The tumor suppressor p53 is a pivotal component of cells that monitors and controls the most important cellular events, including cell cycle arrest, apoptosis, senescence, metabolism, DNA repair and malignant transformation. p53 acts as a switch between life (cell cycle arrest and attendant DNA repair) and death (apoptosis) and thus determines the fate of cells in response to cellular stress (Carvajal and Manfredi, 2013). The mouse double minute 2 (MDM2) homolog oncoprotein, an E3 ubiquitin ligase encoded by the proto-oncogene MDM2, is the primary negative regulator of p53. MDM2 represses p53-dependent transcription via a triple mechanism: nuclear or cytoplasmic ubiquitin-dependent proteasomal degradation of p53, reversible cytoplasmic p53 translocation and direct abrogation of p53-dependent transactivation. MDM2 deficiency leads to p53-driven embryonic lethality, which can be fully rescued by simultaneous deletion of p53 (Jones et al., 1995). In contrast, overexpression of MDM2 impairs p53-mediated regulation of cell growth, with the substantial accumulation of DNA damage, which supports tumorigenesis. Wade et al. (2013) reported that amplification of the MDM2 gene or altered expression of the MDM2 protein is a feature of many tumors. In general, the expression of MDM2 is dependent on two promoters: the upstream P1 promoter in the first exon of the MDM2 gene, which controls basal constitutive expression in normal living cells, and the downstream P2 promoter in the first intron, which initiates inducible expression in response to stimuli such as p53, RAS and estrogen receptor α under stress conditions (Barak et al., 1994). Transcripts derived from the P2 promoter exhibit enhanced translational efficiency that is approximately eight times that of transcripts derived from the P1 promoter (Landers et al., 1997). A naturally occurring single-nucleotide polymorphism (SNP) in the P2 promoter, MDM2 T309G (rs2279744, a thymine-to-guanine change at the 309th nucleotide in the first intron), enhances binding of the transcriptional activator Sp1 to the P2 promoter and stimulates P2-driven inducible expression of MDM2 under stress, with subsequent attenuation of p53 signaling (Bond et al., 2004). Several clinical investigations have indicated that MDM2 T309G is related to increased risk, early onset, high malignancy and poor prognosis in cancers (Boersma et al., 2006; Bougeard et al., 2006; Lind et al., 2006; Ohmiya et al., 2006; Gryshchenko et al., 2008). Moreover, Post et al. (2010) corroborated the correlation between MDM2 T309G and increased tumor risk using two cohorts of genetically engineered mice carrying either the humanized MDM2 SNP309 G allele or T allele.

Proliferative vitreoretinopathy (PVR) is the major cause of failure after rhegmatogenous retinal detachment (RD) surgery and is responsible for 5–10% of all RD cases (Machemer et al., 1991; de la Rua et al., 2008). PVR is generally characterized by postoperative growth and contraction of fibrocellular membranes within the vitreous cavity and on both retinal surfaces and, in many cases, a fibrotic process of the retina itself (Pastor et al., 2002). These periretinal membranes create tangential traction force on the retina, and the distorted retina develops into fixed retinal folds, causing vision-compromising RD (Figure 1). Therefore, identifying and following individuals at high risk of developing PVR after RD surgery for early intervention have great practical significance. SNPs are important to genetic susceptibility and can serve as risk factors for multifactorial diseases in humans. For example, a case–control study by Pastor-Idoate et al. (2013a); Pastor-Idoate et al. (2013b) showed that the Pro variant of the p53 codon 72 polymorphism (rs1042522) and the MDM2 SNP309 G allele are associated with a higher risk of PVR among European subjects undergoing RD surgery. Given that rs1042522 and MDM2 T309G synergistically attenuate the proapoptotic function of p53 in direct and indirect ways, respectively, they produce an additional effect on the risk of PVR in carriers harboring both SNPs (Pastor-Idoate et al., 2013b). This finding highlights the association of MDM2 T309G and individual genetic susceptibility to PVR.

Epimacular membranes (EMMs) are relatively common sight-threatening conditions featuring fibrocellular proliferation along the surface of the internal limiting membrane (ILM) of the retina. These membranes preferentially grow in the macular region. Primary/idiopathic EMMs are strongly associated with the development of posterior vitreous detachment (PVD) and vitreomacular traction induced by age-related changes in the extracellular matrix (ECM) of the vitreoretinal interface (Bu et al., 2014). Other EMMs are secondary to various predisposing conditions, including diabetic retinopathy, retinal vein occlusion, rhegmatogenous RD, high myopia, ocular trauma, vitreous hemorrhage, retinal photocoagulation or cryotherapy, uveitis and cataract surgery. In the initial stage of EMM development, which is termed cellophane maculopathy, thin, translucent membranes increase the foveal light reflex under ophthalmoscopy, although patients are usually asymptomatic. In the progressive stage, known as macular puckers or preretinal macular fibrosis, the membranes become thickened and semitranslucent with incremental degrees of inflammation and fibrosis. Contractile EMMs generate retinal wrinkling and distort the macula, causing macular edema, ischemia or even macular holes (Figure 2). Typical manifestations are decreased visual acuity, central scotoma, metamorphopsia, micropsia/macropsia and monocular diplopia. According to previous histological studies, cellular components in EMMs include hyalocytes, retinal pigment epithelium (RPE) cells, Müller cells (MCs), astrocytes, microglia, macrophages, fibroblasts and myofibroblasts (Smiddy et al., 1989; Zhao et al., 2013; Vishwakarma et al. (2020) demonstrated that retinal glial cells are activated under oxidative stress and interact with other cells to cause neuroinflammation and neurodegeneration, which underlie the formation of EMMs under different pathological conditions. As the activation of oxidative stress and proinflammatory signaling as well as fibrocellular proliferation are, to some extent, modulated by the MDM2-p53 axis in these cells, we sought to determine whether MDM2 T309G is related to EMMs. To the best of our knowledge, no studies have yet examined this issue. In this study, we aimed to investigate the role of MDM2 T309G in EMM formation by comparing the genotype distribution and consistency of the polymorphism in paired membrane-blood samples between the PVR, EMM and healthy Chinese donor groups.

In the present study, we investigated the genotype distribution of MDM2 T309G in fibrocellular membranes, ILMs and blood samples from PVR and EMM patients. For both groups, the proportion of MDM2 SNP309 G allele carriers and the frequency of the G allele (% G) in blood samples were significantly higher than those in blood samples from the healthy donor group. Furthermore, between-group comparisons of MDM2 T309G consistency in paired membrane-blood samples revealed that EMMs are predisposed toward somatic mutation at the MDM2 T309G locus but that PVRMs are not, in contrast to PVR-ILMs and EMM-ILMs, which undergo mutation at similar ratios. Another notable finding was that EMMs and ILMs from EMM patients with preoperative macular holes had a greater mutation rate (% M) than those from patients without preoperative macular holes. Hence, we propose for the first time that MDM2 T309G is associated with the development of EMMs in the Chinese population. That is, carriers of the MDM2 SNP309 G allele are at a higher risk for developing EMMs. Moreover, EMMs and ILMs, especially those complicated with macular holes, are genetically unstable at the MDM2 T309G locus. We hypothesize that MCs, which participate in reactive gliosis against various retinal stresses, or retinal progenitor/stem cells derived from dedifferentiated MCs are susceptible to oxygen free radical-induced single-base substitutions at the MDM2 T309G locus. These cells may be responsible for the genomic instability in EMMs and ILMs, although this hypothesis needs to be further confirmed.

Our results are consistent with a previous report showing that age is a risk factor for EMMs but do not support female sex as a risk factor (Xiao et al., 2017). The average age of the EMM group was 8.87 years older than that of the PVR group, possibly due to the rapid worsening of visual function in PVR patients or the prolonged development of EMMs. Nevertheless, this study is not a large-scale epidemiological survey, and the above results are for reference only. We believe that the difference in average age is mainly attributed to the intrinsic properties of the two diseases and hardly affects our results, as such a difference is far from enough to have a statistically significant impact on overall genomic stability.

PVR is a pathological vitreoretinal fibrosis condition induced by vitreal growth factors, cytokines and inflammatory mediators produced by RPE cells, MCs, microglial cells and macrophages, including platelet-derived growth factor (PDGF), TGF-β2, EGF, FGF2, and TNF-α. Stimulated by these vitreal factors, detached RPE cells undergo epithelial to mesenchymal transition (EMT), acquiring enhanced proliferation and migration capacities, resistance to apoptosis and the ability to produce ECM (Chiba, 2014). The transformed cells then migrate into the subretinal space or, through retinal breaks, the vitreous cavity. They proliferate and transdifferentiate into fibroblasts and myofibroblasts together with other cells to form subretinal or epiretinal PVRMs (Agrawal et al., 2007; Pastor et al., 2016). Therefore, researchers employ PVR vitreous from patients or animals to induce EMT in RPE cells for experiments. In recent years, multiple studies have confirmed that MDM2 is involved in PVR. Initially, chronic activation of Akt and suppression of p53 were identified by Lei et al. (2011) as a signature pathway downstream of the PDGF receptor α (PDGFR-α) signaling cascade in PVR. However, stabilizing intracellular p53 levels by Nutlin-3, a small-molecule antagonist of the MDM2-p53 interaction, abrogated the Akt-p53 pathway and prevented human PVR vitreous-induced contraction of cells isolated from a PVRM (Lei et al., 2012). In fact, PVR vitreous induces ERK/Sp1-dependent MDM2 upregulation and subsequent p53 downregulation in RPE cells. The resultant enhanced abilities of RPE cells to proliferate, survive, contract and migrate are intrinsic to PVR. Inspired by previous works by Pastor-Idoate et al. (2013b), Lei’s team introduced MDM2 T309G into RPE cells to create RPE cells with MDM2 SNP309 T/T and MDM2 SNP309 T/G. Importantly, PVR vitreous-induced cellular responses were enhanced in T/G RPE cells compared with T/T RPE cells, but this was abrogated by genetic silencing of aberrant P2-driven expression of MDM2 caused by MDM2 T309G (Duan et al., 2016; Zhou et al., 2017; Chen et al., 2019). MDM2 T309G also promoted the development of experimental PVR in an in vivo rabbit model (Zhou et al., 2017). Our results show a larger proportion of G allele carriers in the PVR group than in healthy donors and a higher % G in blood samples from PVR patients than from healthy donors. This finding is consistent with the established notion that MDM2 T309G promotes PVR. It has been proposed that aberrant P2-driven inducible expression of MDM2 facilitates EMT in RPE cells by targeting E-cadherin, a cell adhesion protein and a key regulator of EMT, among others, for ubiquitin-dependent proteasomal degradation (Yang et al., 2006; Liu et al., 2019). Thus, carriers of the MDM2 SNP309 G allele exhibit a greater increase in MDM2 levels in response to RD-induced stress. Such an overabundance of MDM2 induces RPE cells to undergo EMT, thereby contributing to PVRM formation. Most importantly, we show that G allele carriers were at a higher risk of developing EMMs than non-G allele carriers (p = 0.0479; OR = 2.047, 95% CI, 1.027–4.149) and that EMM patients exhibited a significantly higher % G than healthy donors (p = 0.0256; OR = 1.567, 95% CI, 1.046–2.335), indicating the role of the MDM2 SNP309 G allele in promoting EMMs (Figure 5; Table 2).

Surprisingly, of all 124 pairs of matched membrane-blood samples, 21 pairs were inconsistent in genotype. Moreover, possible operational errors were excluded by duplicate examination of both forward and reverse sequencing. Since peripheral blood leukocytes are typically genetically stable, such inconsistency arises from somatic mutations in membrane cells under pathological conditions. A likely explanation for this finding is that mutation is a consequence of overproduced reactive oxygen species in the vitreal inflammatory response. Reactive oxygen species are constantly produced through normal cellular oxygen respiration or stress responses to various environmental mutagens and carcinogens, such as ionizing radiation, chemicals and ultraviolet light. Excessive oxygen free radicals attack DNA and its precursor deoxyribonucleotides, leading to DNA damage and mutations. The most studied oxidative DNA lesion, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG)/8-hydroxy-2′-deoxyguanosine (8-OH-dG, an enol tautomer of 8-oxo-dG), has been strongly implicated in cancers due to its capacity to induce the G/C → T/A transversion (Moriya, 1993; Valko et al., 2006). Furthermore, free nucleotides are oxidized more efficiently than DNA, and oxidized dNTPs cause potent genomic instability when they are incorporated into daughter strands during DNA replication (Maki, 2002). 8-Hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP, an equivalent of 8-oxo-dGTP) and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP, an equivalent of 2-oxo-dATP), the oxidized forms of dGTP and dATP, respectively, induce A/T → C/G and G/C → T/A transversions, respectively, in newly synthesized DNA (Inoue et al., 1998). These single-base substitutions are elicited by human DNA polymerase η-, ζ- or REV1-mediated misincorporation of 8-OH-dGTP opposite template adenine and 2-OH-dATP opposite template guanine (Maki and Sekiguchi, 1992; Inoue et al., 1998; Hidaka et al., 2008; Satou et al., 2009). Correspondingly, of the 21 membrane samples with heterozygous mutations in this study, 6 and 15 harbored an A/T → C/G transversion and a G/C → T/A transversion at the MDM2 T309G locus, respectively.

An interesting finding is that % M varied between PVRMs and EMMs (p = 0.0097; OR = 10.18, 95% CI, 1.594–112.4) but not between PVR-ILMs and EMM-ILMs (p = 0.6855; OR = 1.853, 95% CI, 0.3435–10.12), suggesting that EMMs and ILMs are intrinsically connected in terms of genomic instability at the MDM2 T309G locus (Figure 6; Table 3). Histologically, Müller glial cells are a major cellular component of EMMs (Zhao et al., 2013). These cells are a major type of macroglia that maintain retinal homeostasis and provide structural, metabolic and trophic support. Their radial processes extend across the entire neurosensory retina, serving as living optical fibers. Above all, MCs also play a pivotal role in retinal gliosis. Reactive MC gliosis is a protective process against retinal injury that insulates the retina from vitreal pathogenic factors, promotes retinal tissue regeneration and limits tissue remodeling (Bringmann and Wiedemann, 2012). Under diverse pathological conditions, MCs in the mature retina rapidly proliferate and dedifferentiate to pluripotent retinal progenitor/stem cells, which migrate along MC processes to the damaged retinal layer and redifferentiate to replenish lost neurons (Reichenbach and Bringmann, 2013). Any retinal or vitreal insult might trigger this reparative reaction, and common initiating factors are RD, retinal ischemia, PVD, vitreous hemorrhage, endophthalmitis and trauma. However, excessive gliosis leads to glial scars, which are aberrant tissue repairs in response to retinal stress. In particular, various EMMs (primary and secondary) are a distinct type of glial scar attached to the retina by hypertrophied MC fibers (Bringmann and Wiedemann, 2012). In response to stress, retinal glial cells, notably MCs, migrate to the vitreoretinal interface through an intact or defective ILM, proliferating and transdifferentiating, along with hyalocytes, RPE cells, macrophages, fibroblasts and myofibroblasts, on the scaffold of the ILM (collagen IV) to form early EMMs. In brief, EMMs essentially constitute progressive fibrocellular proliferation on the vitreal side of the ILM and are inextricably linked to the ILM. As routine clinical procedures, vitrectomy and the removal of EMMs along with ILM peeling clear the hazardous local microenvironment and relieve macular traction, thus favoring postoperative anatomic and visual recovery of the retina. The ILM is the innermost layer of the neurosensory retina that adheres the vitreous to the retina. It is a thin basement membrane formed by MC endfeet. ECM proteins that make up the ILM, including collagen IV, laminins, nidogens and heparan sulfate proteoglycans, are believed to be produced and secreted by MCs (Peynshaert et al., 2019; Zhang and Johnson, 2021). Based on the above facts, it is reasonable to speculate that MCs are responsible for the propensity of EMMs and ILMs toward somatic mutation at the MDM2 T309G locus.

Last but not least, the heightened % M observed in EMMs (p = 0.0053; OR = 8.250, 95% CI, 1.668–29.20) and ILMs (p = 0.0233; OR = 14.40, 95% CI, 1.560–175.8) from EMM patients with preoperative macular holes compared with those without holes (Figure 7; Table 4) further corroborates this speculation, as oxidative stress and aberrant preretinal gliosis intensify with retinal injury, giving rise to genomic instability at the MDM2 T309G locus in highly proliferating MCs. We hypothesize that the blood–retinal barrier adjacent to macular holes is disrupted and that leukocytes emigrate to the preretinal space or vitreous cavity, releasing cytokines and inflammatory mediators. Inflammatory infiltration produces vast amounts of oxygen free radicals that attack proliferating MCs in EMMs and ILMs and cause somatic mutation at the MDM2 T309G locus. Admittedly, further research is needed to confirm our conjecture.

The major limitations of this study include the following: 1) An insufficient sample size. A substantial number of compound membranes consisting of fibrocellular membranes and ILMs and their matched blood samples were unqualified for this study and excluded from the cohorts. The association between MDM2 T309G and EMMs may need to be further validated with a larger sample size. 2) A lack of blood samples from healthy donors. The genotype distribution of MDM2 T309G varies with race (Wilkening et al., 2007; Knappskog et al., 2011). Therefore, the statistics of genotype distribution for healthy Chinese donors (Knappskog et al., 2011) were selected for comparison to minimize racial/ethnic bias. 3) This study was conducted in only the Chinese population, and it is unclear whether these conclusions can be extended to other ethnicities. Hence, replication studies in larger independent cohorts of different ethnicities are required.

In summary, this study highlights the role of the MDM2 SNP T309G in the development of EMMs. Herein, we report for the first time that the MDM2 SNP309 G allele is an associated factor for EMMs in a Chinese population. This observation provides new insights into the molecular mechanism of these pathologies, which may be beneficial for developing novel approaches and enabling early intervention for high-risk populations to improve visual prognosis.