The Role of EZH2 in Ocular Diseases: A Narrative Review

Abstract

EZH2, acting as a catalytic subunit of PRC2 to catalyze lysine 27 in histone H3, induces the suppression of gene expression. EZH2 can regulate cell proliferation and differentiation of retinal progenitors, which are required for physiological retinal development. Meanwhile, an abnormal level of EZH2 has been observed in ocular tumors and other pathological tissues. This review summarizes the current knowledge on EZH2 in retinal development and ocular diseases, including inherited retinal diseases, ocular tumors, corneal injury, cataract, glaucoma, diabetic retinopathy and age-related retinal degeneration. We highlight the potential of targeting EZH2 as a precision therapeutic target in ocular diseases.

Plain language summary

EZH2 is a protein that helps to regulate the activity of genes in cells. It works as a part of a complex called PRC2 to control a chemical group called lysine 27 in histone H3 and then inhibit the expression of genes. EZH2 is important for the normal development of the retina. Abnormal levels of EZH2 are associated with various eye diseases. This review summarizes the role of EZH2 in different ocular diseases and the potential mechanisms. Targeting EZH2 may be a novel way to treat or prevent ocular diseases.

Tweetable abstract

Review discussing the role of EZH2 in retinal development and ocular diseases to highlight the potential of EZH2 as a precision therapeutic target for treating ocular diseases.

Keywords::

• age-related retinal degeneration
• cataract
• corneal injury
• diabetic retinopathy
• EZH2
• glaucoma
• inherited retinal diseases
• ocular diseases
• ocular tumors
• retinal development

Ocular diseases can lead to visual impairment (VI) and greatly affect the quality of life. A survey in the USA indicated that 1.02 million adults suffered from vision loss and 3.22 million had VI in 2015, with the numbers expected to double by 2050 [1]. Moreover, patients with VI showed higher mortality in a 10-year study in China [2]. A deeper understanding of the pathological mechanisms and better management of eye diseases have been achieved in the last few decades. However, current therapies can seldom fully restore vision or arrest disease progression.

Epigenetics is characterized by heritable alterations of gene expression which lead to phenotype changes, without alteration in DNA sequences [3]. Epigenetic modifications including DNA methylation, noncoding RNA and histone modifications are critical components in regulating various biological processes [4]. EZH2, acting as a core component of PRC2, promotes transcriptional silencing through methylating lysine 27 in histone H3 (H3K27me3) [5,6]. As a key regulator in cell fate determination, DNA damage repair, cell proliferation and differentiation [7], EZH2 has been identified to be implicated in many diseases, including ocular diseases.

Accumulating evidence shows that EZH2 is required for retinal development [8]. Abnormal expression of EZH2 was observed in eye tumors [9–11], ocular fibrotic tissues [12,13] and diabetic retinopathy (DR) [14,15]. Genetic or pharmacological suppression of EZH2 was shown to protect photoreceptor cells from dying and delay the pathological progression of various eye diseases [16–18]. Targeting EZH2 to prevent ocular disorders or delay disease progression could be a promising therapeutic approach. In this article we review the current knowledge of EZH2 in retinal development and a variety of ocular diseases.

EZH2 structure & its functional models

Structure of EZH2

Encoded by a gene located on chromosome 7q35, EZH2 is composed of regulatory segment domains and catalytic domains. The activity of histone methyltransferase is implemented by the C-terminal SET domain and CXC domain. The SET domain is the predominant component for EZH2 methyltransferase function, where methyl groups of S-adenosyl methionine are transferred to histones [19]. N-terminal domains, including the EID domain, domain I and domain II, act as binding sites to promote assembly with other PRC2 subunits, including EED and SUZ12, to ensure the proper functioning of PRC2 [6].

EZH2 functional models

As shown in Figure 1, upstream mediators including noncoding RNAs can regulate EZH2 expression. Canonically, EZH2, in the form of a catalytic subunit, interacts with two other PRC2 subunits, EED and SUZ12, to catalyze H3K27me3 and thus suppresses gene expression and regulates biological processes in several signaling pathways, including the Wnt/β-catenin pathway and PI3K/AKT pathway; however, mounting evidence suggests that EZH2 also has noncanonical epigenetic functions [20,21]. EZH2, in a PRC2-dependent or -independent manner, can methylate nonhistone molecules such as DNA [22] and STAT3 [23] to inhibit or activate gene transcription. In melanoma cells, EZH2 was involved in histone H3 deacetylation by maintaining HDAC1 at the CDKN1A site, thus repressing p21 expression during tumor progression [24]. EZH2 can also function as a coactivator with transcription factors such as E2F1 or as a chaperone to promote the cleavage of the transposable element Alu RNA for transactivation [25,26].

Figure 1. The mechanism of EZH2 in ocular diseases.

Upstream mediators including noncoding RNAs and other factors (orange) can regulate EZH2 expression. (A) Canonically, EZH2, in the form of a catalytic subunit, interacts with two other PRC2 subunits (EED and SUZ12) to catalyze lysine 27 in histone H3 (H3K27me3) and thus suppresses gene expression. EZH2 regulates biological processes (green) in several signaling pathways (blue), including the Wnt/β-catenin pathway and PI3K/AKT pathway. (B) Noncanonically, EZH2, in the context of the PRC2 complex, methylates DNA through recruiting DNA methyltransferase (DNMT). (C) EZH2 is involved in histone 3 deacetylation by maintaining HDAC1, thus repressing p21 expression during tumor progression. (D) EZH2 acts as a chaperone to promote the cleavage of Alu RNA. (E) EZH2 functions as a coactivator with transcription factors such as E2F1. (F) EZH2 methylates nonhistone molecules including STAT3 to regulate the expression of downstream genes. EZH2 is overexpressed in pathological tissues in ocular tumors, corneal injury, cataract and diabetic retinopathy. However, the significance of the EZH2 level in inherited retinal diseases, glaucoma and age-related macular disease is unclear.

EZH2&retinal development

During retinal development, the responses of progenitor cells to extrinsic and intrinsic stimuli are critical to regulate cell proliferation and differentiation. Genetic and epigenetic regulation enables accurate and robust developmental transitions. Mounting evidence suggests that EZH2, as a methyltransferase for H3K27me3, is a key mediator in retinal development (Table 1).

Table 1. Summary of EZH2 inhibitors in retinal development.

Author	EZH2 inhibitors	Models	Effects	Ref.
Rao et al. (2010)	DZNep	Mice, primary RGCs	EZH2 was highly expressed from E14 to P0, but declined in the adult retina Promoted RGC apoptosis	[27]
Rapicavoli et al. (2011)	–	Mice	Six3OS bound to EZH2 family members to regulate retinal cell specification	[28]
Aldiri et al. (2013)	EZH2 MO	Xenopus	EZH2 was detected in retinal progenitors in both the embryonic retina and in the CMZ Reduced eye size and inhibited cell proliferation Did not affect retinal progenitor specification but regulated proneural gene expression Increased the differentiation of Müller glial cells	[29]
Iida et al. (2014)	–	Mice	EZH2 expression was evident in the whole retinal area at E14 and E17 EZH2 was detected in the GCL and weakly in some INL cells at P5 and was only observed in the outer half of the INL at P8 The expression of EZH2 declined after birth and was negligible at about 2 weeks when retinal development was complete	[30]
Iida et al. (2015)	–	Ezh2 knockout mice	Reduced the thickness of ONL and INL Resulted in microphthalmia Accelerated the differentiation of Müller glia and rod photoreceptors Impaired postnatal cell proliferation	[31]
Zhang et al. (2015)	GSK126	Ezh2 knockout mice	EZH2 was highly observed in the GCL and neuroblastic layer by P0 and was largely restricted to the GCL and INL by P10 Impaired retinal development and inhibited late retinal progenitor proliferation Decreased the number of RGCs postnatally Increased photoreceptor and Müller glia differentiation in the early postnatal retina	[8]
Watanabe et al. (2016)	–	Ezh2 knockout mice	EZH2 was strongly expressed in embryonic retina and decreased during the postnatal period EZH2 participated in the differentiation of subsets of bipolar cells	[32]
Fujimura et al. (2018)	–	Ezh2 knockout mice	Enhanced Müller glia differentiation	[33]
Andrews et al. (2019)	–	Human RPE cells, RPE-19 cell line	EZH2 participated in retinoic acid-mediated differentiation of NT2 neuroepithelial progenitor cells Smad-EZH2 complex regulated TGF-β-mediated dedifferentiation of neuroretinal epithelial cells Smad3 and EZH2 cooperated to regulate cellular plasticity in retinal epithelial cells	[34]

CMZ: Ciliary marginal zone; ERG: Electroretinogram; EZH2 MO: Morpholino for EZH2; GCL: Ganglion cell layer; INL: Inner nuclear layer; ONL: Outer nuclear layer; RGC: Retinal ganglion cell; RPE: Retinal pigment epithelium.

EZH2 has been reported to be dynamically expressed in different stages of retinal development, with high levels in retinal progenitors and weak expression in adult retinas in mice [27]. At embryonic stages E14 and E17, EZH2 was evident in all layers in murine retinas, especially in the peripheral retina. After birth, EZH2 expression was restricted to the inner nuclear layer and ganglion cell layer, and negligible in adult retinas [31]. This pattern may suggest that EZH2 actively participates in retinogenesis. Moreover, EZH2 also has an impact on cell differentiation. Rhodopsin was first detected at P2 and Müller glia started to differentiate at P8 in Ezh2 knockout mice, which was not observed in the control group [31]. This shows the accelerated differentiation of Müller glia and rod photoreceptors after EZH2 inhibition. In addition, the number of retinal ganglion cells (RGCs) was significantly reduced at P3 and amacrine cells were damaged in Ezh2 knockout mice [8]. Therefore, EZH2 may act as a critical regulator in the timing of the differentiation of retinal progenitors.

The proliferation of progenitors is also critical for the development of the retina. Suppression of EZH2 could reduce the proportion of cycling cells and growth fraction in the optic vesicle, and contribute to a 15% reduction in eye diameter in embryos [29]. However, no difference in the proportion of S-phase cells was observed, and there was no change in the proportion of cycling cells, in E15 Ezh2 knockout mice [8]. However, reduction in the proliferation of retinal progenitors was observed since P0 [8], revealing that EZH2 regulates the proliferation of progenitors after birth, but not in embryonic stages. The mechanism of the regulation of EZH2 in retinal cell proliferation may be associated with upregulation of cycle-related genes, including CDKN2A and CDKN2B [8], although the underlying mechanism remains to be elucidated.

EZH2&ocular diseases

EZH2&inherited retinal diseases

EZH2 is involved in controlling cell fate during retinal development, especially in photoreceptors, and thus may provide a novel target for inherited retinal diseases (IRDs) (Table 2). Retinitis pigmentosa is the most common form of IRD, characterized by the loss of photoreceptors [35]. In the Rd1 mouse, which is a well-known animal model of retinitis pigmentosa, the thickness of the outer nuclear layer increased from 25.38 ± 6.48 μm to 43.21 ± 3.78 μm after injection of the EZH2 inhibitor DZNep [36]. At P21, when nearly all rods die, immunostaining of retinas showed that there were still at least three layers of photoreceptors with DZNep treatment, while only one layer of photoreceptors was observed in the control group. Additionally, the electroretinogram results showed the visual functions were significantly preserved in the Rd1 eyes with DZNep treatment, indicating that EZH2 inhibition can suppress the death of rods and partially restore visual functions. In agreement with this finding, a reduction of about 70% in apoptotic photoreceptor cells was observed in Rd1 retinas when treated with an EZH2 inhibitor [16].

Table 2. Summary of EZH2 inhibitors in inherited retinal diseases.

Author	EZH2 inhibitors	Models	Effects	Ref.
Ueno et al. (2016)	–	Ezh2 knockout mice	Increased the number of photoreceptor-related genes	[37]
Yan et al. (2016)	–	Ezh2 knockout mice	Reduced the ONL thickness and promoted photoreceptor degeneration Decreased ERG response Regulated the expression of photoreceptor-related genes	[38]
Zheng et al. (2018)	DZNep	Rd1 mice	Increased ONL thickness and delayed photoreceptor loss Improved ERG responses Reduced the calpain activity, attenuated the photoreceptor degeneration	[36]
Mbefo et al. (2021)	UNC1999, EPZ6438	Rd1 mice, Fam161a knockout mice	Inhibited cell apoptosis Prevented photoreceptor degeneration and delayed photoreceptor cell death	[16]

ERG: Electroretinogram; ONL: Outer nuclear layer.

However, Yan et al. reported that embryonic knockdown of Ezh2 induced progressive photoreceptor degeneration and decreased the thickness of the outer nuclear layer by regulating photoreceptor-related genes in postnatal eyes [38], which is in disagreement with the aforementioned studies. Given the complex mechanism of disease development, further studies of the function of EZH2 in photoreceptors are needed. Besides, the role of EZH2 in other forms of IRD is also worthy of investigation.

EZH2&ocular tumors

The importance of EZH2 in the pathophysiology of cancer is now widely recognized, and EZH2 has been a promising target. Inhibitors of EZH2 have been investigated in clinical trials to evaluate their efficacies and safety in malignant pleural mesothelioma, follicular lymphoma, advanced epithelioid sarcoma, B-cell non-Hodgkin lymphomas and solid tumors [39–47]. The study of EZH2 in ocular tumors is still preliminary and there has been no clinical trial for EZH2 inhibitors yet. Table 3 comprises a list of the related studies.

Table 3. Summary of EZH2 inhibitors in ocular tumors.

Author	Ocular tumor	EZH2 inhibitors	Models	Effects	Ref.
Lu et al. (2017)	UM	EZH2 siRNA	UM cell lines	Attenuated cell proliferation and invasion through upregulation of p21 expression and interaction with lncRNA HOXA11-AS	[48]
Jin et al. (2020)		shEZH2, GSK126	UM cell lines, UM human tissues, subcutaneous xenografted mouse model	Elevated expression of EZH2 was detected in the UM tissues, predicting a worse prognosis Suppressed tumor cell migration and invasion Induced apoptosis and abrogated outgrowth of UM tumor	[9]
Li et al. (2021)		EZH2 siRNA	UM cell lines	EZH2 was highly expressed in UM cell lines Inhibited cell proliferation	[49]
Zhang et al. (2021)	UM	–	UM cell lines, UM human tissues	EZH2 was highly expressed in UM human tissues and UM cells EZH2 was related to the tumor prognosis Participated in the anti-UM functions of miR-137	[50]
Chen et al. (2022)		GSK126, GSK503, UNC1999, EPZ6438	UM OMM1 cells, UM human tissues	Overexpression of EZH2 was observed in UM Suppressed tumor cell growth and affected the cell cycle Induced cell apoptosis and ferroptosis	[51]
Cao et al. (2018)	CM	GSK503, UNC1999, shEZH2	CM cell lines, CM human tissues, zebrafish	EZH2 was overexpressed in CMs and was correlated with worse overall survival Inhibited cell proliferation and colony formation Suppressed tumor growth	[10]
Avedschmidt et al. (2016)	IM	–	IM human tissues	Overexpression of EZH2 was observed in IM patients, especially in less differentiated tumor cells	[5]
Khan et al. (2015)	Rb	GSK126, SAH-EZH2	Rb human tissues, primary fetal RPE, human Rb cell lines	EZH2 was highly positive in Rb cells and related to cell differentiation state Reduced ATP production and cell viability in Rb cell lines	[11]
Wen et al. (2022)		GSK503	Human Rb cell lines, orthotopic xenograft model	Restored RB protein expression Reduced tumor proliferation and colony formation Caused a significant reduction in tumor size and weight	[17]
Zhao et al. (2023)		UNC1999, GSK503	Rb human tissues, human Rb cell lines, orthotopic xenograft model	Upregulation of EZH2 was observed in Rb tissues and positively related to poor prognosis Inhibited Rb cell proliferation, migration, invasion and EMT Suppressed tumor growth	[52]

CM: Conjunctival melanoma; EMT: Epithelial–mesenchymal transition; IM: Intraocular medulloepithelioma; Rb: Retinoblastoma; RPE: Retinal pigment epithelium; UM: Uveal melanoma.

Uveal melanoma

Uveal melanoma (UM) is a malignant neoplasm that originates from melanocytes from the choroid, ciliary body and iris [53]. Even if UM patients receive radiation or surgery to remove the primary tumor, almost half of them will develop metastatic cancer eventually [54]. Therefore, better therapies for UM are needed. In 89 primary UM patients, upregulated expression of EZH2 in UM tissues was noted, and the higher EZH2 level was related to an increased risk of metastases and a worse prognosis [55]. Similar results were also observed in studies by Zhang et al. and Wu et al. [50,56]. Additionally, pharmacological suppression of EZH2 induced apoptosis and inhibited the proliferation of UM cells, but did not affect the noncancerous cells [9,57]. The results were further confirmed in vivo. After treatment with the EZH2 inhibitor GSK126, a significant decrease in the volumes and weights of tumors was noted in the subcutaneous xenografted mouse model [9]. EZH2 may act as a target of epigenetic mediators such as miR-124a, miR-26a and miR-137 and therefore epigenetically inhibit the development of UM [49,50,57]. However, the association between EZH2 and UM progression is still unclear.

Conjunctival melanoma

Conjunctival melanoma (CM) is an uncommon aggressive tumor, accounting for 5% of all ocular melanomas [58]. EZH2 mutations are abundant in this invasive melanoma [59]. Treatment with EZH2 inhibitors inhibited cell proliferation and colony formation in CM cell lines [10]. EZH2 ablation mainly increased the proportion of cells in the G2/M phase and promoted cell death by activating the transcription of tumor suppressor gene p21/CDKN1A. The same results were also shown in a zebrafish xenograft model. Inhibition of EZH2 suppressed the proliferation and migration of tumor cells [10]. Given the tumor suppressive effects of EZH2 inhibitors, EZH2 may offer a novel therapeutic target for CM patients. However, reports of EZH2 in CM are limited, and more studies are needed.

Retinoblastoma

Retinoblastoma (Rb), with an incidence rate of 1:16,000, is the most common intraocular malignant tumor in children [60,61]. Biallelic loss of the tumor suppressor gene RB1 causes over 95% of all Rb cases [62]. After RB1 inactivation, tumors develop rapidly. EZH2 has been reported to be downstream of RB1, producing repetitive DNA sequences to promote cancer development [63]. Therefore, EZH2 may be relevant to Rb tumorigenesis. Increased expression of EZH2 was observed in Rb tissues, especially in patients with orbital and choroidal invasion and subretinal and vitreous seeding [64]. Pharmacological suppression of EZH2 inhibited tumor proliferation and colony formation, and also reactivated RB1 expression to regulate the tumorigenesis [17]. Importantly, the effects of EZH2 inhibitors were specific to Rb cells, as there were no signs of suppression on the viability of nontumor primary retinal pigment epithelium (RPE) cells [11].

Intraocular medulloepithelioma

Intraocular medulloepithelioma (IM) is a congenital tumor originating from the neuroepithelial progenitor cells, mostly arising from the nonpigmented ciliary epithelial body [65]. Current therapy includes brachytherapy, tumor resection, enucleation and exenteration, depending on the cancer stages and clinical manifestations [66]. Recently, precision medicine based on epigenetic regulation has been explored. Studies showed that EZH2 staining was exclusively positive in IM tumor cells, especially in poorly differentiated cells [5], suggesting EZH2 may be a potential diagnostic biomarker to evaluate tumor invasion. Besides, given that EZH2 was exclusively expressed in IM tumor cells, targeting EZH2 seems an attractive approach to treat this intraocular neoplasm.

EZH2&corneal injury

Corneal transparency is important to maintain clear vision. When the cornea is damaged by external stimuli, such as inflammation, infection and traumatic injury, corneal wound healing is initiated [67,68]. Unlike the repair processes in other tissues, wound healing of the cornea is a complex procedure involving migration and differentiation of limbal stem cells, cross-talk among different corneal cell types and remodeling of extracellular matrix [69,70]. After corneal scarring is formed, it largely decreases corneal transparency and the reduction cannot be reversed [71]. During this process, myofibroblast transdifferentiation induced by TGF-β plays an important role [72–74]. Inhibiting the dysfunction of transdifferentiation to corneal myofibroblasts is a promising approach to prevent corneal opacity. An increased expression of EZH2 was observed in a corneal injury mouse model [12]. Genetic or pharmacological suppression of EZH2 caused a reduction of COL1A1, FN1 and ACTA2 expressions, which are classical fibrotic markers in TGF-β1-induced corneal myofibroblasts. Meanwhile, EZH2 inhibitor EPZ-6438 alleviated cell migration and collagen contraction, the hallmarks of activated fibroblasts [12]. Corneal neovascularization is another important process for wound healing. EZH2 expression was upregulated in a corneal neovascularization mouse model, and the release of proangiogenesis factors, including VEGF, was reduced following treatment with EZH2 inhibitors [75]. These studies suggested EZH2 actively participated in the process of corneal injury repair and it may serve as a new avenue for preventing corneal scarring (Table 4).

Table 4. Summary of EZH2 inhibitors in corneal injury.

Author	EZH2 inhibitors	Models	Effects	Ref.
Wan et al. (2020)	DZNep, EZH2 siRNA	HUVECs, CoNV model mice	EZH2 expression was upregulated in CoNV model mice Reduced VEGF level induced by alkali burn in the cornea Inhibited VEGF, ROS and H2O2 production induced by H/R in HUVECs	[75]
Liao et al. (2021)	EZH2 siRNA, EPZ6438	Human primary corneal fibroblasts, corneal injury model mice	Suppressed corneal myofibroblast activation and ECM protein synthesis Inhibited cell migration and gel contraction	[12]

CoNV: Corneal neovascularization; ECM: Extracellular matrix; H/R: Hypoxia/reoxygenation; HUVEC: Human umbilical vein endothelial cell; ROS: Reactive oxygen species.

EZH2&cataract

Cataract is a globally widespread, vision-threatening disorder of which aging is the primary cause [76]. During aging, the anterior epithelial cells lose their functions and undergo degeneration, finally leading to cataract [77]. Anti-aging therapy may help to prevent the development of cataract. It has been reported that the suppression of HSF4 could impair lens differentiation and cause cataracts to develop [78,79]. In lens epithelial cells (LECs), HSF4 colocalized with EZH2 in the nucleus. Recruitment of EZH2 by HSF4 downregulated p21 expression and prevented senescence. Conversely, knocking out EZH2 increased the level of p21 and promoted senescence [80].

Till now, opaque lens removal followed by artificial intraocular lens implantation has been the primary treatment modality for cataract [81]. Nevertheless, a wound healing response is initiated by residual LECs after surgery and therefore leads to secondary vision loss, which is known as posterior capsule opacification (PCO) [82–84]. Inhibiting fibrotic responses of LECs is worth investigating to prevent or suppress VI resulting from PCO. Overexpression of EZH2 was observed in human PCO-attached LECs, indicating that EZH2 may be involved in the pathogenesis of PCO [85]. Moreover, the central mediator in fibrosis, EGF, could induce expression of fibrotic markers via a miR-26b-dependent pathway and in turn upregulated EZH2 levels to promote the progression of PCO [85].

Epithelial–mesenchymal transition (EMT) of LECs is an important process in anterior subcapsular cataract [86]. Previous studies showed that MYPT1/PP1 specifically phosphorylated EZH2 at S21, and then enhanced H3K27Me3 expression via the AKT–EZH2 axis and contributed to the prevention of LEC fibrosis [13]. These results provide important insights into the functions and regulation of EZH2 in aging and the fibrotic process of LECs, not only as an enzymatic subunit of the PRC2 complex but also as an independent nonhistone methyltransferase to regulate other transcription factors (Table 5).

Table 5. Summary of EZH2 inhibitors in cataract.

Author	Cataract	EZH2 inhibitors	Models	Effects	Ref.
Dong et al. (2018)	Posterior subcapsular cataract	EZH2 siRNA	Patient LECs, HLE cell lines	Enhanced expression of EZH2 was detected in human PCO-attached LECs Attenuated the downregulation of E-cadherin and upregulation of fibronectin induced by EGF	[85]
Cui et al. (2021)	Cataract	EZH2 siRNA	LEC cell line, UVB-induced cellular senescence cell model	HSF4 downregulated p21 expression by recruiting EZH2 and prevented cellular senescence Increased the p21 levels	[80]
Zhang et al. (2022)	Anterior subcapsular cataract	–	Human LEC cell line, injury-induced ASC mouse model, human ASC lens capsular samples, HLE cell lines with EZH2-S21A mutant, EZH2-silenced zebrafish	Higher phosphorylation of EZH2 at S21 was detected in human ASC plaques and ASC mice Inhibiting AKT activity attenuated EZH2 phosphorylation at S21 in ASC, leading to downregulation of fibronectin and α-SMA expression The dephosphorylation of EZH2 S21 suppressed TGF-β-induced EMT in HLE cells	[13]

ASC: Anterior subcapsular cataract; EMT: Epithelial–mesenchymal transition; HLE: Human lens epithelium; LEC: Lens epithelial cell; PCO: Posterior capsule opacification.

EZH2&glaucoma

Loss of RGCs is an important pathology in glaucoma, and preventing RGC death has been an intense research direction [87,88]. The roles of EZH2 in RGC protection are still not fully understood (Table 6). In the N-methyl-d-aspartate-induced acute RGC death model, treatment with the EZH2 inhibitor DZNep prevented the reduction of RGCs and inner nuclear layer thickness after N-methyl-d-aspartate-induced damage [89]. However, another study showed EZH2 silencing had little impact on the maturation and functions of RGCs, nor did it have an influence on injury reaction induced by elevated intraocular pressure or optic nerve damage [90]. In a glaucoma animal model established by translimbal laser photocoagulation, inhibition of EZH2 even promoted the apoptosis of RGCs, while upregulation of EZH2 reduced the RGC degeneration rate [91,92]. There are thus substantial variations about the roles of EZH2 in RGCs. More investigations are needed.

Table 6. Summary of EZH2 inhibitors in glaucoma.

Author	EZH2 inhibitors	Models	Effects	Ref.
Cheng et al. (2018)	–	Ezh2 knockout mice	Did not affect RGC survival and light-induced ERG responses Did not affect RGC susceptibility to the damage induced by elevated IOP or optic nerve injury	[90]
Zhou et al. (2019)	ShEZH2	Glaucoma rat models, primary rat RGCs	Promoted RGC apoptosis	[91]
Xiao et al. (2021)	DZNep	NMDA-induced mice	Alleviated the cell death in GCL and INL and alleviated the retinal function loss Inhibited microglial activation	[89]

ERG: Electroretinogram; GCL: Ganglion cell layer; INL: Inner nuclear layer; IOP: Intraocular pressure; NMDA: N-methyl-d-aspartate; RGC: Retinal ganglion cell.

EZH2&DR

Activation of MMP9 is a trigger factor in DR, causing angiogenesis, mitochondrial injury and the apoptosis of capillary cells [93]. MMP9 has been proven to be the downstream gene of EZH2 [94,95]. Targeting EZH2 may epigenetically regulate MMP9 and therefore alleviate the progression of DR. High expression of H3K27me3 in the MMP9 promoter region and an approximately twofold increase in EZH2 levels were detected after high glucose stimulation in human retinal endothelial cells (HRECs). EZH2 inhibition reduced the levels of H3K27me3, alleviated the activity of MMP9 and prevented cell apoptosis [18]. Moreover, EZH2 could regulate DNA methylation through the recruitment of DNMT1 and TET2 to control the transcription of MMP9: similar results were observed in the animal model and DR patient retinas, where a significant increase in H3K27me3 and EZH2 levels at the MMP9 promoter and higher expression of DNMT1 and TET2 were noted [18], supporting the fact that EZH2 regulates MMP9 by different epigenetic mechanisms to maintain the cellular integrity in DR.

As well as its regulation of MMP9, EZH2 can also interact with lncRNAs to actively participate in DR. A lncRNA microarray analysis showed that the lncRNA ANRIL was one of the most differentially expressed genes in a normal-glucose-treated group versus a high-glucose-treated group in human retinal endothelial cells [14]. Cooperation between ANRIL and EZH2 in regulating disease development has been reported previously [96,97]. In the diabetic mice, significant elevation of EZH2 and VEGF was detected, while no changes to the levels of EZH2 and VEGF were found in ANRIL knockout diabetic mice. In addition, pharmacological suppression of EZH2 resulted in significant reductions in VEGF and ANRIL levels [14], indicating the interaction between ANRIL and EZH2 in the VEGF signaling axis. Given that VEGF is a key player in angiogenesis in DR [98–101], the role of EZH2 in regulating VEGF and angiogenesis-related diseases may be worth further investigating (Table 7).

Table 7. Summary of EZH2 inhibitors in diabetic retinopathy.

Author	EZH2 inhibitors	Models	Effects	Ref.
Ruiz et al. (2015)	DZNep, EZH2 siRNA	HRECs, diabetic rats	EZH2 was highly expressed in high-glucose-induced HRECs and the retinas of diabetic rats Decreased VEGF level and tube formation	[102]
Thomas et al. (2017)	DZNep	HRECs, diabetic mice	Upregulated expression of EZH2 in high-glucose-induced HRECs and in the retinas of diabetic animals Blockade of EZH2 activity reduced VEGF level	[14]
Duraisamy et al. (2017)	DZNep, EZH2 siRNA	HRECs, diabetic rats, human DR tissues	High level of EZH2 was observed in high-glucose-induced HRECs, retinal microvessels of diabetic rats and human DR retina Ameliorated glucose-induced MMP9 accumulation and prevented mitochondrial damage	[18]
Biswas et al. (2018)	DZNep	HRECs	Increased expression of EZH2 was detected in high-glucose-induced HRECs and in the diabetic animals’ retinas Resulted in downregulation of TNF-α but upregulation of IL-6	[103]
Di et al. (2022)	–	Diabetic rats	Involved in the molecular and cellular mechanisms of Yiqi Tongluo Fang in DR treatment	[104]

DR: Diabetic retinopathy; HREC: Human retinal endothelial cell.

EZH2&age-related macular degeneration

Epigenetic regulation, which is potentially conducive to the aging process, has been widely recognized to be involved in the pathology of age-related macular degeneration (AMD).

As shown in Table 8, EZH2, along with miRNAs, co-regulates AMD-associated genes such as ELL2 and ENTPD1 [105]. Dysfunction of RPE cells is the primary feature of AMD, and EMT of RPE cells is implicated in disease development. In TGF-β1-induced EMT in RPE cells, EZH2 inhibition caused a drastic decrease in the expression of EMT markers, including α-smooth muscle actin, fibronectin and collagen-1 [106]. The level of the tight junction protein ZO-1 was upregulated. Suppression of EZH2 also inhibited the proliferative and migratory abilities of RPE cells, enhancing transepithelial electrical resistance and barrier functions [106]. However, the effect of EZH2 inhibitors in vivo is still unclear.

Table 8. Summary of EZH2 inhibitors in age-related macular degeneration.

Author	EZH2 inhibitors	Models	Effects	Ref.
Peng et al. (2022)	EZH2 siRNA	Human primary RPE cells	Downregulated the expressions of α-SMA, fibronectin and collagen-1 Inhibited cell migration and cell proliferation Alleviated TGF-β1-induced EMT and enhanced barrier functions of RPE cells	[106]
Olivares et al. (2017)	–	miRNA microarray analysis	EZH2, along with miRNAs, co-regulates AMD-associated genes such as ELL2 and ENTPD1	[105]

AMD: Age-related macular degeneration; EMT: Epithelial–mesenchymal transition; RPE: Retinal pigment epithelium.

The importance of Alu RNA accumulation in AMD has been widely recognized in recent years. As a transposable element, Alu RNAs actively participate in gene translation and modify human genomes [107]. An abundance of Alu elements was found to be present in RPE cells from patients with atrophic AMD; Alu inhibition increased cell viability and the barrier function of RPE cells in vitro and invivo [108]. Alu RNA could also promote retinal degeneration through the activation of the NLRP3 inflammasome and therefore facilitate inflammatory responses [109,110]. Interestingly, studies showed that EZH2 could enhance the activity of Alu RNA by accelerating its cleavage [25], suggesting a nonmethylase activity of EZH2. Nevertheless, whether EZH2 interacts with Alu RNAs in AMD pathogenesis remains to be elucidated.

EZH2&other ocular diseases

Studies about EZH2 and its relevance to uveitis and dry eye diseases are lacking. Limited data in one study showed that EZH2 expression was higher in samples from patients with Sjögren’s syndrome and its level paralleled the pathological damage in salivary glands [111]. In addition, EZH2 is closely related to the activity of CD4⁺ T cells in Sjögren’s syndrome. A significant reduction of cell activation and the release of proinflammatory cytokines from CD4⁺ T cells was noted after EZH2 inhibition [111,112]. Sjögren’s syndrome is an important cause of dry eye diseases; thus EZH2 inhibitors may be a potential avenue for Sjögren’s syndrome-related dry eye. Meanwhile, because of the vital role of CD4⁺ T cells in inflammatory reactions, the regulation of EZH2 in uveitis is worth investigating.

Conclusion & future perspective

Previous studies suggested that EZH2 was highly elevated in ocular lesion tissues and participated in many types of eye diseases. However, the findings were mainly from experimental models. Moreover, although current studies demonstrate the benefits of EZH2 inhibitors in different eye diseases, their potential risks – such as damage to cognitive ability, autoimmunity and heart muscles – should be noted [113], and there has been no clinical trial yet. Due to the complex function of EZH2, there remains much that we still do not fully understand. Further investigations to fully explain the EZH2 network, including the PRC2-dependent and -independent effects, will provide insights into a variety of ocular diseases. Meanwhile, the development of efficient, selective and safe EZH2 inhibitors and the realization of translational preclinical pharmacology should be an important goal for its clinical application in ophthalmology.

Executive summary

EZH2&retinal development

• EZH2 is suggested to actively regulate retinal proliferation and differentiation.

• EZH2 is dynamically expressed in different stages of retinal development, with high levels in embryonic retinas but weak expression after birth.

EZH2&inherited retinal diseases

• The effect of EZH2 inhibitors on photoreceptor degeneration in inherited retinal diseases is still controversial, with some studies showing a delay in photoreceptor loss but one other study showing an induced progression.

EZH2&ocular tumors

• EZH2 was shown to be overexpressed in ocular tumors and related to a worse prognosis.

• EZH2 suppression reduced the growth and invasion of tumors through interactions with noncoding RNAs and tumor suppressor genes.

EZH2&corneal injury

• EZH2 inhibition prevented the formation of corneal scarring in a corneal injury animal model by suppressing extracellular matrix synthesis and corneal myofibroblast activation.

EZH2&cataract

• EZH2 is suggested to be related to the aging process and involved in the development of cataract.

• EZH2 inhibition halted the epithelial–mesenchymal transition process and interacted with other mediators, such as miRNAs and transposable elements, to co-regulate disease progression.

EZH2&glaucoma

• The effect of EZH2 inhibitors on retinal ganglion cells is still unclear, with substantial variations in research on the roles of EZH2 in prevention and promotion of retinal ganglion cell loss.

EZH2&diabetic retinopathy

• Upregulated expression of EZH2 was detected in high-glucose-induced human retinal endothelial cells and diabetic animal models.

• Blockade of EZH2 activity reduced MMP9 and VEGF levels in both in vivo and in vitro studies.

EZH2&age-related macular degeneration

• EZH2 inhibition suppressed epithelial–mesenchymal transition and enhanced the barrier functions of retinal pigment epithelium cells.

• The potential interactions between EZH2 and Alu RNA in the pathogenesis of age-related macular degeneration require investigation.

Conclusion & future perspective

• EZH2, as an important epigenetic regulator, acts in both PRC2-dependent and -independent ways to participate in various ocular diseases.

• Despite the benefits of EZH2 inhibitors in different eye diseases, their potential risks, including damage to cognitive ability, autoimmunity and heart muscles, should be noted.

• The development of efficient, selective and safe EZH2 inhibitors and the realization of translational preclinical pharmacology should be an important goal for its clinical application in ophthalmology.

Conceptualization: J Yam. Writing (original draft preparation): Y Peng, C Bui and X Zhang. Writing (review and editing): C Bui, J Chen, C Tham, W Chu, L Chen, C Pang and J Yam. All the authors read and approved the final manuscript.

Financial & competing interests disclosure

This study was supported in part by the CUHK Jockey Club Children’s Eye Care Programme; the CUHK Jockey Club Myopia Prevention Programme; and Health and Medical Research Fund (HMRF), Hong Kong (07180306 and PR-HKCH-8). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

This study was supported in part by the CUHK Jockey Club Children’s Eye Care Programme; the CUHK Jockey Club Myopia Prevention Programme; and Health and Medical Research Fund (HMRF), Hong Kong (07180306 and PR-HKCH-8). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.