS: At the end of the 12th-month, K1, K2, and Kmax were decreased in both groups. In comparison to baseline, there was a decline in the HPMC group in the 3rd- month Kmax change, an increase was observed in the VE-TPGS group. In the 12th-month KVb change, an increase was observed in the HPMC group compared to the baseline, while a decrease was observed in the VE-TPGS group. The other parameters did not show a statistically significant difference between the groups (p > 0.05). CONCLUSION: At the end of 12 months, both riboflavins were effective in stopping the progression of keratoconus and were safe for endothelium. Although both riboflavins provide a decrease in keratometry values, it can be said that VE-TPGS is superior to HPMC in correcting the ectasia on the posterior corneal surface.

Collagen Crosslinking for Keratoconus: Cellular Signaling Mechanisms.

Collagen crosslinking (CXL) is a widely used treatment to halt the progression of keratoconus (KC). Unfortunately, a significant number of patients with progressive KC will not qualify for CXL, including those with corneas thinner than 400 µm. The present study aimed to investigate the molecular effects of CXL using in vitro models, mirroring the normal, as well as thinner corneal stroma seen in KCs. Primary human corneal stromal cells were isolated from healthy (HCFs) and keratoconus (HKCs) donors. Cells were cultured and stimulated with stable Vitamin C resulting in 3D self-assembled extracellular matrix (ECM), cell-embedded, constructs. CXL was performed on (a) thin ECM with CXL performed at week 2 and (b) normal ECM with CXL performed at week 4. Constructs without CXL served as controls. All constructs were processed for protein analysis. The results showed modulation of Wnt signaling, following CXL treatment, as measured by the protein levels of Wnt7b and Wnt10a, correlated to the expression of α-smooth muscle actin (SMA). Further, the expression of a recently identified KC biomarker candidate, prolactin-induced protein (PIP), was positively impacted by CXL in HKCs. CXL-driven upregulation of PGC-1 and the downregulation of SRC and Cyclin D1 in HKCs were also noted. Although the cellular/molecular impacts of CXL are largely understudied, our studies provide an approximation to the complex mechanisms of KC and CXL. Further studies are warranted to determine factors influencing CXL outcomes.

Unravelling the Impact of Cyclic Mechanical Stretch in Keratoconus-A Transcriptomic Profiling Study.

Biomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFß1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFß1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70-90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFß1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| ≥ 1.5, FDR ≤ 0.1, CPM ≥ 10 in ≥1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFß1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ≤ 0.05). Using multi-factorial ANOVA analyses, 479 DEGs were identified in HKCs vs. HCFs including TGFß1 treatment and CMS as cofactors. Among these DEGs, 199 KC-altered genes were responsive to TGFß1, thirteen were responsive to CMS, and six were responsive to TGFß1 and CMS. Pathway analyses using PANTHER and DAVID indicated the enrichment of genes involved in numerous KC-relevant functions, including but not limited to degradation of extracellular matrix, inflammatory response, apoptotic processes, WNT signaling, collagen fibril organization, and cytoskeletal structure organization. TGFß1-responsive KC DEGs were also enriched in these. CMS-responsive KC-altered genes such as OBSCN, CLU, HDAC5, AK4, ITGA10, and F2RL1 were identified. Some KC-altered genes, such as CLU and F2RL1, were identified to be responsive to both TGFß1 and CMS. For the first time, our multi-factorial RNA-Seq study has identified many KC-relevant genes and pathways in HKCs with TGFß1 treatment under CMS, suggesting a potential role of TGFß1 and biomechanical stretch in KC development.

Non-allergic eye rubbing is a major behavioral risk factor for keratoconus.

Since the environmental, behavioral, and socioeconomic factors in the etiology of keratoconus (KTCN) remain poorly understood, we characterized them as features influencing KTCN phenotype, and especially affecting the corneal epithelium (CE). In this case-control study, 118 KTCN patients and 73 controls were clinically examined and the Questionnaire covering the aforementioned aspects was completed and then statistically elaborated. Selected KTCN-specific findings were correlated with the outcomes of the RNA-seq assessment of the CE samples. Male sex, eye rubbing, time of using a computer after work, and dust in the working environment, were the substantial KTCN risk factors identified in multivariate analysis, with ORs of 8.66, 7.36, 2.35, and 5.25, respectively. Analyses for genes whose expression in the CE was correlated with the eye rubbing manner showed the enrichment in apoptosis (TP53, BCL2L1), chaperon-related (TLN1, CTDSP2, SRPRA), unfolded protein response (NFYA, TLN1, CTDSP2, SRPRA), cell adhesion (TGFBI, PTPN1, PDPK1), and cellular stress (TFDP1, SRPRA, CAPZB) pathways. Genes whose expression was extrapolated to the allergy status didn't contribute to IgE-related or other inflammatory pathways. Presented findings support the hypothesis of chronic mechanical corneal trauma in KTCN. Eye-rubbing causes CE damage and triggers cellular stress which through its influence on cell apoptosis, migration, and adhesion affects the KTCN phenotype.

The effect of accelerated pulsed high-fluence corneal cross-linking on corneal endothelium; a prospective specular microscopy study.

BACKGROUND: Corneal collagen cross-linking (CXL) is a procedure utilized for halting keratoconus progression with different approved protocols. The current study aimed to assess the corneal endothelial changes following the relatively new accelerated pulsed high-fluence protocol of epithelium-off corneal cross-linking for the treatment of mild to moderate keratoconus. METHODS: This prospective case series study enrolled 45 eyes of 27 patients with mild to moderate progressive keratoconus who underwent accelerated pulsed high-fluence CXL (pl-ACXL, 30 mW/ cm2 UVA at 365 nm wavelength, 8 min pulsed mode 1 s on / 1 s off with a total energy of 7.2 J/ cm2). The main outcome measures were corneal endothelial changes assessed by specular microscopy at 3 and 6 months postoperatively including endothelial cell density (ECD), coefficient of variation, percentage of hexagonal cells, average, minimum and maximum endothelial cell sizes. Demarcation line depth was assessed 1 month following surgery. RESULTS: The mean age of the studied sample was 24.89 ± 7.21. The mean preoperative ECD (2944.6 ± 247.41 cell/mm2) showed non-significant reduction at 3 and 6 months postoperatively (2931.03 ± 253.82 and 2924.7 ± 224.88 cell/mm2, respectively, P-value = 0.361). There were no significant changes in the mean coefficient of variation, percentage of hexagonal cells, average, minimum and maximum endothelial cell sizes at 3 and 6 months following pl-ACXL (P-value > 0.05). The mean demarcation line depth 1 month after pl-ACXL was 214 ± 17.43 µm. CONCLUSIONS: Corneal endothelial changes following accelerated pulsed high-fluence CXL were minimal with stability of endothelial cell count and non-significant morphological changes. TRIAL REGISTRATION: Clinicaltrials.gov: NCT04160338 (13/11/2019).

The Impaired Wound Healing Process Is a Major Factor in Remodeling of the Corneal Epithelium in Adult and Adolescent Patients With Keratoconus.

Purpose: Keratoconus (KTCN) is the most common corneal ectasia, characterized by pathological cone formation. Here, to provide an insight into the remodeling of the corneal epithelium (CE) during the course of the disease, we evaluated topographic regions of the CE of adult and adolescent patients with KTCN. Methods: The CE samples from 17 adult and 6 adolescent patients with KTCN, and 5 control CE samples were obtained during the CXL and PRK procedures, respectively. Three topographic regions, central, middle, and peripheral, were separated toward RNA sequencing and MALDI-TOF/TOF Tandem Mass Spectrometry. Data from transcriptomic and proteomic investigations were consolidated with the morphological and clinical findings. Results: The critical elements of the wound healing process, epithelial-mesenchymal transition, cell-cell communications, and cell-extracellular matrix interactions were altered in the particular corneal topographic regions. Abnormalities in pathways of neutrophils degranulation, extracellular matrix processing, apical junctions, IL, and IFN signaling were revealed to cooperatively disorganize the epithelial healing. Deregulation of the epithelial healing, G2M checkpoints, apoptosis, and DNA repair pathways in the middle CE topographic region in KTCN explains the presence of morphological changes in the corresponding doughnut pattern (a thin cone center surrounded by a thickened annulus). Despite similar morphological characteristics of CE samples in adolescents and adults with KTCN, their transcriptomic features were different. Values of the posterior corneal elevation differentiated adults with KTCN from adolescents with KTCN and correlated with the expression of TCHP, SPATA13, CNOT3, WNK1, TGFB2, and KRT12 genes. Conclusions: Identified molecular, morphological, and clinical features indicate the effect of impaired wound healing on corneal remodeling in KTCN CE.

Vitamin D Supplementation Impacts Systemic Biomarkers of Collagen Degradation and Copper Metabolism in Patients With Keratoconus.

Purpose: To evaluate the impact of vitamin D (Vit D) supplementation on systemic biomarkers of collagen degradation, inflammation, oxidative stress, and copper metabolism in adolescent patients with keratoconus (KC). Methods: This was a prospective observational pilot study. Twenty patients (age range, 16-19 years) presenting KC and Vit D insufficiency (<30 ng/mL) were included. Vit D supplementation was prescribed by their general practitioner as per the standard of care. Patients were followed up for 12 months. At each visit, best spectacle-corrected visual acuity (BSCVA), maximal keratometry (Kmax), and thinnest corneal thickness (TCT) were evaluated. The primary outcome of the study was the proportion of patients with Kmax progression of less than 1 D throughout the 12-month follow-up time. Blood samples were collected at different time points to evaluate Vit D levels and systemic markers of collagen degradation, inflammation, oxidative stress, and copper metabolism by ELISA or RT-PCR. Results: Lower Vit D levels in the plasma were correlated with higher levels of systemic biomarkers of collagen degradation. Vit D supplementation increased the cell availability of copper. Moreover, stabilization of KC progression was found in 60% of patients (72% of eyes) after 12 months with Vit D supplementation. BSCVA, Kmax, and TCT rates remained stable during the observation period. Conclusions: Our findings support that Vit D administration could affect ocular and systemic biomarkers in KC and illuminate a possible mechanism that can be used to develop new treatment alternatives. Translational Relevance: Although KC therapy currently relies exclusively on surgical procedures, Vit D supplementation may offer a non-invasive and inexpensive alternative with minimal associated side effects.