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Review Article

Biochemical Markers and Alterations in Keratoconus

Shetty, Rohit FRCS, PhD; D'Souza, Sharon MS, FCE; Khamar, Pooja FCRS, PhD; Ghosh, Arkasubhra PhD; Nuijts, Rudy M.M.A. MD, PhD; Sethu, Swaminathan PhD

Author Information
Asia-Pacific Journal of Ophthalmology: November-December 2020 - Volume 9 - Issue 6 - p 533-540
doi: 10.1097/APO.0000000000000332
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Abstract

Keratoconus (KC) is a corneal ectatic condition characterized by focal structural changes, resulting in progressive thinning, biomechanical weakening, and steeping of the cornea that can lead to worsening visual acuity due to irregular astigmatism and corneal scarring in more advanced cases.1,2 KC often presents as a bilateral condition; however, in some instances unilateral or asymmetric presentation have also been reported.1 The prevalence of KC worldwide ranges between 0.3 and 4790 cases per 100,000 population,3 with a calculated global prevalence of 1.38 cases per 1000 population.4 Higher prevalence of KC was observed in certain ethnic populations such as Middle Eastern, South Asian, and Maori.3 The incidence will continue to rise due to advances in, and access to imaging technology, both pivotal in the diagnosis of KC. Typically, the disease onset is during the second decade of life and progresses until the fourth.3 KC can be progressive in some patients and spontaneously stabilized in others. In the early stages of the disease, glasses or soft contact lenses are effective in correcting the refractive error. However, with progressive weakening of the cornea and increase in irregular astigmatism, glasses would no longer be as effective. Different types of contact lenses have been found to be useful in visual rehabilitation of KC patients in different stages. With advances in contact lens technology and the availability of specialty lenses for irregular corneas, even advanced cases of KC can be visually rehabilitated to good vision. Corneal cross-linking procedures have been shown to be effective in slowing or halting the progression of the disease. Additional management strategies that have been reported to be effective include intracorneal ring segments or topography-guided treatments. Corneal transplant would be required to restore vision in cases of very advanced KC with extreme ectasia and scarring, where other strategies to improve vision will not be possible. Despite the advances made in the management of KC, there are some unmet clinical needs such as predicting progression and treatment outcomes, disease monitoring, specific therapeutic targets, and additional information to support personalized/patient-specific treatment. Harnessing the knowledge of biochemical changes in the KC would aid in addressing these needs.

PATHOBIOLOGY OF KC

KC is characterized by global ultrastructural and molecular changes, resulting in a focal macro–structural change in the form of steepening with consequent decrease in visual acuity. Critical structural changes are observed in corneal epithelium, epithelial basement membrane, Bowman layer, and stroma in KC.5 Epithelial changes include thinning particularly over the cone with thickening in the periphery.6,7 Expression of extracellular matrix (ECM) components such as collagens, fibrin, and laminin in epithelial basement membrane are altered in KC.5 Discontinuity or defects in Bowman layer, at times with fibrotic changes in these interrupted sections of the layer, were observed.2,6 Key structural changes in the KC stroma include alterations in the lamellar organization, lateral fiber alignment, fibril density, fibril diameter, and proteoglycan density, along with fewer keratocytes.5,8 However, the specificity, sequence, and proportion of contribution of these ultrastructural changes reported in KC cornea in the development of biomechanical weakening, typical conical protrusion, or steeping resulting in loss of visual acuity is yet to be ascertained. The structural and ECM component changes observed in KC range from ectatic to fibrotic changes based on the stage of the disease process. The scarring or fibrotic changes in KC suggest the dynamic and reparative nature of ECM remodeling process attempting to compensate for the degenerative events that lead to disease initiation and progression. Therefore, understanding the role of stage-specific molecular factors that contribute to ultrastructural changes in KC cornea is necessary. Figure 1 outlines the key plausible events in the pathogenesis of KC. It illustrates the relationship between biochemical factors and corneal tissue in contributing toward KC. Hence, it is essential to determine the various local and systemic biochemical alterations in KC.

FIGURE 1
FIGURE 1:
Proposed keratoconus pathogenesis. Structural and molecular evidence indicates that keratoconus is a stromal condition with epithelial contribution and/or involvement. Schematic outlines the various plausible events involved in the pathogenesis of keratoconus. (i) Physical and/or biological stimuli including environmental insults, injury, atopic conditions, eye rubbing, and hormonal imbalances can induce changes in the corneal epithelial status, which includes changes in epithelial thickness pattern and secretion of various active biochemical factors (indicated as small squares) including inflammatory factors and MMPs. These bioactive factors can further induce changes in barrier function and cellular metabolism in the epithelium. (ii) These bioactive factors produced by epithelium can collectively act on the stromal cells (keratocytes), resulting in their altered functional status and density. The altered stromal cells affect the ECM remodeling processes, leading to aberrant collagen synthesis, arrangement (by reduced eCXL activity and associated cross-links), and degradation. (iii) This results in altered stromal biomechanics and biochemical milieu that sets in a feed-forward mechanism, in both the stroma and epithelium with amplification of the detrimental bioactive factors and structural weakening, resulting keratoconus initiation and progression. BL indicates Bowman layer; DM, Descemet membrane; ECM, extracellular matrix; eCXL, endogenous cross-linking enzyme; MMP, matrix metalloproteinase.

ALTERED BIOCHEMICAL FACTORS IN KC

To unravel the intricacies involved in KC pathogenesis, efforts are being focused on determining the biochemical changes in the stroma, epithelium, tear film, aqueous humor, serum, saliva, and cultured primary corneal keratocytes of KC patients. ECM remodeling events are significantly influenced by multiple biochemical factors such as ECM components, mediators of cellular homeostasis, inflammatory factors, hormones, metabolic products, and chemical elements (Fig. 2).

FIGURE 2
FIGURE 2:
Altered biochemical factors in keratoconus. The schematic summarizes the various biochemical factors either increased or decreased in keratoconus patients. The biochemical factors have been broadly grouped as metabolic factors, hormones, factors associated with cellular homeostasis, chemical elements, inflammatory factors, and extracellular matrix proteins that include collagens, proteoglycans, glycosaminoglycans, cross-links, and proteolysis enzymes. The arrows are indicative of the combined potential of these factors in regulating the various alterations in the extracellular matrix remodeling components and events in keratoconus patients. IFNγ indicates interferon-γ; IgE, immunoglobulin E; IL, interleukin; MMP, matrix metalloproteinase; TNF-α, tumor necrosis factor-alpha.

ECM-Related Factors

Dysregulation in the formation and assembly of core ECM proteins, mainly collagens, are implicated in KC pathogenesis. Decreased total collagen and deficient interlacing have been reported in KC cornea.7,9 Specifically, the expressions of collagen I, VI, VII, XII, and XIII were decreased in KC epithelium.10–12 Collagen I, III, IV, V, VI, and XII levels were lower in the stroma of KC cornea.10–12 Prolidase activity that facilitates collagen turnover or synthesis was also reduced in the tear fluid13 and serum13,14 of KC patients. A distinct and varied pattern was observed in the expressions of collagen I and IV in epithelium and stroma of KC patients with and without breaks in Bowman's layer.15 Furthermore, the expressions of collagen I and IV were different in the epithelium between ectatic and nonectatic regions.15 The levels of collagen IV, fibronectin, and laminin-1 were lower in nonscarred region of the epithelial basement membrane, but increased expressions of collagen IV, VII, fibronectin, fibrillin-1, laminin-1, and laminin-5 were observed in the scarred epithelial basement membrane region of KC cornea.16 Fibronectin and vimentin were also higher in KC stroma.17–19 Variations in the expression patterns of core ECM proteins could be based on the stage of disease and there could also be cellular reparative response to compensate for the ongoing pathology. Hence, correlation of the expression profile of ECM proteins with the stage of disease and ultrastructural details are required.

Proteoglycans and glycosaminoglycans are another group of important components of the ECM, as they interact with fibrillary collagens and facilitate their organization, contributing to the biomechanical and optical properties of the cornea. Altered proteoglycans along the collagen fibrils have been observed in KC cornea compared with controls.20,21 Expressions of biglycan, lumican, osteoglycin, syndecan-1, syndecan-2, perlecan, and keratan sulfate were lower in KC.11,22–25 Other proteoglycans such as decorin, tenascin, keratocan, and dermatan sulfate were increased in KC cornea.17–19,23,25 Furthermore, studies are required to determine the specific contributions of these altered proteoglycans and glycosaminoglycans profile in KC pathogenesis.

Collagen cross-links are critical contributors toward the biomechanical characteristics of any tissue, including cornea. The most dominant cross-link type, lysinonorleucine was decreased in KC cornea compared with controls.26 Lysyl oxidase (LOX) is a key enzyme that facilitates endogenous cross-linking between collagen and elastin fibrils. The expressions of LOX and LOX-like isoforms (LOXL2, LOXL3, LOXL4) were reduced in KC.10,27,28 The expression of LOX in the ectatic zone was lower than in the nonectatic zone15 and was associated with corneal cross-linking outcomes.29 In addition, the enzymatic activity of LOX in the tear fluid exhibited a declining trend with increasing grades of KC.10 These observations clearly emphasize the relevance of endogenous cross-linking enzyme in KC pathogenesis.

Proteolysis enzymes play a crucial role in ECM remodeling by modulating collagen degradation. Imbalance in these factors, as observed in KC, would result in structural and biomechanical weakening of the tissue. Activity of proteolytic enzymes including collagenase, gelatinase, peptidase, and heparanase was increased in KC.30–32 Cathepsins, a class of proteolytic enzymes, were higher in the cornea (cathepsin-B,33 -G,33 -V/L234) and tear fluid (cathepsin-S30) of KC patients. Matrix metalloproteinases (MMPs), another class of proteolytic enzymes, were also increased in the cornea (MMP2,35 MMP910,15,36), tear fluid (MMP9,30,32,36–43 MMP1330,41,44,45), serum (MMP2,46 MMP947), and corneal fibroblasts (MMP948) of KC patients. On the contrary, major proteinase inhibitors (endogenous regulators of these proteolytic enzymes) such as alpha 1-proteinase inhibitor,49 alpha 2-macroglobulin,50 and tissue inhibitor of matrix metalloproteinase-134 were significantly lower in KC.

The expression of MMP9 was higher in the ectatic region compared with nonectatic region in KC cornea.15 MMP9 levels were positively associated with the severity of KC and pathologically with the various corneal metrics in KC patients.10,36,37,39,40 A higher percentage of KC patients with associated allergy tested positive for elevated tear fluid MMP9 (using point-of-care diagnostic test) compared with those KC patients without allergy.37 An increased proportion of KC patients with allergy who tested positive for elevated tear fluid MMP9 exhibited disease progression.37 MMP9 levels were shown to be decreased in the tear fluid after topical cyclosporine treatment36 and corneal cross-linking procedure38 in patients, along with cessation in disease progression. These observations clearly suggest the pivotal role of MMP9 in KC pathogenesis. Similarly, elevated MMP13 level in the tear fluid of KC patients was associated with severity and progression of the disease.30,41,44,45 It is important to note that eye rubbing, a known risk factor in KC pathogenesis, leads to an increase in collagenase activity and MMP13 level in the tear fluid of normal subjects.51 Cumulatively, these findings strongly suggest the imbalance in the status of proteolysis enzymes and their endogenous regulators in KC, thus contributing toward disease pathogenesis. Hence, it would be useful to have strategies to reduce the elevated levels of proteolysis enzymes to improve KC prognosis.

Cellular Homeostasis–Associated Factors

Biochemical factors associated with antioxidative stress response, tissue healing, protein degradation, and host defences were altered in KC. Oxidative stress is a known etiological factor associated with KC pathogenesis. The stress is due to the imbalance between pro-oxidant and antioxidant factors. Oxidative stress in KC was evident with the increase in reactive oxygen species in tear fluid,52 8-oxo-2’-deoxyguanosine in corneal tissue,53 and total oxidant status in the serum54 of KC patients. The antioxidant factors such as glutathione,52,55 nuclear factor erythroid 2-related factor 2 (a transcription factor that plays a crucial role in antioxidant process, including regulation of glutathione production),56 hemeoxygenase,57 superoxide dismutase,58 and heat shock protein-2719 were reduced in KC. Galectin-1 and galectin-3, important for cellular and healing response in cornea and known to mediate antiinflammatory response and reepithelialization process, were observed to be higher in the epithelium and cultured keratocytes of KC patients.43 Lactoferrin, a key contributor to host defence against infection, was reduced in KC tear fluid59,60 and epithelium,11 along with reduction in sIgA (secretory immunoglobulin A) in the tears of KC patients.59 There also seems to be an altered protein degradation machinery in KC cornea with increased levels of ubiquitin19 and decreased autophagy (lower levels of light chain 3-II and lysosomal-associated membrane protein 1) in KC.61 Furthermore, KC keratocytes have exhibited an increase in apoptosis and endocytosis-related proteins suggestive of potential degenerative changes in them and their function.11 A more comprehensive differential status of cellular homeostasis regulators can be determined from high-throughput transcriptomic and proteomic profiling of KC tissues (such studies are listed in a later section of the article), to better understand KC pathogenesis.

Hormones

KC has been associated with conditions related to hormonal imbalance. It has been reported that the prevalence of thyroid gland dysfunction among KC patients was higher than in the normal population.62,63 KC was reported in patients suffering from thyrotoxicosis and Hashimoto disease.64,65 Furthermore, the levels of thyroxine was higher in tear fluid63 and aqueous humor66 of KC patients. Increased expression of thyroxine receptors was also observed in the epithelium and stroma of KC cornea.63 Pregnancy is now considered a risk factor for KC progression due to coincidental, unfavorable corneal biomechanics changes, possibly because of the hormonal alterations during pregnancy.67 The observation was substantiated by other clinical reports stating progression of stable KC after estrogen- and/or progesterone-based hormone replacement therapy for different indications.68,69 Increased expressions of estrogen alpha receptors and androgen receptors along with decreased progesterone receptors were observed in KC corneal epitheilum.70 A decrease in the level of serum and salivary estriol and estrone, and raised dehydroepiandrosterone sulfate was reported in KC patients.55,71 Prolactin and prolactin-induced protein were reported to be reduced in the aqueous humor, tear fluid, blood, and/or saliva of KC patients.66,71 Vitamin D, a steroid hormone well known for its function in various cellular processes including modulation of inflammation and stress response, was observed to be lower in the serum of KC patients compared with controls and the normal range.72 It is also reported that the percentage of vitamin D-deficient subjects were higher in KC group compared with controls and vitamin D deficiency was associated with increased predisposition to KC.73

Metabolic Factors

A distinct set of metabolites was identified to be altered in KC cornea compared with healthy controls after metabolomics profiling using gas chromatography and mass spectrometry.74 The key class of metabolites that was downregulated in KC cornea compared with controls include carboxylic acids, fatty acids, sterols, and hexadecanol.74 The altered metabolites were associated with cellular response pathways related to cellular energetics, oxidative stress, inflammation, and tissue damage.74 Altered hormone-induced cellular bioenergetics or metabolism was observed in cultured corneal fibroblasts from KC patients.55,75,76 A recent study reported altered metabolite or organic acid profile in the tear fluid post–corneal cross-linking77, indicating metabolic factor changes after cross-linking. With currently available metabolomic profile in KC, such as the one reported recently,74 it is imperative to determine causal relationship of metabolic alterations with aberrant ECM remodeling in KC, to determine disease-modifying strategy in the management of KC.

Chemical Elements

Chemical element imbalance has been implicated in the pathogenesis of KC. Copper is a key element, which is implicated in KC due to its essential regulatory role in mediating LOX activity, necessary for endogenous collagen cross-linking process. The levels of copper have been reported to be lower in the serum of KC patients.72,78 It is hypothesized that there could be a copper-deficient state in the center of cornea due to impaired copper ion transition caused by altered pH.79 This copper-deficient state could result in compromised LOX activity leading to initiation of KC.79 This thought process was corroborated by higher copper deposition in the Fleischer ring zone compared with other areas of the KC cornea.79 Similarly, there was an increase in the deposition of iron in the Fleischer ring zone compared with other areas of KC cornea.79 Altered iron metabolism was hypothesized to be contributing to KC, as they function as a cofactor for the formation of hydrolysine essential for the regulation of collagen fiber diameter. However, no difference was observed in the levels of iron in the serum of KC patients compared with controls.78 Incidentally, lactoferrin, an iron-binding protein, was reduced in KC tear fluid59,60 and epithelium,11 which implicates altered iron homeostasis in KC cornea. Levels of other elemental constituents essential for cellular homeostasis including zinc,46,72 selenium,72,78 and magnesium78 were lower in the serum of KC patients. It is essential to explore specific functional status of these elements in KC corneal tissues to bring out the causal association of these elements in KC pathogenesis.

Inflammatory Factors

Inflammatory mediators including cytokines and chemokines are known to modulate ECM remodeling process in both health and disease. Despite KC being defined as a noninflammatory condition, it has been frequently associated with an increase in proinflammatory factors such as interleukin-1 alpha/beta (IL-1α/β), IL-6, and tumor necrosis factor-alpha (TNF-α). IL-1α/β is increased in the tear fluid,30,80–82 cornea,19,83 and serum47 of KC patients. Studies have reported an increase in the levels of IL-6 in either tear fluid,30,36,40 serum,47 corneal sections,84 epithelium,36 or cultured fibroblasts85 of KC patients. Importantly, the expression of IL-6 was higher in epithelium over ectatic zone compared with nonectatic zone.15 Furthermore, eye rubbing was also shown to increase the IL-6 level in tear fluid in study subjects.51 The concentration of IL-8 was higher in the tear fluid30,43,86 and saliva55 of KC patients as well. An increase in the IL-17 family of cytokines including IL-17A,80,87 IL-21,80 and IL-2380 in the tear fluid was observed in KC. It is relevant to note that IL-17 is known to induce the levels of MMP9, thus implicating IL-17 in KC pathogenesis. TNF-α, elevated in tear fluid,81 serum,84 cornea tissue,84 and epithelium36 of KC patients, exhibited a positive association with Belin/Ambrosio deviation display, mean keratometry, and deformation amplitude; and a negative relationship with the thinnest corneal thickness.15 Increased expression of TNF-α was observed in KC patients with defects in the Bowman layer than those without.15 Eye rubbing raised the level of TNF-α in the tear fluid of the test subjects.51 Increased interferon-γ (IFNγ) level in the tear fluid of KC patients was observed to be directly associated with disease progression45 and inversely correlated with central corneal thickness.81 Elevated serum and tear fluid IgE levels88 in KC patients with and without atopy or other allergic conditions provide the molecular association between KC and allergic conditions. Other allergy-associated cytokines such as IL-4,30,80 IL-5,30,80 and IL-1380 were also elevated in the tear fluid of KC patients. The levels of transforming growth factor-beta, an antiinflammatory and profibrotic factor, were higher in tear fluid80 and corneal sections19 of KC patients, which may contribute to the compensatory or reparative responses as seen with ECM changes discussed earlier. These evidences suggest that inflammatory mediators could be key contributing factors in KC pathobiology.

High-Throughput Molecular Profiling

Microarray and ribonucleic acid sequencing–based high-throughput transcriptomic profiling have shown close to a thousand differentially expressed genes in cornea,56 epithelium,61 and stroma89 of KC compared with non-KC controls. Another array-based study revealed that more than 10 micro–ribonucleic acids were differentially expressed in KC epithelium compared with controls.90 Mass spectrometry-based high-throughput proteomic profiling demonstrated differential regulation of hundreds of proteins unique to KC cornea,91 epithelium,92 stroma,92 tear fluid,60 and aqueous humor.93 Proteomic profiling of cultured KC stromal cells has also shown a distinct profile compared with controls.11 Transcriptomic and proteomic studies revealed that genes/proteins related to the regulation of ECM remodeling, inflammation, and cellular stress response were dysregulated in KC.

Comorbidities, Genetic, and Epigenetic Predisposition

Atopy and allergic conditions are major comorbidities that have long been associated with KC pathogenesis. Biomolecular alterations such as elevated IgE and itch factors in atopy, allergy and eye rubbing, and their underlying mechanisms related to KC pathobiology were extensively reviewed by Ahuja et al.88 The associations between KC and other key comorbidities have been listed elsewhere.3 Genome-wide association studies, linkage studies, single-nucleotide polymorphisms, and candidate gene analysis across various ethnic populations have reported associations between KC and genetic variants in ECM remodeling, inflammation, and cellular homeostasis–associated genes.94,95 In addition, epigenetic alterations such as DNA (deoxyribonucleic acid) hypomethylation and hypermethylation have also been reported in KC cornea.96 It is important to associate the genetic/epigenetic variations and biochemical factor alteration in KC to further the understanding of the causal relationship.

FUTURE DIRECTION

Despite the knowledge on ultrastructural changes and biochemical perturbations in KC, the mechanism underpinning the focal nature of the condition remains a mystery. It is quite apparent that KC is a multifactorial condition and the contributing biochemical factors are quite varied among the patient population. As illustrated in Figure 3, the various stimuli, biological pre-dispositions, and comorbidities could contribute to the diversity of these biochemical factors and disease progression dynamics in KC. Despite the continued efforts being made to determine the various biochemical changes in KC, the lack of knowledge integration impedes our understanding of the disease. Comprehensive network analysis of the various biochemical factors would help identify critical molecular pathways that result in altered ECM remodeling that drives KC pathogenesis. An integrated strategy that is designed to study the ultrastructure, biomechanics, and biomolecular profile in the same corneal tissue would improve our understanding of pathogenic events in KC. Furthermore, the knowledge would enable the clinical utility of altered biochemical factors in disease monitoring, prevention, and management of KC.

FIGURE 3
FIGURE 3:
Multifactorial contribution in keratoconus pathogenesis. The schematic suggests that various environmental or external stimuli, in addition to oxidative stress, can damage ocular surface tissues. The cellular protective responses to restore homeostasis are influenced by genetic/epigenetic status, nutritional deficiencies, comorbidities, hormonal therapy, eye rubbing, and major physiological events such as pregnancy. The external stimuli in the background of various predispositions listed could contribute to unique and altered biochemical profile reported in keratoconus. The unique biochemical milieu would influence the extracellular matrix remodelling processes in the cornea with consequent initiation, stabilization, or progression of keratoconus. Therefore, determining the biochemical alterations specific to a patient would provide critical cues in understanding keratoconus pathogenesis, disease monitoring, and possibly, additional therapeutics to prevent disease progression.

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Keywords:

biochemical; extracellular matrix; hormones; inflammation; keratoconus

Copyright © 2020 Asia-Pacific Academy of Ophthalmology. Published by Wolters Kluwer Health, Inc. on behalf of the Asia-Pacific Academy of Ophthalmology.