Autophagy-related pathway dysregulation and apoptosis suppression in pterygium: Role of key biomarkers Beclin-1,ATG5,LC3,p62,and Bcl-2 in pathogenesis and potential nonsurgical targets

Abstract

Objectives

To investigate the roles of apoptosis and autophagy in the pathogenesis of primary pterygium and to evaluate potential biomarkers that could lead to nonsurgical therapeutic approaches.

Methods

In this prospective, observational, controlled study, patients diagnosed with primary pterygium were included. Excised pterygium tissues (study group) were compared with normal conjunctival tissues obtained from autografts (control group). Immunohistochemical staining was used to assess the expression of autophagy-related proteins (Beclin-1, LC3A/B, ATG5, and p62) and apoptosis-related proteins (Bcl-2 and Caspase-8). Quantitative immunohistochemical evaluation was performed using H-score analysis with Image Tool Software.

Results

The study group exhibited significant increases in the expression of Beclin-1 (97.65 ± 2.54 vs. 25.53 ± 0.4), LC3A/B (97.88 ± 5.35 vs. 61.73 ± 5.08), ATG5 (100.73 ± 1.06 vs. 35.35 ± 0.3), p62 (84.18 ± 3.59 vs. 59.54 ± 2.29), and Bcl-2 (107.36 ± 1.60 vs. 53.56 ± 1.38) compared with the control group (p < 0.001 for all). Although Caspase-8 expression was also increased (65 ± 1.53 vs. 49 ± 0.2), the difference was not statistically significant (p = 0.270). These findings suggest altered regulation of autophagy-related pathways accompanied by suppression of apoptotic mechanisms, which may contribute to the persistence, fibrovascular proliferation, and progression of pterygium tissue.

Conclusion

The progression of pterygium appears to be associated with dysregulation of autophagy-related pathways and inhibition of apoptotic mechanisms. These findings suggest that modulation of these cellular mechanisms may provide novel pharmacological therapeutic options, potentially reducing the need for surgical intervention.

Keywords

Apoptosis autophagy Beclin-1 pterygium immunohistochemistry ATG5 LC3A/B

Key summary points

Why carry out this study?

Pterygium is a common degenerative condition with limited nonsurgical treatment options. The roles of autophagy activation and apoptosis suppression in its pathogenesis remain incompletely understood. Therefore, key biomarkers (Beclin-1, ATG5, LC3, p62, and Bcl-2) were investigated to identify potential pharmacological targets.

What was learned from the study?

Pterygium tissues demonstrated significantly higher expression of the autophagy markers Beclin-1, LC3A/B, ATG5, and p62, as well as the antiapoptotic protein Bcl-2, compared with autologous normal conjunctival tissues (all p < 0.001). No significant difference was observed in Caspase-8 expression (p = 0.270). These findings suggest dysregulation of autophagy-related pathways that favor cellular survival and suppress apoptosis, potentially contributing to the progression and persistence of pterygium.

Introduction

Pterygium is a degenerative fibrovascular ocular condition characterized by the extension of the bulbar conjunctiva onto the cornea, typically originating from the nasal side and manifesting as a continuation of the conjunctiva.^1,2 Recent studies have increasingly associated degenerative diseases with the “hallmarks of aging” paradigm, in which impaired or dysregulated macroautophagy represents a central mechanism contributing to cellular dysfunction and chronic tissue remodeling. Dysfunction of autophagy-related pathways may impair cellular homeostasis, increase oxidative stress, and promote the persistence of damaged cells, thereby contributing to degenerative ocular surface disorders such as pterygium.³ Ultraviolet radiation is thought to play a significant role in its pathogenesis, which may account for the higher prevalence of pterygium in equatorial regions. The condition is more frequently observed in countries with hot, sunny climates, particularly among farmers who do not use protective eyewear, and occurs at twice the rate in men compared to women.^4–6 The correlation between sun exposure, ultraviolet radiation, and other risk factors for pterygium has been demonstrated in several studies.^4–8

Pterygium can lead to astigmatism and, in advanced stages, may result in significant visual impairment by encroaching upon the visual axis. Although symptomatic treatment is applicable in the early stages for patients presenting with various complaints, it does not provide an adequate solution in later stages. Successful treatment is achievable only through the excision of the lesion.^1,2,9–11 Surgical interventions for pterygium typically involve the “bare sclera” technique and “simple conjunctival closure” techniques. Although these procedures are relatively straightforward and feasible, with brief surgical durations; however, postsurgical recurrence rates of approximately 70%–90% have been reported.⁹ The removal of the pterygium followed by tissue grafting reduces the likelihood of recurrence. In conjunctival autograft surgery, a segment of conjunctival tissue, along with limbal tissue is harvested from another area of the patient's eye in one piece and transplanted to the site of pterygium excision. An alternative tissue grafting method involves the use of an amniotic membrane graft, in which donor amniotic membrane is attached to the residual limbus and exposed sclera after pterygium excision.⁹

The etiology of pterygium is not fully understood; therefore, its pathogenesis is always a subject of interest. In terms of etiology, limbal stem cell changes due to ultraviolet rays are prioritized.¹⁰ Recent evidence has focused on genetic factors, antiapoptotic mechanisms, cytokines, growth factors, genes involved in DNA repair such as the tumor suppressor gene p53, cell proliferation, migration, and angiogenesis. Evidence implicating the extracellular matrix has also been reported. In addition, human papillomavirus (HPV) infection is among the factors that trigger pterygium development.¹⁰ The mechanisms (s) of pterygium formation are not fully understood, and no single factor has been definitively established as causative. Programmed cell death is an evolutionarily conserved mechanism throughout evolution in multicellular organisms and plays a role in many basic functions, including morphogenesis, tissue homeostasis, and defense against pathogens.^11,12 Apoptosis and autophagy are recognized as the primary mechanisms of programmed cell death. Apoptosis facilitates the elimination of damaged, transformed, infected tissues, as well as healthy tissues that have reached the end of their life span throughout the organism's life cycle. Research indicates that autophagy, characterized by the self-digestion of the cell, unlike apoptosis, contributes to the maintenance of homeostasis by facilitating the recycling of intracellular molecules in the absence of nutrients or under cellular stress conditions.¹³ Although evidence suggests that these processes are regulated through distinct molecular mechanisms, some studies have identified a connection and communication between these two forms of programmed cell death.¹⁴ The predominance of autophagy as a programmed cell death mechanism in scenarios where apoptosis is inhibited further supports the existence of this interrelationship.^15–17

Antiapoptotic members of the Bcl-2 protein family have been shown to interact with Beclin-1, a fundamental autophagy protein, thereby inhibiting autophagy.^15,16 This indicates that Bcl-2 proteins possess both antiapoptotic and anti-autophagic functions, facilitating the coordination of autophagy and apoptosis. Recent studies have demonstrated that Beclin-1 undergoes cleavage and inactivation by caspases during apoptosis.¹⁶ Conversely, Caspase-8 has been identified as a target of selective autophagy and is subject to degradation through autophagic processes.¹⁷ These findings suggest intricate interconnections between autophagy and apoptosis. Both processes are activated by similar stress stimuli, share certain regulatory proteins, and coordinate their activities through these shared elements. The intricate interplay of enzyme–substrate interactions, such as the cleavage of various autophagy proteins by Caspase-8, which itself is a target of autophagy, alongside protein–protein interactions such as Atg5-FADD binding and competitive dynamics between proteins, such as the competition for Bcl-2 binding between the autophagy protein Beclin-1 and pro-apoptotic Bcl-2 proteins containing BH3, is crucial. Although limited research has explored the association between pterygium and apoptosis, some scholars have posited that the development of pterygium may result from disruption of normal apoptotic processes within the conjunctiva.¹⁸ Studies using pterygium cell cultures have demonstrated significant alterations in several markers associated with apoptosis and autophagy. However, the precise mechanisms underlying autophagy and apoptosis in pterygium remain to be fully elucidated.^19–21 These relationships are illustrated in Figure 1.

Figure 1.

Illustration of the functional roles of apoptosis and autophagy markers.

This study aimed to comprehensively investigate the roles of apoptosis and autophagy in the pathogenesis of primary pterygium using tissue samples obtained from affected individuals. By elucidating the involvement of these key cellular processes in pterygium formation and progression, we sought to identify potential molecular targets that may facilitate the development of novel therapeutic strategies and contribute to the existing body of knowledge in this field.

Methods

This study was conducted in accordance with the ethical principles of the Declaration of Helsinki of 1975, as revised in 2024. Written informed consent was obtained from all participants after a thorough explanation of the nature of the study and its potential consequences. The reporting of this study conforms to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines²². Following approval by the ethics committee, consecutive patients who met the inclusion criteria and agreed to participate were recruited from individuals presenting to the Mugla Sıtkı Koçman University Training and Research Hospital Ophthalmology Outpatient Clinic between August 2024 and August 2025. In this prospective, observational, controlled study, participants diagnosed with primary pterygium constituted the case (pterygium) group. Both pterygium tissue and adjacent healthy conjunctival tissue were obtained from these individuals during surgical excision. The adjacent healthy conjunctival tissue (control group) served as an intra-individual control for comparative analyses of cellular processes, including apoptosis and autophagy, within the same participant. Sample size determination and power analysis were performed using G*Power software.

The study included two groups. Group 1 (pterygium group; n = 30) comprised patients with primary pterygium who underwent surgical excision with conjunctival autografting. Group 2 (control group; n = 30) consisted of autologous healthy conjunctival tissue samples (1 × 1.5 mm) obtained from the superior temporal bulbar conjunctiva of the ipsilateral eye during the same procedure.

Preoperative assessments and inclusion/exclusion criteria

Best-corrected visual acuity, slit-lamp biomicroscopic examination, intraocular pressure (IOP) measurement using Goldmann applanation tonometry, and a detailed fundus examination were performed preoperatively in all participants.

To minimize confounding factors that could influence apoptosis- or autophagy-related pathways, participants underwent comprehensive screening for ocular and systemic conditions known to affect these cellular processes. Exclusion criteria included diabetes mellitus, diagnosis of glaucoma, recurrent pterygium, a history of previous corneal surgery, and refusal to provide informed consent or participate in the study. Patients meeting any of these exclusion criteria were not enrolled. Strict inclusion and exclusion criteria, together with the use of autologous conjunctival controls, were applied to minimize selection bias and interindividual variability.

In all cases, excision with free conjunctival autograft surgery was performed. All procedures were conducted by the same surgeon under local anesthesia. The operation commenced with separation of the pterygium tissue from the sclera under topical and subconjunctival anesthesia, followed by appropriate site preparation. The conjunctival autograft was excised from the superior temporal conjunctival region in a manner sufficiently wide to cover the excised pterygium tissue and to procure adequate normal tissue for the control group. After the conjunctival autograft had been excised and a portion measuring1 × 1.5 mm had been separated for the control group, it was secured to the bare sclera area using 8-0 absorbable polyglactin suture with the epithelial side oriented upward. The excised pterygium tissue and the normal conjunctival tissue separated from the conjunctival autograft were subsequently compared utilizing immunohistochemistry.

The samples were processed according to standard tissue-tracking protocols and embedded in paraffin blocks. Subsequently, 0.5-µm sections were obtained and mounted on lysine–coated slides. A 3% hydrogen peroxide (H₂O₂) solution was applied to the samples and allowed to react at room temperature for 10 min. Following the standard staining procedure, the diluted primary antibody was applied, and the samples were incubated overnight at +4°C. On the following day, a 3,3′-diaminobenzidine (DAB) mixture was prepared according to the number of samples, consisting of 27 µL of DAB (3,3'-diaminobenzidine) substrate in 1000 µL of DAB dilution. At the end of the incubation period, the DAB mixture was applied to the slides and left for 2 min. The preparations were then rinsed with distilled water. The smear preparations were subsequently washed with distilled water and passed through a series of alcohol and xylene solutions without drying, and the adhesion of the coverslip to the slide was achieved using Entellan liquid. Immunohistochemical staining was performed using antibodies against Bcl-2, Beclin-1, ATG5, and Caspase-8. The primary objective of immunohistochemical staining was to identify and visualize cellular macromolecules known to be present within the cell through staining techniques. During the immunohistochemical assessment in both study groups, 10 areas were randomly selected from each sample. Within these 10 distinct areas, all cells, both positive and negative, were enumerated, and their percentages were calculated.

The staining intensity of positively stained cells was categorized into three levels: weak (+), medium (++), and strong (+++) in accordance with the criteria of the “H-score method.” The percentage of cells exhibiting weak (+) positive staining was determined, and this percentage was multiplied by 1 to yield a corresponding value. Similarly, the percentage of cells with medium (++) positive staining was calculated, and this result was multiplied by 2 to obtain a second value. For cells with strong (+++) positive staining, the percentage was calculated and multiplied by 3 to derive a third value. These three values were then summed to calculate an “H-score value” for each patient. The following formula was employed for the calculation of the H-score. In the assessment of H-scores, two blinded observers independently evaluated all H-scores. For each sample, H-scores obtained from 10 randomly selected microscopic fields were averaged, and the final results were expressed as mean ± standard error of the mean (SEM).

H-score = 1 × (percentage of cells stained with weak (+) positivity) + 2 × (percentage of cells stained with medium (++) positivity) + 3 × (percentage of cells stained with strong (+++) positivity)

Public transcriptomic validation was performed using publicly available Gene Expression Omnibus (GEO) datasets containing transcriptomic data from pterygium and normal conjunctival tissues. Expression patterns of autophagy- and apoptosis-related genes corresponding to the immunohistochemical markers examined in this study, including BECN1, MAP1LC3A/B, ATG5, SQSTM1/p62, BCL2, and CASP8, were evaluated to provide external molecular validation of the immunohistochemical findings.

Statistical analyses were performed using SPSS version 30 (SPSS Inc., Chicago, IL, USA). Morphometric measurements were carried out using UTHSCSA Image Tool software (University of Texas Health Science Center at San Antonio, USA). In addition to descriptive statistical methods, including mean and SEM, one-way analysis of variance was used for intergroup comparisons. Tukey’s multiple-comparison test was applied for subgroup analyses, the paired Student’s t-test was used for intra-individual group comparisons where appropriate, and the chi-square test was used for qualitative data comparisons. To reduce the risk of inflated type I error due to multiple biomarker comparisons, p-values were adjusted using the Benjamini–Hochberg false-discovery rate (FDR) correction method. Adjusted p-values (q-values), effect sizes (Cohen's d), and 95% confidence intervals (CIs) were calculated and reported for all biomarker comparisons. The primary outcomes were predefined as the H-score expression levels of Beclin-1, LC3A/B, ATG5, p62, Bcl-2, and Caspase-8. The results were assessed at a significance level of p < 0.05.

Results

The study included 30 right eyes from 30 patients who underwent conjunctival autograft surgery for primary pterygium. The mean age of the patients was 46.5 ± 5.3 years (range, 38–60 years). Fourteen patients were male (46.6%), and 16 were female (53.4%).

The study conducted a histological analysis of pterygium and healthy conjunctival tissue samples, employing immunohistochemical staining techniques. The samples were examined under a light microscope, and H-scores were calculated. The immunohistochemical evaluation focused on the staining results of Beclin-1, LC3A/B, ATG5, Bcl-2, p62, and Caspase-8 antibodies. The findings for the control and study groups, respectively, were as follows: Beclin-1expression, 25.53 ± 0.4 and 97.65 ± 2.54 (p < 0.001); LC3A/B expression, 61.73 ± 5.08 and 97.88 ± 5.35 (p < 0.001); ATG5 expression, 35.35 ± 0.3 and 100.73 ± 1.06 (p < 0.001); p62 expression, 59.54 ± 2.29 and 84.18 ± 3.59 (p < 0.001); Bcl-2 expression, 53.56 ± 1.38 and 107.36 ± 1.60 (p < 0.001); and Caspase-8 expression, 49 ± 0.2 and 65 ± 1.53 (p = 0.270). These results are presented in Table 1. Data are expressed as mean ± SEM. A graphical comparison of biomarker H-score expression levels between the control and pterygium groups is additionally presented in Figure 2.

Figure 2.

Comparison of H-score expression levels of apoptosis- and autophagy-related biomarkers between control and pterygium groups.

Table 1.

H-score evaluation obtained after staining using apoptosis antibodies (Bcl-2, Caspase-8) and Beclin-1, LC3 A/B, ATG-5 and P62 antibodies.

	Control group mean ± SEM (Min–Max)	Study group mean ± SEM (Min–Max)	p^a
Bcl-2	53.56 ± 7.5 (40.3–74.13)	107.36 ± 8.76 (97.3–133)	<0.001
Caspas-8	49.25 ± 1.24 (46.32–51.54)	65.92 ± 8.55 (50.21–80.0)	0.270
Beclin-1	25.53 ± 2.24 (22.31–29.01)	97.6 ± 13.91 (67.68–125.35)	<0.001
LC3A/B	61.73 ± 5.08 (80.02–90.00)	97.88 ± 5.35 (88.00–109.00)	<0.001
ATG5	35.35 ± 1.71 (28.01–38.12)	100.73 ± 5.82 (89.06–120.0)	<0.001
p62	59.54 ± 2.29 (57.01–66.12)	84.18 ± 3.59 (80.02–90.00)	<0.001

Values are presented as mean ± SEM. Benjamini–Hochberg false-discovery rate (FDR) correction was applied for multiple comparisons.

Data presented as standard error of the mean (SEM). Comparisons between groups were performed using one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

After Benjamini–Hochberg FDR correction for multiple comparisons, the increased expression levels of Beclin-1, LC3A/B, ATG5, p62, and Bcl-2 remained statistically significant (adjusted p < 0.05 for all), with large effect sizes and narrow 95% CIs. Caspase-8 expression did not show a statistically significant difference after correction.

Public transcriptomic validation using GEO datasets demonstrated differential expression patterns of autophagy- and apoptosis-related genes between pterygium and normal conjunctival tissues. Increased expression trends were observed for BECN1, MAP1LC3A/B, ATG5, SQSTM1/p62, and BCL2, whereas CASP8 expression showed less prominent differences between the groups. These findings were generally consistent with the immunohistochemical results and supported the dysregulation of autophagy- and apoptosis-related pathways in pterygium tissue.

Figure 3 presents light microscopic histological images acquired following immunohistochemical staining with antibodies against Bcl-2, Beclin-1, ATG5, and Caspase-8 at 20× magnification.

Figure 3.

Histological images obtained after immunohistochemical staining (20× magnification).

Discussion

The primary histological feature of pterygium is hyperproliferative fibrovascular tissue, suggesting that fibrotic processes contribute significantly to its progression. Recent studies have demonstrated that fibrosis is integral to the development of pterygium. These fibrotic processes result in alterations of normal tissue architecture, abnormal cellular proliferation, and degradation of collagen structures.²¹ Autophagy, a programmed cellular process, is involved in maintaining the equilibrium between apoptosis and fibrosis.²³ Although cells may survive through their autophagic capabilities, apoptosis, another programmed cell death mechanism, may be initiated when autophagy is inadequate.²⁴

In the context of pterygium, the progression of fibrosis has been associated with dysregulation of autophagy-related pathways and suppression of apoptosis, both of which may contribute to abnormal fibrovascular proliferation and persistence of pterygium tissue. Apoptosis, a process of programmed cell death, is crucial for maintaining tissue homeostasis by ensuring the orderly cell demise. The dynamic equilibrium between cell death and renewal has been well documented in various tissues. Previous studies have indicated that, in pterygium tissue, apoptosis is either suppressed or certain cell types are dysregulated when compared with normal conjunctiva. This reduction in apoptosis, together with cellular alterations, may lead to abnormal proliferation and the formation of invasive fibrovascular tissue in conjunctival fibroblasts and epithelial cells.^24,25

Exposure to UVB radiation is known to induce DNA damage through increased oxidative stress, which subsequently affects the regulation of pro-apoptotic proteins, including Bcl-2. This activation suppresses apoptosis and contributes to the proliferation of abnormal pterygium tissue.^26,27 In the present study, we observed a significant increase in Bcl-2 expression in pterygium tissue, which is classified as abnormal conjunctival tissue, in comparison to normal conjunctival tissue. The Bcl-2 gene is recognized for its antiapoptotic properties, inhibiting the process of apoptosis.²⁷ Our findings demonstrated elevated Bcl-2 expression in pterygium tissues, suggesting that suppression of apoptosis facilitates pterygium formation. The objective of this study was to investigate the role of Beclin-1 in pterygium tissue through immunohistochemical analysis, with particular focus on the mechanisms of apoptosis- and autophagy. Beclin-1 is a pivotal protein involved in the regulation of autophagy, a process that degrades and recycles damaged organelles and proteins, thereby maintaining cellular health. Within this mechanism, Beclin-1 plays a central role in the formation of double-membrane structures known as autophagosomes, which sequester damaged organelles and proteins within the cell.²⁷

ATG5 is a critical autophagy marker involved in autophagosome formation. It plays a pivotal role during the early stages of autophagy by forming a complex with ATG12 and ATG16, thereby facilitating elongation and closure of the autophagosomal membrane. Proper functioning of this process is essential for cellular survival under stress conditions. Beclin-1, an autophagy-inducing gene, initiates autophagosome formation and regulates autophagosome maturation. In addition, LC3A/B (microtubule-associated protein 1 light chain 3A/B) is integral to autophagosome membrane formation, and the conversion of LC3-I to LC3-II through lipidation is commonly used as a marker associated with autophagosome formation and autophagy-related activity. The adaptor protein p62/SQSTM1 mediates the recognition and transport of ubiquitinated protein aggregates to autophagosomes by binding LC3, whereas accumulation of p62 indicating impaired autophagic flux. Collectively, the coordinated activities of Beclin-1, ATG5, LC3, and p62 reflect the dynamic regulation of autophagy machinery and their crucial role in maintaining cellular homeostasis, particularly under oxidative and inflammatory stress.^20,21,27 In our study, we observed elevated expression of Beclin-1 in pterygium tissues, suggesting dysregulation of autophagy-related pathways in these tissues. Autophagy can lead to either cell death or survival. The substantial increase in Beclin-1 may suggest that autophagy-related responses that favor cellular survival pathways, as evidenced by the concurrent rise in ATG-5 levels. Specifically, the increased expression of Beclin-1, LC3A/B, and ATG5 in pterygium tissues suggests enhanced autophagosome-related signaling and altered regulation of autophagy-related pathways. Therefore, the combined increase in Beclin-1, LC3A/B, ATG-5, and Bcl-2 is associated with suppression of apoptosis together with enhanced autophagosome-related signaling, suggesting that autophagy-related responses may favor cellular survival rather than cell death in pterygium tissue.

There is a paucity of studies in the literature investigating programmed cell death mechanisms in pterygium, particularly concerning the role of autophagy in pterygium tissues, which remains an area of ongoing interest. A comprehensive study employing immunohistochemical analyses of rabbit conjunctival fibrosis and markers of apoptosis, autophagy, and inflammation, both in vitro and in vivo, reported elevated levels of Beclin-1, the ATG5/12 conjugate, and LC3B in pterygium tissue. This study aimed to modulate dysregulated autophagy- and apoptosis-related pathways in pterygium tissue through the application of pirarubicin and demonstrated increased levels of Beclin-1, the ATG5/12 conjugate, and LC3B following treatment. These findings suggest that pirarubicin exerts an antifibrotic effect on pterygium tissue by regulating impaired autophagy, which favors survival over cell death. Specifically, pirarubicin may enhance autophagy marker expression in pterygium tissue, thereby suppressing fibroblast proliferation and inhibiting pterygium growth. In addition, pirarubicin treatment induced mitochondria-mediated apoptosis and modulated impaired autophagy mechanism. This process involved activation of the mitochondria-mediated apoptotic pathway, as evidenced by activation of caspase-3, caspase-7, and caspase-9 following high-dose pirarubicin treatment (15 µmol/L).²³

Consistent with these previous findings, our study also demonstrated increased expression of Beclin-1 and ATG5 in pterygium tissues, supporting dysregulation of autophagy-related pathways in fibrovascular conjunctival tissue. However, unlike pirarubicin-based experimental models in which apoptosis-related caspase activation was observed, Caspase-8 expression did not show a statistically significant increase in our study. This finding may suggest that autophagy-related pathways in pterygium preferentially support cellular survival mechanisms rather than apoptosis-mediated cell death. Increased Bcl-2 expression together with elevated Beclin-1, LC3A/B, ATG5, and p62 levels further supports altered regulation of autophagy- and apoptosis-related pathways in pterygium tissue.²⁰ In a separate study, a fibrosis model was developed to investigate autophagy-related pathways in pterygium tissue. It was observed that intact autophagy activation attenuated the fibrotic process, whereas inhibition of autophagy exacerbated fibrosis. Consequently, regulation of autophagy may represent a potential therapeutic target for the treatment of pterygium.²³ In our study, we observed significant upregulation of Beclin-1, ATG5, LC3A/B, and p62 expression in pterygium tissues, supporting dysregulation of autophagy-related pathways in these tissues. Autophagy can result in either cellular survival or cell death, depending on the cellular context and the extent of activation. The concurrent upregulation of Beclin-1 and ATG5 suggests an enhancement in autophagosome formation and altered regulation of early autophagy-related processes, thereby promoting a cytoprotective (pro-survival) response rather than cell death. Furthermore, the elevation of LC3A/B supports increased autophagosome formation, whereas accumulation of p62 may indicate partial blockage or overload of autophagic flux, potentially resulting from chronic oxidative or inflammatory stress within the conjunctival microenvironment. In addition, increased expression of Bcl-2, an antiapoptotic protein, suggests that apoptosis is suppressed while the autophagic machinery remains active up to the autophagosome formation stage. Collectively, the upregulation of Beclin-1, ATG5, LC3A/B, p62, and Bcl-2 suggests that autophagy favors cellular survival over cell death in pterygium tissue, thereby contributing to the persistence and progression of the lesion. Another study reported that pterygium formation and cellular proliferation increased when apoptosis was inhibited and cellular proliferation was stimulated, respectively.¹⁸ Previous studies evaluated the expression of p53, Bcl-2, and Ki-67 were assessed in both primary and recurrent pterygium tissues and compared these findings with normal conjunctival tissue samples.^25,26 The findings indicated that the expression levels of p53, Bcl-2, and Ki-67 were markedly elevated in the pterygium tissue samples relative to the control group. The authors noted that pterygium has been characterized by tumor-like properties, with antiapoptotic mechanisms and cellular proliferation playing a significant role in its etiopathogenesis. Previous research has explored autophagy- and apoptosis-related markers in pterygial tissues.

One study demonstrated increased Beclin-1 and LC3B expression in pterygium tissue compared with normal conjunctiva, suggesting altered autophagy-related activity as a potential cellular response to oxidative damage.¹⁴ Similarly, another study reported upregulation of ATG5 and p62/SQSTM1, indicating that autophagy is initiated but that autophagic flux may be incomplete, potentially resulting in the accumulation of damaged proteins.²¹ In another study, elevated Bcl-2 levels were observed together with reduced caspase activity, suggesting suppression of apoptosis and promotion of cellular survival. These findings align with our results, in which increased expression of Beclin-1, LC3A/B, ATG5, p62, and Bcl-2 collectively supports dysregulation of autophagy-related pathways together with inhibition of apoptosis, supporting the hypothesis that autophagy serves a cytoprotective role in the pathogenesis and progression of pterygium.²⁸ In our study, the increased Beclin-1 expression observed in pterygium tissues suggests that this protein plays an active role in the initiation of autophagy. Beclin-1 is a fundamental protein involved in autophagosome formation and is a key regulator of autophagy. Beclin-1 expression was significantly higher in pterygium tissues than in normal conjunctival tissue. This increase may represent the autophagic response of cells to oxidative stress and UV exposure. The elevated Beclin-1 levels observed in our study are consistent with these findings and may reflect altered autophagy-related responses contributing to cellular survival in pterygium tissue.

LC3A/B (microtubule-associated protein 1 light chain 3A/B) serves as a morphological marker of autophagy and is integral to the formation of autophagosomal membranes. The conversion of LC3-I to LC3-II conversion is commonly used as a marker associated with autophagosome formation and autophagy-related activity. In pterygium tissues, elevated LC3B levels suggest altered autophagy-related activity during the early stages of autophagosome formation. Similarly, our study demonstrated increased LC3A/B expression, suggesting altered autophagosome-related activity in pterygium tissue. This observation supports the hypothesis that autophagy facilitates cellular survival in pterygium under chronic stress conditions.^14,20,27 ATG5, which is crucial during the early stages of autophagy, is involved in the elongation and closure of autophagosomal membranes.

The ATG5–ATG12 complex is crucial for the proper progression of autophagy.²⁹ One study demonstrated that increased ATG5 expression in the pterygium epithelium indicates initiation of autophagy, although autophagic flux may not be fully completed.²⁰ In our study, elevated ATG5 expression similarly suggested altered regulation of early autophagy-related processes, supporting the notion that the cell develops a protective response through autophagic mechanisms.²⁰

p62/SQSTM1 interacts with LC3 by recognizing ubiquitinated proteins and facilitating the transport of these complexes to autophagosomes for degradation. During active autophagy, p62 is degraded; however, when autophagic flux is impaired, it accumulates within cells.³⁰ It has been reported that p62 expression is elevated in pterygium tissues, suggesting that autophagy is initiated but not fully completed. In our study, increased p62 levels similarly suggest dysregulation of autophagy-related pathways, with autophagic flux potentially being partially impaired, resulting in the accumulation of damaged proteins. This implies an increased autophagic burden under chronic oxidative stress. Bcl-2, an antiapoptotic protein, plays a pivotal role in regulating the balance between autophagy and apoptosis by directly interacting with Beclin-1. Overexpression of Bcl-2 suppresses apoptosis by inhibiting mitochondrial cytochrome-c release and caspase activation, thereby promoting cellular survival. Conversely, Caspase-8 functions as an initiator caspase in the extrinsic apoptotic pathway and interacts with autophagy-related proteins. Recent studies have demonstrated that Caspase-8 can be selectively degraded through autophagy, thereby establishing a regulatory feedback loop between the two processes.³¹ In our study, increased Bcl-2 expression, together with decreased Caspase-8 activity, suggests suppression of apoptosis and a cellular shift toward autophagy-mediated survival in pterygium tissues. These findings indicate that dysregulation of the Bcl-2–Beclin-1–Caspase-8 axis may play a significant role in the persistence and progression of pterygium by preventing programmed cell death and promoting autophagy-related maintenance of damaged cells. Consequently, our study suggests that balancing the development of apoptosis and autophagy in pterygium tissue is crucial in controlling fibrotic progression within the tissue. In light of our results, we observed significant differences in the primary markers of autophagy- and apoptosis-related markers compared with normal conjunctival tissue. These findings may contribute to the development of novel therapeutic strategies new therapeutic strategies targeting autophagy- and apoptosis-related pathways. We propose that comprehensive study, particularly one that examines the entire pathway in the autophagy mechanism and identifies the stage at which deterioration occurs, will be beneficial in developing alternative treatment methods for pterygium tissue, where surgery is currently the sole treatment option. Furthermore, we believe that detailed evaluation of the autophagy and apoptosis mechanisms, which form a complex structure, using different techniques, will pave the way for potential treatment options for pterygium by regulating these mechanisms.

One important limitation of the present study is that autophagy was evaluated using static immunohistochemical expression of autophagy-related proteins, including Beclin-1, LC3A/B, ATG5, and p62. Although these markers provide evidence of dysregulation of autophagy-related pathways, they do not directly measure dynamic autophagic flux. In particular, increased p62 accumulation may also indicate impaired autophagic degradation or incomplete autophagic flux. Therefore, our findings should be interpreted with caution and not considered definitive evidence of functional autophagy activation. Future studies using LC3-II/p62 turnover assays, lysosomal inhibition experiments, and additional molecular techniques are needed to clarify the precise status of autophagic flux in pterygium tissue. In addition, transmission electron microscopy-based evaluation of autophagosome ultrastructure may provide further evidence regarding autophagy-related phenotypes in pterygium tissue.

Conclusion

This study demonstrates that dysregulation of apoptosis and autophagy-related pathways may play an important role in the progression of pterygium. Pterygial samples showed significant differences in key pathway markers compared with normal conjunctival tissues. Our findings highlight the importance of these mechanisms in pterygium pathogenesis and suggest potential nonsurgical therapeutic strategies. Future research should evaluate the entire autophagy pathway, identify stages of dysregulation, and investigate the interplay between apoptosis and autophagy to uncover regulatory networks and develop targeted therapies that may contribute to future nonsurgical therapeutic approaches.

Footnotes

Acknowledgments

We thank the participants of the study.

ORCID iDs

Nazlı Can

Aylin Karalezli

Murat Kaşıkcı

Göksu Alaçamlı

Ethical approval

The study protocol was approved by the Ethical Committee of Mugla Sıtkı Koçman University (E-72855364-050.01.04-238041) and was conducted in accordance with the ethical principles of the Declaration of Helsinki of 1975, as revised in 2024. Written informed consent was obtained from all participants. Due to delays in reagent procurement and laboratory processing, patient recruitment and tissue analyses were completed during the 2024–2025 study period under the scope of the originally approved protocol.

Author contributions

“G.K. was responsible for the design of the study. G.K. and N.C. undertook the data collection. The data analysis was conducted by A.Y. and K.Y. The interpretation of the data was carried out by K.Y. and G.A. The drafting of the manuscript was the responsibility of K.Y. and G.A. M.K. performed the critical revision of the manuscript and revised the paper in accordance with the journal guidelines. All authors reviewed and approved the final version of the paper.”

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

This article reports original research and has not been published previously nor is it under consideration for publication elsewhere. This manuscript has not been submitted to any other journal.

The contents of this manuscript are original, and there is no plagiarism.

All authors agree to pay the Article Processing Charges if the manuscript is accepted for publication.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Prior presentation

This research has not been previously presented at any conference or symposium.

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