Mechanisms and biomarker candidates in pterygium development

This content is licensed under a Creative Commons Attributions 4.0 International License. ABSTRACT | Pterygium pathogenesis has been mainly asso­ ciated with UV light exposure; however, this association remains quite controversial. The complete mechanism of pterygium also remains to be clarified. Factors such as inflammation, viral infection, oxidative stress, DNA methylation, inflammatory mediators, extracellular matrix modulators, apoptotic and oncogenic proteins, loss of heterozygosity, microsatellite instability, lymphangiogenesis, epithelial­mesenchymal cell transition, and alterations in cholesterol metabolism have been identified as causes. Several studies aimed to clarify the molecular mechanisms underlying the growth and proliferation of pterygium. Understanding its molecular basis provides new potential therapeutic targets for its prevention and treatment. A comprehensive search of the databases, namely, MedLine, EMBASE, and LILACS, was conducted with the following key words: pterygium, epidemiology, pathogenesis, biomarkers, and review. This review describes the epidemiology, clinical presentation, and current investigation of biological mediators involved in pterygium development.


INTRODUCTION
Pterygium is a nonneoplastic elastotic degeneration of subepithelial growth, originating from the bulbar con junctiva that extends to the corneal surface, and even reached the visual axis in some cases. It is a common ocular surface disorder, especially in geographical areas near the equator. The exact cause of pterygium remains unclear; however, some risk factors are identified as causes, with longterm ultraviolet radiation exposure as the most important (1,2) . Although pterygium is generally regarded as a benign and cosmetic problem, it may result in significant visual morbidity or even potential blindness in severe cases if not properly treated (3) . It usually occurs in the nasal area, but can develop temporally or in both directions and may even occur bilaterally. Pterygium surgery is generally considered when symptoms do not respond to conservative treatment, when it induces visual disturbances, disability, or for cosmetic purposes (4,5) .
With regard to the mechanisms, genetic factors were suggested in some studies, noting that some genes asso ciated with DNA repair play a crucial role in pterygium development. However, studies on genetic variant contributions are limited in sample size and should be cautiously interpreted. Chronic irritation with actinic damage is likely responsible for the typical fibrovascular reaction of pterygium (6) . Growth factors, cytokines, and matrix metalloproteinase are involved in the pathoge nesis of pterygium and, along with UV exposure, may trigger the proinflammatory aspects (7) .
This review describes the epidemiology, clinical pre sentation, and investigation of biological modulators found in recent literatures. Therefore, the following keywords were searched: pterygium, epidemiology, an giogenesis, proliferation, inflammation, gene, protein, pathogenesis, and tight junction proteins.

Epidemiology
The prevalence of pterygium has been investigated in several populationbased studies. Rates widely vary de pending on the studied population, ranging from 2.8% to 38.7%, as recent studies in South Korea and China found prevalences of 8.8% (8) and 9.84% (9) , in contrast to 16% in Arizona (USA) (10) and 38.7% in Northwest Ethiopia (11) .
In Brazil, no population data were conducted on pterygium prevalence throughout the country, and only a few studies investigated this subject even for specific regions. A survey in riverside communities at the Soli moes and Japura rivers, in Amazonas, showed that its prevalence in the general population was 21.2% (12) . Ano ther survey, still in the Brazilian Amazon, reported that up to 36.6% prevalence of pterygium was observed in the indigenous population (13) . In the southeast region, a study in Botucatu City revealed a prevalence of 8.12% (14) .
A largescale survey on the rate of pterygium and other ocular diseases has not been conducted yet. A metaanalysis published in 2013, covering a total of 20 studies involving 12 countries with 900,545 samples, showed a combined pterygium prevalence rate of 10.2% (95% confidence interval [CI]: 6.3% to 16.1%), in the general population (15) . Table 1 summarizes the epidemio logical information.
Pterygium more frequently occurs in young adults, rarely before aged 15 years. It is believed to have some inherited patterns; however, several risk factors have been reported, such as dust exposure, heat, inflamma tion, and eye surface infection, rural residency, advan ced age, low educational levels, and outdoor activity (16) .
The prevalence of pterygium has been described as higher in males (16) ; however, other studies have shown that both genders have the same proportions (17) or even predominantly occurred in women (18) . Its relationship with smoking was also investigated, but remains inconclusive (15) .

Clinical presentation
Pterygium is a conjunctival fibrovascular tissue that extends to the cornea and can lead to irritating symp toms, visual disturbance, recurrent inflammation, and aesthetic alterations in the ocular surface. Its diagnosis is confirmed with slitlamp examination, in which the pterygium can be classified as grade 1, when the fibro vascular tissue reaches the limbus; grade 2, when it co vers the cornea in approximately 2 mm; grade 3, when it reaches the pupil margin; and grade 4, when it exceeds the pupil ( Figure 1). As regards its morphological featu res, pterygium can be classified as involutive or atrophic when it allows visualization of structures immediately below the lesion and as inflamed once the fibrovascular tissue is fleshy and prevents visualization of the structures below (19,20) . Recently, a functional classification using the corneal topographic data based on corneal higherorder irregularity was proposed by Miyata et al. to objectively evaluate pterygium severity. Hence, pterygium was gra ded based on corneal irregularity within the three zones: 1.0, 3.0, and 5.0 mm diameters. Thus, increased corneal irregularity within a 1.0mm diameter was considered to highly result in the risk for visual function impact, and increased corneal irregularity within a 5.0mm diame ter was considered as mild severity with visual function influences (21) .
Visual impairment induced by pterygium growth occurs due to induced astigmatism and opacification in the visual axis, requiring surgical treatment, as well as recurrent inflammation that does not improve with topical treatment (20) .
Its surgical procedure consists of dissecting the head of the pterygium from the cornea and resection of the conjunctiva and Tenon's capsule. Several surgical techni ques have been used, with excision of the pterygium followed by autologous conjunctival grafts as the most common, showing lower recurrence rates. Recurrence after a surgical treatment can be identified by the growth of conjunctival vessels toward the limbal edge inducing fibrous tissue growth into the cornea and representing poor outcomes (22) . However, clinical features such as ex tensive size, inflammation, and recurrent lesions remain challenges during the surgical treatment (23,24) .

UV radiation exposure
UV radiation from the sunlight is divided into three categories: 1. UVA (wavelength, 320400 nm) has the longest wave length and maximum penetration power; thus it is not attenuated by the ozone layer. Is an important inducer of pigmentation and contributes to premature skin aging, immunosuppression, and carcinogenesis (25) .   2. UVB (wavelength, 280320 nm) is absorbed by the ozone layer and comprises approximately 1%10% of the total UV radiation that reaches the Earth's surface. It is responsible for various biological events, including sunburn, immunosuppression, and carcinogenesis (25) . 3. UVC (wavelength, 200280 nm) has the highest energy among the three UV rays and possesses strong muta genic properties. It is almost completely absorbed by the ozone layer, thereby imposing negligible effects to human eyes (26) . UVB light exposure has been attributed as a major cause of pterygium. This kind of radiation can poten tially harm and alter cells and tissues through direct phototoxic effects on the cellular DNA and generation of reactive oxygen species, which damage the cellular DNA. Wavelengths below 300 nm have been known as the most biologically active forms and are absorbed by the cornea. Exposure to UVB radiation causes oxidative stress, which may lead to upregulation of many potential mediators of pterygium growth (2729) as shown in figure 2.

Viral infections
The polymerase chain reaction technique allowed examination of the alleged involvement of viral infec tions in the process of pterygium pathogenesis. Some reports demonstrated the presence of herpes simplex virus and human papilloma virus (HPV) in pterygium samples (30,31) . Viruses encode proteins that inactivate p53, leading to chromosomal instability and increasing the likelihood of cell progression to malignancy. HPV is most frequently found in the pterygium, with variable pre valence rates (32) . Its involvement as a cofactor in the pterygium pathogenesis is suggested, but remains controversial. If indeed HPV is involved in pterygium pa thogenesis or recurrence, antiviral medications or vac cination may be new options in pterygium therapy (30,33) .

Molecular mechanisms
Many studies have proposed possible mechanisms of pterygium development, including oxidative stress, extracellular matrix modulators, apoptotic and onco genic proteins, loss of heterozygosis, DNA methylation, inflammatory mediators, lymphangiogenesis, transition from mesenchymal epithelial cells, and cholesterol me tabolism alterations. These studies show evidences that several molecules, such as matrix metalloproteinases (MMPs), growth factors, and interleukins (ILs), are re lated to proliferation, inflammation, angiogenesis, and fibrosis, as shown in figure 3 and detailed below (3436) .

Tumor suppressor genes
Tumor suppressor genes prevent cells from conver ting into cancer cells and regulate cell growth along with protooncogenes (37) . One of the tumor suppressor genes that have been extensively studied is p53. A survey (38) showed that >20% of all pterygium samples were positive for p53 expression. Another immunohis tochemical study (37) evaluated 13 pterygium samples and 2 normal conjunctiva samples, which showed that 54% of pterygium were positive for p53 aberrant expression, whereas no pathological staining was observed in the normal conjunctiva. Therefore, the aberrant expression of p53 is suggested to promote cell proliferation and slow down apoptosis, thereby accelerating the deve lopment of pterygium; besides, the possible growth of limbal tumors is also suggested to be caused by cellular DNA damage that causes mutations in other genes (39) . In addition to p53, other tumor suppressor genes, such as p63, p16, and p27, were possibly involved in the develop ment of pterygium. P63 is more expressed in the basal and parabasal layers in primary pterygium and in the total thickness of the epithelium in recurrent pterygium. Increased expression of p16 protein was also observed in pterygium. Both p63 and p16 appeared to be rarely ex pressed in the normal conjunctiva (40) . P27 gene showed low nuclear immuno reactivity in pterygium tissues, differing from other tumor suppressor genes (41) .

Apoptosis-related proteins
Survivin is a protein encoded by the BIRC5 gene in humans; it is a member of the apoptosis inhibitory gene family and is expressed in the pterygium epithelium (42) . The molecular mechanisms of survivin regulation are still not well understood; however, survivin regulation seems to be associated with the p53 protein. Oxidative stress has been demonstrated to be caused by the acti vation of survivin leading to pterygium growth (43) . In addition, survivin has been found to be highly expressed in all pterygium tissues, but not in the normal human conjunctiva. Survivin was found to be closely related with COX2 in primary pterygium, suggesting an antia poptotic mechanism (44) .
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulatory proteins, which can induce or inhibit apoptosis. It is encoded by the Bcl-2 gene in humans (45,46) . Bcl-2 expression was noted in the basal epithelial layer of all pterygium epithelial cells, whereas the normal conjunctiva showed no evidence of the pro tein (39) . Decreased miR122 expression in the pterygium can result in cell apoptosis abnormalities due to its regulation of Bcl-w expression, also a gene of the Bcl-2 family, antiapoptotic, and subsequently contribute to the development of pterygium (47) .
Rapamycin complex 1 (mTORC1) is a central regu lator of cell growth, proliferation, protein synthesis, autophagy, and transcription. The role of mTORC1 is to activate the protein translation. mTOR signaling is highly activated; therefore, aberrant apoptosis and cell prolife ration were observed in pterygium samples. Activation of mTORC1 has been shown to inhibit apoptosis in pterygium by regulating Beclin-1dependent autophagy by targeting Bcl-2. mTORC1 also negatively regulates the fibroblast growth factor receptor 3 (FGFR3) through the inhibition of p73, thereby stimulating cell proliferation in pterygium. This demonstrates that mTORC1 signaling is highly activated in pterygium and provides new pa thways on its pathogenesis and progression (48) .

Cell adhesion molecules
Cell adhesion molecules play an important role in various physiological and pathological phenomena. These proteins are located on the cell surface and are intrinsically involved in cell binding and other extracellu lar matrix related to cell adhesion, including selectin, and integrin (49) . The expression of intercellular adhesion molecule1 (ICAM1) is found to be present in pterygium and absent in the epithelium of a normal conjunctiva (50) . Ecadherin and betacatenin have also been suggested to be concentrated in the pterygium tissue and are possibly involved in the epithelial proliferation and adhesion (51) .

Proliferation-related proteins
Proliferationrelated proteins such as Ki67, cyclin D1, and nuclear proliferation antigen play a key role in the cell cycle. Ki67 is an important marker of cell prolife ration. An abnormal expression of ki67 was found in pterygium samples when compared to a normal conjunc tiva (52) . Proliferating cell nuclear antigen (PCNA) is a nuclear nonhistone protein necessary for DNA synthesis, and its expression may be used as a marker of cell prolifera tion. The expression of PCNA was significantly higher in pterygium than that in a normal conjunctiva (53) . Cyclin D1 is a wellknown cell cycle control gene that pro motes cell cycle progression. A study found that PCNA and cyclin D1 were overexpressed in the limbal part of pterygium epithelial cells as compared with normal conjunctiva samples, which might lead to hyperprolife ration of epithelial cells (54) . Cyclin D1 protein expression in fleshy pterygium was found to be significantly higher than that in the atrophic ones. Another study indicated that βcatenin expressed in the nuclei/cytoplasm could increase cyclin D1 protein expression, which favors the proliferation of pterygium cells (55) .

Heat shock proteins
Heat shock proteins (HSP) are a protein family produ ced by cells in response to exposure to stressful condi tions. They were first described in relation to heat shock, but are recently known to be expressed during other stresses, including exposure to cold temperatures, UV light, and during wound healing or tissue remodeling (52) . The expression of HSPs, i.e., Hsp27, Hsp70, and Hsp90, and hypoxiainducible factor1α (HIF1α) were increa sed in pterygium. The expression of Hsp27 was detec ted in the epithelial, endothelial, and vascular smooth muscle cells in pterygium, but only in the epithelium in normal conjunctiva (56) . Changes in HIF1α and HSP le vels in pterygium are believed to represent an adaptive process for cell survival under stressful conditions (57) .

Tight junction proteins
Tight junction proteins represent a form of celltocell adhesion in the epithelial or endothelial cell layers, forming continuous seals around the cells and also ser ving as a physical barrier to prevent solutes and water from passing freely through the paracellular space. Claudin family proteins are an important part of this functional and structural barrier and dysregulation on its expression may result in various diseases including cancer (58) . In normal cornea and conjunctiva, claudin1 and claudin4 positivity were demonstrated immunohis tochemically (59) .
Claudins are indispensable proteins for the forma tion and maintenance of tight junctions. A strong immu nohistochemical expression of claudin1 was found in epithelium conjunctiva samples, whereas its expression in the pterygium samples was low. The significant decrease in claudin1 expression in the pterygium compared to the normal conjunctiva seems to be involved in the pa thogenesis of pterygium (60) .

Extracellular matrix proteins
The extracellular matrix (ECM) is a collection of ex tracellular molecules secreted by support cells that provide structural and biochemical support to the sur rounding cells (61) .
The aberrant expression of extracellular matrix proteins is believed to may be directly associated with the proliferative growth of pterygium, because it is a fi brovascular tissue characterized by an excessive depo sition of extracellular matrix and vascular growth. The extracellular matrix proteins contain keratin, elastin, collagen, and fibrin, among others. K8, K16, K14, and AE3 have been known to be present throughout the thickness in the pterygium epithelium but are absent in the nor mal conjunctiva (62) . In fact, pterygium samples showed a higher mRNA level and tropoelastin expression than the conjunctival tissue. Type II collagen expression was positive only in pterygium, whereas collagen types I, III, and IV were detected in both the pterygium and normal conjunctiva (63) .

Matrix metalloproteinases and tissue inhibitors of metalloproteinases
Matrix metalloproteinases (MMPs), also known as ma trixins, hydrolyze components of the extracellular matrix. These proteinases play a central role in several biological processes, such as embryogenesis, normal tissue remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration (64) . MMPs are a multigene family of >25 secreted and cell surface enzymes that process or de grade various extracellular matrices (65) , which can be di vided into five subgroups based on substrate preference: collagenases (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP10), mem braneassociated MMPs (MT1MMP, MT2MMP), and others (e.g., MMP12, MMP19, MMP20). Tissue inhibitors of metalloproteinases (TIMPs) bind to and prevent the activities of most MMPs. The relationship between pterygium and these two groups of proteins in the pathogenesis of pterygium has been studied (39) . MMP1, MMP2, MMP3, TIMP1, and TIMP3 were detected in greater amounts in pterygium tissues, epithelial cells, and fibroblasts as compared to normal conjunc tiva (66,67) . MMP3 was positively regulated and located in the pterygium epithelium, which may help explain the various pterygium phenotypes (68) . A study showed that cyclosporin A can reduce MMP3 and MMP13 expres sions in the pterygium fibroblast culture (69) .
MMP and TIMP expressions vary at the different stages of pterygium. The balance break between MMPs and TIMPs may be considered to be responsible for the progression or recurrence of pterygium (39) .

ILs
ILs are a group of cytokines, secreted proteins, and signal molecules first seen to be expressed by the white blood cells (leukocytes). These cells play vital roles in the inflammation process; thus, ILs can be closely related to pterygium (66) .
The expression of IL1α, IL1β RA, and IL1β precur sor proteins in primary pterygium and normal conjunc tival epithelium were detected via immunofluorescence. Enhanced levels of IL1 family proteins were present in pterygium only. Likewise, IL1α was found to be highly expressed not only in primary but also in recurrent pterygium (67) .
IL6 and IL8 were strongly expressed in the pterygium epithelium as compared to the normal cornea, conjunc tiva, and limbus. In addition, IL6 and IL8 proteins were significantly elevated in pterygium treated with UVB, suggesting that UVB could induce the secretion of these two ILs (70) . IL8 can also induce corneal vascularization directly (71) . IL10 had also been reported to be expressed more in pterygium than that in the normal conjunctiva. Recently, IL17 was found to be upregulated in the ocular surface in inflammatory pathologies, such as pterygium (72) .

Growth factors
A growth factor is a natural substance capable of sti mulating cellular growth, proliferation, healing, and cellu lar differentiation. They are important in the regula tion of various cellular processes, such as mitosis (73) .
Numerous growth factors are thought to have a role in pterygium pathogenesis, such as the vascular endo thelial growth factor (VEGF), transforming growth fac torbeta (TGFβ), basic fibroblast growth factor (bFGF), insulinlike growth factor, nervous growth factor, and connective tissue growth factor (CTGF) (7) . The VEGF fa mily has been extensively investigated in ophthalmology, because of its role in pathological angiogenesis and in increasing the vascular permeability in ocular diseases, such as pterygium and retinal diseases (74) .
Increased expression of VEGF leads to angiogenesis and lymphangiogenesis, which may influence the normal metabolism of the connective cells and promote vascu lar growth. When compared to the normal conjunctiva, pterygium showed higher VEGF levels (75,76) . TGFβ re gulates various processes common to tissue repair and disease, including fibroblast proliferation, angiogenesis, synthesis, and degradation of extracellular matrix pro teins (77) . TGFβ1 and TGFβ2 were found to be positively regulated, whereas transforming growth factorbeta receptor 1,2 (TGFRβ1, β2) was negatively regulated in pterygium (78,79) .
AntiVEGF drugs such as ranibizumab and bevacizumab have been widely used for the treatment and control of ocular diseases associated with vascular proliferation (80) . Although some studies suggest the use of antiVEGF as an adjuvant therapy for surgery, studies conducted to charac terize its use for the treatment of pterygium are lacking (81) .
Understanding the etiopathogenesis and most rele vant factors involved in pterygium may allow advances on strategies to prevent its onset and progression, which may even prevent surgical procedures in the future. Although various studies have already been conducted, important genes and proteins have probably not yet been disco vered. In this sense, performing additional research to better understand the etiopathogenic mechanisms and, thus, promote more targeted and effective treatment options, especially in recurrent cases, may be interesting.