Understanding cataract development in axial myopia: The contribution of oxidative stress and related pathways

Abstract

Myopia is an evolving global health challenge, with estimates suggesting that by 2050 it will affect half of the world's population, becoming the leading cause of irreversible vision loss. Moreover, myopia can lead to various complications, including the earlier onset of cataracts. Given the progressive aging of the population and the increase in life expectancy, this will contribute to a rising demand for cataract surgery, posing an additional challenge for healthcare systems. The pathogenesis of nuclear and posterior subcapsular cataract (PSC) development in axial myopia is complex and primarily involves intensified liquefaction of the vitreous body, excessive production of reactive oxygen species, impaired antioxidant defense, and chronic inflammation in the eyeball. These factors contribute to disruptions in mitochondrial homeostasis, abnormal cell signaling, lipid peroxidation, protein and nucleic acid damage, as well as the induction of adverse epigenetic modifications. Age-related and oxidative processes can cause destabilization of crystallins with subsequent protein accumulation, which finally drives to a lens opacification. Moreover, an altered redox status is one of the major contributors to the pathogenesis of PSC. This review aims to summarize the mechanisms known to be responsible for the accelerated development of cataracts in axial myopia and to enhance understanding of these relationships.

1. Introduction

Myopia is a refractive error that can be classified qualitatively into three types: axial myopia (resulting from an above-average axial length (AL) of the eyeball), refractive myopia (caused by changes in the structure or location of the eye's optical components), and secondary myopia (caused by specific conditions that are not considered general risk factors for myopia). Quantitatively, myopia is classified as low myopia (LM), where the spherical equivalent refractive error of the eye is ≤ −0.5 diopters (D) but > −6.0 D after cycloplegia, and high myopia (HM), where the refractive error is ≤ −6.0 D after cycloplegia, along with pre-myopia [1]. While genetic factors undeniably play a role in the development of myopia, environmental factors have a greater influence [[2], [3], [4], [5]]. However, the pathogenesis of this refractive defect is still not fully understood. Among the mechanisms and signaling pathways associated with myopia, several have been identified, including hormones (dopamine, insulin, insulin-like growth factor 1 (IGF-1), glucagon, prostaglandins), neurotransmitters (acetylcholine, nitric oxide (NO), gamma-aminobutyric acid (GABA)), and other biomolecules such as retinoic acid, transforming growth factor β (TGF-β), homolog sonic hedgehog (SHH), cyclic adenosine monophosphate (cAMP), matrix metalloproteinase 2 (MMP-2), and hypoxia-inducible factor 1α (HIF-1α). These molecules influence the reduction of collagen content, extracellular matrix integrity, scleral remodeling, and AL elongation, ultimately leading to the thinning of the choroid and retina [[5], [6], [7]].

In 2000, myopia affected approximately 1.4 billion individuals (22.9 % of the population), with HM present in 163 million (2.7 %) people worldwide. It is estimated that by 2050, the number of individuals with myopia will rise to 4.8 billion (49 % of the global population), while 938 million (9.8 %) will develop HM. The extremely rapid increase in this refractive error classifies it as a civilization disease [8]. In 2015, the global potential loss of productivity due to uncorrected myopia-related visual impairment was estimated at 244 billion USD [9]. Myopia can also lead to several complications, including open-angle glaucoma, macular degeneration, and retinal detachment, which can significantly reduce visual acuity and even lead to blindness, creating additional social and economic burdens on healthcare systems [[10], [11], [12]]. Population-based studies have confirmed that myopia is a risk factor for the development of nuclear cataracts (NC) and posterior subcapsular cataracts (PSC) [[12], [13], [14], [15]]. However, the mechanisms behind these associations remain an active area of research.

A cataract is a condition characterized by lens opacification, resulting in a significant reduction in visual acuity. The most common type is age-related cataract (ARC). Its pathogenesis is complex and involves various processes, including cellular aging, increased apoptosis, mitochondrial dysfunction, excessive production of reactive oxygen species (ROS), impaired antioxidant defense, cell signaling defects, loss of proteostasis, water-electrolyte imbalances, genetic and epigenetic instability. These factors contribute to lipid peroxidation, DNA damage, protein modification and aggregation, all of which reduce the lens's transparency [16,17]. Cataracts remain the leading cause of blindness and the second most common cause (after uncorrected refractive errors) of moderate to severe visual impairment in individuals aged ≥50 years worldwide [18]. The age-standardized pooled prevalence estimate for cataracts is 17.20 % [19]. As the global population ages, life expectancy increases, and the prevalence of myopia rises, cataract incidence continues to grow. The increasing demand for cataract surgery, along with its associated costs, will place an even greater strain on healthcare systems and socioeconomic resources [18].

This review aims to summarize the current knowledge regarding the potential mechanisms by which axial myopia contributes to the accelerated development of NC and PSC.

2. Liquefaction of the vitreous body

The vitreous body (VB) plays a crucial role in the distribution and elimination of molecular oxygen from the vascular structures of the eyeball [20,21]. This process is largely facilitated by the presence of ascorbic acid (AsA), which is found in extremely high concentrations in the healthy VB, reaching an average of 2 mM, compared to a blood level of just 50–60 μM [22]. The reaction of AsA with molecular oxygen produces dehydroascorbic acid (dAsA) and water. Additionally, AsA eliminates hydrogen peroxide (H₂O₂) from the VB, offering retinal protection against oxidative stress (OS) and lipid peroxidation [20,23]. The gel-like state of the VB, which slows the migration of dissolved gases and maintains intraocular gradients, is essential for antioxidant defense. Oxygen diffuses from the retinal blood vessels into the VB, with partial pressure at the retinal surface around 22.0 mmHg, where it is utilized by the inner layers. Further diffusion is mediated by AsA, maintaining partial pressure at 7.0 mmHg in the VB center and 9.0 mmHg near the posterior lens capsule [20,21,24] (Fig. 1). These biochemical and physical properties allow richly vascularized ocular structures like the retina and choroid to remain highly oxygenated, while sensitive structures like the lens are kept in a relatively hypoxic environment.

Fig. 1.

(A) Intact vitreous body (VB) with preserved oxygen (O₂) partial pressure gradient and undisturbed diffusion of glutathione (GSH) and glutathione disulfide (GSSG) between the superficial, metabolically active zone and the lens nucleus. (B) Liquified VB with subsequent impaired O₂ distribution affecting nuclear cataract development.

As aging progresses, the VB loses its homogeneity and undergoes liquefaction, a degenerative process triggered by OS and increased levels of proteolytic enzymes. The loss of proteoglycans and collagen IX, along with the detachment of hyaluronic acid from collagen, leads to the aggregation of collagen II fibers, previously dispersed in the center of the VB. This reorganization creates free fluid spaces (lacunae) between collagen fibers, allowing them to bond [25]. This change, along with eye movements, facilitates convection and circulation of fluids, leading to increased oxygen transport from the retina toward the lens. As oxygen mixes with the liquefied VB, the chances of interaction with AsA increase, resulting in the depletion of both oxygen and AsA. When the active transport of AsA into the VB remains stable, its cumulative depletion reduces oxygen consumption, leading to increased intraocular oxygen partial pressure and greater exposure of the lens to oxygen and ROS [20,21,26]. Moreover, since the human body cannot synthesize AsA de novo, it must be obtained exogenously through the diet [20,22]. Computational models show that mild VB liquefaction can lead to a twofold increase in oxygen levels near the posterior surface of the lens, with up to a fourfold increase in cases of significant liquefaction [26]. Faster diffusion in liquefied VB may also enhance passive diffusion of sodium ions into the lens through the posterior capsule, disrupting lens function [27].

The reduced form of glutathione (GSH), synthesized in lens epithelial cells (LECs) and immature lens fibers, is one of the most important components of the lens's antioxidant defense. It is delivered to the lens center via gap junctions and the microcirculatory system. As the lens ages, cytoplasmic stiffening in the deeper layers and oxidative damage to transport proteins limit both GSH movement to the center and the transport of its oxidized form, glutathione disulfide (GSSG), from the lens nucleus to the surface cells. Increased oxidation of lens proteins and lipids worsens in the lens center, creating a feedback loop that makes the nucleus more vulnerable to oxidative damage and the accumulation of abnormal proteins, leading to cataract development [28]. Loss of the gel-like structure of the VB and its antioxidant potential exacerbates NC formation. Among patients aged 51 to 70, the degree of VB liquefaction is a stronger predictor of lens nucleus opacification than age [29]. Structural degeneration of the VB is also associated with posterior vitreous detachment (PVD), which can enhance oxidative changes. However, isolated PVD has not been shown to increase lens oxygen concentration [26].

Patients undergoing pars plana vitrectomy (PPV) under general anesthesia with 100 % oxygen inhalation showed an immediate and significant increase in oxygen partial pressure compared to those operated on under local anesthesia with 21 % oxygen [24]. Animal studies also demonstrate that increased oxygen diffusion into the lens following VB oxygenation raised oxygen content in the lens nucleus [30]. In hyperbaric oxygen therapy (HBO), where 100 % oxygen is inhaled at 2–3 atm of pressure, oxygen transfer to tissues increases up to 20-fold, impairing antioxidant defense with continued treatment [31,32]. After 15 sessions of HBO, oxidative stress levels increased, with a simultaneous rise in ROS [32]. Palmquist et al. [33] reported that half of the study participants who attended 150–850 HBO sessions developed NC, and all, except a 24-year-old man, exhibited early-stage lens nucleus opacification. Additionally, NC developed earlier in caisson workers exposed to elevated oxygen levels for extended periods [34].

With the growing accessibility and popularity of HBO, it is increasingly applied in the treatment of both acute conditions (such as decompression sickness, crush syndrome, acute traumatic ischemia, carbon monoxide poisoning, gas embolism, and sudden idiopathic hearing loss) and chronic conditions (including non-healing wounds, necrotic soft tissue infections, skin grafts and flaps at risk of necrosis, radiation-induced tissue damage, and chronic osteomyelitis). Certain factors and conditions that indicate the use of HBO (e.g., prior radiotherapy, uncontrolled diabetes) may accelerate cataract development, which is why patients should be informed of potential complications. Nevertheless, in most cases, the benefits of this therapy outweigh the risk of exacerbating cataract formation [31,32]. The results from PPV patients provide compelling evidence that an intact VB protects the lens from NC progression. Oxygen partial pressure in the eye was significantly higher after PPV, and the oxygen gradient between the lens and the VB center was reduced [24]. Among patients over 50 years old, 60–95 % developed NC requiring surgery within two years of PPV [[35], [36], [37], [38]], while those under 50 showed less than 10 % incidence, likely due to more efficient oxygen metabolism and greater resistance to oxidative damage in younger lenses [38]. Notably, patients undergoing epiretinal membrane (ERM) peeling without VB removal did not show increased cataractogenesis [39]. Furthermore, rabbit lens analyses six months after PPV under systemic hyperoxia revealed reduced levels of water-soluble proteins, AsA, catalase (CAT), and sodium-potassium pump activity, alongside increased levels of malondialdehyde (MDA) and TGF-β. These changes were most prominent in the lens nucleus [40]. Observational studies and Mendelian randomization indicate that an above-average AL increases the risk of VB degeneration, including earlier liquefaction [25,41,42]. This may partly explain the accelerated development of NC in individuals with myopia. PVD usually occurs when more than 60 % of the VB is liquefied [43]. Hayashi et al. [44] found that among patients aged 50–59 and 60–69, total PVD occurred in 54 % and 73.9 % of eyes with an AL > 26.0 mm, respectively, compared to only 14 % and 44 % of eyes with an AL ≤ 26.0 mm. Morita et al. [45] demonstrated that in eyes with an AL > 26.0 mm, PVD occurred about 20 years earlier than in those with LM, estimating that each additional diopter of myopia reduces the age of PVD onset by 0.91 years from the average of 61 years [46]. As the eyeball elongates, the volume of the VC also increases. Animal studies of deprivational myopia suggest that VB gel formation occurs only during the embryonic phase, while postnatal eyeball growth is supported by the production of a low-protein fluid that fills the enlarging VC [47]. Postmortem analysis of myopic eyes confirmed lower levels of proteins, collagen, and hyaluronic acid in the VB compared to emmetropic controls [48]. In longer eyes, structural and biochemical changes in the retinal pigment epithelium (RPE) may affect the VB. Mouse models of myopia show lower levels of potassium, sodium, chlorine, and proteins essential for homeostasis and recovery in the VB, alongside elevated levels of inflammation-related proteins [49]. Levels of MMPs, particularly MMP-2, which degrade type IX collagen crucial for maintaining the VB's gel-like state, are about 50 % higher in myopic eyes compared to controls [[50], [51], [52]]. As the AL increases, levels of pro-inflammatory cytokines, OS, and lipid metabolism intensify, further accelerating VB degeneration [[53], [54], [55]]. Peng et al. [56] also noted elevated levels of Dickkopf glycoprotein 1 (DKK1) in the VB of myopic subjects, correlating strongly with AL. DKK1 inhibits the Wnt/β-catenin pathway, which is responsible for collagen accumulation and extracellular matrix protein release, but its role in VB liquefaction is not yet fully understood [56,57].

3. Disrupted mitochondrial homeostasis

LECs serve as the metabolic center of the lens, and maintaining mitochondrial homeostasis within these cells is crucial to ensure proper function and preserve lens transparency. Mitochondria are the primary source of ROS in LECs. When oxygen levels in the ocular microenvironment increase, oxygen metabolism intensifies, leading to greater ROS production due to electron leakage in the mitochondrial respiratory chain [58]. Mitochondrial DNA (mtDNA), located in close proximity to ROS generation sites, is particularly vulnerable to oxidative damage because it lacks the protective histones present in nuclear DNA. Damage to mtDNA exacerbates ROS production, creating a self-perpetuating cycle of mitochondrial dysfunction and oxidative stress [59]. In experiments on rat lenses, administration of a mitochondrial uncoupler worsened H₂O₂-induced lens opacities, highlighting the sensitivity of mitochondrial homeostasis [60]. Conversely, the application of mitochondrial-targeted antioxidants significantly inhibited cataract formation [61].

Sun et al. [60] investigated mitochondrial homeostasis, including biogenesis, dynamics, and autophagy in the LECs of patients with high-myopic cataracts (HMC) and ARC. In patients with HMC, there was a marked reduction in mtDNA copy numbers and elevated levels of oxidative DNA damage markers, such as 15A3. These markers colocalized with the mitochondrial marker translocase of outer mitochondrial membrane 20 (TOM20), indicating greater oxidative damage to mtDNA compared to patients with ARC. The study also revealed lower levels of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in HMC patients. PGC-1α and TFAM, through their interaction, regulate mitochondrial function and energy metabolism, which are essential for maintaining overall cell health [62]. Additionally, PGC-1α modulates the antioxidative activity of nuclear factor erythroid 2-related factor 2 (NRF2), which is vital in sustaining redox balance. NRF2 binds to antioxidant response elements (AREs) in the promoter regions of antioxidant enzyme genes, thereby inducing their expression [63].

Mitochondrial dynamics, which include both fission and fusion, are influenced by environmental factors such as oxidative stress. In response to oxidative stress, mitochondria often accelerate their division (fission) to remove damaged organelles—a process mediated by dynamin-related protein 1 (DRP1) [64]. On the other hand, mitochondrial fusion, controlled by mitofusin 1 (MFN1) and mitofusin 2 (MFN2), allows for the repair of moderately damaged mitochondria [65]. In patients with high-myopic nuclear cataracts (HMNC), there is an observed increase in DRP1 levels coupled with a reduction in fusion proteins, suggesting that impaired mitochondrial dynamics contribute to cataract formation by favoring excessive fission and limiting fusion, thereby reducing mitochondrial repair potential [60].

Mitochondrial autophagy, or mitophagy, helps degrade damaged or malfunctioning mitochondria [66]. In the LECs of patients with HMC, there was a significant increase in the colocalization of mitophagy-related proteins, such as microtubule-associated protein 1A/1B-light chain 3 (MAP1A/1B LC3) and TOM20, alongside a noticeable reduction in mitochondrial numbers. In contrast, mitochondrial autophagy was less frequently observed in the ARC group. Of the two primary pathways for mitochondrial labeling in mitophagy, the phosphatase and tensin homolog-induced kinase 1 (PINK1)/Parkin signaling pathway was predominantly activated in the LECs of HMC patients [60]. These findings demonstrate significantly greater impairment of mitochondrial homeostasis in the LECs of HMC patients compared to those with ARC, suggesting that this disruption contributes to the faster progression of cataracts in individuals with myopia (Fig. 2).

Fig. 2.

Disrupted mitochondrial homeostasis in lens epithelial cell (LEC) caused by reactive oxygen species (ROS) activity. ATP: adenosine triphosphate; Ca²⁺: calcium ion; DRP1: dynamin-related protein; LC3: light chain 3; LEC: lens epithelial cell; MFN1: mitofusin 1; mtDNA: mitochondrial deoxyribonucleic acid; PINK1: phosphatase and tensin homolog induced kinase 1; ROS: reactive oxygen species.

4. Lipid peroxidation

The retina has an extremely high metabolic demand, with the highest mitochondrial density and oxygen consumption of any tissue in the body, making it particularly vulnerable to hypoxia [67]. In cases of high myopia, the elongation of the eyeball is often accompanied by impaired retinal vascularization [68]. Studies have shown that the greater the degree of myopia, the lower the ocular pulse amplitude, a parameter that reflects choroidal blood flow. This reduction in blood flow due to eyeball elongation has also been confirmed in animal models [69,70]. Such circulatory impairments lead to repeated episodes of transient microischemia, followed by retinal reperfusion, which are major contributors to chronic OS. The initial phase of ROS production originates from mitochondria during ischemic conditions, followed by xanthine oxidase activation in response to oxygen and glucose deprivation. The final phase, triggered during reperfusion, involves calcium-dependent activation of NADPH oxidase, further increasing ROS production [71]. Moreover, the retina is continually exposed to ultraviolet (UV) radiation, which exacerbates ROS generation and oxidative stress. Accumulated ROS can activate nuclear factor κB (NF-κB), promoting the release of proinflammatory cytokines [72]. This chronic inflammation leads to endothelial cell activation and dysfunction, causing reduced vasodilation and impaired leukocyte recruitment. This cascade worsens microcirculatory disturbances, further reducing oxygen availability and amplifying oxidative stress [73].

The outer segments of retinal photoreceptors are rich in polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA), which is highly susceptible to peroxidation due to its numerous double bonds. This process results in the formation of MDA, a toxic byproduct of lipid peroxidation [74]. Studies on animals with induced HM have revealed elevated MDA levels in their retinas and scleras, as well as reduced activity of the antioxidant enzyme superoxide dismutase (SOD) compared to controls [75]. Proteomic analysis of chick retinas with myopia also showed increased levels of glutathione S-transferase Mu 2 (GSTM2), an enzyme important in the degradation of lipid peroxides, reflecting a compensatory response to increased oxidative damage [76].

Higher concentrations of MDA have also been observed in ARC compared to clear lenses, with the highest levels found in HMC [[77], [78], [79]]. Additionally, patients with HMC showed increased GSH oxidation and reduced SOD levels compared to emmetropic individuals [78,79]. These findings suggest that elevated MDA levels in ARC are linked to age-related declines in antioxidant defenses. However, the significantly higher levels of MDA and oxidative protein damage in HMC highlight the intensified oxidative stress in high myopia. This may not only result from lipid peroxidation in lens fiber membranes but also from the migration of toxic byproducts from other ocular regions. Elevated MDA levels in the VB of myopic patients point to a retinal origin and imply its potential role in cataract formation [79]. A correlation has also been found between the degree of myopia and the concentration of lipid peroxidation products, such as thiobarbituric acid (TBA), in the subretinal fluid of patients with retinal detachment [80].

Babizhayev et al. [81] and Goosey et al. [82] demonstrated that the injection of lipid peroxidation products into the vitreous cavity led to their accumulation in the lens, a reduction in GSH levels, and PSC formation in animal models. Their studies revealed that even small amounts of oxidized lipids are sufficient to damage lens fibers and initiate cataractogenesis. MDA's toxic effects are largely due to its high propensity for forming cross-links with proteins. Furthermore, MDA can induce aggregation of soluble lens proteins, with its accumulation levels correlating with cataract severity. Lipid peroxidation, along with membrane protein oxidation, can also disrupt membrane permeability, leading to impaired water-electrolyte balance. This imbalance rapidly compromises cell hydration, resulting in lens opacification [77,81]. The relationship between increased retinal-derived lipid peroxidation and PSC formation is also observed in individuals with tapetoretinal degenerations such as retinitis pigmentosa (RP) [[81], [82], [83]]. These findings suggest that excessive retinal lipid peroxidation, often found in elongated eyeballs, contributes to lens damage and plays a role in the development of NC and PSC in myopic individuals.

5. Oxidative stress

OS has been found not only to play a significant role in the progression of ocular AL but also to induce several abnormalities associated with myopia [84,85]. Total antioxidant capacity (T-AOC) is an indicator of all antioxidant substances and reflects the organism's ability to neutralize ROS. A decrease in T-AOC implies increased OS, which leads to damage to DNA, proteins, and lipids [86]. Additionally, MDA, the main product of lipid peroxidation, is often used in studies as a biological marker of OS [40,75,[77], [78], [79],84,[87], [88], [89]].

The oxidative damage in the course of myopia can disrupt the neuromodulation of dopamine and NO throughout ocular growth and development [85]. In pathological conditions such as under the influence of cytokines, inducible nitric oxide synthase (iNOS) is activated, which catalyzes the synthesis of the free radical i.e. NO from l-arginine [90]. Metabolomic analysis of aqueous humor (AH) from the anterior chamber of the eyeball demonstrated elevated level of arginine and citrulline, which are associated with NO formation, among subjects with HM [91]. NO, produced at high levels, can be further converted to subsequent reactive nitrogen species, as well as enhance ROS production in the mitochondria. Elevated level of nitrite/nitrate, the more stable forms of NO, reflects the degree of nitrosative and, to some extent, oxidative stress [90]. Increased nitrite level has also been reported in the lenses of cataract patients (the highest for PSC) compared to translucent lenses, but this was not a statistically significant difference [92]. On the other hand, it was observed that when α-crystallins were incubated with nitrite, lens proteins were modified similar to those in cataract development, while aminoguanidine, an iNOS inhibitor, caused inhibition of cataractogenesis [93,94]. The oxidative damage in the course of myopia can disrupt the neuromodulation of dopamine and NO throughout ocular growth and development [85]. In pathological conditions, such as under the influence of cytokines, inducible nitric oxide synthase (iNOS) is activated, catalyzing the synthesis of the free radical NO from l-arginine [90]. Metabolomic analysis of aqueous humor (AH) from the anterior chamber of the eyeball demonstrated elevated levels of arginine and citrulline, which are associated with NO formation, among subjects with HM [91]. NO, produced at high levels, can be further converted to subsequent reactive nitrogen species, as well as enhance ROS production in the mitochondria. Elevated levels of nitrite/nitrate, the more stable forms of NO, reflect the degree of nitrosative and, to some extent, oxidative stress [90]. Increased nitrite levels have also been reported in the lenses of cataract patients (the highest for PSC) compared to translucent lenses, though this was not a statistically significant difference [92]. On the other hand, it was observed that when α-crystallins were incubated with nitrite, lens proteins were modified similarly to those in cataract development, while aminoguanidine, an iNOS inhibitor, caused inhibition of cataractogenesis [93,94].

Evaluation of the AH taken during cataract surgery in emmetropic, LM (AL < 26.0 mm), and HM (AL ≥ 26.0 mm) patients showed reduced T-AOC in both myopic groups, more pronounced in those with HM. A negative correlation between T-AOC and AL was also noted. Moreover, total nitrite levels were significantly higher in the group with HM compared to those with standard AL. No significant differences were observed between LM and HM. However, there was a positive correlation between total nitrite levels and AL [95]. Completely opposite results of analysis of AH taken during cataract removal were reported by Kim et al. [87]. They showed a reduced level of 8-hydroxydeoxyguanosine (8-OHdG), a product of oxidative DNA damage and a biomarker of cellular OS, in subjects with myopia (AL ≥ 26.0 mm) compared to controls. Additionally, the level of 8-OHdG was negatively correlated with AL. Furthermore, there was no correlation between the level of MDA and AL. These findings suggest the presence of a lower oxidative status in the AH of patients with myopia. This, according to the authors, may be due to reduced metabolism resulting from degeneration of RPE in the above-average long eyeballs. The difference between the AL correlation with 8-OHdG and MDA is likely due to the fact that 8-OHdG is more stable and has a longer half-life than MDA [96,97]. 8-OHdG also has a higher molecular weight, which is thought to make it more difficult to flow through the trabecular meshwork and consequently prolong the time it remains in the anterior chamber [96]. However, it should be kept in mind that in the above-mentioned study, AL was assessed using ultrasound biometry, which was likely related to lower accuracy of measurements, and the biomarkers were estimated only in the AH, and the presence of cataracts in these patients may have affected the results.

The analysis of AH acquired during phakic lens implantation in subjects aged 18–45 years, without ophthalmic or general diseases that somehow simulates an AH state close to the physiological one, seems to have a lower potential for error. It was proven that among patients with ocular AL ≥ 28.0 mm, T-AOC was lower compared to the control group with AL ≤ 26.0 mm. T-AOC showed a negative correlation with AL, while MDA level in AH was positively correlated with AL. There were no differences in NO concentration, which the authors assigned to the possible NO break-down upon sample storage [88]. Additionally, examination of the anterior lens capsule obtained during cataract surgery indicated that T-AOC was lower and MDA concentration higher in the HMC compared to ARC group (AL ≤ 24.5 mm) [89]. The above results support the link between myopia and impaired oxidative balance. Metabolomic analysis which compared the blood serum of subjects with LM (spherical equivalent (SE) > −3.0 D and SE < 0.0 D) and HM (SE < −6.0 D) found the presence of 20 metabolites that could be potential biological markers of HM. In the second group, higher level of aminomalonic and oleopalmitic acids was noted, indicating an increase in OS. Aminomalonic acid reflects oxidative damage to proteins caused by free radicals and/or ionizing radiation, whereas OS induced by carbon tetrachloride can be confirmed by an increase in oleopalmitic acid [98,99]. Moreover, higher concentrations of three metabolites: shikimic acid, 4-hydroxyphenylacetic acid and anandamide, which exhibit antioxidant properties and/or protect against ROS-induced damage, were observed in the HM group [[100], [101], [102]]. Furthermore, increased energy metabolism in HM was evidenced by the in-creased level of its intermediate products, i.e. citric and oxaloacetic acid [103]. Studies on VB, which is a repository of molecules actively secreted or diffusing from surrounding tissues, as well as blood vessels, appear to be the most relevant. Proteomic analyses of the VB of animals with induced myopia have shown increased amounts of ovotransferrin, which has antioxidant properties and a function similar to SOD [55,104]. Its increased level may be a response to the rising OS that comes with eyeball elongation [55]. Proteomic analysis of VB acquired during PPV showed significantly lower levels of NRF2, a key transcription factor regulating antioxidant protection in HM patients (AL ≥ 26.5 mm and choroidal-retinal degeneration present) compared to controls (AL < 26.5 mm, no choroidal-retinal degeneration). Its level decreased with in-creasing AL and the lowest values were recorded in patients with AL > 29.0 mm. However, the levels of prostaglandin H2 d-isomerase (PGDS) and glutathione peroxidase 3 (GPX3) were higher among patients with ocular AL ranging from 26.5 to 29.0 mm than those with AL > 29.0 mm or AL < 26.5 mm [54]. PGDS plays an important role in modulation of immune and inflammatory responses, apoptosis, neuroprotection and support of antioxidant protection [105]. Its expression is induced by NRF2 [106]. The increase in PGDS levels in AL patients between 26.5 and 29.0 mm is likely related to antioxidant mobilization and inhibition of neuronal apoptosis. The main source of PGDS in the eyeball is the RPE. This is probably why an increase in AL > 29.0 mm, associated with more intense RPE atrophy, results in a reduction in PGDS levels [54]. Oxygen-induced damage to lens proteins leads to increased aggregation and progression of cataract development. The severity of OS can be demonstrated by an elevation of proteins with carbonyl groups, as well as a reduction in the amount of thiol groups in proteins (P-SHs) [107]. The loss of P-SHs in a reduced form is caused by the limitation of GSH availability. In lenses with cataracts among myopic patients, both GSH and SOD levels were lower compared to emmetropic patients [78,79]. Moreover, proteomic comparison of ARC and HMC showed increased phosphorylation of proteins in the lens nucleus in the latter group, including glutathione synthetase (GSS) and glutathione disulfide reductase (GSR), enzymes important for the maintenance of high GSH levels [108]. Furthermore, GSH content varies between types of cataracts. PSC, especially when coexisting with NC, is characterized by a particularly rapid and pronounced decline in GSH [109].

The comparison of the lenses of myopic (AL ≥ 24.0 mm) and emmetropic individuals found that the content of proteins with carbonyl groups increases with age, while the level of P-SHs decreases. In addition, oxidative damage to lens proteins occurs significantly faster in myopic subjects. The greatest decline in P-SHs content among ARC patients is observed between the 7th and 8th decades of life, whereas among myopic patients, it tends to appear 10 years earlier. Likewise, analysis of translucent lenses showed a lower amount of P-SHs in myopic individuals compared to controls [110].

Increased levels of cytokines in cultured human LECs induced by H₂O₂ or UVB radiation demonstrate that OS can also enhance the inflammatory response in the lens [111,112]. For a more detailed evaluation of this relationship, Thompson et al. [113] conducted a study on mouse lenses with an inducible absence of the glutamylcysteine ligase catalytic subunit (GCLC), an enzyme involved in GSH biosynthesis. Deletion of Gclc was thought to enhance OS, resulting in increased production of 42 cytokines in the lens. Potential mechanisms by which OS induces inflammation in LECs include posttranslational modification of histones, mtDNA damage, increased levels of interferon regulatory factor 1 (IRF1), activator protein 1 (AP-1), signal transducer and activator of transcription 3 (STAT3), NOD-like receptor 3 (NLR3), as well as activation of the mitogen-activated protein kinase (MAPK) signaling pathway [111,[114], [115], [116], [117], [118], [119]].

6. Inflammation

It is postulated that HM is a disease associated with inflammation, and there is a proinflammatory microenvironment in eyes with above-average AL, which predisposes them to developing a number of abnormalities. Animal studies support an increased inflammatory status in eyes with myopia, showing elevated levels of interleukin (IL)-6, IL-8, NF-κB, and tumor necrosis factor α (TNFα) [120]. However, extracting material from the human eyeball for analysis is difficult, as it requires surgical intervention. To date, studies are usually based on evaluating AH obtained during cataract removal [[121], [122], [123]].

A comparison of cytokine content in the AH of people with HMC and ARC (AL 21.0–25.0 mm) showed increased levels of several proinflammatory factors in the anterior chamber of the former group [121]. A subsequent study also confirmed higher levels of IL-6 and MMP-2 in the AH of individuals with cataracts and myopia (AL > 25.0 mm) compared to a control group (AL ≤ 25.0 mm) with cataracts. Additionally, both IL-6 and MMP-2 demonstrated a strong positive correlation with AL. For every millimeter of ocular AL growth, IL-6 levels increased by 1.85 pg/mL, and MMP-2 levels rose by 0.57–0.86 ng/mL [122]. IL-6 is a key inflammatory mediator, closely linked to MMP-2 production in neurodegenerative and neuroinflammatory conditions [124]. It is believed that IL-6 also triggers MMP-2 activity in the retina, contributing to scleral thinning and elongation of the eyeball [122,125]. Furthermore, IL-6 promotes the migration of various cell types in malignancies and is thought to play a role in the abnormal migration and adhesion of lens cells [126]. Moreover, decreased levels of the IL-1 receptor antagonist (IL-1RA), which is believed to block the IL-1β receptor, may enhance the inflammatory response. IL-1β induces the production of IL-6 and IL-8 and stimulates LEC proliferation and collagen synthesis [123,127,128]. Additionally, ROS produced by mitochondria contribute to elevated levels of IL-1β by activating NLR-3 [119]. Increased levels of monocyte chemoattractant protein 1 (MCP-1), which recruits monocytes to sites of inflammation, have also been observed in HMC patients [123,129]. The evaluation of LECs collected during cataract removal in patients with ocular AL ≥ 26.0 mm revealed higher levels of IL-6 and IL-8 compared to controls with AL ≤ 24.0 mm. These findings suggest that changes in protein configuration in the AH not only reflect LEC activity but may also affect LECs as ocular AL elongates [130]. These studies highlight the connection between myopia and chronic moderate inflammation in the anterior chamber. However, it is important to note that the presence of cataracts may have influenced the results.

The previously mentioned analysis of AH taken during phakic lens implantation in healthy subjects seems to be less prone to error. It demonstrated that in the group of patients with AL of the eyeballs ≥28.0 mm, the levels of IL-6, IL-1β, and MMP-2 were significantly higher compared to the control group with AL ≤ 26.0 mm. The concentrations of IL-6 and MMP-2 in AH were positively correlated with AL, and this linear relationship was observed in both the HM and LM groups as well [88]. Additionally, the levels of MMP-1, MMP-2, MMP-9, and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the AH of phakic implanted patients were positively correlated with AL [131]. Moreover, the HMC group, compared to the ARC group (AL < 26.0 mm), showed not only increased levels of IL-6 and MCP-1 in AH but also a higher weight (estimated from cytokine concentrations in AH and VB) of IL-6, IL-8, IL-10, MCP-1, and interferon-inducible protein 10 (IP-10) in VB. Furthermore, the weight of IL-8 and IP-10 increased with the severity of myopia [132].

While the concentration of drugs or cytokines in the AH tends to increase or decrease in line with the VB, the VB, which is in direct contact with the retina, seems to better reflect the posterior ocular microenvironment [133]. In the VB (acquired during PPV due to serous retinal detachment, myopia-related retinoschisis, idiopathic ERM, or macular hole) of patients with HM, higher levels of IL-6, MCP-1, IP-10, interferon-γ (IFN-γ), eotaxin, and macrophage inflammatory protein-1α (MIP-1α) were found compared to the emmetropic group [53]. IFN-γ and IL-6 are cytokines strongly related to the inflammatory process, while IP-10 exhibits pleiotropic properties, including the regulation of cell growth and promotion of inflammation [134]. Eotaxin recruits eosinophils to the site of inflammation [135]. Additionally, in response to IL-1β stimulation, MIP-1α, produced mainly by macrophages, activates granulocytes and induces the synthesis and release of other proinflammatory cytokines, such as IL-1, IL-6, and TNF-α [136]. Importantly, TNF-α may contribute to cataract development by increasing mRNA levels of laminin and type IV collagen, as well as promoting the proliferation and migration of LECs [137,138].

Samples taken during PPV carried out for retinal detachment or macular hole in patients with HM (AL ≥ 26.0 mm and SE ≤ −6.0 D) compared to a control group (SE ± 0.5 D) operated on for idiopathic macular hole or ERM showed elevated levels of MMP-2 in the former group. TIMP-2 levels were also compensatorily increased but not sufficiently to neutralize MMP-2 activity, resulting in an elevated MMP-2/TIMP-2 ratio [51]. MMPs and TIMPs regulate the extracellular matrix and tissue vascularization during the inflammatory response. The MMP-2/TIMP-2 imbalance shows a local effect in the eyeball, leading to scleral remodeling. These findings support the presence of an inflammatory microenvironment in myopic eyeballs. However, it should be noted that VB must be taken surgically, and pathological conditions that are an indication for PPV may affect the final profile of inflammatory factors, interfering with the full assessment of the correlation between myopia and inflammatory status.

Numerous studies have shown a link between chronic inflammation and the development of PSC. Gwon et al. [139] were the first to establish this connection by injecting the inflammatory agent concanavalin A into the VB of rabbits, leading to the formation of cataracts similar to those seen in humans. Another instance is the significantly higher occurrence of PSC in patients with uveitis associated with Fuchs' syndrome. In these patients, the AH exhibited much higher levels of proinflammatory cytokines, including basic fibroblast growth factor (bFGF), IL-6, IL-8, and IL-10, compared to those with ARC. Additionally, IL-6 and IL-8 levels were positively correlated with the severity of PSC [138]. A study on rats with hereditary retinal degeneration found increased proinflammatory factors in the VB. This increase could potentially disrupt the basement membrane complex, leading to abnormal migration and differentiation of LECs, which may contribute to PSC development [126]. In patients with RP, PSC was the most common cataract type, affecting about 41–53 % of individuals [83]. A comparison of the VB between patients with RP and those with idiopathic ERM showed significantly higher levels of proinflammatory cytokines and chemokines in the RP group, which may be involved in cataract formation. Some of these cytokines, including IL-1β, IL-6, IL-8, IL-10, MCP-1, MMP-2, IP-10, and IFN-γ, were also found at elevated levels in myopic patients [140].

7. Epithelial-mesenchymal transition of LECs

PSC, characterized by asymmetric lens opacity, is less influenced by aging than NC. Richardson et al. [141] proposed a two-stage process for PSC formation. In the first phase, known as the initiating phase, various risk factors (OS, inflammation, abnormal water-electrolyte balance, and metabolic disturbances) disrupt the function and damage LECs. This leads to the proliferation and migration of LECs along the inner surface of the posterior capsule, from the lens equator to the posterior pole, where they aggregate. During this migration, the cells undergo abnormal differentiation, resulting in the formation of dysplastic Wedl cells, also referred to as bladder cells. The first stage of PSC formation can be partially or completely reversed with the removal of the initiating factors [141,142]. In contrast, the second phase, known as the maturation phase, is irreversible. It involves increased OS and inflammation related to biological aging, which further progresses PSCs, leading to the disintegration of lens fibers and the accumulation of vacuoles and/or dense lamellar opacities [141].

TGF-β, particularly TGF-β2, mediates the epithelial-to-mesenchymal transition (EMT) of LECs, causing these epithelial cells to adopt a mesenchymal-like phenotype and produce extracellular matrix proteins [143]. This transition is associated with decreased levels of epithelial cadherins, which are responsible for cell adhesion and intercellular connections. Simultaneously, levels of proteins that promote cell migration, such as α-smooth muscle actin (α-SMA) and vimentin, increase. A positive correlation exists between TGF-β levels in the AH and the severity of posterior capsule opacification (PCO) and anterior subcapsular cataract (ASC) [144]. Additionally, the TGF-β/SMAD signaling pathway has been implicated in the formation of congenital PSC [145].

Although the exact mechanisms underlying PSC formation in adults are not completely understood, it is suggested that LECs lose intercellular connections and gain transient mobility through a process similar to EMT [141]. Evidence indicates that exposing LECs to moderate OS and low levels of reduced GSH activates the TGF-β/SMAD and Wnt/β-catenin signaling pathways, leading to EMT [146,147]. Moreover, elevated cytokines resulting from OS are linked to increased α-SMA levels in LECs [113]. Studies of rat lenses have shown that the antioxidative properties of GSH and catalase (CAT) significantly prevent TGF-β-induced subcapsular lens opacities [148]. Furthermore, FGF, which initiates and maintains LEC differentiation into lens fibers, is also important in PSC formation [149]. In the final stage, a mesenchymal-to-epithelial transition (MET) may occur, leading to partial or complete restoration of the original cell type [141].

It has been shown that the level of TGF-β2 in the AH collected during cataract or clear lens removal is positively correlated with ocular AL, reaching the highest values in patients with an AL of 29.0 mm or greater [150]. Additionally, an analysis of LECs obtained during cataract surgery revealed increased levels of α-SMA in patients with LM (AL 24.0–26.0 mm) and HM (AL ≥ 26.0 mm) compared to a control group (AL ≤ 24.0 mm). Furthermore, the level of α-SMA increased with the elongation of the eyeball, but there were no significant differences in its concentration between LM and HM patients. Further evaluation of LECs from individuals with an AL greater than 24.0 mm confirmed the presence of an EMT-related phenotype, which was also positively correlated with AL [130]. These findings suggest that OS, inflammation, the formation of lipid peroxidation products, and disturbances in the water-electrolyte balance of the lens, exacerbated by increasing AL, initiate the formation of PSC and sustain its progression. These relationships may explain the higher incidence and earlier diagnosis of this type of cataract among people with myopia.

8. α-crystallins and posttranslational modifications

Proteins make up 33 % of the human lens weight, with 80 % of that being water-soluble in young individuals. This water-soluble fraction primarily consists of proteins called crystallins, which can be divided into two main groups: α-crystallins and β/γ-crystallins. The α-crystallins are the largest proteins, with a molecular weight of 600–800 kDa, and account for about one-third of the lens proteins. There are two α-crystallin subunits, αA and αB, encoded by the CRYAA and CRYAB genes, respectively [151].

α-crystallins, being more than just structural proteins, also belong to the family of small heat shock proteins (sHSPs) and act as chaperones. They bind to unfolded proteins (UPs) and, by forming stable complexes, prevent uncontrolled aggregation, which would otherwise lead to the formation of large, light-scattering particles. This function is crucial for maintaining lens transparency, as the lens does not exchange proteins, meaning they remain in the lens for life. The primary function seems to be carried out by αA-crystallins, which additionally protect αB-crystallins from heat-induced aggregation, among other things [151,152]. Furthermore, α-crystallins protect cells from stress-induced apoptosis, regulate the cell cycle and growth, enhance genetic stability, and physically and functionally interact with cell membranes and the cytoskeleton [153].

Studies in Cryaa- and/or Cryab-knockout (KO) animals have confirmed the key role of α-crystallins in maintaining protein homeostasis and preserving lens transparency [[153], [154], [155], [156]]. Point mutations in CRYAA and CRYAB, associated with non-syndromic, hereditary forms of cataract, have also been identified [151,157]. Additionally, certain mutations that cause subtle structural abnormalities, in combination with environmental factors, may reduce the protective effects of α-crystallins, leading to earlier development of NC [158]. CRYAA expression has been shown to be reduced in ARC patients compared to an age-matched control group without cataracts [159]. Similarly, longitudinal studies demonstrate that α-crystallin levels decrease over time, possibly due to their depletion as chaperone proteins, and the loss of free α-crystallins accelerates cataractogenesis [160].

As aging progresses, crystallins gradually lose their ability to maintain lens proteins in their native form due to various posttranslational modifications, including racemization [161]. This process, common among long-lived proteins, involves the conversion of amino acids from L-to D-configuration, which can result in abnormal folding and protein aggregation, contributing to cataract formation. Higher levels of d-amino acids have been observed in the lens proteins of ARC patients compared to an age-matched control group without cataracts. Racemization particularly affects the aspartic acid (Asp) residues of crystallins, specifically modifying L-Asp 58 to D-isoAsp 58 [162,163]. In contrast, the lenses of patients with HMC showed a significantly higher conversion of L-Asp 58 to D-Asp 58 in αA-crystallins compared to ARC subjects. The total number of racemized amino acids was also higher in αA-crystallins from HMC lenses (31.89 % vs. 35.44 %). This atypical conversion of αA-crystallin amino acid residues may be significant in the earlier formation and distinct phenotype of cataracts in myopic patients. Additionally, the level of D-Asp 58 racemization has been shown to increase with longer AL, suggesting a link between higher oxidative stress in myopic eyes and the earlier, more severe posttranslational modifications [164].

Moreover, proteomic analysis of HMC by Zhang et al. [108] revealed a significantly increased phosphorylation of lens nucleus proteins compared to ARC. Phosphorylation, another type of posttranslational modification, was found to occur at an elevated rate in α- and β-crystallins in HMC. Increased phosphorylation activity was also observed in filensin and phakinin, intermediate filaments present in lens fibers. These filaments are key components of the cytoskeleton and, like crystallins, play a crucial role in maintaining lens transparency [165]. The elevated levels of phosphopeptides in the lens nucleus of high myopic individuals may contribute to the accelerated development of NC in this patient group.

9. Unfolded protein response

Impaired balance in the endoplasmic reticulum (ER), caused by hypoxia or OS, leads to the accumulation of UPs and ER stress. To maintain cell survival and restore homeostasis, the adaptive unfolded protein response (UPR) is activated. This process involves activating enzyme and transcription factor systems that enhance the ER's capacity to fold proteins, reduce the synthesis of most proteins, and promote the degradation of abnormal proteins. The UPR can be divided into three distinct signaling pathways, each involving a different ER transmembrane protein: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). Under normal conditions, these proteins are bound to immunoglobulin-binding protein (BIP) within the ER lumen, which prevents their activation. However, during ER stress, BIP (acting as a chaperone protein) binds to UPs, thereby releasing PERK, IRE1, and ATF6.

After being released from its complex with BIP, PERK undergoes dimerization and is activated through autophosphorylation (p-PERK), which leads to the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). In its phosphorylated form (p-eIF2α), the formation of the translation initiation complex is inhibited, reducing the efficiency of AUG start codon recognition and thereby suppressing protein synthesis in cells under ER stress. However, molecules with internal ribosome entry site (IRES) sequences can bypass this PERK-dependent translation block. Consequently, the phosphorylation of eIF2α results in the preferential synthesis of specific proteins, including ATF4. Additionally, p-PERK activates the transcription factor NRF2.

Autophosphorylation of IRE1 activates its ribonuclease activity, enabling the excision of an intron from the mRNA encoding X-box binding protein-1 (XBP1) through alternative splicing, producing the spliced form of X-box binding protein-1 (XBP1s). This creates a stable transcription activator that binds to ER stress-response elements (ERSEs) in the promoters of many UPR pathway genes. Similarly, the transcription factor ATF6, upon dissociation from BIP, translocates to the Golgi apparatus, where it is activated by proteases. Like XBP1s, the activated ATF6 protein binds to ERSEs within DNA, regulating the expression of UPR-related genes [166,167] (Fig. 3).

Fig. 3.

Mechanism of unfolded protein response (UPR) activation triggered by ultraviolet (UV) radiation and oxidative stress. ATF6: activating transcription factor 6; BAX: BCL-2-associated X protein; BBC3: BCL-2-binding component 3; BCL-2: B-cell lymphoma 2; BIM: BCL-2 interacting mediator of cell death; BIP: immunoglobulin binding protein; CHOP: CCAAT/enhancer-binding protein homologous protein; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; IRE1: inositol requiring enzyme 1; KLF6: Krüppel-like factor 6; mRNA: messenger ribonucleic acid; P: phosphate group; PERK: protein kinase RNA-like ER kinase; UP: unfolded protein; UV: ultraviolet; XBP1: X-box binding protein 1; XBP1s: spliced form of X-box binding protein 1.

Yang et al. [168] analyzed LECs of individuals with HMC (SE ≥ −10.0 D), ARC and HM without cataract (collected during traumatic lens removal surgery within 12 h from ocular injury). Samples of LECs taken from cadavers (up to 6 h after death) of emmetropic subjects without cataracts were used as the control group. In both the ARC and HMC groups, the protein level of p-EIF2α, p-IRE1, ATF6, as well as gene expression of BIP, ATF4, ATF6, XBP1s, were significantly elevated compared to the control group. However, the level of mRNA and proteins of αA- and αB-crystallin were lower compared to the control group. There were no differences in the LECs of subjects with HM and without cataracts compared to the HMC and ARC groups. This result indicates that the increased activation of the UPR (including the PERK/EIF2α/ATF4 pathway) in LECs through translational inhibition may reduce α-crystallins concentration and contribute to cataract development. On the other hand, the reduction of chaperone proteins promotes the accumulation of UPs, thereby enhancing ER and UPR stress.

Meanwhile, Tian et al. [169] noted higher levels of Krüppel-like factor 6 (KLF6) and ATF4 in the LECs of HM subjects compared to emmetropic subjects. KLF6 is a tumor suppressor. It belongs to a family of transcription factors that regulate cell cycle, proliferation and apoptosis [170]. Research involving UV radiation, designed to simulate daily sunlight exposure and associated with OS, found that KLF6 overexpression in LECs potentiated their susceptibility to ER stress when compared to normal cells. In addition, the first group had a significantly higher number of cells that underwent apoptosis. The accumulation of UPs results in the activation of the PERK/EIF2α/ATF4 pathway. Subsequently, ATF4 activates ATF3, resulting in an increase in the level of the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) [169]. The mechanism of CHOP induction of apoptosis is associated with downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2) protein; upregulation of proapoptotic proteins: BCL-2-binding component 3 (BBC3), BCL-2-associated X protein (BAX), and BCL-2 interacting mediator of cell death (BIM) [171]. Activation of BAX and inactivation of BCL-2 has been demonstrated to be further enhanced when KLF6 is overexpressed in LECs, and therefore the BAX/BCL-2 ratio will be increased [169]. CHOP also enhanced ROS secretion and decreased the amount of GSH in the cell [171]. Moreover, elevated CHOP levels are associated with activation of growth arrest and DNA damage inducible protein 34 (GADD34), leading to eIF2α dephosphorylation and restoration of previously inhibited translation. This results in even greater ER stress when stress triggers are still present [172].

During conditions of moderate ER stress in LECs, the UPR plays a proadaptive role. However, chronic ER stress can override the regulatory potential of the UPR pathway and lead to its proapoptotic form, a common element in the pathogenesis of various types of cataracts [166,167]. Furthermore, increased levels of KLF6 and activation of the ATF4/ATF3/CHOP pathway in LECs from HM subjects through intensified vulnerability of these cells to ER stress and increased induction of apoptosis may contribute to accelerate cataract development.

10. β/γ-crystallins and the pathological lens growth

Analysis of orbital magnetic resonance imaging data has shown a larger equatorial diameter and a flatter anterior lens surface in HM patients compared to emmetropic individuals [173]. This finding may explain the higher incidence of incorrect postoperative intraocular lens positioning in HM patients [174,175]. Additionally, an evaluation of LECs obtained during refractive lens replacement in HM patients revealed increased expression of genes encoding β/γ-crystallins compared to a control group (AL 22.0–24.5 mm), where lens replacement was performed due to presbyopia. Higher levels of β/γ-crystallins were positively correlated with lens size, and similar results were reported in two independent mouse models of myopia. β-Crystallins are a complex group of oligomers consisting of polypeptides encoded by seven genes, with molecular weights ranging from 23 to 32 kDa. In their native state, individual polypeptides form dimers and higher-order complexes. In contrast, γ-crystallins are the smallest group of crystallins, with molecular weights of about 20 kDa or less, encoded by four genes. Native γ-crystallins do not interact with each other or with other proteins. It has been suggested that chronically elevated β/γ-crystallin gene expression may contribute not only to lens dislocation but also to faster NC development in myopic eyes, as a result of increased lens fiber compression [173]. During the embryonic stage, lens development is controlled by transcription factors, including the musculoaponeurotic fibrosarcoma oncogene homolog (MAF) [176]. However, in adults with HM, MAF remains active (unlike in emmetropic eyes) and, by directly regulating the promoter regions of β/γ-crystallin genes, contributes to the accumulation of these proteins in the lens. Furthermore, in LECs of HM patients, elevated levels of TGF-β1 have been observed among 40 selected growth factors. Additionally, the levels of TGF-β1 receptor (TGF-β1R) and effector molecules such as SMAD2/3, its phosphorylated form (p-SMAD2/3), and SMAD4 (which interacts with p-SMAD2/3 to activate transcription of target genes), are higher in the lenses of both mice and humans with HM. It has been found that MAF not only directly regulates β/γ-crystallin gene expression but also indirectly does so by increasing the autocrine secretion of TGF-β1, leading to activation of the SMAD signaling pathway. TGF-β1 also promotes the proliferation of primary LECs [173]. Lai et al. [177] identified that an imbalance in the MAPK and calcium signaling pathways is crucial in pathological lens growth in HM patients, possibly because TGF-β, in addition to the SMAD-dependent pathway, can also interact with MAPK [178].

Yao et al. [179] demonstrated that the number of lens fibers is significantly higher in HM mice compared to controls. Additionally, they revealed that in the LECs of HM mice, significant inhibition of the Notch signaling pathway is accompanied by enhanced differentiation of LECs into lens fibers. The Notch pathway is critical for determining cell lineage fate, promoting progenitor and stem cell proliferation, and preventing premature differentiation [180,181]. After binding to a ligand located on the surface of neighboring cells, the Notch receptor undergoes degradation, leading to the release of the Notch intracellular domain (NICD), which is then translocated to the nucleus. There, it forms a complex with transcription factors, such as recombination signal binding protein for immunoglobulin kappa J region (RBPJ), which controls the expression of target genes [180]. Negative regulation of the Notch2 receptor plays a key role in determining LEC fate and controlling lens size, even in adulthood. This is supported by observations of increased lens fiber numbers in Notch2-KO mice [182]. In both mice and humans with HM, inhibition of NOTCH2 expression in LECs resulted in decreased repression by HES1 on cyclin-dependent kinase inhibitor 1C (CDKN1C or p57Kip2) and MAF. CDKN1C is an initiator of differentiation, while MAF increases β/γ-crystallin gene expression directly and indirectly, leading to the accumulation of these structural proteins. Together, these mechanisms contribute cooperatively to excessive lens growth [179] (Fig. 4).

Fig. 4.

Mechanisms in lens epithelial cell (LEC) resulting in pathological lens growth in the course of axial myopia. CDKN1C: cyclin-dependent kinase inhibitor 1C; LEC: lens epithelial cell; MAF: musculoaponeurotic fibrosarcoma oncogene homolog; NECD: Notch extracellular domain; NICD: Notch intracellular domain; P: phosphate group; Rbpj: recombination signal binding protein for immunoglobulin kappa J region; TGFβ1: transforming growth factor β 1; TGFβ1R: transforming growth factor β receptor 1.

Comparative proteomic analyses of AH extracted from HMC and ARC subjects have aimed to expand our understanding of the mechanisms underlying lens opacification and identify potential biomarkers. Xiang et al. [183], using isobaric tags for relative and absolute quantitation (iTRAQ), identified 146 proteins with differential regulation in eyes with HMC, 49 of which were downregulated and 97 of which were upregulated, including 6 crystallins: αA, αB, βA3, βA4, βB1, and βB2. Slightly different results were presented by Ji et al. [184], who indicated that cataract development in the context of HM is associated with the downregulation of 87 proteins, including crystallins βA3, βB1, βB2, βS, γC, and γD. Additionally, Zhou et al. [185] analyzed lenses diagnosed with NC obtained during extracapsular cataract extraction. Of the 40 proteins selected, six showed axial length-dependent expression. Connexin 46 (α3 gap junction protein) and βB2-crystallin levels were lower in eyes with above-average axial length. Although these studies report differences in the set of proteins in HMC eyes compared to controls, further research is required to investigate the network of potential links to lens transparency due to the complexity and variability of proteome analysis.

11. The epigenetic regulation of genes expression

Epigenetic regulation of gene expression induced by environmental stress can occur through DNA methylation, histone modification, or the action of microRNAs (miRNA) [[186], [187], [188]]. Methylation typically involves the addition of a methyl group to a cytosine within CpG dinucleotides (cytosine-phosphate-guanine). When CpG islands in promoter regions become methylated, the electrostatic properties of chromatin change, leading to gene silencing [186,187]. Hypermethylation of gene promoters, such as MGMT, OGG1, and LOXL1, has been associated with accelerated cataract development [[189], [190], [191]]. In LECs, hypermethylation of CpG islands in the CRYAA promoter reduces the binding ability of transcription factors, particularly specificity protein 1 (SP1), to DNA, thereby decreasing CRYAA transcription and downregulating αA-crystallin [192]. Treatment of LECs with the DNA demethylating agent zebularin, a cytosine analog, has been shown to reverse this process [192,193]. Reduced αA-crystallin expression via DNA methylation has been observed in patients with age-related nuclear cataract (ARNC) and nuclear cataract secondary to PPV, confirming the importance of this mechanism in cataract formation and progression [[192], [193], [194]]. Another study showed that in the LECs of patients with HMC, compared to those with ARC with the same cataract grade based on the Lens Opacities Classification System III (LOCS III), the CRYAA promoter exhibited greater CpG island methylation, leading to reduced αA-crystallin expression. Notably, the HMC patients in this study were significantly younger than the ARC patients [195].

Zhu et al. [194] demonstrated that DNA methyltransferase 1 (DNMT1) expression was higher in HMC patients than in ARC subjects (AL ≤ 24.5 mm). DNMT1 is the primary methyltransferase in LECs, and its expression was elevated in the HMC group at both the mRNA and protein levels, indicating that DNMT1 mediates and/or maintains CpG hypermethylation. Furthermore, treatment of cultured LECs with a DNMT1 inhibitor (5-aza-2′-deoxycytidine) significantly increased mRNA and protein levels of α-crystallin [89]. Methylation of CpG islands in the promoters of glutathione S-transferase Pi 1 (GSTP1) and thioredoxin reductase 2 (TXNRD2) genes in LECs from HMC subjects was significantly higher than in controls, leading to reduced expression of these genes. To verify that OS played a role, LEC cultures were treated with H₂O₂ to simulate the hyperoxic conditions in myopic eyes. Initially (from days 2–6), GSTP1 and TXNRD2 levels increased to protect cells from oxidative damage. However, by day 8, their levels significantly decreased, likely due to promoter hypermethylation mediated by DNMT1 [89].

GSTP1 catalyzes the conjugation of hydrophobic and electrophilic compounds to GSH, plays a role in transthiolation, and exhibits strong antioxidant activity, particularly in high-oxygen environments [196,197]. Its levels and activity are significantly reduced in cataractous lenses compared to clear lenses [198]. Chen et al. [199] reported the presence of epigenetic modifications in GSTP1 that affect its expression in LECs and the lens cortex. They indicated that methylation in two GSTP1 promoter regions may be associated with greater ARNC severity. Methylation of the GSTP1 promoter was even higher in HMC compared to ARC [89].

TXNRD is crucial for reducing thioredoxin (TRX) to its active form. Downregulation of its mitochondrial form, TXNRD2, impairs the mitochondrial TRX system, leading to oxidative damage in LECs [200]. Reduced TRX system activity has been observed in all cataract types [201]. Both the TRX system and GSH can reduce protein disulfides (PSSPs), but TXNRD activity has been shown to be more critical than GSH for maintaining normal LEC growth under hyperbaric oxygen conditions [202]. TXNRD is also more effective than GSH in protecting LECs from UVA radiation [203]. This is likely due to the TRX system's role in activating transcription factors that regulate cell growth and participating in deoxyribonucleotide formation needed for DNA synthesis in UVA-damaged cells [200,204].

In myopic patients, early-onset VB liquefaction and chronic oxidative stress lead to increased expression of DNMT1 and hypermethylation of the promoter regions of CRYAA, GSTP1, and TXNRD2. The failure of αA-crystallins to provide adequate protection disrupts protein homeostasis, impacting both structural proteins and antioxidant enzymes. DNA methylation and oxidative stress interact in a vicious cycle, accelerating cataract formation in individuals with higher-than-average AL [89,195] (Fig. 5).

Fig. 5.

The vicious cycle between oxidative stress and epigenetic regulation of genes expression in the development of cataract in the course of axial myopia A: adenine; C: cytosine; –CH₃: methyl group; CpG: cytosine-phosphate-guanine; DNMT1: DNA methyl transferase 1; G: guanine; GSTP1: glutathione S-transferase Pi 1; O₂: molecular oxygen; T: thymine; TXNRD2: thioredoxin reductase 2.

An important role in epigenetic modifications is also played by miRNA, which regulates gene expression by inhibiting translation and destabilizing the transcripts with which it interacts [188]. One of the mechanisms regulating miRNA activity involves binding by circular RNA (circRNA), which reduces the availability of free miRNA in the cell. A growing body of research indicates that circRNA, as a posttranscriptional regulator of gene expression, participates in cataract progression through its effects on the cell cycle, proliferation, apoptosis, autophagy, and OS response. Additionally, circRNA can interact with proteins in various forms, thereby affecting their functions [188,[205], [206], [207], [208]].

Ma et al. [209] were the first to evaluate the effect of circRNA in patients with HMC. Sequencing of the entire transcriptome of LECs from patients with HMNC; AL > 26.0 mm and age-related cortical cataract (ARCC; AL 21.0–24.5 mm) revealed the presence of 40.1 % (3687) uniquely occurring circRNA in HMC. Among this pool, 1163 circRNA exhibited significantly different levels between the two groups. CircRNA specific to HMC appeared to be involved in chromatin and protein modification and bound to the TGF-β, Wnt, and mTOR signaling pathways, which are known to be implicated in cataract initiation and progression [[210], [211], [212]].

Of the four circRNA selected for further analysis, circRNA from the AF4/FMR2 family member 1 gene (circAFF1) was found to be over three times more abundant in LECs of HMC patients. Furthermore, LECs transfected with increased levels of circAFF1 compared to controls showed enhanced proliferation and migration, as well as reduced apoptosis. This may potentially lead to an increased accumulation of lens fibers and earlier onset of lens nucleus sclerosis. Additionally, circAFF1 is likely to positively regulate tropomyosin 1 (TPM1) through binding with miR-760 [209]. Although TPM1 has been shown to be positively regulated in LECs isolated from cataract lenses compared to translucent lenses, its role in the pathogenesis of HMC remains to be fully elucidated [213,214].

12. Therapeutic approaches for oxidative stress in cataract

OS plays a crucial role in the pathophysiology of many ophthalmic diseases. It affects ocular surface (dry eye syndrome, pterygium), the cornea (keratoconus, Fuchs endothelial corneal dystrophy, diabetic keratopathy), the optic nerve (glaucoma, Leber's hereditary optic neuropathy, anterior ischemic optic neuropathy), and retina (age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion, RP, retinopathy of prematurity) [215]. The undeniable role of OS in cataract development has led to numerous studies on antioxidant supplementation as a means of slowing cataractogenesis. Although several studies have shown a correlation between the intake of vitamin C and other antioxidant molecules and a reduced risk of lens clouding, long-term clinical trials seem to contradict this relationship [216,217]. The Age-Related Eye Disease Study found that daily oral intake of antioxidant tablets (vitamin C, 500 mg; vitamin E, 400 IU; beta-carotene, 15 mg) did not reduce the risk of cataract formation [218]. Similarly, the Roche European American Cataract Trial, conducted in the UK and the US, demonstrated that daily intake of antioxidants (vitamin C, 750 mg; vitamin E, 600 mg; beta-carotene, 18 mg) slightly reduced cataract progression in the American cohort but showed no significant effect in the British cohort [219]. Likewise, in a randomized trial by Christen et al. [220], daily consumption of 500 mg of vitamin C among Americans had no impact on the incidence of cataracts or the need for cataract surgery after eight years of observation. Conversely, a study on the Swedish Mammography Cohort of women revealed that vitamin C supplementation for more than 10 years increased the risk of cataract surgery by 25 % [221]. Another Swedish study found that multivitamin supplementation, when combined with vitamin C, was not associated with cataract development, but high doses of vitamin C might increase the risk [222]. This could be due to the observation that high levels of vitamin C exhibit strong pro-oxidant effects on mammalian lenses, as its metabolites may enhance OS [223]. Findings from studies on dietary habits suggest that a well-balanced diet rich in fruits and vegetables, which naturally contain vitamin C, may be a more optimal to slow lens opacification process. The European Eye Study showed that high daily consumption of fruits and vegetables was associated with a significant reduction in the incidence of cataracts and the need for cataract surgery [224]. Similarly, a study conducted in Greece confirmed the protective properties of an antioxidant-rich diet. An increase in vitamin C intake by 185 mg/day halved the risk of cataract development [225]. Additionally, daily dietary selenium intake may aid in cataract prevention [226]. A study among American women showed that a high Healthy Eating Index score was the strongest modifiable factor reducing the risk of NC, suggesting that antioxidant-rich food, rather than supplementation alone, has protective effects [227]. However, it is important to note that longitudinal dietary studies involve numerous uncontrollable variables affecting cataract progression, which can significantly impact results and obscure actual relationships.

Among new therapeutic approaches, NRF2 activation stands out, as it regulates the activation of various antioxidant genes. NRF2 inducers, such as the acetyl ester of the trimethylated amino acid l-carnitine, hyperoside or flavonoids have been shown to reduce cataract development by stimulating antioxidant defenses [228]. Additionally, melatonin, through its regulation of NRF2 pathways, may also delay ARC [229].

13. Conclusions

Oxidative imbalance during the course of axial myopia is a major contributor to the accelerated development of NC and PSC. Excessive production of ROS, coupled with diminished antioxidant protection and chronic inflammation in individuals with above-average AL eyeballs, triggers a series of signaling pathways and mechanisms. These processes lead to modifications in lens proteins, resulting in loss of function and aggregation, as well as epithelial-to-mesenchymal transition of LECs. Additionally, excessive lipid peroxidation, nucleic acid damage, and epigenetic modifications contribute to the cataractogenesis process by creating a vicious cycle of dependency. Given the steadily rising prevalence of myopia, alongside the aging population and increasing life expectancy, understanding the underlying processes of premature cataract formation in individuals with axial myopia is crucial. This knowledge is key to developing therapeutic approaches that could potentially prevent or slow the progression of cataracts.

CRediT authorship contribution statement

Marta Świerczyńska: Writing – review & editing, Writing – original draft, Visualization, Investigation, Data curation, Conceptualization. Agnieszka Tronina: Writing – review & editing. Adrian Smędowski: Writing – review & editing, Supervision.

Funding sources

This research received no external funding.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Adrian Smędowski was employed by the company GlaucoTech Co. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

8-OHdG	8-hydroxydeoxyguanosine
α-SMA	α-smooth muscle actin
AH	aqueous humor
AL	axial length
AP-1	activator protein 1
ARC	age-related cataract
ARE	antioxidant response element
ARNC	age-related nuclear cataract
AsA	ascorbic acid
ASC	anterior subcapsular cataract
Asp	aspartic acid
ATF6	activating transcription factor 6
BAX	BCL-2-associated X protein
BBC3	BCL-2-binding component 3
BCL-2	B-cell lymphoma 2
bFGF	basic fibroblast growth factor
BIM	BCL-2 interacting mediator of cell death
BIP	immunoglobulin binding protein
cAMP	cyclic adenosine monophosphate
CAT	catalase
CDKN1C	cyclin-dependent kinase inhibitor 1C
CHOP	CCAAT/enhancer-binding protein homologous protein
circAFF1	circRNA of the AF4/FMR2 family member 1 gene
circRNA	circular RNA
CpG	cytosine, phosphate group, guanine
D	diopter
dAsA	dehydroascorbic acid
DHA	docosahexaenoic acid
DKK1	Dickkopf glycoprotein 1
DNA	deoxyribonucleic acid
DNMT1	DNA methyltransferase 1
DRP1	dynamin-related protein 1
eIF2α	α subunit of eukaryotic translation initiation factor 2
EMT	epithelial to mesenchymal transition
ER	endoplasmic reticulum
ERM	epiretinal membrane
ERSE	ER stress-response element
GABA	gamma-aminobutyric acid
GADD34	growth arrest and DNA damage inducible protein 34
GCLC	glutamylcysteine ligase catalytic subunit
GPX3	glutathione peroxidase 3
GSH	glutathione
GSR	glutathione disulfide reductase
GSS	glutathione synthetase
GSSG	glutathione disulfide
GSTM2	glutathione S-transferase Mu 2
GSTP1	glutathione S-transferase Pi 1
H2O2	hydrogen peroxide
HBO	hyperbaric oxygen therapy
HIF-1α	hypoxia-inducible factor 1α
HM	high myopia
HMC	high-myopic cataract
HMNC	high-myopic nuclear cataract
IFN-γ	interferon γ
IGF-1	insulin-like growth factor 1
IL	interleukin
IL-1RA	IL-1 receptor antagonist
iNOS	inducible nitric oxide synthase
IP-10	interferon-inducible protein 10
IRE1	inositol requiring enzyme 1
IRES	internal ribosome entry site
IRF1	interferon regulatory factor 1
iTRAQ	isobaric tag for relative and absolute quantitation
KLF6	Krüppel-like factor 6
KO	knockout
LEC	lens epithelial cell
LM	low myopia
LOCS III	Lens Opacities Classification System III
MAF	musculoaponeurotic fibrosarcoma oncogene homolog
MAP1A/MAP1B LC3	microtubule-associated protein 1A/1B-light chain 3
MAPK	mitogen activated protein kinase
MCP-1	monocyte chemoattractant protein 1
MDA	malondialdehyde
MET	mesenchymal to epithelial transition
MFN1	mitofusin 1
MIP-1α	macrophage inflammatory protein-1α
miRNA	microRNA
MMP-2	matrix metalloproteinase 2
mRNA	messenger RNA
mtDNA	mitochondrial DNA
NADPH	reduced nicotinamide adenine dinucleotide phosphate
NC	nuclear cataract
NF-κB	nuclear factor κB
NICD	Notch intracellular domain
NLR3	NOD-like receptor 3
NO	nitric oxide
NRF2	nuclear factor erythroid 2-related factor 2
OS	oxidative stress
PCO	posterior capsule opacification
p-eIF2α	phosphorylated form of eIF2α
PERK	protein kinase ER RNA-like kinase
PGCα	peroxisome proliferator-activated receptor-γ coactivator 1α
PGDS	prostaglandin H2 d-isomerase
PINK1	phosphatase and tensin homolog induced kinase 1
p-PERK	phosphorylated form of PERK
PPV	vitrectomy through the pars plana
PSC	posterior subcapsular cataract
P-SH	thiol group in protein
PSSP	protein disulfide
PVD	posterior vitreous detachment
RBPJ	immunoglobulin kappa J region
ROS	reactive oxygen species
RP	retinitis pigmentosa
RPE	retinal pigment epithelium
SE	spherical equivalent
SHH	homolog sonic hedgehog
sHSP	small heat shock protein
SOD	superoxide dismutase
SP1	specificity protein 1
STAT3	signal transducer and activator of transcription 3
T-AOC	total antioxidant capacity
TBA	thiobarbituric acid
TFAM	mitochondrial transcription factor A
TGF-β	transforming growth factor β
TGF-β1R	TGF-β1 receptor
TIMP-1	tissue inhibitor of metalloproteinase 1
TNFα	tumor necrosis factor α
TOM 20	translocase of outer mitochondrial membrane 20
TPM1	tropomyosin 1
TRX	thioredoxin
TXNRD2	thioredoxin reductase 2
UP	unfolded protein
UPR	unfolded protein response
UV	ultraviolet
VB	vitreous body
VC	vitreous chamber
XBP1	X-box binding protein 1
XBP1s	spliced form of XBP1

Data availability

No data was used for the research described in the article.