Mechanical Strain of Corneal Epithelium Influences the Expression of Genes Implicated in Keratoconus

Loren Moon; Pritpal Kaur; Jiangxia Wang; Akrit Sodhi; Charles Eberhart; Uri Soiberman

doi:10.1167/iovs.66.1.52

. 2025 Jan 23;66(1):52. doi: 10.1167/iovs.66.1.52

Mechanical Strain of Corneal Epithelium Influences the Expression of Genes Implicated in Keratoconus

Loren Moon ¹, Pritpal Kaur ¹, Jiangxia Wang ², Akrit Sodhi ¹, Charles Eberhart ¹, Uri Soiberman ^1,^✉

PMCID: PMC11758933 PMID: 39847367

Abstract

Purpose

Although mechanical injury to the cornea (e.g. chronic eye rubbing) is a known risk factor for keratoconus progression, how it contributes to loss of corneal integrity is not known. Here, we set out to determine how eye rubbing can influence keratoconus progression by exploring the expression of known disease markers in mechanically stressed corneal epithelial cells.

Methods

To explore the effects of mechanical stress on the expression of genes implicated in keratoconus (e.g. WNT10A, COL12A1, and TGFB1), we measured their expression using an in vitro model that simulates eye rubbing by cyclic stretching of an immortalized human corneal epithelial cell line (hTCEpi) for 16 hours. We further examined the influence of WNT10A expression in hTCEpi cells using loss-of-function approaches.

Results

Mechanical strain led to a marked reduction in WNT10A mRNA and protein expression, as well as decreased collagen XII mRNA and protein expression, in hTCEpi cells. Reduced expression of WNT10A protein in WNT10A knockdown cells resulted in reduced protein expression of collagens I and XII, and reduced mRNA expression of MMP9 and TGFB1. Conversely, primary keratocytes treated with recombinant WNT10A protein increased TGFB1 mRNA expression.

Conclusions

We provide a molecular explanation for how mechanical strain results in reduced expression of WNT10A in the corneal epithelium, which, in turn, leads to depletion of collagen type I and XII, and TGFβ1 expression. These results provide a molecular link among mechanical strain, WNT10A expression, and the biomechanical failure of the keratoconus cornea.

Keywords: keratoconus, corneal biomechanics, corneal epithelium, collagen, Wnt-signaling

Keratoconus is a common corneal ectasia that leads to visual impairment starting early in life.¹ Currently there are no US Food and Drug Administration (FDA) approved pharmacological treatments for this disease, and surgical treatments are costly, invasive, and may not be available to all patients in need, highlighting the importance of ongoing studies designed to identify new therapeutic avenues. Given its elasticity, Bowman's layer plays an important role in establishing the biomechanical stability of the cornea.² Bowman's layer is known to be fragmented in keratoconus, and recent evidence associates thinning in this structure with keratoconus, implicating Bowman's layer dysfunction in the disease process.³ Indeed, Bowman's layer onlay grafting has recently been shown to promote corneal flattening in severely keratoconic eyes.⁴ Of note, collagen type XII localizes to Bowman's layer and plays an important role in maintaining the integrity of stress bearing structures.⁵^–⁹ Our previous transcriptomic studies have shown reduction in WNT10A and COL12A1 mRNA transcripts in keratoconus corneal epithelium. Both proteins have also been reported to be markedly reduced or absent in Bowman's layer in keratoconus corneas.⁵^,¹⁰ Although it has previously been reported that WNT10A positively regulates expression of collagen I, another major component of the corneal extracellular matrix which localizes to Bowman’s layer, whether collagen XII is also regulated by WNT10A in corneal epithelial cells is not known. Furthermore, what triggers downregulation of WNT10A (and collagen XII) expression is not clear.¹⁰

In this regard, eye rubbing is a known risk factor for the initiation and progression of keratoconus, and cessation of eye rubbing leads to disease stability; however, the molecular changes induced in the corneal epithelium by chronic eye rubbing are unclear.¹¹^–¹² Here, we set out to determine whether mechanical strain influences WNT10A and collagen XII expression in corneal epithelial cells, and if these proteins regulate TGFβ1 signaling in the adjacent stromal keratocytes.

Materials and Methods

Cell Culture

Human corneal epithelial (hTCEpi) cells gifted by the Shukti Chakravarti laboratory¹³ were confirmed to be mycoplasma negative using a LookOut Mycoplasma PCR Detection Kit (Sigma-Aldrich, St. Louis, MO, USA). The cells were cultured in epithelial growth media (KGM Gold Keratinocyte Growth Medium, Lonza, Basel, Switzerland). TLA293T cells were grown in mammalian basal media (Dulbecco's Modified Eagle Medium; Gibco, Waltham, MA, USA) and prepared for lentiviral creation in 10 cm cell culture plates.

Primary human keratocytes were extracted from research-approved limbal corneal ring remnants used for transplantation.¹⁴ Briefly, epithelium and endothelium were removed, then the stromal tissue was finely diced and digested with collagenase type I (Life Technologies, Grand Island, NY, USA) in low-glucose serum-free (LGSF) media within a water bath at 37°C. Extracted primary keratocytes were initially cultured and allowed to proliferate in 10% FBS DMEM/F-12 (Thermo Fisher, Franklin, MA, USA). For experimentation, only keratocytes of passages two to four were used, and these cells were kept under low-glucose conditions in order to maintain their quiescent phenotype and to avoid transformation to activated fibroblasts.¹⁴

Mechanical Stimulation

To recapitulate the effects of eye rubbing in vitro, we used a model previously utilized to study the influence of mechanical stretch on stromal keratocytes in keratoconus.¹⁵ Briefly, 25,000 hTCEpi cells were plated in 3 individual 144 mm² Cytostretcher chambers (Curi Bio, Seattle, WA, USA) and grown to 100% confluence. The chambers were placed in a Cytostretcher machine (Curi Bio, Seattle, WA, USA) and uniaxial strain stimulation was applied to the cells. The strain pattern consisted of a cyclical ramp pattern that stretched the cells to a maximum of 0.6 mm, or 3% of the baseline, for a duration of 1 second, followed by 1 second of relaxation, and repeated over a total duration of 16 hours. For comparison, control cells were similarly seeded in 144 mm² chambers but cultured under normal static conditions. All samples were maintained at 37°C with 5% CO₂. After stretching, the cells were collected for RNA and protein, as described below.

In Vitro Creation of WNT10A Loss of Function in Corneal Epithelial Cells

The WNT10A locus was targeted in hTCEpi cells using two different CRISPR approaches, generating two separate WNT10A depleted hTCEpi cell lines.¹⁴ All cell populations were maintained at 37°C and 5% CO₂.

Approach A – Electroporation

A lentivirus encoding a combined Cas9 and WNT10A specific guide RNA, VB201126–1208bqw (Supplementary Table S1; VectorBuilder, Chicago, IL, USA) was used to transduce wild type hTCEpi cells. Cas9 expression was verified by Western blot (WB; Supplementary Fig. S1). An additional synthetic WNT10A specific guide RNA (see Supplementary Table S1; Synthego, Redwood City, CA, USA) was used to electroporate the cells using a Neon Electroporation system (Thermo Fisher, Franklin, MA, USA). Clonal selection was performed in a 96-well cell culture plate using serial dilutions. Clones with at least 80% reduction in collagen XII expression were selected for assays. The WNT10A electroporated knockdown clones are identified as such throughout the study.

Approach B – Lentiviral Transduction

A lentivirus encoding a blasticidin resistant Cas9 plasmid, CAS10138 (Horizon, Cambridge, UK) was used to transduce hTCEpi cells. Cas9-expressing cells were selected with blasticidin. Cas9 expression was verified by WB (see Supplementary Fig. S1).

An additional plasmid encoding the sequence of the synthetic guide RNA used in approach A, alongside another WNT10A-specific guide RNA, together with a hygromycin resistance sequence, VB230207-1390rev (see Supplementary Table S1; VectorBuilder, Chicago, IL, USA). The plasmid was packaged into a lentiviral vector in TLA293T cells with packaging and enveloping plasmids VSVG, RRE, and REV (Addgene, Watertown, MA, USA) and the virus was collected 48 hours post-transfection. Virus-containing media was centrifuged at 1000 rpm for 5 minutes at room temperature and filtered through a 0.58 nm filter. Then, 75,000 Cas9-expressing hTCEpi that were plated in a six-well culture plate were transduced with the lentivirus, and then selected with blasticidin and hygromycin for 2 weeks. Clonal selection was performed by dilution in a 96-well culture plate, and 3 individual clones with reduction of WNT10A transcript and protein expression (minimum of 2% expression in the transcript) were identified and used for assays. The individual WNT10A lentiviral suppressed clones are labeled as such.

Creation of Control (Scramble) Population

Using a lentivirus coding for a scramble guide RNA sequence, VB230207-1345jsw (see Supplementary Table S1; VectorBuilder, Chicago, IL, USA), we transduced the Cas9-expressing human corneal epithelial cells to create a control population. These cells underwent selection with hygromycin and blasticidin over 2 weeks and then clonal selection in a 96-well culture plate.

Gain of Function Co-Culture Assay

A WNT10A overexpression plasmid, VB221021-1162dcz (see Supplementary Table S1; VectorBuilder, Chicago, IL, USA) was packaged into a lentiviral vector using TLA293 cells, as described above. The hTCEpi cells were transduced with the lentivirus and selected with blasticidin. Quantitative PCR (qPCR) and WB were performed to verify WNT10A overexpression compared with scramble controls.

A gain of function assay was created using a co-culture in individual transwells (Corning Life Sciences, Kennebunk, ME, USA). Then, the 100,000 WNT10A-overexpressing epithelial cells or scramble controls were seeded in the upper chamber to create a monolayer, whereas 30,000 primary human keratocytes/activated fibroblasts were placed in the lower chamber and grown in KGM, which contains very low serum (0.04%). The activated fibroblasts grown in the lower chambers were collected for qPCR after 72 hours of exposure.

In addition, 30,000 primary keratocytes in their second passage, sourced from 2 different biological human donors were seeded in 3 individual wells and treated with exogenous recombinant WNT10A protein (Aviva Systems, San Diego, CA, USA) at 1 ng/mL. LGSF media with recombinant WNT10A protein was changed every 24 hours. The concentration was established through a dynamic treatment curve ranging from 0 to 2 ng/mL. Keratocytes were collected for qPCR after 72 hours of treatment. The control group consisted of keratocytes grown in LGSF without supplemental recombinant WNT10A. In all cases, the experiments were performed under standard cell culture conditions (i.e. 37°C and 5% CO₂).

Quantitative PCR

Cells were lysed using RLT Plus buffer and RNA was extracted using a RNEasy Plus kit (Qiagen, Valencia, CA, USA). A total of 500 ng RNA was reverse transcribed to cDNA using the iScript cDNA synthesis kit (Thermo Fisher, Franklin, MA, USA), as per the manufacturer's protocol. The qPCR was performed in duplicates with Taqman probes for targets WNT10A, COL1A1, COL12A1, MMP9, AXIN2, TGFB1, and SMAD3, with RPL13A serving as the housekeeping gene for epithelial cells and POLR2A for keratocytes/activated fibroblasts (Supplementary Table S2). The ΔΔCt values were used to calculate relative gene expression.

Western Blot

After reaching 80% confluence, the cells were washed with ice cold PBS, collected, and lysed with RIPA buffer (Merck, Darmstadt, Germany) and protease-phosphatase inhibitors P0044, P5726, and P8340 (Sigma Aldrich, Darmstadt, Germany). The resulting pellet was spun down at 7000 revolutions per minute (rpm) at 4°C for 5 minutes, and the supernatant was collected. Protein concentration was quantified using a Pierce BCA Protein assay (Thermo Fisher, Franklin, MA, USA) and run on a Novex 4 to 12% Wedgewell Tris-Glycine mini gel (Thermo Fisher, Franklin, MA, USA). Gel electrophoresis was performed, and the proteins were transferred to a polyvinylidene fluoride membrane. The membrane was blocked for an hour at room temperature with 5% bovine serum albumin (Thermo Fisher, Franklin, MA, USA) in 0.1% Tween 20–Tris-buffered saline, then incubated overnight at 4°C with target antibodies for WNT10A (GTX111191, 1:1000; Genetex, Lubbock, TX, USA), collagen XII (1:500, NBP1-88062; Novus Biologicals, Centennial, CO, USA), collagen I (AF6220, 1:1000; R&D Systems, Minneapolis, MN, USA), and TGFβ1 (MA5-15065, 1:1000; Invitrogen, Chicago, IL, USA). After incubation and successive washing of the primary antibodies, appropriate secondary antibodies were added to the membrane. Imaging was performed with ECL Western Blotting substrate (Cytiva, Buckinghamshire, UK) and an iBright Imaging System (Thermo Fisher, Franklin, MA, USA). Glyceraldehyde 3-phosphate dehydrogenase (G8795, 1:5000; Sigma-Aldrich, Darmstadt, Germany) was used as a loading control and densitometry was performed on the resulting images using ImageJ (National Institute of Health, Bethesda, MD, USA).

Statistical Analysis

All samples were calculated against an average of the controls, and the Mann-Whitney U test was used for statistical comparisons between groups using the statistical analysis software Stata version 17.0 (StataCorp, College Station, TX, USA). A P value of less than 0.05 was considered statistically significant. Medians were used to calculate percent change of protein or mRNA level for comparison of under- or overexpression between groups. Data are presented as medians with 95% confidence intervals and graphed using Prism version 10.03 (GraphPad, Boston, MA, USA).

Results

Mechanical Strain Causes Downregulation of WNT10A in Corneal Epithelial Cells

To evaluate the effect of mechanical strain from chronic eye rubbing on the expression of WNT10A in corneal epithelium, a monolayer of hTCEpi cells was grown on 3 separate 12 mm × 12 mm Cytostretcher chambers and cyclically stretched to a maximum of 0.6 mm for a duration of 1 second, followed by 1 second of relaxation over a total duration of 16 hours. Cells were carefully inspected by light microscopy to confirm that mechanical strain did not cause any discernible changes to cell confluence or survival (Fig. 1A). The qPCR analysis showed a 94% reduction of WNT10A mRNA (P < 0.05) in stretched cells compared with controls (Fig. 1B). WB analysis extended these results (Fig. 1C, left), demonstrating an 86% reduction in protein level of WNT10A (P < 0.05; Fig. 1C, right).

Figure 1. — Mechanical strain stimulates a downregulation of WNT10A in corneal epithelial cells. Mechanical strain was applied to immortalized corneal epithelial (hTCEpi) cells mounted on cytostretcher chambers. (A) Light microscopy imaging of the cells showed no difference in confluence between stretched and control (static) cells. Cell lysates were then analyzed for transcript and protein levels of WNT10A (panels B and C, respectively) for three stretched groups and three control groups (N = 6). * Indicates statistical significance (P < 0.05).

Expression of WNT10A Regulates Collagens Type I and XII in Corneal Epithelial Cells and Leads to Dysregulation of Other Keratoconus Markers

To evaluate whether the reduction of WNT10A expression in corneal epithelial cells previously reported in human keratoconus tissue, and reproduced in our in vitro epithelial cell stretch experiments, influences the expression of collagens I and XII, we targeted the WNT10A locus in hTCEpi cells using CRISPR/Cas9.¹⁰ WNT10A protein expression was markedly reduced (−79%, P < 0.05) following knockdown of WNT10A (Fig. 2A). These WNT10A deficient cells demonstrated a corresponding 100% decrease in collagen XII expression and 59% decrease in collagen I (P < 0.05 for both) compared with scramble controls (Fig. 2B).

Figure 2. — WNT10A knockdown in Cas9 expressing hTCEpi cells using CRISPR. The WNT10A locus was targeted in Cas9-WNT10A guide RNA expressing hTCEpi cells using an additional specific WNT10A guide RNA through electroporation. Cas9 cells transduced with a nontargeting sequence were used as controls. (A) Cell lysates were collected for WB analysis of WNT10A, collagen XII, and collagen I, with GAPDH serving as a loading control. Knockdown populations are demarcated as KD. (B) Quantification of WB densitometry in knockdown cells (KD) for WNT10A, collagen XII, and collagen I. Comparisons were performed in six suppressed clones compared to three controls (N = 9). * Indicates statistical significance (P < 0.05).

The long-term viability of these WNT10A-deficient cells beyond three passages was poor. To overcome limitations in the survival of WNT10A-deficient hTCEpi cells, we set out to reproduce the partial knockdown of WNT10A observed in keratoconus corneal epithelium in patients in an in vitro model.¹⁰ For that purpose, we used lentiviral transduction of hTCEpi cells to knockdown WNT10A using CRISPR/Cas9. In our transduced cell populations, we observed a significant reduction in WNT10A mRNA expression compared with controls (−96%, P < 0.05; Fig. 3A), resulting in a 27% reduction in WNT10A protein expression by WB (Fig. 3B). In addition, COL12A1 mRNA expression was reduced (−85%, P < 0.05; see Fig. 3B), resulting in an 87% reduction of collagen XII protein by WB (P < 0.05 for both; see Fig. 3B). Similar results were observed for collagen I (−100% at the transcript level and −81% at the protein level, P < 0.05 for both; see Fig. 3B). A reduction in the WNT target AXIN2 expression was also observed in the WNT10A under-expressing group (−94%, P < 0.05; see Fig. 3A), confirming the expected suppression of canonical Wnt signaling as a result of this manipulation (Lim, 2016 and Jho, 2002). Other targets previously implicated in keratoconus also had reduced mRNA levels in the WNT10A suppressed groups, including MMP9 (−98%, P < 0.05), TGFB1 and SMAD3 (−99% and −91%, respectively, P < 0.05; Fig. 3C). WB analysis confirmed a reduction in TGFβ1 protein expression by 55% (P < 0.05; Fig. 3D).

Figure 3. — WNT10A regulates collagen XII production and TGFB1 pathway in corneal epithelial cells. Cas9-expressing hTCepi cells were transduced with a lentivirus coding for two individual WNT10A guide RNA sequences and compared to Cas9 cells transduced with a nontargeting sequence. Cell lysates were analyzed for transcript and protein levels of WNT10A, collagen XII, and collagen I (panels A and B, respectively). Transcript levels for MMP9 as a marker of keratoconus, and AXIN2 as a marker of Wnt pathway activation were also measured (panel A). In order to evaluate the TGFβ pathway, mRNA, and protein levels of TGFβ1 (panels C and D, respectively) and mRNA levels of SMAD3 (panel C) were measured. Comparisons were done in three lentiviral transduced clones against three scramble controls (N = 6). * Indicates statistical significance (P < 0.05).

Mechanical Strain Leads to a Downregulation of Collagens and Other Markers of Keratoconus in Corneal Epithelial Cells

In order to assess whether WNT10A deficiency induced by mechanical strain regulates collagen XII and other genes previously implicated in keratoconus, hTCEpi cells were placed on a cell stretch apparatus, as described above. Along with WNT10A, we also found a decrease in transcript levels of COL12A1 (−87%), COL1A1 (−77%), AXIN2 (−94%), and MMP9 (−59%, P < 0.05 for all comparisons; Fig. 4A). WBs demonstrated marked reductions in collagen XII (−64%, P < 0.05 for all comparisons; Fig. 4B).

Figure 4. — WNT10A downregulation as a result of mechanical strain recapitulates the keratoconus corneal epithelial molecular phenotype. Mechanical strain was applied to corneal epithelial cells mounted on cytostretcher chambers. (A) Cell lysates were analyzed for transcript levels of COL12A1, COL1A1, AXIN2, and MMP9. (B) Protein analysis was also performed via WB to determine collagen XII expression in the stretched and control populations (*left panel* = Western blot and *right panel* = densitometry). Comparisons were performed in three stretched groups against three control groups (N = 6). * Indicates statistical significance (P < 0.05).

WNT10A Promotes TGFB1 mRNA Expression in Primary Activated Fibroblasts

TGFβ signaling in keratocytes has previously been implicated in keratoconus pathogenesis.¹⁶^–²⁰ We therefore set out to determine if WNT10A deficiency in the corneal epithelium was associated with reduction in the transcript levels of TGFB1 and SMAD3 in adjacent keratocytes. For these studies, we used two independent but complimentary assays. In the first assay, primary activated fibroblasts grown in co-culture with WNT10A overexpressing hTCEpi cells demonstrated a 48% increase in TGFB1 transcript level (P < 0.05; Fig. 5A). A similar trend was observed using a second assay, treating primary keratocytes with exogenous recombinant WNT10A protein, which led to a 66% and 75% increase in TGFB1 mRNA expression in 2 separate biological keratocyte cell lines (P < 0.05; Fig. 5B).

Figure 5. — WNT10A overexpression results in increase in TGFB1 mRNA expression. (A) Schematic of co-culture experiment, with WNT10A-overexpressing hTCepi cells in the upper chamber and primary activated fibroblasts in the lower chamber. WB demonstrates WNT10A overexpression (O/E) in epithelial cells transduced with a lentivirus coding for a WNT10A overexpression plasmid. The qPCR shows increased TGFB1 transcript levels in fibroblasts grown in co-culture with WNT10A overexpressing corneal epithelial cells. (B) Increased TGFB1 mRNA expression in two primary fibroblast cell lines sourced from different biological donors treated with 1 ng/mL of recombinant WNT10A. * Indicates statistical significance (P < 0.05).

Discussion

Atopic disease has been associated with an increased risk for keratoconus, and eye rubbing has been reported to be a risk factor for both keratoconus initiation and for disease progression.¹² However, the molecular mechanisms that lead to the corneal biomechanical failure seen in keratoconus as a result of eye rubbing remains unclear. Our previous studies have shown reduced expression of WNT10A and collagen XII in the corneal epithelium and Bowman's layer of corneas with keratoconus.⁵^,¹⁰ By mimicking cyclical eye rubbing in vitro using stretch assays, a model previously utilized to study the influence of mechanical stretch on stromal keratocytes in keratoconus,¹⁵ we demonstrate here that corneal epithelial cells exposed to mechanical strain also have reduced WNT10A mRNA and protein expression. We also show that the downregulation of WNT10A expression caused by mechanical strain is associated with a reduction in collagen XII expression. Thus, mechanical strain, a known upstream regulator of mechanotransduction and Wnt signaling, could play a major role in regulating gene expression in the corneal epithelium of keratoconus eyes (Fig. 6).²¹

Figure 6. — Our findings suggest that alterations in corneal epithelial expression of WNT10A in keratoconus lead to reduced protein levels of collagens XII and I, which can then lead to reduction in corneal strength, as seen in keratoconus.

Although mechanical stretch has been suggested as a potential trigger for the development of keratoconus, the specific mechanism in which eye rubbing induces the molecular changes seen in keratoconus is unclear.²¹ Furthermore, it is unclear which layer of the cornea is most affected by eye rubbing: while the corneal stroma and Descemet's membrane are rigid structures, Bowman's layer is a highly elastic structure²² and therefore can be affected by stretch induced by eye rubbing; in addition, previous confocal microscopy studies have shown morphological changes in keratoconus basal epithelium that may be the result of stretch,²³ and these findings suggest that both epithelium and Bowman layer can be affected by eye rubbing. Here, we show that mechanical strain has significant effects on the expression of multiple additional genes in corneal epithelial cells which regulate tissue structural integrity. In addition to COL12A1, we further report that by downregulating WNT10A mRNA expression, mechanical stretch also results in a decrease in the mRNA expression of COL1A1 and MMP9 genes, also previously implicated in keratoconus.²⁴^–²⁵ These observations demonstrate the broad changes in the expression of structural proteins that may occur in patients following eye rubbing.

Indeed, collagen XII is a critical component in maintaining the structure of the corneal extracellular matrix, where it interacts with other collagen fibrils, including collagen types I, V, and proteoglycans. Collagen type I, V, and XII localize to Bowman's layer where they help maintain its mechanical properties.⁶^,⁸^,²⁸^,²⁹ Genetic variations in collagen type V have been reported in patients with keratoconus, whereas COL12A1 variants were associated with reduced corneal thickness, a hallmark of keratoconus.²⁸^,²⁹ Collagen XII plays a significant role in stromal architecture and promotes tight adhesions of the epithelium to its basement membrane.³⁰ COL12A1 mRNA expression has been reported to be reduced in keratoconus corneas, suggesting that it may play a role in the pathogenesis of this disease.¹⁰^,²⁴ We have previously shown that collagen XII is absent from Bowman's layer in keratoconus.⁵^,¹⁰ A role for collagen XII in keratoconus is further supported by studies using a knockout model of COL12A1 in mice, which demonstrate a reduction in corneal biomechanical strength that is remarkably similar to that observed in patients with keratoconus.³¹

Although Bowman's layer is not thought to play a significant mechanical role in healthy corneas, we hypothesize that it is crucial for maintaining the integrity of biomechanically failing corneas, as occurs in keratoconus.³²^,³³ This hypothesis is consistent with histopathological findings implicating Bowman's layer in the pathogenesis of keratoconus. Fragmentation of Bowman's layer is a histopathological hallmark of keratoconus, and it is thinner in keratoconus corneas compared with healthy controls.³⁴^–³⁶ Traditionally, the corneal epithelium is not considered to provide any mechanical strength to the cornea, yet recent studies using ultrahigh-speed Scheimpflug camera have shown a reduction in corneal stiffness after corneal epithelial removal.³⁷ Therefore, even the small amounts of extra-cellular matrix produced by the epithelium may impact corneal biomechanics, especially in keratoconus. Our findings add to our knowledge by demonstrating how WNT10A is an upstream regulator of collagen XII expression. Therefore, a reduction in WNT10A in the corneal epithelium could lead to reduced expression of collagen XII in the epithelium as well as adjacent structures, including the epithelial basement membrane and Bowman's layer. This, in turn, may lead to reduced biomechanical strength of the anterior cornea and, eventually, corneal ectasia.

We also demonstrate how WNT10A expression in corneal epithelial cells could influence TGFβ pathway dysregulations in adjacent stromal keratocytes/activated fibroblasts. Previous studies have reported TGFB1 abnormalities in keratoconus: keratoconus stromal keratocytes were shown to have an aberrant response to TGFβ1 stimulation, such as a decrease in SMAD6 and SMAD7, which are negative regulators of the TGFβ pathway.¹⁶^–¹⁸ The mRNA levels of SMAD2 and TGFB2 are elevated in epithelium of severe keratoconus cases,¹⁹ and RNA sequencing has revealed dysregulation of core TGFβ pathway elements in keratoconus corneal buttons.²⁰ A review of single cell RNA-sequencing data (i.e. from the UCSC Cell Browser), revealed that the vast majority of WNT10A mRNA in the cornea is produced by the corneal epithelium. We hypothesized that reduced expression of WNT10A mRNA in keratoconus could have significant implications on neighboring cells that are dependent on this paracrine interaction. Using our corneal epithelium-keratocyte co-culture model, we demonstrate that WNT10A produced by the corneal epithelium modestly regulates TGFB1 mRNA expression in activated stromal fibroblasts. A similar result was noted when primary keratocytes were treated with recombinant human WNT10A protein. In addition, WNT10A deficiency was associated with reduced TGFβ1 protein expression and SMAD3 mRNA expression. Given the putative role that TGFβ1 plays in stromal collagenesis, these findings link Wnt signaling abnormalities in the corneal epithelium with stromal dysregulation of the TGFβ pathway and the collagen depletion seen in keratoconus. Although it is unclear how WNT10A affects the TGFβ pathway in the keratoconus corneal epithelium, previous studies have suggested a complex crosstalk between Wnt and TGFβ signaling pathways.³⁸ Based on existing literature, we hypothesize that WNT10A may affect TGFβ signaling primarily through β-catenin interactions with SMAD proteins (SMAD3), modulation of SMAD phosphorylation via non-canonical pathways, and alterations in the ECM that impact TGFβ receptor signaling.³⁹

Collectively, these observations highlight the possibility that early events seen in keratoconus may be initiated by the effects of eye rubbing on the corneal epithelium, which later lead to structural changes in Bowman's layer and the anterior stroma as a result of gradual collagen XII depletion. Collagen XII dysregulation in the corneal stroma, in turn, may have additional effects on TGFβ signaling as collagen XII has previously been reported to play a regulatory role on TGFβ activity.⁴⁰

Limitations of our study include the challenge of establishing a stable cell line with a complete knock-out of WNT10A mRNA expression. Although we were successful in downregulating WNT10A mRNA expression using two different approaches, it was difficult to maintain the viability of isolated clones, suggesting that WNT10A underexpression may result in a proliferative (or survival) disadvantage compared with wild type cells. However, because WNT10A underexpression in primary corneal epithelial samples derived from patients with keratoconus is only modest, our partial WNT10A knockdown models may better recapitulate this keratoconus phenotype.¹⁰ Another limitation of our study was that our experiments were performed in vitro. However, our initial observations demonstrating downregulation of WNT10A and COL12A1 mRNA were performed using an unbiased approach to assess gene expression in corneal tissue samples from dozens of patients with keratoconus with different disease stages, providing supportive clinical data and lending strength to the current results. Finally, whereas eye rubbing plays an important role in the development of keratoconus, it is one of many mechanisms which contribute to this complex disease; additional studies examining how changes in gene expression in response to biomechanical changes are influenced by other factors, including oxidative stress, apoptosis, differential cytokine expression and immunomodulation, growth factors, and vitamin and hormonal deficiencies.⁴¹^–⁵⁰ Some of our experiments were conducted in immortalized corneal epithelial cells that retain their limbal phenotype; in-vivo, limbal epithelial cells repopulate the corneal epithelium after corneal transplant procedures and can therefore interact with the underlying stromal tissue.¹³ Since keratoconus recurrence after corneal transplant procedures has been reported by many groups, and is specific to keratoconus only, the use of these immortalized cells in our study is advantageous and may be consistent with the biology of keratoconus in-vivo.⁵¹

In conclusion, we demonstrate how mechanical strain could be a molecular driver for disease progression in keratoconus by reducing WNT10A mRNA expression, leading to downregulation of multiple structural (e.g. collagen I, collagen XII, and MMP-9) and signaling (e.g. TGFβ) molecules in the keratoconus cornea. This, in turn, may result in the biomechanical instability of Bowman's layer and the anterior stroma that characterizes keratoconus. These findings provide additional support to the hypothesis that epithelial dysregulations and epithelium-keratocyte crosstalk are at the center of the mechanism that trigger the development of this vision threatening disease.

Supplementary Material

Supplement 1

iovs-66-1-52_s001.pdf^{(206.9KB, pdf)}

Supplement 2

iovs-66-1-52_s002.pdf^{(136.3KB, pdf)}

Acknowledgments

The authors thank the laboratory of Ian Pitha at the Wilmer Eye Institute for the usage of his Cytostretcher.

Supported by a Physician Scientist Award from Research to Prevent Blindness and philanthropic grants from Debbie Colson and Jeffrey Williams, Ellen A. Cherniavsky, Hymowitz Family Foundation, Tyrone and Jennifer Throop, the Kahn Foundation, and Donald Jump.

Disclosure: L. Moon, None; P. Kaur, None; J. Wang, None; A. Sodhi, None; C. Eberhart, None; U. Soiberman, Physician Scientist Award from Research to Prevent Blindness (F)

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8. Marchant JK, Zhang G, Birk DE.. Association of type XII collagen with regions of increased stability and keratocyte density in the cornea. Exp Eye Res. 2002; 75(6): 683–694. [DOI] [PubMed] [Google Scholar]
9. Wessel H, Anderson S, Fite D, et al.. Type XII collagen contributes to diversities in human corneal and limbal extracellular matrices. Invest Ophthalmol Vis Sci. 1997; 38(11): 2408–2422. [PubMed] [Google Scholar]
10. Foster JW, Parikh RN, Wang J, et al.. Transcriptomic and immunohistochemical analysis of progressive keratoconus reveal altered wnt10a in epithelium and Bowman's layer. Invest Ophthalmol Vis Sci. 2021; 62(6): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Mazharian A, Flamant R, Elahi S, et al.. Medium to long term follow up study of the efficacy of cessation of eye-rubbing to halt progression of keratoconus. Front Med. (Lausanne). 2023; 10: 1152266. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Jaskiewicz K, Maleszka-Kurpiel M, Michalski A, et al.. Non-allergic eye rubbing is a major behavioral risk factor for keratoconus. PLoS One. 2023; 18(4): e0284454. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Robertson DM, Li L, Fisher S, et al.. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci. 2005; 46(2): 470–478. [DOI] [PubMed] [Google Scholar]
14. Foster JW, Gouveia RM, Connon CJ. (2015). Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions. Sci Rep . 2015; 5: 10839. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Akoto T, Cai J, Nicholas S, et al.. Unravelling the impact of cyclic mechanical stretch in keratoconus-a transcriptomic profiling study. Int J Mol Sci. 2023; 24(8): 7437. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Priyadarsini S, McKay TB, Sarker-Nag A, et al.. Keratoconus in vitro and the key players of the TGF-β pathway. Mol Vis. 2015; 21: 577–588. [PMC free article] [PubMed] [Google Scholar]
17. Foster J, Wu WH, Scott SG, et al.. Transforming growth factor β and insulin signal changes in stromal fibroblasts of individual keratoconus patients. PLoS One. 2014; 9(9): e106556. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Lyon D, McKay TB, Sarkar-Nag A, et al.. Human keratoconus cell contractility is mediated by transforming growth factor-beta isoforms. J Funct Biomater. 2015; 6(2): 422–438. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Engler C, Chakravarti S, Doyle J, et al.. Transforming growth factor-β signaling pathway activation in keratoconus. Am J Ophthalmol. 2011; 151(5): 752–759.e2. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Kabza M, Karolak JA, Rydzanicz M, et al.. Collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways in keratoconus corneas. Eur J Hum Genet. 2017; 25(5): 582–590. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Dou S, Wang Q, Zhang B, et al.. Single-cell atlas of keratoconus corneas revealed aberrant transcriptional signatures and implicated mechanical stretch as a trigger for keratoconus pathogenesis. Cell Discov. 2022; 8(1): 66. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Last JA, Thomasy SM, Croasdale CR, Russell P, Murphy CJ.. Compliance profile of the human cornea as measured by atomic force microscopy. Micron. 2012; 43(12): 1293–1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Hollingsworth JG, Bonshek RE, Efron N.. Correlation of the appearance of the keratoconic cornea in vivo by confocal microscopy and in vitro by light microscopy. Cornea. 2005; 24(4): 397–405. [DOI] [PubMed] [Google Scholar]
24. Chaerkady R, Shao H, Scott SG, et al.. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J Proteomics. 2013; 87: 122–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Shetty R, Ghosh A, Lim RR, et al.. Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A. Invest Ophthalmol Vis Sci. 2015; 56(2): 738–750. [DOI] [PubMed] [Google Scholar]
26. Gordon MK, Foley JW, Birk DE, et al.. Type V collagen and Bowman's membrane. Quantitation of mRNA in corneal epithelium and stroma. J Biol Chem. 1994; 269(40): 24959–24966. [PubMed] [Google Scholar]
27. Chen S, Mienaltowski MJ, Birk DE.. Regulation of corneal stroma extracellular matrix assembly. Exp Eye Res. 2015; 133: 69–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Bryant G, Moore P, Sathyamoorthy M.. The association of a single nucleotide variant in col5a1 to early onset keratoconus and pectus excavatum-convergence of extracellular matrix pathologies. Medicina. (Kaunas). 2024; 60(6): 974. [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Iglesias AI, Mishra A, Vitart V, et al.. Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nat Commun. 2018; 9(1): 1864. [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Cheng EL, Maruyama I, SundarRaj N, et al.. Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas. Curr Eye Res. 2001; 22(5): 333–340. [DOI] [PubMed] [Google Scholar]
31. Hemmavanh C, Koch M, Birk DE, et al.. Abnormal corneal endothelial maturation in collagen XII and XIV null mice. Invest Ophthalmol Vis Sci. 2013; 54(5): 3297–3308. [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Torres-Netto EA, Hafezi F, Spiru B, et al.. Contribution of Bowman layer to corneal biomechanics. J Cataract Refract Surg. 2021; 47(7): 927–932. [DOI] [PubMed] [Google Scholar]
33. Wilson SE. The cornea: no difference in the wound healing response to injury related to whether, or not, there's a Bowman's layer. Biomolecules. 2023; 13(5): 771. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Naderan M, Jahanrad A, Balali S.. Histopathologic findings of keratoconus corneas underwent penetrating keratoplasty according to topographic measurements and keratoconus severity. Int J Ophthalmol. 2017; 10(11): 1640–1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Sawaguchi S, Fukuchi T, Abe H, et al.. Three-dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol. 1998; 116(1): 62–68. [DOI] [PubMed] [Google Scholar]
36. Sykakis E, Carley F, Irion L, et al.. An in depth analysis of histopathological characteristics found in keratoconus. Pathology. 2012; 44(3): 234–239. [DOI] [PubMed] [Google Scholar]
37. Ziaei M, Gokul A, Vellara H, Lu LM, Patel DV, McGhee CNJ.. Measurement of in vivo biomechanical changes attributable to epithelial removal in keratoconus using a noncontact tonometer. Cornea. 2020; 39(8): 946–951. [DOI] [PubMed] [Google Scholar]
38. Shah R, Amador C, Chun ST, et al. Non-canonical Wnt signaling in the eye. Prog Retin Eye Res. 2023; 95: 101149. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Cao X, Wang X, Zhang W, et al. WNT10A induces apoptosis of senescent synovial resident stem cells through Wnt/calcium pathway mediated HDAC5 phosphorylation in OA joints. Bone. 2021; 150: 116006. [DOI] [PubMed] [Google Scholar]
40. Sun M, Koudouna E, Cogswell D, et al.. Collagen XII regulates corneal stromal structure by modulating transforming growth factor-β activity. Am J Pathol. 2022; 192(2): 308–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Kim WJ, Rabinowitz YS, Meisler DM, et al.. Keratocyte apoptosis associated with keratoconus. Exp Eye Res. 1999; 69(5): 475–481. [DOI] [PubMed] [Google Scholar]
42. Krok M, Wróblewska-Czajka E, Łach-Wojnarowicz O, et al.. Analysis of cytokine and chemokine level in tear film in keratoconus patients before and after corneal cross-linking (CXL) treatment. Int J Mol Sci. 2024; 25(2): 1052. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Liu J, Gao J, Xing S, et al.. Bioinformatics analysis of signature genes related to cell death in keratoconus. Sci Rep. 2024; 14(1): 12749. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Jun AS, Cope L, Speck C, et al.. Subnormal cytokine profile in the tear fluid of keratoconus patients. PLoS One. 2011; 6(1): e16437. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Joseph R, Boateng A, Srivastava OP, et al.. Role of fibroblast growth factor receptor 2 (fgfr2) in corneal stromal thinning. Invest Ophthalmol Vis Sci. 2023; 64(12): 40. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Wu X, Deng Q, Han Z, et al.. Screening and identification of genes related to ferroptosis in keratoconus. Sci Rep. 2023; 13(1): 13956. [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Chen X, Liu C, Cui Z, et al.. Integrative transcriptomics analysis and experimental validation reveal immunomodulatory patterns in keratoconus. Exp Eye Res. 2023; 230: 109460. [DOI] [PubMed] [Google Scholar]
48. Peris-Martínez C, Piá-Ludeña JV, Rog-Revert MJ, et al.. Antioxidant and anti-inflammatory effects of oral supplementation with a highly-concentrated docosahexaenoic acid (DHA) triglyceride in patients with keratoconus: a randomized controlled preliminary study. Nutrients. 2023; 15(5): 1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Lasagni Vitar RM, Fonteyne P, Knutsson KA, et al.. Vitamin D supplementation impacts systemic biomarkers of collagen degradation and copper metabolism in patients with keratoconus. Transl Vis Sci Technol. 2022; 11(12): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Van L, Bennett S, Nicholas SE, et al.. Prospective observational study evaluating systemic hormones and corneal crosslinking effects in keratoconus. Ophthalmol Sci. 2023; 4(2): 100364. [DOI] [PMC free article] [PubMed] [Google Scholar]
51. Yoshida J, Murata H, Miyai T, et al. Characteristics and risk factors of recurrent keratoconus over the long term after penetrating keratoplasty. Graefes Arch Clin Exp Ophthalmol. 2018; 256(12): 2377–2383. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1

iovs-66-1-52_s001.pdf^{(206.9KB, pdf)}

Supplement 2

iovs-66-1-52_s002.pdf^{(136.3KB, pdf)}

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[bib7] 7. Gordon MK, Foley JW, Lisenmayer TF, et al.. Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development. Dev Dyn. 1996; 206(1): 49–58. [DOI] [PubMed] [Google Scholar]

[bib8] 8. Marchant JK, Zhang G, Birk DE.. Association of type XII collagen with regions of increased stability and keratocyte density in the cornea. Exp Eye Res. 2002; 75(6): 683–694. [DOI] [PubMed] [Google Scholar]

[bib9] 9. Wessel H, Anderson S, Fite D, et al.. Type XII collagen contributes to diversities in human corneal and limbal extracellular matrices. Invest Ophthalmol Vis Sci. 1997; 38(11): 2408–2422. [PubMed] [Google Scholar]

[bib10] 10. Foster JW, Parikh RN, Wang J, et al.. Transcriptomic and immunohistochemical analysis of progressive keratoconus reveal altered wnt10a in epithelium and Bowman's layer. Invest Ophthalmol Vis Sci. 2021; 62(6): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11. Mazharian A, Flamant R, Elahi S, et al.. Medium to long term follow up study of the efficacy of cessation of eye-rubbing to halt progression of keratoconus. Front Med. (Lausanne). 2023; 10: 1152266. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12. Jaskiewicz K, Maleszka-Kurpiel M, Michalski A, et al.. Non-allergic eye rubbing is a major behavioral risk factor for keratoconus. PLoS One. 2023; 18(4): e0284454. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13. Robertson DM, Li L, Fisher S, et al.. Characterization of growth and differentiation in a telomerase-immortalized human corneal epithelial cell line. Invest Ophthalmol Vis Sci. 2005; 46(2): 470–478. [DOI] [PubMed] [Google Scholar]

[bib14] 14. Foster JW, Gouveia RM, Connon CJ. (2015). Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions. Sci Rep . 2015; 5: 10839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15. Akoto T, Cai J, Nicholas S, et al.. Unravelling the impact of cyclic mechanical stretch in keratoconus-a transcriptomic profiling study. Int J Mol Sci. 2023; 24(8): 7437. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16. Priyadarsini S, McKay TB, Sarker-Nag A, et al.. Keratoconus in vitro and the key players of the TGF-β pathway. Mol Vis. 2015; 21: 577–588. [PMC free article] [PubMed] [Google Scholar]

[bib17] 17. Foster J, Wu WH, Scott SG, et al.. Transforming growth factor β and insulin signal changes in stromal fibroblasts of individual keratoconus patients. PLoS One. 2014; 9(9): e106556. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18. Lyon D, McKay TB, Sarkar-Nag A, et al.. Human keratoconus cell contractility is mediated by transforming growth factor-beta isoforms. J Funct Biomater. 2015; 6(2): 422–438. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19. Engler C, Chakravarti S, Doyle J, et al.. Transforming growth factor-β signaling pathway activation in keratoconus. Am J Ophthalmol. 2011; 151(5): 752–759.e2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20. Kabza M, Karolak JA, Rydzanicz M, et al.. Collagen synthesis disruption and downregulation of core elements of TGF-β, Hippo, and Wnt pathways in keratoconus corneas. Eur J Hum Genet. 2017; 25(5): 582–590. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21. Dou S, Wang Q, Zhang B, et al.. Single-cell atlas of keratoconus corneas revealed aberrant transcriptional signatures and implicated mechanical stretch as a trigger for keratoconus pathogenesis. Cell Discov. 2022; 8(1): 66. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22. Last JA, Thomasy SM, Croasdale CR, Russell P, Murphy CJ.. Compliance profile of the human cornea as measured by atomic force microscopy. Micron. 2012; 43(12): 1293–1298. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23. Hollingsworth JG, Bonshek RE, Efron N.. Correlation of the appearance of the keratoconic cornea in vivo by confocal microscopy and in vitro by light microscopy. Cornea. 2005; 24(4): 397–405. [DOI] [PubMed] [Google Scholar]

[bib24] 24. Chaerkady R, Shao H, Scott SG, et al.. The keratoconus corneal proteome: loss of epithelial integrity and stromal degeneration. J Proteomics. 2013; 87: 122–131. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25. Shetty R, Ghosh A, Lim RR, et al.. Elevated expression of matrix metalloproteinase-9 and inflammatory cytokines in keratoconus patients is inhibited by cyclosporine A. Invest Ophthalmol Vis Sci. 2015; 56(2): 738–750. [DOI] [PubMed] [Google Scholar]

[bib26] 26. Gordon MK, Foley JW, Birk DE, et al.. Type V collagen and Bowman's membrane. Quantitation of mRNA in corneal epithelium and stroma. J Biol Chem. 1994; 269(40): 24959–24966. [PubMed] [Google Scholar]

[bib27] 27. Chen S, Mienaltowski MJ, Birk DE.. Regulation of corneal stroma extracellular matrix assembly. Exp Eye Res. 2015; 133: 69–80. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib28] 28. Bryant G, Moore P, Sathyamoorthy M.. The association of a single nucleotide variant in col5a1 to early onset keratoconus and pectus excavatum-convergence of extracellular matrix pathologies. Medicina. (Kaunas). 2024; 60(6): 974. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29. Iglesias AI, Mishra A, Vitart V, et al.. Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases. Nat Commun. 2018; 9(1): 1864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30. Cheng EL, Maruyama I, SundarRaj N, et al.. Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas. Curr Eye Res. 2001; 22(5): 333–340. [DOI] [PubMed] [Google Scholar]

[bib31] 31. Hemmavanh C, Koch M, Birk DE, et al.. Abnormal corneal endothelial maturation in collagen XII and XIV null mice. Invest Ophthalmol Vis Sci. 2013; 54(5): 3297–3308. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib32] 32. Torres-Netto EA, Hafezi F, Spiru B, et al.. Contribution of Bowman layer to corneal biomechanics. J Cataract Refract Surg. 2021; 47(7): 927–932. [DOI] [PubMed] [Google Scholar]

[bib33] 33. Wilson SE. The cornea: no difference in the wound healing response to injury related to whether, or not, there's a Bowman's layer. Biomolecules. 2023; 13(5): 771. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34. Naderan M, Jahanrad A, Balali S.. Histopathologic findings of keratoconus corneas underwent penetrating keratoplasty according to topographic measurements and keratoconus severity. Int J Ophthalmol. 2017; 10(11): 1640–1646. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35. Sawaguchi S, Fukuchi T, Abe H, et al.. Three-dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol. 1998; 116(1): 62–68. [DOI] [PubMed] [Google Scholar]

[bib36] 36. Sykakis E, Carley F, Irion L, et al.. An in depth analysis of histopathological characteristics found in keratoconus. Pathology. 2012; 44(3): 234–239. [DOI] [PubMed] [Google Scholar]

[bib37] 37. Ziaei M, Gokul A, Vellara H, Lu LM, Patel DV, McGhee CNJ.. Measurement of in vivo biomechanical changes attributable to epithelial removal in keratoconus using a noncontact tonometer. Cornea. 2020; 39(8): 946–951. [DOI] [PubMed] [Google Scholar]

[bib38] 38. Shah R, Amador C, Chun ST, et al. Non-canonical Wnt signaling in the eye. Prog Retin Eye Res. 2023; 95: 101149. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib39] 39. Cao X, Wang X, Zhang W, et al. WNT10A induces apoptosis of senescent synovial resident stem cells through Wnt/calcium pathway mediated HDAC5 phosphorylation in OA joints. Bone. 2021; 150: 116006. [DOI] [PubMed] [Google Scholar]

[bib40] 40. Sun M, Koudouna E, Cogswell D, et al.. Collagen XII regulates corneal stromal structure by modulating transforming growth factor-β activity. Am J Pathol. 2022; 192(2): 308–319. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] 41. Kim WJ, Rabinowitz YS, Meisler DM, et al.. Keratocyte apoptosis associated with keratoconus. Exp Eye Res. 1999; 69(5): 475–481. [DOI] [PubMed] [Google Scholar]

[bib42] 42. Krok M, Wróblewska-Czajka E, Łach-Wojnarowicz O, et al.. Analysis of cytokine and chemokine level in tear film in keratoconus patients before and after corneal cross-linking (CXL) treatment. Int J Mol Sci. 2024; 25(2): 1052. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 43. Liu J, Gao J, Xing S, et al.. Bioinformatics analysis of signature genes related to cell death in keratoconus. Sci Rep. 2024; 14(1): 12749. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 44. Jun AS, Cope L, Speck C, et al.. Subnormal cytokine profile in the tear fluid of keratoconus patients. PLoS One. 2011; 6(1): e16437. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib45] 45. Joseph R, Boateng A, Srivastava OP, et al.. Role of fibroblast growth factor receptor 2 (fgfr2) in corneal stromal thinning. Invest Ophthalmol Vis Sci. 2023; 64(12): 40. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib46] 46. Wu X, Deng Q, Han Z, et al.. Screening and identification of genes related to ferroptosis in keratoconus. Sci Rep. 2023; 13(1): 13956. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib47] 47. Chen X, Liu C, Cui Z, et al.. Integrative transcriptomics analysis and experimental validation reveal immunomodulatory patterns in keratoconus. Exp Eye Res. 2023; 230: 109460. [DOI] [PubMed] [Google Scholar]

[bib48] 48. Peris-Martínez C, Piá-Ludeña JV, Rog-Revert MJ, et al.. Antioxidant and anti-inflammatory effects of oral supplementation with a highly-concentrated docosahexaenoic acid (DHA) triglyceride in patients with keratoconus: a randomized controlled preliminary study. Nutrients. 2023; 15(5): 1300. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib49] 49. Lasagni Vitar RM, Fonteyne P, Knutsson KA, et al.. Vitamin D supplementation impacts systemic biomarkers of collagen degradation and copper metabolism in patients with keratoconus. Transl Vis Sci Technol. 2022; 11(12): 16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib50] 50. Van L, Bennett S, Nicholas SE, et al.. Prospective observational study evaluating systemic hormones and corneal crosslinking effects in keratoconus. Ophthalmol Sci. 2023; 4(2): 100364. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 51. Yoshida J, Murata H, Miyai T, et al. Characteristics and risk factors of recurrent keratoconus over the long term after penetrating keratoplasty. Graefes Arch Clin Exp Ophthalmol. 2018; 256(12): 2377–2383. [DOI] [PubMed] [Google Scholar]

PERMALINK

Mechanical Strain of Corneal Epithelium Influences the Expression of Genes Implicated in Keratoconus

Loren Moon

Pritpal Kaur

Jiangxia Wang

Akrit Sodhi

Charles Eberhart

Uri Soiberman

Abstract

Purpose

Methods

Results

Conclusions

Materials and Methods

Cell Culture

Mechanical Stimulation

In Vitro Creation of WNT10A Loss of Function in Corneal Epithelial Cells

Approach A – Electroporation

Approach B – Lentiviral Transduction

Creation of Control (Scramble) Population

Gain of Function Co-Culture Assay

Quantitative PCR

Western Blot

Statistical Analysis

Results

Mechanical Strain Causes Downregulation of WNT10A in Corneal Epithelial Cells

Figure 1.

Expression of WNT10A Regulates Collagens Type I and XII in Corneal Epithelial Cells and Leads to Dysregulation of Other Keratoconus Markers

Figure 2.

Figure 3.

Mechanical Strain Leads to a Downregulation of Collagens and Other Markers of Keratoconus in Corneal Epithelial Cells

Figure 4.

WNT10A Promotes TGFB1 mRNA Expression in Primary Activated Fibroblasts

Figure 5.

Discussion

Figure 6.

Supplementary Material

Acknowledgments

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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