October 2016
Volume 57, Issue 13
Open Access
Cornea  |   October 2016
Differential Molecular Expression of Extracellular Matrix and Inflammatory Genes at the Corneal Cone Apex Drives Focal Weakening in Keratoconus
Author Affiliations & Notes
  • Natasha Pahuja
    Cornea Department, Narayana Nethralaya, Bangalore, India
  • Nimisha R. Kumar
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
  • Rushad Shroff
    Cornea Department, Narayana Nethralaya, Bangalore, India
  • Rohit Shetty
    Cornea Department, Narayana Nethralaya, Bangalore, India
  • Rudy M. M. A. Nuijts
    Cornea Clinic, Department of Ophthalmology, Maastricht University Medical Center, the Netherlands
  • Anuprita Ghosh
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
  • Abhijit Sinha-Roy
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
  • Shyam S. Chaurasia
    Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
  • Rajiv R. Mohan
    Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri, United States
    Mason Eye Institute, School of Medicine, University of Missouri, Columbia, Missouri, United States
    Harry S. Truman Veterans' Memorial Hospital, Columbia, Missouri, United States
  • Arkasubhra Ghosh
    GROW Research Laboratory, Narayana Nethralaya Foundation, Bangalore, India
    Singapore Eye Research Institute, Singapore
  • Correspondence: Arkasubhra Ghosh, GROW Research Laboratory Narayana Nethralaya Foundation, Narayana Health City, # 258/A, Bommasandra, Hosur Road, Bangalore 560 099, India; [email protected]
  • Rajiv R. Mohan, University of Missouri, 1600 E. Rollins Road, Columbia, Missouri, 65211, USA; [email protected]
  • Footnotes
     NP and NRK contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science October 2016, Vol.57, 5372-5382. doi:https://doi.org/10.1167/iovs.16-19677
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      Natasha Pahuja, Nimisha R. Kumar, Rushad Shroff, Rohit Shetty, Rudy M. M. A. Nuijts, Anuprita Ghosh, Abhijit Sinha-Roy, Shyam S. Chaurasia, Rajiv R. Mohan, Arkasubhra Ghosh; Differential Molecular Expression of Extracellular Matrix and Inflammatory Genes at the Corneal Cone Apex Drives Focal Weakening in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2016;57(13):5372-5382. https://doi.org/10.1167/iovs.16-19677.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: In this study, we elucidated the differential expression of a set of local molecular factors in ectatic cone area of the cornea to uncover a functional cause for focal corneal weakening characteristic of the keratoconus (KC) disease.

Methods: All human corneal samples were collected after approval of Institutional Ethics Committee and informed consent. Keratoconus patients were classified based on clinical parameters, topographical features, and structural deformity. Epithelial cells were collected from KC patients (n = 66) undergoing corneal cross-linking procedures from cone apex and periphery. Nonectatic refractive surgery patients (n = 23) served as controls. The ratio of epithelial gene expression in cone and periphery of each eye was estimated by quantitative PCR and correlated with clinical data. Similar cone versus periphery analysis was done from the KC stroma and from KC patients with Bowman's layer (BL) breach observed by anterior segment optical coherence tomography (OCT).

Results: Epithelium from the cone apex of KC patients had elevated levels of inflammatory factors TNF-α, IL-6, and matrix metalloproteinase 9 (MMP-9) but reduced Lysyl oxidase (LOX) and Collagen IVA1, which also demonstrated correlation with corneal curvature and deformity parameters. Stromal gene expression from KC patients showed trends similar to epithelium. Epithelium collected from the cone apex of BL breached KC patients showed significantly elevated MMP-9, TNF-α, and IL-6 levels but reduced IL-10, tissue inhibitor of metalloproteinases 1 (TIMP-1), and Collagen IVA1 expression.

Conclusions: This study provides the first evidence that altered corneal epithelial and stromal expression of specific genes at the corneal cone apex drives focal structural weakness in KC.

Keratoconus (KC) is a corneal ectatic disease characterized by progressive focal thinning and irregular astigmatism that leads to reduced visual acuity.1,2 The disease is highly prevalent in South Asia including Indian subcontinent suggesting underlying genetic predisposition.1,3 The disease progression is often variable although it typically manifests in the second decade of life in the majority of the patients. The primary clinical features of KC are corneal steepening with an acentric, typically inferior, thin-cone apex resulting in visual distortion, central corneal scarring in severe disease, Fleischer's ring composed of circular deposits of iron in corneal epithelium, and presence of Vogt's striae in posterior corneal stroma.46 The disease is bilaterally asymmetrical without a sex bias.2,7 Mechanical factors such as eye rubbing and contact lens wear have been associated with KC incidence.8 Other conditions, such as connective tissue disorders, Down's syndrome, genetic factors, and atopy have also been reported to have a higher incidence of KC.3,9 Recent data suggest involvement of several inflammatory factors, enzymes, and cytokines such as matrix metalloproteinase 9 (MMP-9), IL-6, TNF-α, and Cathepsins that were found to be increased in tear samples of KC patients.1015 Major treatment strategies for KC patients includes customized contact lens or surgical procedures such as topography guided photo refractive keratectomy (T-PRK), corneal collagen cross-linking (CXL), and intrastromal corneal ring segments (ICRS).1620 Because disease progression is marked by focal corneal weakening that increases in diameter with time, most of the treatment modalities focus on strengthening of the cornea. 
The triggering factors and etiology of KC is still poorly understood. A number of studies have reported the deregulation of genes, cytokines, enzymes, and other biomarkers in KC patients.14,21 We have previously demonstrated elevation of MMP-9 and IL-6 expression in KC tears as well mRNA expression in corneal epithelium.22 Conversely, we observed a severity-dependent reduction of Lysyl oxidase (LOX), Collagen IA1 (COL IA1), and Collagen IVA1 (COL IVA1) expression at both the mRNA and protein levels.22,23 In KC, the changes in collagen content and enzymes involved in maintaining corneal structure are deregulated.23,24 Matrix metalloproteinases are a set of enzymes that respond to injury or stress leading to remodeling of the extracellular matrix.25 Matrix metalloproteinase-9 is secreted by the cornea26 and has been implicated in KC22,23 apart from other diseases. Matrix metalloproteinase-9 activity is upregulated by inflammatory cytokines TNF-α and IL-6,22 which consequently causes degradation of type I and IV collagen in eye disease.27 Therefore, factors affecting alteration in collagen structure of the KC cornea24,2830 could be targets to manage this disease, which currently does not have any medicines or eye drops for treatment. The copper-amine oxidase, LOX,31,32 cross links collagen and elastin33 into insoluble fibers via oxidation of adjacent peptidyl lysines (epsilon amino group) to reactive aldehydes. Reduction of LOX levels in KC has been reported.34 Additionally, a linkage study of familial and case-control KC patients suggests that LOX gene containing genomic loci may be associated with KC35 although pathogenic mutants were not found.36 We observed LOX to be reduced in KC23 epithelium in a severity-dependent manner at both the mRNA expression and enzymatic activity levels. Keratoconus is thus characterized by an altered corneal gene expression21 profile resulting in biomechanical changes24,28,30 affecting the normal corneal structure.29 Bone morphogenetic proteins (BMP) are a family of signaling factors belonging to the TGF-β superfamily and play important roles in embryonic development, extracellular matrix synthesis, tissue repair, and homeostasis.37 Among these, BMP-7 was identified as a major signaling molecule for the development of mammalian eye and kidney.38 Bone morphogenetic proteins-7 is a potent regulator of TGF-β signaling39 by counteracting the epithelial mesenchymal transition pathways, which is used in therapy for tissue remodeling in corneal opacity.40 Therefore, it is important to understand its role in the tissue remodeling process in KC. 
In addition, the KC cornea as a whole is treated as a diseased cornea, however, the thinning and protrusion that occurs in the disease is a localized phenomenon. The topographically defined characteristic zone around the cone apex has the maximum deformity, which progressively increases with increasing severity of disease. Further, the periphery of the ectatic area is often thicker, a “water-bed” effect. Despite this, the management options currently in practice for KC do not take into account such local phenomena. Therefore, with the advent of corneal cross-linking, a method to stabilize the KC cornea from progressing further, it is now imperative to understand how the ectatic and nonectatic areas of the KC cornea behave at a molecular level. We therefore analyzed, in a group of KC patients at a single center, the expression of various inflammation related (TNF-α, IL-6, IL-10), collagen structure–related (COL IVA1, COL IA1, and LOX) and extracellular matrix remodeling–related (MMP-9, TIMP-1, BMP-7) genes within the ectatic and nonectatic zones of KC patients. 
Materials and Methods
Clinical Study Design
This study was approved by the Narayana Nethralaya institutional review board (Bangalore, India) and carried out as per Indian Council of Medical Research and institutional human ethics guidelines in accordance to ARVO's policies and the tenets of the Declaration of Helsinki. All samples were collected after informed written consent. The study group was selected from patients who reported to the cornea clinic at Narayana Nethralaya. Keratoconus patients of Indian origin, undergoing standard-of-care surgical intervention for correction of visual defects were included in the study. 
A total of 66 KC patients undergoing transepithelial photorefractive keratectomy (T-PRK) or corneal cross-linking were included for collection of corneal epithelium intraoperatively. Additionally, 23 subjects with normal corneal topography, undergoing photorefractive keratectomy (PRK) for correction of refractive errors were included as controls for the collection of corneal epithelium samples. Patients using contact lenses or any type of anti-inflammatory systemic medications (e.g., antiallergic, anti-inflammatory drugs and steroids) were excluded from the study. Further, those who had undergone any prior ocular surgical intervention (e.g., penetrating keratoplasty/corneal collagen cross-linking and cataract surgery) for either eyes were excluded from the study. Patients with recent (<3 months) allergic or infection history either in the eye or systemic were also excluded. All subjects underwent a dry eye evaluation using Schirmer's test, tear break-up time (T-BUT), and corneal fluorescein staining. Subjects with concurrent symptoms of dry eye as per Dry Eye Work Shop (DEWS) classification were excluded. Patients with systemic inflammatory or autoimmune diseases were also excluded. There were no safety endpoints because there were no experimental interventions or any deviation from standard of care. 
Patient Diagnosis, Clinical Data Acquisition, and Grading
Patients were diagnosed for KC using retinoscopy, slit-lamp biomicroscopy, and corneal refraction measurements. Corneal topography of all the patients was acquired with Pentacam (OCULUS Optikgeräte GmbH, Wetzlar, Germany) upon first presentation at the clinic and used for diagnosis and grading of the KC patients.4,41 Keratoconus grades were determined from cumulative analysis of biomicroscopy data, slit-lamp, spherical and cylindrical refraction change, mean central keratometry measures, and corneal thickness as defined by the Amsler–Krumeich classification,42,43 but patients with grades III and IV were defined as severe KC group reported earlier.44 Corneal topography flat-axis keratometry (K1), steep-axis keratometry (K2), maximum keratometry (K-max), mean keratometry (Km), and central corneal thickness (CCT) were measured using Scheimpflug imaging (Pentacam; OCULUS Optikgeräte GmbH). 
Corneal deformation was assessed with Corvis-ST (OCULUS). Briefly, an air puff was applied on the cornea and images of one cross-section of the deforming cornea were captured over a period of 30 ms. Deformation amplitude (or the displacement of the corneal apex during applanation in millimeters) was recorded. Belin-Ambrosio enhanced ectasia display overall deviation index (BAD-D) of the eyes was recorded from Pentacam. The BAD-D index is a measure of severity of keratoconus (i.e., greater the magnitude of BAD-D, greater the severity of the disease observed in these patients). 
For Bowman's layer imaging, seven sequential 3-mm axial scans with a high-resolution Spectral-Domain Optical Coherence Tomography (SD-OCT) device (Envisu; Bioptigen Inc., Morrisville, NC, USA) was performed. The device has an axial digital resolution of 1.93 μm with a lateral digital resolution of 3 μm and an optical resolution of 2.4 μm in tissue. The same examiner performed the OCT imaging on all subjects. Every scan was performed along the nasal-temporal direction. 
Patient-Derived Corneal Epithelium
Epithelial cells were collected from epithelial debridement procedure routinely performed during ocular surface surgeries, such as corneal collagen cross-linking (CXL) or topography-guided photo refractive keratectomy (T-PRK) in KC patients. Control epithelial cells were collected from subjects undergoing PRK who demonstrated no corneal surface distortions and had no clinical signs of KC. The corneal epithelium study group had a total size of 23 controls and 66 KC patients (Grade 1 = 44, Grade 2 = 29, and Grade 3 = 17). Surgical procedures for all patients and controls were performed under topical anesthesia with Proparacaine 0.5% ophthalmic solution. In KC eyes undergoing CXL or T-PRK, the location of the cone was determined using the elevation map taken from corneal topography. A 4.5 mm trephine was used to mark the diameter of cornea centered at the KC cone apex referred to as “cone” leaving a superficial demarcation. The epithelium overlying this area was scraped with a mechanical scraper, followed by separate scraping of the surrounding epithelium over the peripheral 9-mm cornea referred to as “periphery” (Fig. 1). The underlying Bowman's layer was kept hydrated by moist Merocel sponges. The remainder of the surgical or cross-linking procedures were done as per normal clinical procedures for CXL or T-PRK. In controls undergoing PRK, a pupil centered 4.5-mm diameter was marked using trephine as ‘cone' and removed by mechanical scraping, whereas the surrounding epithelium extending approximately to 9 mm was obtained as ‘periphery' sample. Debrided epithelial cells were immediately transferred to −80°C for storage used later for RNA extraction. 
Figure 1
 
Method for obtaining epithelial samples from ectatic and nonectatic zones in cornea. (A) A representative en face photograph of the epithelial debridement from the patient cornea superimposed on the corresponding topographic scan indicating collection of epithelium from the ectatic cone area and nonectatic periphery. (B) Schematic representation of the surgical procedure used for collecting differential epithelium from the “cone” and “periphery” regions from the subjects.
Figure 1
 
Method for obtaining epithelial samples from ectatic and nonectatic zones in cornea. (A) A representative en face photograph of the epithelial debridement from the patient cornea superimposed on the corresponding topographic scan indicating collection of epithelium from the ectatic cone area and nonectatic periphery. (B) Schematic representation of the surgical procedure used for collecting differential epithelium from the “cone” and “periphery” regions from the subjects.
In patients with advanced KC, for vision correction, a full-thickness penetrating keratoplasy or corneal transplant procedure was performed. In these patients, a full thickness corneal resection is done followed by the placing a donor corneal graft with running sutures placed in radial fashion. From five such patients, we obtained matched epithelium and stromal samples of the disease cone and periphery as per the procedure mentioned earlier by ectasia zone marking using a trephine ring. Samples obtained intraoperatively were immediately frozen and stored at −80°C. 
Isolation of RNA, cDNA Synthesis, and Real-time PCR
Isolation of mRNA and analysis of gene expression have been described previously.22 Briefly, total RNA was extracted from patient's cone and peripheral regions of epithelia using 500 μL TRIZOL reagent treatment according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA) for 3 minutes followed by addition of 50 μL of chloroform and vigorous mixing. The mixture was then centrifuged and the clear aqueous layer was added to 600 μL of isopropyl alcohol followed by precipitation. The washed and resuspended RNA pellet was then quantified and quality assessed. Form stromal samples, the tissue was chopped for 1 minute in 50 μL of TRIZOL reagent followed by addition of remaining 250 μL and incubated for a total time of 3 minutes. Quantitative real-time PCR was performed as previously reported.45 The quantitative real-time PCR cycle includes preincubation at 95°C for 5 minutes, 40 amplification cycles at 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 30 seconds using a CFX Connect real-time PCR detection system (Bio-Rad, Philadelphia, PA, USA). Total levels of LOX, MMP-9, IL-6, IL-10, BMP-7, TNF-α, TIMP-1, COL IA1, and COL IVA1 were estimated after normalization to actin. Primer sequences are available on request. 
Statistical Analysis
All statistical analysis was performed with MedCalc v16.2.0 (MedCalc, Inc., Ostend, Belgium) and GraphPad Prizm 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). If the test was statistically significant, post hoc comparison of the group means was performed. One-way ANOVA was performed for comparison of groups. The data value of the individual groups was reported as average ± SEM. Linear regression analyses were performed among gene expression ratios for each gene as well as clinical parameters in this study. For smaller study group of the keratoplasty and Bowman's break subjects, the Mann-Whitney U test was done. 
Results
Clinical Analysis of Patient Study Group
The study group clinical characteristics are summarized in Table 1. The group consisted of 23 control, 29 KC Grade 1, 26 KC Grade 2, and 11 KC Grade 3 subjects. The corneal curvature increased with each grade of KC compared with controls as measured by keratometry; mean K values were controls: 44.38 ± 0.33; Grade 1: 46.14 ± 0.5, Grade 2: 49.44 ± 0.34, and Grade 3: 55.56 ± 0.67 (P value < 0.01). Manifest refraction spherical equivalent (MRSE) recorded were controls: −2.32 ± 0.19; Grade 1: −1.95 ± 0.23, Grade 2: −3.66 ± 0.5, and Grade 3: −9.21 ± 0.99 (P value < 0.01). Corneal measurement for thinnest corneal thickness (TCT) using Pentacam reduced across the grades as follows: controls: 503.17 ± 7.84; Grade 1: 467.65 ± 4.49, Grade 2: 456.12 ± 6.02, and Grade 3: 436 ± 8.34 (P value < 0.01). Consequently, corneal biomechanical properties of KC patients increased with each grade compared with controls; Corvis deformation amplitude (mm) measured showed controls: 1.16 ± 0.02; Grade1: 1.23 ± 0.02, Grade 2: 1.27 ± 0.02, and Grade 3: 1.35 ± 0.04 (P value < 0.01). BAD-D scores were control: 1.86 ± 0.13; Grade 1: 6.53 ± 0.44, Grade 2: 8.84 ± 0.47, and Grade 3: 10.71 ± 0.65 (P value < 0.01). Therefore, the clinical gradation correlates strongly with corneal curvature and vision loss. 
Table 1
 
Summary Statistics Table Showing Demographics and Clinical Spectrum of the Patient Group
Table 1
 
Summary Statistics Table Showing Demographics and Clinical Spectrum of the Patient Group
LOX and COL IVA1 mRNA Expression Is Reduced in the Ectatic Areas of the KC Corneal Epithelium
In order to address the question of focal changes in gene expression at the ectatic zone of the cornea, we reported the ratios of gene expression of the samples collected from cone to periphery for each subject. This will help us to minimize inherent, patient-specific gene expression differences that may be influenced by the genetic makeup, environmental stimuli, and clinical history. This ratio reflected the ectatic cone specific gene expression differences across the study group. In Figure 1A, we illustrate a schematic diagram of the surgical procedure performed to obtain the corneal epithelium from the ectatic and nonectatic areas. In Figure 1B, a representative KC eye is demonstrated with its corresponding topographic map with the superimposed image demonstrating the zone considered as cone and as periphery. 
In Figure 2A, we observed a significant reduction (P = 0.005) in LOX expression in the epithelial cells collected from KC patients (0.81 ± 0.09) compared with controls (1.62 ± 0.41). Next, we observed that cone specific LOX levels were reduced across the KC grades (Fig. 2B) suggesting its role in disease initiation and progression. The data also revealed a significant reduction (P = 0.023) in COL IVA1 levels in the KC cone area (0.85 ± 0.08) compared with controls (1.5 ± 0.41; Fig. 2C). Such a reduction in cone specific expression was observed across the grades as well (Fig. 2D). However, COL IA1 showed no significant differences in the gene expression ratio between the cone and periphery zones in KC subjects when compared with controls (Fig. 2E). There was a decrease in the ratio of COL IA1 expression in the higher grade, which is expected (Fig. 2F). Taken together, the data suggests a progressive reduction in LOX and COL IVA1 expression at the ectatic zone in the KC cornea with the progression of disease. 
Figure 2
 
Reduced mRNA expression of LOX, COL IVA1, and COL IA1 in the ectatic areas of the KC corneal epithelium. Ratio of relative mRNA expression values from cone and periphery of control versus KC group (A, C, E) and different grades of KC (B, D, F) for LOX (A, B), COL IVA1 (C, D), COL IA1 (E, F) in KC patients (different grades) compared with controls (*P value < 0.05).
Figure 2
 
Reduced mRNA expression of LOX, COL IVA1, and COL IA1 in the ectatic areas of the KC corneal epithelium. Ratio of relative mRNA expression values from cone and periphery of control versus KC group (A, C, E) and different grades of KC (B, D, F) for LOX (A, B), COL IVA1 (C, D), COL IA1 (E, F) in KC patients (different grades) compared with controls (*P value < 0.05).
Elevated MMP-9 Levels Are Observed in Ectatic Zone of KC Cornea but TIMP-1 and BMP-7 Remain Unchanged
The ratio of gene expressions from cone and peripheral cornea was analyzed for the matrix modulatory genes MMP-9, TIMP-1, and BMP-7. We observed a significant elevation of expression in MMP-9 (P = 0.038; Fig. 3A) in the cones of KC patients (2.45 ± 0.43) compared with controls (0.94 ± 0.17). It is interesting to note that the ratio of expression decreases with the grades of KC (Fig. 3B). This can be attributed to the higher zone of ectasia in these patients and the fact that the inflammation is higher in the severe grades of KC. Hence, because the periphery is also expressing a higher amount of MMP-9 in these patients, the ratio of cone to periphery reduces. However, TIMP-1 (Figs. 3C, 3D) and BMP-7 (Figs. 3E, 3F) do not show such local gene expression differences at the cone. 
Figure 3
 
Ectatic zone of keratoconus patients exhibit high MMP-9, unchanged TIMP-1, and BMP-7. Ratio of relative mRNA expression values from cone and periphery of (A, B) MMP-9, (C, D) TIMP-1, (E, F) BMP-7 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 3
 
Ectatic zone of keratoconus patients exhibit high MMP-9, unchanged TIMP-1, and BMP-7. Ratio of relative mRNA expression values from cone and periphery of (A, B) MMP-9, (C, D) TIMP-1, (E, F) BMP-7 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Inflammatory Gene Expression of TNF-α and IL-6 Is Increased at the Cone in KC Patients
Next, we evaluated transcript levels of TNF-α, IL-6, and IL-10 cytokines were compared between the cone and peripheral zones of the KC and control patients. Tumor necrosis factor-α expression ratios were significantly elevated (P = 0.008) suggesting upregulation at the cone apex in KC patients compared with controls (Fig. 4A) and the similar trend was observed across the KC grades (Fig. 4B). Although IL-6 showed an increase in KC patient cones over controls (Figs. 4C, 4D), the increase was not statistically significant. In contrast, IL-10 expression was reduced in the KC cones of Grade 3 patients compared with controls although the differences were not significant (Fig. 4E). It is interesting to note that cone-specific gene expression of IL-10 and TNF-α are opposite in Grade 3 KC (Figs. 4F, 4B, respectively). The individual cone and periphery data for each gene across the study group are summarized in Supplementary Tables S1 and S2
Figure 4
 
Inflammatory gene expression in ectatic zone of keratoconus patients exhibit high TNF-α, IL-6, and IL-10. Ratio of relative mRNA expression values from cone and periphery of (A, B) TNF-α, (C, D) IL-6, (E, F) IL-10 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 4
 
Inflammatory gene expression in ectatic zone of keratoconus patients exhibit high TNF-α, IL-6, and IL-10. Ratio of relative mRNA expression values from cone and periphery of (A, B) TNF-α, (C, D) IL-6, (E, F) IL-10 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Gene Expression Ratios of Corneal Cone Versus Peripheral Cornea in KC Patients Correlate With Clinical Parameters
The clinical parameters such as the steepest point of the KC cone apex (K-max), BAD-D, TCT (corneal pachymetry), and Corvis deformation amplitude (index of biomechanical characteristics) showed correlation with the progression of disease, and hence validated the patient classification methods used for this study group. We attempted to correlate our mRNA expression ratios obtained from the ectatic and nonectatic areas across the genes with clinical parameters (Table 2). The genes that significantly correlate are COL IA1 with BMP-7 (P = 0.0408) and TNF-α (P = 0.0061), LOX with COL IVA1 (P < 0.0001) and IL-10 (P = 0.0264), IL-10 with IL-6 (P = 0.0444) and MMP-9 (P = 0.0249). The ratio of gene expression values was further correlated with clinical and biomechanical parameters (Table 2). Those which significantly correlates were K-max with COL IVA1 (P = 0.0494); MMP-9 (P = 0.027) and LOX (P = 0.0086). BAD-D correlates with LOX (P = 0.0046); TNF-α (P = 0.0296). Tumor necrosis factor-α correlates with Corvis deformation amplitude (P = 0.0386); Km (P = 0.03) and TCT (P = 0.0102). 
Table 2
 
Correlation of Clinical Parameters With Ratio of Individual Gene Expression Values at the Cone and Periphery of the Subject Corneas
Table 2
 
Correlation of Clinical Parameters With Ratio of Individual Gene Expression Values at the Cone and Periphery of the Subject Corneas
The KC Patient Corneal Stroma Also Demonstrates Cone Versus Periphery Differences Similar to the Epithelium
Next, we wanted to test if the cone area of KC stroma also demonstrates similar local changes in gene expression as observed for the epithelium from the same patients. We therefore obtained cone and periphery samples from both stroma and epithelium of the Grade 4 KC patients (n = 5) undergoing keratoplasty. Figure 5 illustrates individual relative gene expression ratios from cone and peripheral areas of the stroma as well as epithelium from individual patients (relative to the expression value of epithelium periphery of each patient). LOX expression was significantly lower in the cone of the stroma (Fig. 5Aii) while both collagen genes demonstrated high variability in expression (Figs. 5B, 5C), perhaps owing to the advanced disease stage of the disease. We observed that relative MMP-9 levels were much higher in the stroma compared with epithelium in KC corneas and compared with the paired periphery, MMP-9 was increased in the cone in both epithelium and stroma (Fig. 5D). TIMP-1 was also expressed at a higher level in the stroma compared with epithelium, but was reduced in the cone compared with the periphery (Fig. 5E). Bone morphogenic protein-7 expression levels were comparable across stroma and epithelium but were reduced in the cone (Fig. 5F). The data illustrates that the differences in the gene are more prominent focally at the cone apex of the stroma compared with the peripheral stroma and are similar to the observed trend in the epithelium. 
Figure 5
 
Keratoconus patient corneal stroma demonstrates cone and periphery differences. Relative gene expression of cone and periphery of both stroma and epithelium from the same set of patients (n = 5) is plotted individually or as an average for the group. The expression values are shown relative to the value at the epithelial periphery for (Ai, Aii) LOX, (Bi, Bii), COL IVA1, (Ci, Cii) COL IA1, (Di, Dii) MMP-9, (Ei, Eii) TIMP-1, (Fi, Fii) BMP-7, (Gi, Gii) TNF-α, (Hi, Hii) IL-6, (Ii, Iii) IL-10. Statistical significance; *P value < 0.05; between the groups and P value #, <0.05 within the same group.
Figure 5
 
Keratoconus patient corneal stroma demonstrates cone and periphery differences. Relative gene expression of cone and periphery of both stroma and epithelium from the same set of patients (n = 5) is plotted individually or as an average for the group. The expression values are shown relative to the value at the epithelial periphery for (Ai, Aii) LOX, (Bi, Bii), COL IVA1, (Ci, Cii) COL IA1, (Di, Dii) MMP-9, (Ei, Eii) TIMP-1, (Fi, Fii) BMP-7, (Gi, Gii) TNF-α, (Hi, Hii) IL-6, (Ii, Iii) IL-10. Statistical significance; *P value < 0.05; between the groups and P value #, <0.05 within the same group.
KC Patients With Bowman's Layer Defects Demonstrate Distinct Proinflammatory Gene Expression Profile
It is not very well understood what triggers KC. We observed that few KC patients have a breach in their Bowman's layer using a hand-held OCT device. Figure 6 illustrates three representative cases of KC subjects with observed breach and three KC subjects without a breach in Bowman's layer. This is supported by the adjacent topographic map, which illustrates the corneal deformity in each case. For the assessment of Bowman's layer breach, each subject was scanned at seven different areas. We then analyzed the focal gene expression changes at the cone apex and periphery in these subjects with breach (n = 4) and without breach (n = 10). Figure 7 demonstrates the gene expression differences observed among these two groups of KC patients. We observed a reduction in LOX (Fig. 7A) and COL IVA1 (Fig. 7B) in these patients in both cone and periphery coinciding with an increase in TNF-α (Fig. 7D) and reduction in IL-10 (Fig. 7E). The reduction in LOX and COL IVA1 is more pronounced in the cone region of the KC patients with breaks in the Bowman's layer. Interluekin-6 expression was not significant (Fig. 7F). Interestingly, COL IA1 demonstrates an increase in expression in the patients with Bowman's breach (Fig. 7C) suggesting an injury specific response. Matrix metalloproteinase-9 expression was also showed similar differences between the two groups except in the periphery of the Bowman's breach cases (Fig. 7G). TIMP-1 levels were reduced in the periphery of the Bowman's breach samples that correlates to the increased MMP-9 in the same samples (Fig. 7H). Although no statistical differences were observed in BMP-7 expression, the trends show reduction in the cone but slightly elevated in the subjects with breach (Fig. 7I). Together, this data illustrates a distinct gene expression profile in the corneal epithelium from KC patients that have a breach in their Bowman's layer suggestive of an injury-induced inflammation. The reduction in LOX and COL IVA1 could be an indication of aberrant healing processes. 
Figure 6
 
Breach in Bowman's layer observed in some subset patients with KC. Representative cases of KC subjects with observed frank breach in the Bowman's layer indicated by circle (AC) and matched KC subjects without a breach (DF).
Figure 6
 
Breach in Bowman's layer observed in some subset patients with KC. Representative cases of KC subjects with observed frank breach in the Bowman's layer indicated by circle (AC) and matched KC subjects without a breach (DF).
Figure 7
 
Normalized mRNA expression of KC patients with Bowman's break (n = 4) compared with that from age matched KC (controls, n = 10) subjects without Bowman's breach in epithelium differentially collected from cone and periphery region. Statistical significance represented as *P value < 0.05; **<0.01 between the groups and P value #<0.05 within the same group. Normalized gene expression of (A) LOX, (B) COL IVA1, (C) COL IA1, (D) TNF-α, (E) IL-10, (F) IL-6, (G) MMP-9, (H) TIMP-1, (I) BMP-7.
Figure 7
 
Normalized mRNA expression of KC patients with Bowman's break (n = 4) compared with that from age matched KC (controls, n = 10) subjects without Bowman's breach in epithelium differentially collected from cone and periphery region. Statistical significance represented as *P value < 0.05; **<0.01 between the groups and P value #<0.05 within the same group. Normalized gene expression of (A) LOX, (B) COL IVA1, (C) COL IA1, (D) TNF-α, (E) IL-10, (F) IL-6, (G) MMP-9, (H) TIMP-1, (I) BMP-7.
Discussion
The molecular pathogenesis of KC is complex, involving a variety of extracellular matrix remodeling factors, cellular signaling factors, and inflammatory pathways. Recently, local corneal inflammatory response has emerged as a primary driver of disease pathogenesis.1012,14,15,22,23 These pathways drive the structural weakness in the collagen layer of the cornea leading to focal weakening and protrusion of the ectatic cornea observed in KC. In our previous study, we reported elevated levels of MMP-9 and inflammatory cytokines in KC tears.22 However, activity of the collagen cross-linking enzyme LOX was reduced in KC tears.23 In addition, the repair collagen type located in the corneal basement membrane, COL IVA1 was significantly reduced in KC patients.23 However, we do not fully understand why the disease is localized to a small ectatic zone, particularly in the initial stages. 
In an attempt to further understand the molecular processes driving KC, we examined differences in local gene expression in epithelial and stromal cells between ectatic and nonectatic zones of patient corneas. By examining the differences within the same patient, we were able to normalize for inherent genetic, environmental, and individual differences. We observed reduced LOX and COL IVA1 expression whereas MMP-9 and inflammatory cytokines were elevated (Figs. 215524) in the ectatic zones of the corneas in KC patients. This suggests that the focal weakening at the cone apex in KC may be triggered by an inflammation driven mechanism, which eventually results in the matrix disorganization during disease progression. It is interesting to note that both LOX and COL IVA1 gene expression ratios significantly correlate with each other (Table 2) and show negative correlation with the inflammatory cytokines. Similarly, immunomodulatory cytokine, IL-10 correlates significantly with LOX, IL-6, and MMP-9 illustrating the active, abnormal inflammatory milieu being involved in the disease (Table 2). In addition, TNF-α expression levels positively correlate with BAD-D, CORVIS, and Km but negatively with TCT measurements (Table 2) suggesting its expression at the diseased, ectatic area of the cornea is elevated with increasing disease severity. K-max negatively correlates with both LOX and COL IVA1 but positively with MMP-9 (Table 2) indicating a relationship between matrix dysregulation, focal corneal weakening, and disease progression. LOX also negatively correlated with BAD-D (Table 2). Together, these data demonstrate that inflammation-mediated changes in the corneal collagen and LOX expression and regulation locally at the cone apex may drive the focal corneal deformity in KC. 
This focal area specific expression was also observed in the KC stroma when gene expression was measured in paired stroma and epithelium from keratoplasty patients (Fig. 5). The paired epithelium and stroma data demonstrates that expression levels in both epithelium and stroma mimic each other although the levels of individual genes are different in each layer. Although the small sample number in this experiment is a limitation, it should be noted that the data shows concordance with the epithelium data from the larger study group. Despite the advanced disease in these samples, which rendered the ectatic zone much wider, the average trends of the expression ratios held true for key genes like LOX, MMP-9, COL IVA1, and so on. The reduction in BMP-7 in the cone of both stroma and epithelium is observed in these samples (Fig. 5) suggestive of abnormal wound healing response, which favors scarring in advanced disease. In fact, exogenous BMP-7 has been demonstrated to reduce scarring and fibrosis40 in the cornea. Finally, we attempted to quantify and correlate differential gene expression in KC patients with breach in Bowman's layer and compared them with KC patients having intact Bowman's layer (Fig. 6). The focal gene expression patterns in these patients show an increase in the proinflammation proteins TNF-α and MMP-9 while reduced amounts of regulatory cytokine IL-10 (Fig. 7). It has been shown that inflammatory stimulus, particularly, TNF-α treatment reduces collagen synthesis46 and/or deposition.47 In addition, TNF-α treatment also inhibits LOX production.47 Most importantly, we found significantly reduced mRNA expression levels of LOX and COL IVA1 in these subjects. This is indicative of a gene expression profile responding to the ongoing injury or increased stromal epithelial interaction that destabilizes the cornea. It should be noted that the data in this set was compared with a set of KC patients without any detectable Bowman's layer breaks. The small size of BL breach group is a limitation, but the observations are important when taken in context of the epithelium data. These observations suggest that an initial breach in the Bowman's layer barrier function triggering epithelial–stromal interactions may be an initiating event in KC. This may be caused due to a microtrauma, excessive eye rubbing, or inherent deformities; all leading to a sustained inflammatory stimulus. Previous studies have shown that the KC cornea demonstrates irregularities in the Bowman's layer, abnormal stromal collagen structure due to scarring, and focal fibrotic deposits30 suggesting history of microtrauma in the cornea of these patients. Further, scarring due to contact lens and abnormal eye rubbing has also been implicated in KC pathogenesis.8 The CLEK study reports 5-year corneal scarring incidence at 13.7% overall and 16.7% for KC patients wearing contact lens.48 In addition, atopy, connective tissue disorders, and genetic variations is genes such as collagens and TGF-β have also been implicated in KC,3,9 all of which relate to the possibility of corneal injury or matrix-related degeneration. These factors cumulatively support a thesis that the disease may be driven primarily by an underlying inflammatory process initiated in a focal area by a breach in the corneal epithelial–stromal barrier, the Bowman's layer. 
In conclusion, the results of this study demonstrate that KC is driven by inflammatory factors and dysregulated extracellular matrix factors in the cone apex of the cornea. Further, it suggests that matrix remodeling factors such as LOX and MMPs play an important role in this process, while the inflammatory factors may trigger the disease pathogenesis and progressive thinning of the cornea. Therefore, it follows that altered local expression of molecular factors in the ectatic cone area could be driving the focal weakening in KC corneas. We therefore postulate that surgical correction of KC using collagen cross-linking should be concentrated at the cone in KC corneas rather than the entire cornea as currently practiced. 
Acknowledgments
Supported by Narayana Nethralaya Foundation (Bangalore, India) and Allergan (AG and RS; Bangalore, Karnataka, India). This research was also partially supported by the Vision Group in Science and Technology (VGST/GRD-374; AG; Bangalore, Karnataka, India) and Ruth M. Kraeuchi Missouri Endowed Chair Ophthalmology fund (RRM; Columbia, MO, USA). 
Disclosure: N. Pahuja, None; N.R. Kumar, None; R. Shroff, None; R. Shetty, Allergan (F); R.M.M.A. Nuijts, None; A. Ghosh, None; A. Sinha-Roy, None; S.S. Chaurasia, None; R.R. Mohan, None; A. Ghosh, Allergan (F) 
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Figure 1
 
Method for obtaining epithelial samples from ectatic and nonectatic zones in cornea. (A) A representative en face photograph of the epithelial debridement from the patient cornea superimposed on the corresponding topographic scan indicating collection of epithelium from the ectatic cone area and nonectatic periphery. (B) Schematic representation of the surgical procedure used for collecting differential epithelium from the “cone” and “periphery” regions from the subjects.
Figure 1
 
Method for obtaining epithelial samples from ectatic and nonectatic zones in cornea. (A) A representative en face photograph of the epithelial debridement from the patient cornea superimposed on the corresponding topographic scan indicating collection of epithelium from the ectatic cone area and nonectatic periphery. (B) Schematic representation of the surgical procedure used for collecting differential epithelium from the “cone” and “periphery” regions from the subjects.
Figure 2
 
Reduced mRNA expression of LOX, COL IVA1, and COL IA1 in the ectatic areas of the KC corneal epithelium. Ratio of relative mRNA expression values from cone and periphery of control versus KC group (A, C, E) and different grades of KC (B, D, F) for LOX (A, B), COL IVA1 (C, D), COL IA1 (E, F) in KC patients (different grades) compared with controls (*P value < 0.05).
Figure 2
 
Reduced mRNA expression of LOX, COL IVA1, and COL IA1 in the ectatic areas of the KC corneal epithelium. Ratio of relative mRNA expression values from cone and periphery of control versus KC group (A, C, E) and different grades of KC (B, D, F) for LOX (A, B), COL IVA1 (C, D), COL IA1 (E, F) in KC patients (different grades) compared with controls (*P value < 0.05).
Figure 3
 
Ectatic zone of keratoconus patients exhibit high MMP-9, unchanged TIMP-1, and BMP-7. Ratio of relative mRNA expression values from cone and periphery of (A, B) MMP-9, (C, D) TIMP-1, (E, F) BMP-7 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 3
 
Ectatic zone of keratoconus patients exhibit high MMP-9, unchanged TIMP-1, and BMP-7. Ratio of relative mRNA expression values from cone and periphery of (A, B) MMP-9, (C, D) TIMP-1, (E, F) BMP-7 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 4
 
Inflammatory gene expression in ectatic zone of keratoconus patients exhibit high TNF-α, IL-6, and IL-10. Ratio of relative mRNA expression values from cone and periphery of (A, B) TNF-α, (C, D) IL-6, (E, F) IL-10 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 4
 
Inflammatory gene expression in ectatic zone of keratoconus patients exhibit high TNF-α, IL-6, and IL-10. Ratio of relative mRNA expression values from cone and periphery of (A, B) TNF-α, (C, D) IL-6, (E, F) IL-10 in KC patients (different grades) compared with controls or separated by disease grade (*P value < 0.05).
Figure 5
 
Keratoconus patient corneal stroma demonstrates cone and periphery differences. Relative gene expression of cone and periphery of both stroma and epithelium from the same set of patients (n = 5) is plotted individually or as an average for the group. The expression values are shown relative to the value at the epithelial periphery for (Ai, Aii) LOX, (Bi, Bii), COL IVA1, (Ci, Cii) COL IA1, (Di, Dii) MMP-9, (Ei, Eii) TIMP-1, (Fi, Fii) BMP-7, (Gi, Gii) TNF-α, (Hi, Hii) IL-6, (Ii, Iii) IL-10. Statistical significance; *P value < 0.05; between the groups and P value #, <0.05 within the same group.
Figure 5
 
Keratoconus patient corneal stroma demonstrates cone and periphery differences. Relative gene expression of cone and periphery of both stroma and epithelium from the same set of patients (n = 5) is plotted individually or as an average for the group. The expression values are shown relative to the value at the epithelial periphery for (Ai, Aii) LOX, (Bi, Bii), COL IVA1, (Ci, Cii) COL IA1, (Di, Dii) MMP-9, (Ei, Eii) TIMP-1, (Fi, Fii) BMP-7, (Gi, Gii) TNF-α, (Hi, Hii) IL-6, (Ii, Iii) IL-10. Statistical significance; *P value < 0.05; between the groups and P value #, <0.05 within the same group.
Figure 6
 
Breach in Bowman's layer observed in some subset patients with KC. Representative cases of KC subjects with observed frank breach in the Bowman's layer indicated by circle (AC) and matched KC subjects without a breach (DF).
Figure 6
 
Breach in Bowman's layer observed in some subset patients with KC. Representative cases of KC subjects with observed frank breach in the Bowman's layer indicated by circle (AC) and matched KC subjects without a breach (DF).
Figure 7
 
Normalized mRNA expression of KC patients with Bowman's break (n = 4) compared with that from age matched KC (controls, n = 10) subjects without Bowman's breach in epithelium differentially collected from cone and periphery region. Statistical significance represented as *P value < 0.05; **<0.01 between the groups and P value #<0.05 within the same group. Normalized gene expression of (A) LOX, (B) COL IVA1, (C) COL IA1, (D) TNF-α, (E) IL-10, (F) IL-6, (G) MMP-9, (H) TIMP-1, (I) BMP-7.
Figure 7
 
Normalized mRNA expression of KC patients with Bowman's break (n = 4) compared with that from age matched KC (controls, n = 10) subjects without Bowman's breach in epithelium differentially collected from cone and periphery region. Statistical significance represented as *P value < 0.05; **<0.01 between the groups and P value #<0.05 within the same group. Normalized gene expression of (A) LOX, (B) COL IVA1, (C) COL IA1, (D) TNF-α, (E) IL-10, (F) IL-6, (G) MMP-9, (H) TIMP-1, (I) BMP-7.
Table 1
 
Summary Statistics Table Showing Demographics and Clinical Spectrum of the Patient Group
Table 1
 
Summary Statistics Table Showing Demographics and Clinical Spectrum of the Patient Group
Table 2
 
Correlation of Clinical Parameters With Ratio of Individual Gene Expression Values at the Cone and Periphery of the Subject Corneas
Table 2
 
Correlation of Clinical Parameters With Ratio of Individual Gene Expression Values at the Cone and Periphery of the Subject Corneas
Supplement 1
Supplement 2
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