The Proteins of Keratoconus: a Literature Review Exploring Their Contribution to the Pathophysiology of the Disease

Home , AZGP1, BANP, CRB1, IPO5, Keratin 12, MAL (gene)

Adv Ther (2019) 36:2205–2222 https://doi.org/10.1007/s12325-019-01026-0

REVIEW

Eleftherios Loukovitis . Nikolaos Kozeis . Zisis Gatzioufas . Athina Kozei . Eleni Tsotridou . Maria Stoila . Spyros Koronis . Konstantinos Sfakianakis . Paris Tranos . Miltiadis Balidis . Zacharias Zachariadis . Dimitrios G. Mikropoulos . George Anogeianakis . Andreas Katsanos . Anastasios G. Konstas

Received: June 11, 2019 / Published online: July 30, 2019 Ó The Author(s) 2019

ABSTRACT abnormalities primarily relate to the weakening of the corneal collagen. Their understanding is Introduction: Keratoconus (KC) is a complex, crucial and could contribute to effective man- genetically heterogeneous multifactorial agement of the disease, such as with the aid of degenerative disorder characterized by corneal corneal cross-linking (CXL). The present article ectasia and thinning. Its incidence is approxi- critically reviews the proteins involved in the mately 1/2000–1/50,000 in the general popula- pathophysiology of KC, with particular tion. KC is associated with moderate to high emphasis on the characteristics of collagen that myopia and irregular astigmatism, resulting in pertain to CXL. severe visual impairment. KC structural Methods: PubMed, MEDLINE, Google Scholar and GeneCards databases were screened for relevant articles published in English between January 2006 and June 2018. Keyword combi- Enhanced Digital Features To view enhanced digital nations of the words ‘‘keratoconus,’’ ‘‘risk features for this article go to https://doi.org/10.6084/ m9.ﬁgshare.8427200.

E. Loukovitis K. Sfakianakis Hellenic Army Medical Corps, Thessaloniki, Greece Division of Surgical Anatomy, Laboratory of Anatomy, Medical School, Democritus University of E. Loukovitis Á N. Kozeis Á A. Kozei Á E. Tsotridou Á Thrace, University Campus, 68100 Alexandroupolis, M. Stoila Á S. Koronis Á P. Tranos Á M. Balidis Á Greece Z. Zachariadis Á G. Anogeianakis Ophthalmica Eye Institute, Vas Olgas 196 and D. G. Mikropoulos Á A. G. Konstas (&) Ploutonos 27, 54655 Thessaloniki, Greece 1st and 3rd University Departments of Ophthalmology, Aristotle University, Thessaloniki, Z. Gatzioufas Greece Augenklinik, Hornhaut, Katarakt und Refractive e-mail: [email protected] Chirurgie, Universitaetsspital Basel, Mittlere Strasse 91, 4031 Basel, Switzerland G. Anogeianakis Association for Training in Biomedical Technology, A. Kozei Aristogeitonos 6, 54638 Thessaloniki, Greece School of Pharmacology, University of Nicosia, Makedonitissis 46, 2417 Nicosia, Cyprus A. Katsanos Department of Ophthalmology, University of E. Tsotridou Á M. Stoila Ioannina, Ioannina, Greece Faculty of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece 2206 Adv Ther (2019) 36:2205–2222 factor(s),’’ ‘‘genetics,’’ ‘‘genes,’’ ‘‘genetic associa- Scholar and GeneCards databases for articles tion(s),’’ ‘‘proteins’’, ‘‘collagen’’ and ‘‘cornea’’ related to KC. The keywords used were ‘‘kera- were used. In total, 272 articles were retrieved, toconus,’’ ‘‘risk factor(s),’’ ‘‘genetics,’’ ‘‘genes,’’ reviewed and selected, with greater weight ‘‘genetic association(s),’’ ‘‘proteins,’’ ‘‘collagen’’ placed on more recently published evidence. and ‘‘cornea’’ and all their relevant combina- Based on the reviewed literature, an attempt was tions. The search focused on articles written in made to tabulate the up- and down-regulation of English from January 2006 until June 2018. A genes involved in KC and their protein products total of 177 articles were identiﬁed and and to delineate the mechanisms involved in reviewed, and both their text and references CXL. were analyzed. The analysis of these references Results: A total of 117 proteins and protein revealed an additional 95 relevant articles, classes have been implicated in the pathogene- which were also subsequently reviewed. The sis and pathophysiology of KC. These have been current article is based on previously conducted tabulated in seven distinct tables according to studies and does not contain studies on human their gene coding, their biochemistry and their subjects or animals performed by any of the metabolic control. authors; its aim is to summarize current Conclusion: The pathogenesis and pathophys- knowledge on the proteins involved in KC. It iology of KC remain enigmatic. Emerging evi- also represents an attempt to map and catego- dence has improved our understanding of the rize the proteins that are up- or down-regulated molecular characteristics of KC and could fur- in KC according to their corneal stromal ther improve the success rate of CXL therapies. location.

Keywords: Collagen; Corneal biomechanical characteristics; Corneal cross-linking; CXL; RESULTS Ectasia; Keratoconus proteins; Ophthalmology Histopathology, Biochemistry and Biomechanics of KC Corneas INTRODUCTION Histologically, KC is characterized by degrada- Keratoconus (KC) is a corneal dystrophy that tion of the basal membrane of the corneal seriously affects the quality of life of KC patients epithelium, diminishing of the number and [1]. Its prevalence ranges between 1/2000 and density of collagen fibrils, thinning of the cor- 1/50,000 in the general population. The condi- neal stroma, and keratocyte apoptosis with tion is characterized by moderate to substantial necrosis [2–4, 9]. Keratocyte apoptosis and visual impairment due to the development of necrosis involves the central anterior stroma corneal protrusion and thinning [2–6]. In KC, and Bowman’s lamina and, typically, weakens visual acuity decreases due to progressive myo- the corneal tissue [12, 13]. Slit-lamp examina- pia, irregular astigmatism and, often, corneal tion reveals subepithelial and anterior stromal apical opacification [4, 6, 7]. The key underlying scars [14] due to degeneration of epithelial basal factor for these pathological changes is weak- cells, as well as fine folds in the posterior stroma ening of collagen tissue in corneal stroma [6, 8]. at the corneal apex (Vogt’s striae). Endothelial Although KC is generally a bilateral disorder damage is rarely visible. The presence of Fle- [2, 9, 10], rare unilateral cases have also been isher’s ring, which is formed by the accumula- described [11]. tion of hemosiderin around the base of the corneal cone [2, 15], is also frequent. In contrast, breaks of the Descemet membrane with METHODS subsequent leakage of aqueous humor into the corneal stroma, leading to corneal edema and The present review is based on literature search decompensation (acute hydrops), are rare. that utilized the PubMed, MEDLINE, Google Finally, evident corneal nerves together with a Adv Ther (2019) 36:2205–2222 2207 decrease in sub-basal nerve density are promi- hydroxyproline concentrations [23]—an indi- nent features of advanced KC [2, 15, 16]. cation that the alteration of corneal biome- The stroma in KC is characterized by a chanical properties in KC are the result of decrease in the number of collagen lamellae and collagen cleavage into smaller peptides [23]. a reduction in the amounts of microfibrillar and This conclusion is further supported by the fine granular material [17]. Changes are also observation that the solubility of pepsin-treated observed in the arrangement of fibrils of the collagen from KC corneas is greater than that anterior stroma [15, 18], together with abnor- from normal corneas—apparently due to the mal distribution of collagen fibers. This results breakdown of KC corneal collagen into smaller in the decrease of corneal mechanical resistance peptides that has already occurred [23]. [15, 18]. Overall, the thinning of the corneal stroma Collagen itself is associated with keratocyte apoptosis, alterations in the extracellular matrix (ECM), The amount of collagen, which is a major cor- and changes in the activities of several enzymes neal protein [2, 24, 25], is reduced in KC [12, 19] that involve the activation of degrading [12, 26]. The decrease in collagen types I and III enzymes and the promotion of cell death, both is related to alterations in the ECM basement of which are mostly due to oxidative stress membrane [26], while the decrease in collagen [20, 21]. Although the mechanisms of tissue type IV is related to alterations in the basement breakdown remain obscure [21], over the last membranes, of which collagen type IV is the 25 years there has been strong evidence that major structural component [24, 27, 28]. In collagens are susceptible to oxygen-free radical addition to the decrease in the amount of col- damage [20, 22]. In addition, collagen is the lagen in the cornea, the shape and transparency only protein susceptible to fragmentation by of the cornea also change due to re-orientation superoxide anions, a process during which of the collagen molecules and fibrils [29, 30]. small 4-hydroxyproline-containing-peptides are The combination of changes in collagen orien- liberated [20]. In the presence of oxygen, tation and the reduction of total collagen con- hydroxyl radicals cleave collagen into small tribute to corneal thinning in KC [30]. peptides; the cleavage is, apparently, specific to Type IV collagens are major structural com- proline or 4-hydroxyproline residues [20]. In ponents of basement membranes and consist of contrast, hydroxyl radicals, in the absence of six proteins encoded by six genes (COL4A1– oxygen, do not induce fragmentation of colla- COL4A6). Interestingly, these genes are orga- gen molecules, but they trigger the polymer- nized in pairs in a head-to-head conformation ization of collagen through the formation of so that each gene pair shares a common pro- new cross-links such as dityrosine or disulfide moter [31]. Thus, the COL4A6 gene is organized bridges [20]. in a head-to-head conformation with the Thinning of the stroma and alterations of COL4A5 (alpha 5 type IV collagen) gene. Simi- ECM in KC affect the biomechanical properties larly, the gene pairs for COL4A1 and COL4A2 of the cornea. Comparisons of uniaxial tensile and for COL4A3 and COL4A4 are also con- strength between KC and normal corneas have formed head-to-head [32]. This means that shown that maximum load and stress, maxi- deletions in one member of the pair of genes mum stiffness, and relative energy absorption extend into the other; it also means that alter- were smaller in keratoconic corneas than in native splicing results in multiple transcript normal ones [2, 23]. Furthermore, load and variants encoding different isoforms [24, 33]. stress values at corresponding strain values were Collagen type IV a1/a2 chains have been smaller in keratoconic compared to normal reported in KC patients [34], while COL4A1 has corneas both for the initial (exponential) and been shown to be down-regulated in KC corneas for the linear parts of the curves [23]. In addi- [35]. The chromosomal loci of COL4A1 and tion, no differences between KC and normal COL4A2 are close to 13q32 [32], with the corneas have been detected in uronic acid or 2208 Adv Ther (2019) 36:2205–2222

COL4A1 gene consisting of 52 exons and Other Proteins Involved in KC COL4A2 gene of 48 exons [28]. Mutations in COL4A3 and COL4A4 may be related to There are more than 1500 different peptides decreases in collagens I and III which contribute and proteins in the human tears, both of intra- the increase of the KC risk [28]. It is noteworthy and extracellular origin. Their anatomic loca- that there are several reports that COL4A3 has tion, the genes that code for them and their been found to be associated with KC in at least potential implication in KC are summarized in two European populations [28, 36]. Further- Tables 1, 2 and 3. They include cytokines and more, genomic loci that differ between ethnic small molecules and are followed by lipids and groups and are associated with variation in metabolites [44, 45]. Among them, one can central corneal thinning [37, 38] are found in distinguish lactoferrin, secretory immunoglob- the genes that encode COL1A1, COL1A2, ulin A, tear lipocalin, lysozyme, lipophilin, COL8A2 and COL5A1 [37, 38]. proline-rich proteins and serum albumin, all of There are seven polymorphisms (M1327 V, which help maintain the health of the ocular V1516 V and F1644F in COL4A4, and P482S, surface [44–46]. Since abnormal levels of P141L, D326Y and G895G in COL4A3) that are enzymes and inflammatory molecules are found associated with KC under recessive, dominant in KC patients [44, 47, 48], the deregulation of or additive models [28, 39]. Finally, the expres- proteins has been linked to the thinning of the sion of collagen types XII, XIII, XVIII and XV is cornea [14, 49]. Changes in the structural altered in KC, although no relationships have integrity of the cornea including changes in the been identified between mutations in these collagen content (e.g., the reduction in the genes and their expression [40, 41]. It should be number of collagen lamellae) [2, 9], and alter- noted that collagen types XIII, XV, and XVIII ations in enzymes have been reported in KC are mainly expressed in basal corneal cells, and patients [14, 49]. The biochemical abnormali- that these types are involved in the adhesion ties observed in the KC cornea (epithelium and between corneal epithelial cells as well as stroma) include the increase in the level of between corneal epithelial cells and the base- degradative enzymes and the decrease of the ment membrane [35, 41]. protease inhibitors [50]. Thus, changes in collagen have a vital role in Recent clinical studies imply the existence of the progression of KC. Ensuring collagen sta- an inflammatory component in the pathogen- bility is the main target in corneal cross-linking esis of KC [47, 51]. This means that tissue (CXL), a relatively new, minimally invasive degradation in KC involves the expression of outpatient procedure used for the management pro-inflammatory cytokines, inflammatory of KC. Before the advent of CXL, there was no mediators, cell adhesion molecules and matrix treatment to modify the underlying patho- metalloproteinases (MMPs) [47, 51, 52]. physiology and arrest corneal ectasia. The Inflammation in KC is characterized by the introduction of this method to ophthalmology presence of many inflammatory cells and goes back to 1997 [42]. Subjective and objective markers such as IL-1, IL-6, MMP-9, TGF-b, and results following this method seem to be TNF-a [47, 51–54]. Moreover, inflammatory promising. CXL is a photo-induced reaction, factors like IL-4, IL-5, IL-6, IL-8, TNF-a, TNF-b, used to increase the rigidity of the corneal col- MMP-1, MMP-3, MMP-7, MMP-9, MMP-13 and lagen and its resistance to ectasia, ensuring the cathepsins are increased in the tears of KC mechanical and biochemical stability of the patients [47, 51, 52], a finding that suggests the stromal tissue. Ultraviolet light A at 370 lm deregulation of the underlying molecular excites riboflavin (vitamin B2) which functions pathways [55]. Table 4 summarizes the proteins as a photosynthesizer. Free radicals and oxidiz- that have been identified as definitive or ing substance cause the formation of new potential biomarkers in KC together with the covalent bonds between collagen fibrils within genes that are known to code for them. the cornea which stabilize stroma [43]. Adv Ther (2019) 36:2205–2222 2209

Table 1 Proteins (and their genes) of the corneal epithelium and their regulation in keratoconus Up-regulated Down-regulated Protein Gene Protein Gene [reference] [reference] S100 calcium-binding protein A4 S100A4 [44] Calpain small subunit 1 (CAPNS1) CAPNS1 (S100A4) [14, 44] Cytokeratin KRT [44] FTH 1 (ferritin heavy chain protein 1) FTH1 [14, 44] Gelsolin GSN [44] Annexin A2 ANXA2 [14, 44] Alpha enolase (ENO1) ENO1 [44] Heat shock protein beta 1 (HSPB1) HSPB1 [14, 44] Keratin-5 (KRT5) KRT5 [14, 44] Annexin A8 ANXA8 [14, 44] L-lactate dehydrogenase (LDH) LDH [14, 44] Serum albumin ALB [14, 44] SFRP1 (secreted frizzled-related protein 1) SFRP1 [44]

Table 2 Proteins (and their genes) found in both epithelium and stroma and their regulation in keratoconus Up-regulated Down-regulated Protein Gene [reference] Protein Gene [reference] Increased epithelial and stromal Decreased epithelial and stromal Keratin type I cytoskeletal 14 (KRT14) KRT14 [14, 44] Transketolase TKT [14, 44] Keratin type I cytoskeletal 16 (KRT16) KRT16 [14, 44] Pyruvate kinase PKLR [14, 44] Tubulin beta chain (TUBB) TUBB [14, 44] Stratiﬁn (14-3-3 sigma isoform) SFN [14, 44] Lamin-A/C (LMNA) LMNA [14, 44] Phosphoglycerate kinase 1 (PGK 1) PGK1 [14, 44] S100 calcium-binding protein A4 S100A4 [14, 44] NAD (P)H dehydrogenase NQO1 [14, 44] (S100A4) (quinone) 1 Heat shock cognate 71 kDa protein HSPA8 [14, 44] (HSPA8)

Protein Changes in KC and the a2 macroglobulin decrease [14, 50]. Corneal thinning therefore is caused by the up- Changes in proteins that might contribute to regulation of cellular proteases and the down- corneal thinning include the increase of regulation of their inhibitors [14]. Both enzyme degradative enzymes such as acid phosphatases, up-regulation and enzyme inhibitor down-reg- acid esterases and acid lipases [24]. In eyes with ulation involve the mRNA as well as the protein KC, the levels of enzymes such as cathepsin B levels of expression [56]. The increase of corneal and cathepsin G increase, while protease inhi- proteinase activity is also involved in the bitors like the a1 proteinase inhibitor (a1-PI) pathophysiology of KC [50]. As a result, KC may 2210 Adv Ther (2019) 36:2205–2222

Table 3 Proteins (and their genes) exclusive of the stroma and their regulation in keratoconus Up-regulated Down-regulated Protein Gene [reference] Protein Gene [reference] Vimentin VIM [14, 44] b-actin (ACTB) ACTB [14, 49, 71] Keratocan (KTN) KERA [14, 44] TGF-beta (transforming growth factor beta) TGFBI [14, 44] induced ig-h3 (Bigh3) protein Decorin DNC [14, 44] Meprin A-5 protein, and receptor protein-tyrosine 14, 44 phosphatase mu (MAM domain) Keratin 12 KRT12 [14, 44] Serotransferrin TF [14, 44] Haptoglobin HP [14, 44] HuR (human antigen R) ELAVL1 [14, 49, 71] precursor (HP) Apolipoprotein A-IV APOA4 [14, 44] Isoforms 2C2A of collagen alpha-2 (VI) chain COL6A2 [14, 44] precursor (APOA4) ALDH3A1 (aldehyde ALDH3A1 [14, 44] dehydrogenase 3A1) Lipoprotein Gln LPL [14, 44] Prolipoprotein 14, 44 TIMP3 TIMP3 [81, 142] TIMP1 TIMP1 [14] develop because of defective regulation of the biomarker for KC [63]. Analysis of the tear pro- proteinase activity in the cornea [50]. Nitroty- teome shows that the key for the maintenance rosine malonaldehyde, glutathione S trans- of a healthy corneal structure is the quality of ferase, and inducible nitric oxide synthase levels tear fluids [65]. Three-dimensional cultures of increase, while aldehyde dehydrogenase and human KC cells [66, 67] show an increase in superoxide dismutase levels decrease [14]; in oxidative stress levels when compared to nor- addition, there is an accumulation of GAG mal human corneal fibroblast cultures [68]. (glycosaminoglycan) polyanions in keratoconic Other known pathogenic factors are prolactin- corneas [57], while analysis of the KC corneal induced protein (PIP) [69] which is involved in proteome indicated a decrease in decorin, ker- actin binding and the zinc-alpha-2-glycoprotein atocan, lumican, and biglycan [58]. Keratan (AZGP1) [65]. The interplay between these two sulfate (KS) antigenicity appears to decrease in proteins (PIP-AZGP1) is apparently involved in the central, thinned region of the keratoconic KC pathogenesis [63]. cornea [59], while the KS organization in cor- PIP expression is regulated by transforming neas with advanced KC is markedly different growth factor-b (TGF-b), which has a vital role from that in healthy corneas [60]. Interestingly, in corneal wound healing and is implicated in there are molecular alterations in proteoglycans the pathophysiology of KC [65]. The TGF-b in some connective tissue disorders that are signaling pathway is a complex, multibranched associated with KC [61, 62]. signal transduction cascade that may modulate Gross cystic disease fluid protein-15 ECM, making it a potential contributing factor (GCDFP15), a secretory glycoprotein, expressed in KC pathogenesis [21]. However, increases of in the proteome of human tear fluids [63], TGF-b2 levels in the aqueous humor of consists of 14 kDa [64] and is a potential Adv Ther (2019) 36:2205–2222 2211

Table 4 Proteins that serve as biomarkers in keratoconus protein 1 (SP1) characterize KC corneas [74]. and their genes Species-specific tRNA processing (STP1) transcription factor is involved in tRNA maturation; Proteins Gene [reference] STP1 transcription factor belongs to a specificity COL8A1 COL8A1 [40] protein/Kruppel-like factor family, and it shares structural similarities and sequence homology COL8A2 COL8A2 [40] with 25 other members of this family [75, 76]. Basal corneal cells The over-expression of SP1 is associated with Collagen type XII COL12A1 neuro-degenerative diseases such as Hunting- ton’s chorea [77] and tumorigenesis [78], while [35, 41] the promoter activity of the human a1-PI gene Collagen type XIII COL13A1 in corneal cells is suppressed by the up-regula- [35, 41] tion of Sp1 [79, 80]. Other proteins such as secreted protein Collagen type XVIII COL18A1 acidic and rich in cysteine (SPARC), human [35, 41] leukocyte antigen, mitochondrial complex I Collagen type XV COL15A1 genes and 2q21.3 with RAB3 GTPase-activating [35, 41] protein subunit 1 (catalytic) are also involved in KC [81–83]. Luminac, biclycan, keratocan and IL1A IL1A decorin decrease in KC corneas [84], whereas [119, 126, 127] keratocytes express heparan sulfate proteogly- IL1B IL1B cans [85, 86]. In addition, bone morphogenic [119, 126, 127] protein 4 and the gene and protein expression of TGF are increased in KC corneas [21, 87]. SPARC (secreted protein acidic and SPARC [81–83] The epithelial–endothelial IL-1 system is rich in cysteine) vital, not only for the organization of the cor- Human leukocyte antigen 81–83 nea but also for its ability to respond to mechanical injuries and pathogen invasions, by TIMPs TIMP provoking keratocyte apoptosis [88]. The up- [68, 81, 142] regulation of IL-1 in KC induces keratocyte SLC4A11 (sodium bicarbonate SLC4A11 apoptosis [89] and manifests itself as an increase transporter-like protein 11) [91, 92] in IL-1 protein levels and a simultaneous rise in the number of IL-1 binding sites [87, 90]. The AQP5 (water channel protein AQP5 [93] IL-1 family consists of pleiotropic cytokines. aquaporin 5) The genes that encode for the IL-1 cytokines are located on chromosome 2q14 [89] and their transcription is affected by polymorphisms keratoconic patients are not confirmed by located in this region [89]. Finally, cytokines IL- immunofluorescence studies [21]. 1a and IL-1b control the immune response, the The mRNA of human antigen R (HuR) is a pro-inflammatory response and hematopoiesis; very important post-transcriptional regulator of their effects are modulated by the IL-1 receptor gene expression that is expressed in all prolif- antagonist [91]. erating cells [70]. The expression of mRNA and Sodium bicarbonate transporter-like protein protein levels of b-actin and HuR are decreased 11 (SLC4A11) belongs to the SLC4 family of on KC corneal stroma compared to normal bicarbonate transporters [91] and may partici- corneas [14, 49, 71], indicating that the down- pate in KC pathogenesis, because its functional regulation of b-actin and HuR is the result of failure leads to keratocyte apoptosis [91, 92]. their mutual interaction [71]. The SLC4A11 gene is located in the 20p13 KC is also associated with amyloid deposits chromosome and functions as an electrogenic, [72, 73], and increased levels of specificity Na ? coupled, borate co-transporter [91]. Also, 2212 Adv Ther (2019) 36:2205–2222 the water channel protein, aquaporin 5, may be chain are down-regulated [14, 44]. Of the pro- a marker for KC [93]. teins of the corneal epithelium, keratin-5, Some HLA antigens, including HLA-A26, B40 annexin A8, L-lactate dehydrogenase and serum and DR9, have been linked to early onset KC albumin are up-regulated [14, 44], and calpain [82]. In addition, keratoconic corneas manifest small subunit 1, Ferritin heavy chain protein 1 a decrease of IGF-2 transcripts and over-expres- (FTH 1), annexin A2 and heat shock protein sion of IGF-binding proteins 3 and 5 [94]. Pro- beta 1 are down-regulated [14, 44]. teins such as dual-specificity phosphatases or Recently, the role of b-galactoside-binding mitogen-activated protein kinase phosphatases proteins galectin (Gal)-1 and Gal-3 in patients (MKPs) are activated by oxidative stress in KC with KC and postcorneal CXL treatment was [95]. The same proteins regulate the immune investigated in vitro [98]. These proteins are response and dephosphorylate tyrosine and associated with various cellular responses that threonine residues on MKPs [96]. involve inflammation [99]. In KC, increased Proteomic analyses of KC patient epithelia levels of Gal-1 and Gal-3 were detected in con- using two-dimensional gel electrophoresis fol- junctival epithelial cells compared to controls lowed by mass spectrometry [44] identified high [98]. In addition, keratocytes of KC patients over-expression of S100A4, cytokeratin and were found to release significant amounts of gelsolin in keratoconic epithelium [44], while Gal-1 in the stroma. In vitro, CXL promoted the alpha enolase was slightly up-regulated. Other release of Gal-1 from keratocytes but decreased studies, using the same method, suggest that the concentration of inflammatory biomarkers, beta actin and alpha enolase are slightly such as IL-6, IL-8, MMP-2 and MMP-9 [98]. expressed in corneal wing and superficial Overall, these results suggest that CXL exerts an epithelial cells of KC patients [44, 49]. It is immunosuppressive effect on keratocytes by interesting that proteins like gelsolin and inhibiting the release of MMPs and cytokines cytokeratins are implicated in ocular (e.g., vit- and increasing the levels of anti-inflammatory reoretinopathy) and non-ocular (e.g., cystic Gal-1 [98]. fibrosis, cancer, steatohepatitis) disorders [44, 97]. The FAS/FASLG Genes and Apoptosis Corneal epithelial and stromal proteins are expressed differentially in KC [14, 44], and six Apoptosis is considered the primary pathway of epithelial proteins, keratin type I cytoskeletal cell death in KC [13]. Diseases related to defec- 14, keratin type I cytoskeletal 16, tubulin beta tive apoptotic mechanisms are associated with chain, lamin-A/C, S100-A4 and heat shock polymorphisms in the FAS [100] and FASLG cognate 71 kDa protein, were identified using a [101] genes [102, 103]. The FAS-encoded protein label-free nano-ESI-LC–MS/MS method to be belongs to the TNF-receptor superfamily and increased in KC. Five other proteins, transketo- contains a death domain. It has a central role in lase, pyruvate kinase, 14-3-3 sigma isoform, the physiological regulation of programmed cell phosphoglycerate kinase 1 and nicotinamide death, whereby its interaction with its ligand adenine dinucleotide phosphate, and dehydro- leads to the formation of a death-inducing sig- genase (quinone) 1, are decreased [14, 44]. naling complex that includes the FADD (Fas- Stromal proteins, such as vimentin, kerato- associated death domain protein) and caspases can, decorin, keratin 12, haptoglobin precursor, 8 and 10 and is responsible for apoptosis [100]. apolipoprotein A-IV precursor, aldehyde dehy- The FAS isoforms that lack the transmembrane drogenase 3A1 (ALDH3A1), lipoprotein Gln and domain may negatively regulate the apoptosis prolipoprotein, are up-regulated in KC [14, 44]. mediated by the full-length isoform. The FASLG while A-5 protein, TGF-beta (transforming gene also belongs to the TNF superfamily. It growth factor beta) ig-h3 (Bigh3), receptor pro- encodes a transmembrane protein which, when l tein-tyrosine phosphatase- (MAM) domain- it binds to FAS, triggers apoptosis [101]. containing protein 2, serotransferrin, meprin, and isoforms 2C2A of collagen alpha-2 (VI) Adv Ther (2019) 36:2205–2222 2213

The FAS/FASLG system is expressed in the are more penetrant or associated with a more cornea and potentially has an important func- severe outcome [112, 114]. These variants have tion in the physiology of the normal cornea, as been identified in diverse populations and well as the pathophysiology of corneal diseases localities. Thus, KC chromosomal loci [103, 104] through the modulation of kerato- 16q22.3–q23.1 [115] were identified in 20 small cyte apoptosis following epithelial injury. Finnish pedigrees [115]; locus 20q12 was iden- Indeed, IL-1 stimulation induces corneal tified in an isolated population in Tasmania, fibroblasts to produce apoptosis-associated FAS Australia [116]; loci 5q22.23–q24.2 were iden- ligand. The fact that the same cells also express tified in a large Northern Irish family [117]; loci the FAS receptors makes them ideal candidates 5q14.3–q21.1 were identified from a large four- for inducing autocrine suicide of keratocytes in generation white family pedigree [118]; and loci KC corneas. In addition, the development of KC 3p14–q13 were identified in an Italian two- is also characterized by an increase in the cor- generation autosomal dominant pedigree [119]. neal sensitivity to apoptotic cytokines owing to Other loci associated with KC have been abnormalities in the components of the FAS mapped to chromosomes 20p11.21 (KTCN1) pathways [88]. This association between [120], 16q22.3–q23.1 (KTCN2) [115], 3p14–q13 chronic keratocyte apoptosis and ongoing (KTCN3) [119], 2p24 (KTCN4) [121], epithelial injury could be the link to previously 1p36.23–36.21 [122], 4q31 [116, 123], unrecognized risk factors for KC, such as 5q14.3–q21.1 [118], 5q21.2 [124], 5q31 [123], chronic eye rubbing, contact lens wear or atopic 5q32–q33 [124], 8q13.1–q21.11 [123], 8q24 and eye disease [105]. Finally, it is noteworthy that 9q34 [123], 12p12 [123], 13q32 [125], 14p11 apoptosis is a common mechanism between KC [116, 123], 14q11.2 [124], 14q24.3 [123], and Fuchs corneal endothelial dystrophy 15q2.32 [124], 15q22.23–q24, 16q22-q23, [13, 106]. 17q24, 20q12, 21q [115, 117, 126], and 17p13 [123, 127]. Other Genes Involved in KC Pathogenesis Generally, there are KC-associated single- nucleotide polymorphisms (SNPs), as in the KC has been associated with genes and proteins cases of COL4A3 (collagen type IV, alpha 3) at that regulate cellular and extracellular processes 2q36–q37, COL4A4 (collagen type IV, alpha 4) like wound healing, keratocyte proliferation, at 2q35–q37, IL-1A and IL-1B (both members of oxidative damage, differentiation, apoptosis the interleukin 1 cytokine family) and proteolysis [107, 108]. These genes are up- [28, 112, 128, 129]. It should be noted that the regulated in KC and myopic corneas [109]. In protein encoded by IL-1A is a pleiotropic cyto- addition, KC patients present with abnormal kine involved in various immune responses, gene expressions in at least some genes [44] and is proteolytically processed and released in identified using genome-wide scans, genome- response to cell injury, inducing apoptosis wide association studies, studies of descent, and [130]. The protein encoded by IL-1B is an family-based linkage studies [110]. Additionally, important mediator of the inflammatory more than 50 candidate genes have been response, and is involved in a variety of cellular excluded as contributors to KC development activities, including cell proliferation, differen- [111, 112]. Tables 5, 6 and 7 summarize the tiation, and apoptosis [131]. proteins that have been identified as targets of Several other candidate genes have been up- or down-regulation in KC as well as those proposed in KC, including SOD1 (superoxide genes that are known to code for them. dismutase 1 gene) (MIM 147450, locus b KC is characterized by genetic heterogeneity 21q22.11), TG I, DOCK9 (dedicator of cytoki- [2, 4, 8, 112]. Many distinct genetic loci have nesis 9 gene), which is located at 13q32, ZEB1, been mapped for KC, but none has been con- FLG [123, 132–136], LOX (lysyl oxidase) at firmed as a KC-associated genetic factor 5q23.2, VSX1 (visual system homeobox-1 gene) b [113, 114]. Some of those variants apparently (KTCN1, MIM605020, locus 20p11.2), TGF- 1, IL-1A (interleukin 1A gene), IL-1B (interleukin 2214 Adv Ther (2019) 36:2205–2222

Table 5 Up-regulated or down-regulated proteins and their genes (if identified) in keratoconus Up-regulated proteins Gene [reference] Down-regulated Gene [reference] proteins Collagen type XV COL15 [28] Collagen 12, 26 Inflammatory mediators 47, 51, 52 Collagen type I, II, IV COL1A1, COL1A2, COL2A1 [26] Proinflammatory cytokines 47, 51, 52 COL4A1 COL4A1 [35] Cell adhesion molecules 47, 51, 52 a1-PI (a1 proteinase 14, 28 inhibitor) MMPs 47, 51, 52 a2 Macroglobulin A2 M [14, 50] Degradative enzymes 14 Inhibitors of cellular 14 proteases Acid phosphatases 14 Aldehyde ALDH [14] dehydrogenase Acid esterases 14 Superoxide dismutase SOD [14] Acid lipases 14 Decorin DCN [58, 84] Cathepsin B CTSB [14, 50] Keratocan KERA [58, 84] Cathepsin G CTSG [14, 50] Lumican (LUM) LUM [58, 84] Cellular proteases 14 Biglycan BGN [58, 84] Nitrotyrosine malonaldehyde 14 KS (Keratan sulfate) KERA [59] antigenicity- central corneal Glutathione S transferase (GSTs) GST [14] Collagen type IV COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6 [28] iNOS (inducible nitric oxide synthase) NOS1, NOS2, IGF-2 transcripts IGF2 [94] NOS3 [14] GAG (glycosaminoglycan) polyanions 84 IGKC IGKC [44] (immunoglobulin kappa chain) (AZGP1) zinc-alpha-2-glycoprotein AZGP1 [69] Lactoferrin (LF) LTF [44] GCDFP15 (gross cystic disease fluid PIP [64, 69] protein 15)-prolactin-inducible protein (PIP) SP1 (specificity protein 1) SP1 [73] Amyloid deposits 72 BMP4 (bone morphogenic protein 4) BMP4 [21, 86, 87] Adv Ther (2019) 36:2205–2222 2215

Table 5 continued Up-regulated proteins Gene [reference] Down-regulated Gene [reference] proteins

TGF TGFBI [21, 86, 87] HSPGs (heparan sulfate proteoglycans) HSPG2 [83, 85] IL-1 IL1a, IL1b [87, 89] IGFBP (IGF binding proteins) 3 and 5 IGFBP3, IGFBP5 [94] DUSPs (dual-speciﬁcity phosphatases) (DUSP) [95] MKPs (mitogen-activated protein kinase (MAPK) [95, 96] phosphatases) TNF-a TNF [44, 47, 51, 52] ANGPTL7 (angiopoietin-related ANGPTL7 [84] protein 7) TIMP3 TIMP3 [81, 142] MMP2 MMP2 [14] MMP9 MMP9 [55]

Table 6 Proteins (and their genes) associated with kera- Table 7 Proteins (and their genes) found in keratoconus toconus but uncertain as to their role tears but uncertain as to their role Protein Gene [reference] Protein Gene [reference] HLA-A26 HLA-A*26 [82] Lipocalin LCN [44] HLA-B40 HLA-B*40 [82] Lysozyme C LYZ [44] HLA-DR9 HLA-DR*9 [82] Immunoglobulin alpha (IgA) IgA [44] Gelsolin GSN [44, 97] Immunoglobulin kappa (IGKC) IGKC [44] Cytokeratins KRT [44, 97] Precursors to prolactin 44

1B gene) at 2q14, IPO5, STK24 and HGF (hepa- amaurosis and Down syndrome, both of which tocyte growth factor) are associated with KC [133, 140]. [89, 112, 120, 133, 134, 137, 138]. It is of Genes VSX1, SOD1, IL-1B, COL4A3, COL4A4 interest that the protein encoded by FLG is an and LOX are the most probable genetic sub- intermediate ﬁlament-associated protein that strates of KC [28, 112, 120, 133, 141], although aggregates keratin intermediate ﬁlaments in the the role of VSX1 in the pathogenesis of KC mammalian epidermis [139]. It is also of interest remains controversial [142]. The LOX gene that the SOD1 gene, along with the CRB1 gene, encodes an enzyme that initiates the crosslink- have been implicated in Leber congenital ing of collagens and elastin by catalyzing 2216 Adv Ther (2019) 36:2205–2222 oxidative deamination of the epsilon amino Exploring the proteomic changes in KC and group in lysine residues of elastin and lysine analyzing its complex genetics will increase our and hydroxylisine residues of collagen [81]. understanding of its pathophysiology, and, Other genes that have been implicated in KC most importantly, will potentially enable tar- pathogenesis are the microRNA 184 (miR-184) geted genetic treatments in the future. gene which is positioned at 15q22–q25 [143], and the SPARC, MMP-2, MMP-9, COL6A1, COL8A1, and TIMP-3 [81, 112]. The TIMP-3 ACKNOWLEDGEMENTS gene belongs to the tissue inhibitor of metallo- proteinase gene family that are involved in ECM degradation [144]; mutations in these genes Funding. No funding or sponsorship was have been linked to Sorsby’s fundus dystrophy, received for the publication of this article. an autosomal dominant disorder [144]. In addition, the MPDZ-NF1B (rs132183), FOXO1 Authorship. All named authors meet the (rs2721051), RXRA-COL5A1 (rs1536482), International Committee of Medical Journal COL5A1 (rs7044529), FNDC3B (rs4894535) and Editors (ICMJE) criteria for authorship for this BANP-ZNF469 [112, 145, 146] genes have been article, take responsibility for the integrity of implicated in the pathogenesis of KC. the work as a whole, and have given their Finally, in KC patients from Poland, two approval for this version to be published. SNPs in the RAD51 [147] gene have been genotyped [148]. The protein encoded by the Disclosures. Anastasios G. Konstas is a RAD51 gene interacts with BRCA1 and BRCA2, member of the journal’s Editorial Board. Eleft- which are important elements of the cellular herios Loukovitis, Nikolaos Kozeis, Zisis response to DNA damage. BRCA2 regulates both Gatzioufas, Athina Kozei, Eleni Tsotridou, Maria the intracellular localization and the DNA- Stoila, Spyros Koronis, Konstantinos Sfakiana- binding ability of this protein, and its inactiva- kis, Paris Tranos, Miltiadis Balidis, Zacharias tion is thought to lead to genomic instability Zachariadis, Dimitrios G. Mikropoulos, George and tumorigenesis [147]. Anogeianakis and Andreas Katsanos have nothing to disclose.

CONCLUSION Compliance with Ethics Guidelines. This article is based on previously conducted studies The pathophysiology of keratoconus is multi- and does not contain any studies with human factorial and still elusive. Differential expression participants or animals performed by any of the of several corneal proteins in KC [14, 49] results authors. in changes in the structural integrity of the cornea, its collagen content and its morphology Data Availability. Data sharing is not [14, 49]. The biochemical abnormalities applicable to this article as no datasets were observed in corneal epithelium and stroma in generated or analyzed during the current study. KC include increased activity of degradative enzymes and reduced activity of protease inhi- Open Access. This article is distributed bitors [50]. Corneal thinning, therefore, is under the terms of the Creative Commons probably caused by the up-regulation of cellular Attribution-NonCommercial 4.0 International proteases and the down-regulation of their License (http://creativecommons.org/licenses/ inhibitors [14]. The increase of proteinase by-nc/4.0/), which permits any noncommercial activity [50] results in the induction of use, distribution, and reproduction in any degradative process in the cornea [50]. More- medium, provided you give appropriate credit over, oxidative damage and keratocyte apopto- to the original author(s) and the source, provide sis seem to play an important role in the a link to the Creative Commons license, and etiology of KC. indicate if changes were made. Adv Ther (2019) 36:2205–2222 2217

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