Introduction

Pterygium is a common ocular disease [1-3]. The global prevalence of the disease ranges from 1.1% to 53%. Meanwhile, according to the 2010 Basic Health Research data, the prevalence of this disease in Indonesia reached 3.2% affecting both eyes, and 1.9% affecting one eye. Besides, the recurrence rate of this disease is relatively high, ranging from 10-80% globally and 24-89% in Indonesia [4]. The high recurrence rate and progression rate of recurring pterygium remains a challenging clinical problem for ophthalmologists in Indonesia and has not been resolved [5-7]. Therefore, new treatment strategies are needed to address the problem.

A combination of pterygium treatment with adjuvant therapy developed from natural ingredients, with minimal side effects but greater benefits, can be considered in treating eye diseases [8]. One of the natural ingredients is epigallocatechin-3-gallate (EGCG), the extract of green tea. Studies have shown that EGCG can effectively reduce the recurrence and migration rates of pterygium fibroblast cells in laboratory settings, without causing harm to conjunctival cells [9]. As a result, the EGCG administration is proposed as a promising and innovative therapy for primary pterygium treatment [10]. Furthermore, postoperative adjuvant therapy with EGCG holds potential as a successful approach for managing pterygium recurrence. In the future, EGCG could be formulated into eye drops, offering an alternative therapeutic option for this condition.

Results and Discussion

Pterygium

Pterygium is a degenerative condition affecting the wing-shaped conjunctiva [8]. It typically manifests as a triangular growth of fleshy fibrovascular tissue extending from the bulbar conjunctiva onto the cornea, primarily on the nasal side. The primary cause of pterygium is exposure to ultraviolet (UV) radiation (Figure 1) [8]. This condition is among the most prevalent corneal disorders, leading to a decline in visual acuity. To assess pterygium, certain criteria should be considered, including its location, size, vascularity, extent, and the area of corneal involvement (measured using Castroviejo's calipers to determine the chord at the limbus and the degree of corneal encroachment from the limbus). A pterygium consists of three components: the cap, which is found at the leading edge and characterized by an avascular zone resembling a halo; the head, located peripherally to the cap; and the body, the primary portion of the pterygium that connects with the bulbar conjunctiva. Pterygium can be classified as progressive, exhibiting thickness, fleshy appearance, high vascularity, and progressive growth towards the center of the cornea; or atrophic, which is characterized by thinness, attenuation, poor vascularity, and a stationary state [2].

Figure 1: Pterygium. (A) Nasal pterygium and (B) Nasal and temporal (double-headed) pterygium [2]

The recurrence of pterygium is influenced by various factors, including but not limited to the UV radiation exposure, environmental irritants (such as dust and wind), viral agents, genetic factors, as well as immunological and inflammatory factors. Recent investigations have also highlighted additional risk factors, such as the presence of specific gene transcription factors like cAMP response element binding protein (CREB), phospholipase D, cytochrome P450 (CYP) 1A1, aquaporin-1, and aquaporin-3. The pterygium pathogenesis involves a complex interplay of these underlying factors. Nonetheless, it is crucial to emphasize that sunlight exposure remains the predominant risk factor, significantly contributing to the initiation and progression of pterygium [2].

Pterygium development is influenced by hereditary factors and sun exposure (Figure 2). Hereditary factors impact the growth factors involved in fibroblast division. Inflammation-induced fibroblast mitosis generates a large number of fibroblasts that collectively produce collagen [11]. The excess collagen is invaded by fibroblasts, and the stimulation of growth factors and ROS leads to the formation of new blood vessels, contributing to pterygium formation.

Figure 2: The potential role of the UV light in the pterygium pathogenesis. Exposure to the UV light can induce changes in limbal stem cells, initiating the formation of pterygium. Pterygial cells, characterized by increased expression of inflammatory cytokines, growth factors, and MMPs, contribute to inflammation, fibrogenesis, vascularization, and pterygium invasion. The UV radiation or interactions between pterygium cells and limbal fibroblasts in a bFGF/TGFβ-dependent manner activate the fibroblasts, leading to elevated levels of various growth factors and MMPs. This process results in remodelling of the extracellular matrix, dissolution of Bowman's membrane, and pterygium invasion

Collagenolysis facilitates the invasion of new fibroblasts and blood vessels into the stroma. The pterygium occurrence at the surgical limbus is attributed to the abundance of endothelial cells, which generate ROS upon sun exposure [4].

One widely accepted hypothesis regarding pterygium pathogenesis is that exposure to the environmental factors, particularly the UV radiation, can activate various signaling pathways that trigger the production of mediators responsible for pterygium growth [1]. At the molecular level, the UV radiation is associated with the generation of active free radicals that can damage and deactivate macromolecules. DNA methylation can also regulate matrix remodeling and cell adhesion, influencing the formation of epithelial pterygium. Furthermore, pterygium may exhibit resistance to the UV radiation-induced apoptosis, which can be attributed to an imbalance between proapoptotic and antiapoptotic proteins [12].

Basic fibroblast growth factor (bFGF)

Cell proliferation is a prominent characteristic in the development of pterygium. Numerous growth factors have been investigated, and a comprehensive list of these molecules, along with a brief overview of their roles as pathogenic factors, is presented in Table 1. Among the growth factors affected by the UV-B radiation is bFGF, also known as fibroblast growth factor 2 (FGF2) [13]. This factor plays a significant role in angiogenesis, wound healing, and various endocrine signaling pathways [14, 15]. Studies have demonstrated increased expression of bFGF in infiltrating mast cells, epithelial cells, and blood vessels associated with pterygium. Mast cells can also act as an additional source of bFGF. Detorakis et al. conducted qRT-PCR analysis to measure FGF2 mRNA levels in normal pterygium and conjunctiva, revealing higher expression of FGF2 in pterygium compared to normal tissue [16]. This upregulation of FGF2 can induce the expression of cyclooxygenase-2 (COX-2), an enzyme absent in normal conjunctiva, but present in human pterygium fibroblasts. COX-2 expression is crucial for inflammatory cytokine-induced angiogenesis, suggesting that FGF2 contributes to inflammation and angiogenesis in pterygium [16, 17].

Pterygium fibroblast cell apoptosis-associated protein

Survivin, Bcl-2, Bax, and Bcl-w are examples of proteins implicated in the regulation of cell apoptosis. Survivin, a significant member of the apoptosis inhibitor family, plays a role in inhibiting caspase activation, thereby negatively regulating apoptosis. Research by Liu et al. demonstrated strong expression of survivin in pterygium tissue, with localization in both the nucleus and cytoplasm of epithelial cells. In contrast, normal conjunctival epithelium showed weak expression limited to the cytosol. Knockdown of survivin was found to suppress the propagation of pterygium epithelial cells and correlated with the downregulation of p63 expression, as well as the upregulation of p57 and p21 expression. This suggests that oxidative stress can trigger the activation of survivin expression, leading to a hyperproliferative state that may contribute to pterygium growth [17].

Table 1: Growth factor and its role in the pathogenesis of pterygium [18]

therapy

The high recurrence rate associated with surgery remains a problem. Thus, adjuvant medical therapies have been incorporated into the postoperative management of pterygium. Many sources have indicated that the addition of these therapies leads to a dramatic decline in the relapse rate of the disease. It is expected to serve as a highly-effective and reliable alternative treatment in the future.

Mitomycin C (MMC)

Mitomycin C (MMC) is an antimicrobial/anticancer agent derived from Streptomyces caespitosus. Since 1963, it has been proposed as an adjunctive treatment for pterygium surgery. The combination of conjunctival autograft with MMC, irrespective of the dosage or method of application, has demonstrated substantial efficacy in reducing pterygium recurrence. Furthermore, postoperative administration of MMC through topical eye drops is a viable approach. However, it is crucial to consider the potential complications associated with MMC usage, including the development of chronic keratopathy and toxic keratoconjunctivitis. To ensure better control of the dosage, intraoperative administration of MMC is recommended [19].

Some studies have investigated the effectiveness of utilizing preoperative subconjunctival injection of MMC as an adjunct therapy for pterygium surgery. In a prospective study involving 36 patients, a subconjunctival injection of 0.1 ml-0.15 mg/ml MMC was administered into the pterygium head one month prior to surgical excision. The study followed up for a duration of two years, during which a recurrence rate of 6% was observed, and no complications related to wound healing were documented. Another randomized controlled trial (RCT) study involved 50 eyes with recurrent pterygium, which were divided into two groups: a preoperative MMC injection group and a postoperative topical MMC group. In the preoperative group, a 0.1 ml-0.15 mg/ml MMC injection was administered the day before the pterygium excision surgery. The difference in relapse and complication rates between the two groups did not yield statistically significant results. As a result, the authors concluded that preoperative low-dose subconjunctival injection of MMC is an effective approach for managing recurrent pterygium [2].

Epigallocathecin-3-Gallate (EGCG)

Green tea derived from Camellia sinensis contains a diverse range of flavonoids, with catechins being a major class of flavonoids, including epigallocatechin gallate (EGCG) (Figure 3). EGCG is the most abundant catechin and has demonstrated anti-inflammatory and antioxidant properties in various cell types. Recent studies conducted in vivo and in vitro have also highlighted the antivasodilating, anticarcinogenic, and neuroprotective effects of EGCG [9].

Figure 3: Epigallocathecin-3-gallate (EGCG) chemical structure [6]

Pterygium is a condition affecting the eyes and characterized by the development of triangular hyperplastic growth, conjunctivalization, inflammation, and remodeling of connective tissue. The cellular properties of pterygium cells bear a resemblance to those of neoplastic cells. Within green tea, there exists a catechin known as EGCG, which exhibits antitumorigenic properties. The effects of green tea catechins on the survival and migration of human primary pterygium cells differ from their impact on conjunctival cells. Both human primary pterygium cells and conjunctival cells express the EGCG receptor, specifically the 67 kDa laminin receptor. Multiple studies have provided evidence demonstrating the significant inhibitory effect of green tea extract and EGCG on pterygium cell proliferation while having minimal impact on conjunctival cells. These findings hold promise for the development of a potential novel therapeutic approach for the primary treatment of pterygium in the future [10].

Studies have observed that the administration of 50 μg/ml of EGCG can suppress the expression of basic fibroblast growth factor (bFGF), an angiogenic factor, as well as reduce the level of vascular endothelial growth factor (VEGF). These two growth factors play a role in the formation of fibrous tissue [20]. Kapoor et al. demonstrated that the application of 50 μl of EGCG at a concentration of 0.8 mg/ml using a full-thickness skin incision model in rats significantly improved the quality of wound healing and led to the formation of non-excessive fibrotic tissue [21]. The dditional roles of EGCG are presented in Table 2.

This study only evaluates the direct pathway of EGCG effects on bFGF expression and fibroblast cell apoptosis in HPF, which could be considered as a limitation. Further research is required to analyze the specific pathways associated with the effect of EGCG treatment on pterygium recurrence. The detailed pharmacodynamic pathway of EGCG needs to be investigated in future developmental studies. However, despite these limitations, this research serves as a pilot study on the EGCG potential for bFGF expression and fibroblast cell apoptosis in HPF, as compared to the standard adjuvant therapy, namely MMC.

Table 2: Mechanism of Epigallocatechin-gallate (EGCG)

Conclusion

Pterygium is an ocular disease characterized by the growth of fibrovascular tissue on the corneal surface, primarily prevalent in tropical and subtropical regions with high sun exposure. The recurrence of pterygium is influenced by multiple factors. Ultraviolet (UV) radiation, particularly UV-B, indirectly contributes to the inflammatory process, oxidative stress, increased angiogenic factors, and production of growth factors (such as bFGF) in pterygium. These factors are associated with chronic inflammation, DNA damage, and processes involving migration, differentiation, cell proliferation, apoptosis, and angiogenesis, leading to the inflammation of pterygium cells. To minimize pterygium recurrence, treatment strategies can incorporate adjuvant therapy developed from natural ingredients with minimal side effects, such as EGCG. EGCG possesses anti-inflammatory, antioxidant, antifibrotic, and antineoplastic properties, which are relevant concerning the neoplastic characteristics of pterygium. EGCG has the potential to reduce bFGF expression and induce apoptosis in HPF cells, offering a promising avenue for preventing pterygium recurrence.

Disclosure Statement

No potential conflict of interest was reported by the authors.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' Contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

ORCID

Astry Ayunda

https://orcid.org/0009-0000-0809-584X

Luki Indriaswati

https://orcid.org/0009-0000-4904-5013

Muhammad Firmansjah

https://orcid.org/0009-0008-5441-5769

Djoko Agus Purwanto

https://orcid.org/0000-0002-2718-903X

Budi Utomo

https://orcid.org/0000-0002-1147-3263

HOW TO CITE THIS ARTICLE

Astry Ayunda, Luki Indriaswati, Muhammad Firmansjah, Djoko Agus Purwanto, Budi Utomo. Expresion of Basic Fibroblast Growth Factor (bFGF) and Cell Apoptosis in Human Pterygium Fibroblast (HPF) after the Adminstration of Epigallocathecin-3-Gallate (EGCG) and Mitomycin-C (In Vitro Laboratory Experimental Study). J. Med. Chem. Sci., 2023, 6(12) 3159-3166.

DOI: https://doi.org/10.26655/JMCHEMSCI.2023.12.29

URL: https://www.jmchemsci.com/article_177760.html