Journal of Diabetes & Metabolism

Epigenetic Insights: DNA Methylation and Gene Expression in Diabetic Retinopathy–A Mini Review

Shaista¹, Abdul Waris^1*, Sheelu Shafiq Siddiqi², Tanusree Debbarman³, Vivek Ram K.U.¹ and Dimple Thacker^{1 *}Correspondence: Abdul Waris, Junior Resident, Aligarh Muslim University Institute of Ophthalmology, J.N. Medical College and Hospital, Aligarh Muslim University, Aligarh, UP 202002, India, Email:

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Introduction

Epigenetics is change in gene function caused by change in chromosomes without DNA sequence changes. They are stable, static throughout life, hereditable, influenced by environment, lifestyle and disease process. There are three types of epigenetic modification: Methylation, histone modification, non-coding RNA expression. These systems have the ability to independently control particular epigenetic phenomena, but they can also work together to cooperatively control several physiological processes.

DNA methylation is the process of transfer of methyl group to the C-5 position of the cytosine ring by an enzyme DNA Methyltransferase (DNMT) [1]. 98% of DNA methylation in human occur in CpG dinucleotide in somatic cells while some occurs in embryonic cells (Figure 1) [2]. It’s involvement in genomic imprinting, X chromosome inactivation, and the suppression of repetitive elements is critical for normal development and proper cellular functioning. Furthermore, dysregulation of DNA methylation has been connected to a number of illnesses [3].

Figure 1: Effect of DNA methylation on DNA transcription (CpG: High concentration base pair of cytosine guanine nucleotide).

There are three types of DNMT [4]. DNMT 1 is in charge of transmitting the daughter strand's copy of the DNA methylation pattern during DNA replication during S phase of the cell cycle [5]. DNMT 2 instead of methylating DNA, methylates cytosine in the aspartic acid anticodon loop of t-RNA [6]. DNMT 3A AND 3B perform methylation de novo during development. Mice lacking DNMT 3A dies at 4 week of age and DNMT 3B knock out becomes lethal at embryonic age of E 14.5 to E 18.5 [7]. DNMT 3L helps in de novo methyl transfer by increasing the ability of DNA to bind methyl donor [8]. DNMT 3L null mice are viable but heterozygous mice derived from homozygous DNMT null oocyte dies around E 9. In conclusion, DNMT 1 is necessary for de novo methylation of genomic DNA, and DNMT 3A AND 3B support replication-related maintenance methylation [9].

Literature Review

DNA methylation and diabetes retinopathy

In diabetics, the retina's vasculature, DNMT expression is elevated and DNMT is activated. Transcription is impacted by CpG hypermethylation because it prevents certain transcription factors from attaching to the DNA sequence [10]. CG dinucleotide within promoter is protected from methylation. Hypermethylation of CpG dinucleotide recruits Methylated CpG (MeCP) binding protein to bind the promoter region causing repression of transcription [11].

The TET dioxygenase family, consisting of TET1, TET2, and TET3, is essential for DNA demethylation as it transforms 5-methylcytosine into 5-hydroxymethylcytosine [12]. Due to this, certain genes can be activated and expressed and the chromatin can be opened up for transcription factor binding [13]. In people with diabetes, Tets are also activated in the retina and its vascular system. TET2 is the most extensively researched and closely connected subtype in the pathophysiology of DR [14]. Age-related Macular Degeneration (AMD), retinitis pigmentosa, and DR are linked to DNA methylation (Figure 2) [15].

Figure 2: Increased expression on DNMT and TET causing gene expression and repression respectively.

Through three additional pathways—oxidative stress, inflammation, and neovascularization—DNA methylation also plays a role in the pathogenic process of DR.

Oxidative stress

The term "oxidative stress" describes the harmful impact that Reactive Oxygen Species (ROS) can have on tissues, cells, or organs [16]. High levels of ROS can harm lipids, membranes, proteins, and nucleotides, among other cell components. The retina is an excellent target for Reactive Oxygen Species (ROS) due to its high oxygen consumption and metabolic rate. Diabetes-induced cytosolic ROS damage the retina's mitochondria and speed up the death of retinal capillary cells, which is an early phase in the retinal vascular system that leads to Diabetic Retinopathy (DR).

By possibly contributing to the development of DR through oxidative stress phenomenon, aberrant DNA methylation in the following linked genes may be the mechanism.

Matrix Metalloproteinase-9 (MMP-9): The transcription of MMP-9, an enzyme linked to retinal mitochondrial injury, is regulated by the methylation state of its DNA. Hyperglycaemia enhances Dnmt1 and TET2 binding to the MMP-9 gene promoter in retinal endothelial cells. After Dnmt1 adds a methyl group to the cytosine to generate 5Mc, TET2 demethylates the cytosine into 5hmC, which activates MMP-9 transcription. Lastly, elevated MMP-9 gene transcription and activity damages retinal mitochondria and encourage oxidative stress, which may result in the onset of DR [17].

mtDNA-related methylation: POLG1, D-loop, and MLH1: Diabetes causes malfunction in the retina's mitochondria, which damages the DNA (mtDNA) and increases the rate at which capillary cells die. These problems appear prior to any discernible histological alterations, suggesting a major role for mitochondria in the development of diabetic retinopathy [18]. Furthermore, a dysfunctional Electron Transport Chain (ETC) system affects the effective electron transfer that occurs during oxidative phosphorylation and produces Reactive Oxygen Species (ROS). This creates a positive feedback loop wherein ROS stimulate oxidative stress and subsequently produce additional ROS. Ultimately, the process of apoptosis is started when too much cytochrome c from damaged mitochondria seeps into the cytoplasm [19].

DNA methylation, which is frequently seen in Nuclear DNA (nDNA), is also seen in mitochondrial DNA (mtDNA), which has Tet and Dnmt sequences. Diabetes results in the activation of Dnmts expression, hypermethylation of mtDNA in retinal mitochondria, impaired transcription of mtDNA-encoded genes, defective mitochondria, and an acceleration of capillary cell apoptosis. The link between nDNA and mtDNA maintains mitochondrial homeostasis, and mutations in nuclear gene expression brought on by abnormal DNA methylation can jeopardise mitochondrial function. The catalytic subunit of the nuclear-encoded enzyme responsible for mtDNA replication is called Polymerase Gamma 1 (POLG1) [20].

“In addition to binding POLG1 with the Displacement loop (D-loop) to regulate its replication, POLG is involved in repairing mtDNA damage. Due to retinal Dnmts, which are activated in DM, the regulatory site of POLG1 is hypermethylated. This hypermethylation impairs mtDNA biogenesis and its transcription and damages the ETC system, speeding up the deleterious loop of ROS. The D-loop region is a critical control point for mtDNA transcription and replication. Compared with other regions of mtDNA in DM, the D-loop is more highly methylated and disrupts the transcription of mtDNAencoded genes that are indispensable for maintaining the ETC system, which increases the electron leakage of the ETC complexes. These harmful results directly produce excessive superoxide radicals, aggravating the development of DR. In addition, the D-loop is the site where mtDNA binds to the inner mitochondrial membrane and is more vulnerable to damage than other regions of mtDNA.

In addition, the status of mtDNA methylation is positively correlated with increased base mismatches. MutL Homolog 1 (MLH1) is an enzyme important in the DNA Mismatch Repair (MMR) pathway” [21]. Hyperglycaemia conditions reduce MLH1 mitochondrial localization and hyper-methylate its promoter with activated Dnmt1. Finally, the repression of gene transcripts leads to mtDNA mismatches and mitochondrial damage. In conclusion, the aforementioned research indicates that maintaining a state of equilibrium in DNA methylation might have an impact on the prevention of mitochondrial DNA damage and the deceleration or obstruction of retinal disease progression.

Ras-related C3 botulinum toxin substrate 1 (RAC1): RAC1 is an essential component of NADPH Oxidases (Nox), which is an early phase in the formation of DR in which the majority of cytosolic ROS are produced by Noxs in retinal cells. Functional and transcriptional activation of RAC1 in the retina of DM is associated with the promoter's DNA methylation conditions. Although Dnmt1 produces 5mC at the RAC1 promoter when Dnmts and Tets are both activated in DM, a simultaneous rise in TET2 hydroxy methylates it to 5hmC, initiating the transcription of RAC1. This finding stimulates capillary cell apoptosis and Nox2-ROS-mitochondrial damage, which ultimately results in DR [22].

Peroxisome Proliferator-Activated Receptor Alpha (PPARα): PPARα functions as a transcription factor and contributes to the control of oxidative stress. Dnmt1 is highly expressed and interacts with the PPARα promoter to enhance its DNA methylation levels and suppress PPARα expression in Human Retinal Capillary Pericytes (HRCPs) treated with high hyperglycemia. As a result, ROS levels and the quantity of apoptotic cells are sharply increased. Furthermore, PPARα methylation can be inhibited and retinal cell death can be decreased by Dnmts suppression, according to a recent study. These provide compelling proof that Dnmt1-mediated PPARα DNA methylation speeds up ROS production and death, leading to a rise in defective retinal cells [23].

Mitochondrial Superoxide Dismutase (MnSOD): MnSOD is in charge of eliminating mitochondrial malfunction and scavenging mitochondrial superoxide [24]. Reduced MnSOD levels in DM may raise ROS levels and be a major factor in the pericyte loss of DR. Research has indicated that there is a substantial difference in MnSOD transcription in the DR group compared to the NO-DR group, and that the DR group has increased DNA methylation of the MnSOD promoter [25]. This suggests that in the retina of diabetics, hypermethylation of a specific promoter and decreased MnSOD activity occur, which ultimately lead to the development of DR.

Inflammation

Inflammation is a chronic manifestation of raised blood sugar which activates inflammatory response, increased pro-inflammatory cytokines, acute phase proteins, chemokines causing retinal angiogenesis and neurodegeneration [26]. Early DR alterations in the retina include low level chronic leukocyte activation, recurrent capillary blockage and progression, and retinal ischemia. Activated microglia found in the retinal plexiform layer control retinal inflammation [27]. In rats with DR, it has been demonstrated that suppression of proinflammatory chemicals prevents degenerative processes [28].

In DR, DNA methylation of promoter of TNF gene decreases causing increased expression of TNF which is a proinflammatory cytokine resulting in inflammation [29].

Transforming Growth Factor Beta-1 (TGFβ1), Monocyte Chemoattractant Protein-1 (MCP-1), and Tumour Necrosis Factor Superfamily Member 2 (TNFSF2), which together play a role in the development of vascular complications in DM [30-32]. DNA methylation in the TGFβ1, MCP-1 and TNFSF2 genes are also significantly reduced in DR patients. These findings prove that hypomethylation in the promoter of these inflammatory responserelated genes participates in DR development.

An important intergenic Long Non-Coding RNA (lncRNA) known for its influence on cancer's epigenetic mechanisms is Metastatic- Associated Lung Adenocarcinoma Transcript 1 (MALAT1) [33]. MALAT1 may control the inflammatory response via independent and dependent mechanisms, which collectively help to create a complicated inflammatory response that leads to the development of DR [34].

Neovascularization

Capillary non-perfusion, retinal ischemia stimulates pro-angiogenic factors in the PDR stage of DR [35]. The stimulation of intricate cascade signals in DM patients promotes aberrant neovascularization by elevating angiogenic factors and mediating retinal endothelial cell proliferation [36]. A crucial compensatory mechanism for retinal ischemia is angiogenesis induced by VEGF, which stimulates the proliferation of retinal endothelial cells, increases the amount of VEGF released by both endothelium and non-endothelial cells, and promotes abnormal neovascularization [37].

Increased production of VEGF due to hypomethylation of the VEGF promoter suggests that DNA methylation has a role in the neovascularization and development of PDR processes [38]. Reduced transcription of maternally expressed gene 3 (9MEG-30) in DR inhibits the endothelial-mesenchymal transition and VEGF production, hence impeding the development of DR [39-41]. In human retinal microvascular endothelial cells, DNMT promotes DNA methylation of the MEG3 promoter to inhibit MEG3 expression, speeding up proliferation, migration, and neovascularization (Figure 3) [42].

Figure 3: Different genes and process resulting in diabetic retinopathy.

Therapeutic potential of DNA methylation

Diagnosis of DR at a relatively early stage: The clinical signs of diabetic retinopathy occurs long after diagnosis of diabetes mellitus. level of DNA methylation in early stage of the disease can act as a biomarker for early diagnosis of the DR. diabetic patients with retinopathy and those without it have different levels of mtDNA methylation in their peripheral blood [43].

Methylation-specific PCR is a simple and rapid method for quantifying DNA methylation. DNA methylation changes can be noninvasively detected in peripheral blood and can be easily quantifiable using PCR, ELISA, HPLC etc. (Refer Table 1 for different molecular markers in diagnosis of DR).

Table 1. Novel molecular biomarkers related to diabetic retinopathy (SNP: Single Nucleotide Polymorphism).

Predicting progressive stage of DR (PDR): DNA methylation status may possibly be used as a non-invasive biomarker for predicting the progressive stage of DR, known as PDR. It has been demonstrated that the DNA methylation of some genes in peripheral blood can be used as a prospective biomarker of PDR in patients with type 1 diabetes. These genes include the following: (1) TNF; (2) Chitinase 3-Like Protein 1 (CHI3L1), which participates in tissue injury, inflammation, tissue repair, and remodelling responses; (3) Chimerin 2 (CHN2), which may be a key element of proximal insulin signalling, playing a role in insulin resistance and growth deficiency; and (4) Gastric Inhibitory Polypeptide Receptor (GIPR), which encodes glucose-dependent insulinotropic polypeptide receptor [44-46]. Additionally, PDR patients had lower levels of DNA methylation in these genes [47]. Furthermore, it has been noted that PDR patients have hypomethylated versions of a few particular genes that are implicated in the cytotoxicity pathway mediated by natural killer cells [48]. indicating even further that various methylation patterns may function as encouraging indicators of PDR [49-54].

Predicting effectiveness of drug therapy: The potential utility of methylation status as a predictor of VEGF receptor medication efficacy in cancer cells has been established by the analysis of promoter methylation [55]. At present, however, this type of study in DR is insufficient; nonetheless, additional research on the key genes is anticipated to enable the use of DNAme-related markers to predict the efficacy of DR therapy.

Treatment: Targeting DNA methylation: Risk factors DR include lifestyle choices, environmental variables, dyslipidemia, hyperglycemia, obesity, and Hyperhomocysteinemia (HHcy). These factors might change the epigenetic status of many target organs either separately or in combination [56].

Hyperglycemia: Due to the fact that hyperglycemia is the precursor of aberrant DNA methylation in diabetes, glycaemic management is a crucial preventive intervention in diminishing the likelihood of diabetic retinitis and vision impairment [57-59]. First, when mtDNA and nDNA methylation and Dnmts and Tets activation are unaltered throughout the early stages of diabetes mellitus, the preservation of retinal mitochondria is facilitated by the restoration of appropriate glycaemic management. This demonstrates that the DNA methylation mechanism does not harm the retina of diabetes individuals when blood glucose is controlled early and kept at healthy levels. Furthermore, long-term tight glycaemic management can improve aberrant methylation status and ultimately postpone or stop the development of DR, even in patients who are unable to achieve strict glycaemic control during the first phase of diabetes mellitus [60].

Hyperhomocysteinemia (HHcy): HHcy is a significant contributor to retinal microvascular damage and an independent risk factor for the onset of retinopathy [61]. Furthermore, it might have negative synergistic effects with hyperglycemia [62]. In order to preserve normal DNA methylation status under physiological settings, Hcy can generate SAM and boost Dnmts activity. But in DR, elevated levels of circulating Hcy cause a worldwide rise in retinal DNA methylation [63]. Additional impacts on the methylation of genes essential for mtDNA synthesis result in a harmful cycle of reactive oxygen species and mitochondrial malfunction, which exacerbates the development of DR [64]. Thus, in order to prevent DR, we can implement a variety of strategies that regulate the amount of Hcy in the air. For instance, deficiencies in folic acid and vitamin B₁₂ may contribute to Hypochloremia (HCy). These nutrients are necessary for the correct metabolism of Hcy [65]. Thus, using folic acid and vitamin B₁₂ to treat pre-existing HHcy may help lower the likelihood of developing DR [66].

Hyperlipidaemia/obesity: Some cross-sectional and longitudinal studies indicate an association between an increased risk of DR and hyperlipidemia/obesity [67]. In DM rats, hyperlipidemia exaggerates hyperglycemia-induced DNA methylation, increases Dnmts and Tet2, and alters methylation of mtDNA and RAC1. Consequently, mitochondrial damage and retinopathy are accelerated [68].

Dnmt inhibitors: The Dnmt inhibitors 5-azacytidine and 5-aza-20-deoxycytidine have been granted approval by the US Food and Drug Administration for the treatment of myeloid malignancies and cutaneous T-cell lymphoma [69]. Research on Dnmt inhibitors has evolved from non-selective to selective. first-generation Dnmt inhibitors including 5-azacitidine and 5-aza-2′-deoxycytidine inhibit all three DNMTS (1, 3a, and 3b); however, subsequent research revealed that nanaomycin A and GSK3685032 specifically inhibited Dnmt3b and Dnmt1, respectively [70].

Glycaemic management alone cannot instantly improve the DNA methylation-hydroxy methylation pathway in the retina. Direct Dnmt inhibition, on the other hand, may enhance aberrant DNA methylation during this reversal phase, so mitigating persistent mitochondrial dysfunction and delaying or preventing the onset of DR. For example, during times of good glycaemic control after recovery of hyperglycemia, direct suppression of Dnmts can restore DNA methylation and gene transcription of the MLH1 gene. Furthermore, throughout the development of DR, one mechanism that may compromise antioxidant defence systems is DNA methylation. If Dnmt inhibitors are administered prior to the acceleration of apoptosis in retinal capillary cells, they can prevent mtDNA damage by inhibiting further DNA methylation of genes crucial for preserving mitochondrial homeostasis. 5-aza-2′-deoxycytidine, a DNAdemethylating medication, has the potential to treat DR since it can reverse elevated Dnmts and control the expression of antioxidant enzymes to increase antioxidative capability [71]. Despite its nonselective nature and its ability to block three different Dnmts, 5-aza-2′-deoxycytidine has not been observed to cause considerable damage to retinal structure [72]. This finding may be connected to the fact that all Dnmts are more active in a diabetic environment. To increase efficacy and lessen negative effects, selective inhibitors can be studied more as there hasn't been much study done in this field. Dnmt1, for instance, exhibits unique and tissue-specific features [73]. It will be possible to investigate the viability of using a Dnmt1 selective inhibitor to treat DR by learning more about the distribution properties of Dnmt1 in retinal tissue.

Folic acid and vitamin B₁₂: For DNA methylation, Folic Acid (FA) is a dietary methyl donor, and vitamin B₁₂ is a cofactor in the process of moving the methyl group from FA to DNA [74,75]. Although studies indicate that low FA levels can cause both hypomethylation and hypermethylation, misregulating DNA methylation, FA has been utilised extensively in the clinic to treat DR [76]. A blood folic acid/ vitamin B₁₂ deficit is linked to a higher risk of developing diabetes, and taking these nutrients supplements lowers the risk of developing diabetes. Further supporting the importance of FA and vitamin B₁₂ in DR treatment, implies that deficits in these nutrients may influence the equilibrium of DNA methylation and so facilitate the development of DR. Nevertheless, more research is required to fully understand the DNA methylation-related mechanism behind its therapeutic action.

Synergistic therapy with current therapies: We think that Dnmts inhibitors can be used in addition to existing medicines, based on the results of the present research. The restrictions and side effects of traditional therapy can be reduced with this synergistic therapy, and the therapeutic outcome for DR can be enhanced.

Anti-VEGF therapy is mainly used in PDR patients, and has a significant effect on neovascularization and diabetic macular edema, which can improve visual acuity [77]. The main problems are as follows: A large part of the population is not sensitive to anti-VEGF therapy [78]. Studies have demonstrated that the therapeutic efficacy of the

• A large part of the population is not sensitive to anti-VEGF therapy [78]. Studies have demonstrated that the therapeutic efficacy of the VEGF-targeted medicines can be influenced by the DNA methylation status of the VEGF receptor promoter. Therefore, the DR patients who are not sensitive to anti-VEGF therapy may be associated with the abnormal DNA methylation of VEGF receptor promoter caused by diabetes. Anti-VEGF therapy in combination with Dnmt inhibitors may be a potential approach to improve the therapeutic effects of those special DR patients.
• Anti-VEGF therapy is mainly used in the PDR stage with obvious ocular symptoms, and Dnmt inhibitors are expected to prevent entering the PDR stage. As mentioned previously, DNA methylation in peripheral blood is expected as a diagnostic biomarkers of early DR. With close follow-up before the diagnosis of PDR, Dnmt inhibitors can be used to reverse epigenetic abnormalities caused by hyperglycemia in a short period of time. In conjunction with effective blood glucose management in the later stages, it is extremely encouraging to postpone or even avert the progression of DR to PDR.
• Considering the chronic course of DR and the short-term efficacy of anti-VEGF therapy, PDR patients should be regularly and frequently injected with anti-VEGF for a period of time. If this treatment is interrupted, it could be disastrous and result in irreparable eyesight loss [79]. However, many patients find it challenging to attain adequate compliance due to the inconvenience of follow-up and the significant financial load. Dnmts inhibitors can be utilised as adjunctive therapy to increase patient compliance by extending the duration of effect and lowering associated costs.

Discussion

In a nutshell, methylation studies on whole genome and specific genes are very limited on diabetic retinopathy. Maghbooli Zhila, et al. [80] in a case control study done on 168 patients using high pressure liquid chromatography concluded that whole DNA methylation levels are significantly higher in DR patients. Zhuo Chen, et al. compared DNA methylation profile in genomic DNA of whole blood from 32 cases with 31 controls without retinopathy [81]. 153 loci depicted hypomethylation and 225 depicted hypermethylation and also concluded DNA methylation persist at certain loci associated with glycemia for several years supporting and epigenetic basis for metabolic memory.

Shikha Tewari, et al. conducted an animal study to understand the mechanism of DNA methylation status of POLG1 promoter in streptozocin induced diabetic retinopathy rat models [82]. Isolated retinal endothelial cells exposed to high glucose compared to those exposed to normal glucose showed continued POLG1 downregulation and CpG island hypermethylation, which continued to remain same even after three months of glycemia control following poor glycemic control supporting metabolic memory. Manisha Mishra and Renu Kowluru conducted yet another animal study to investigate possible crosstalk between mtDNA methylation and base mismatch in the development of diabetic retinopathy [83]. The effect of inhibition of DNMT by 5 aza-2’-deoxycytidine on glucose induced mt-DNA base mismatch was investigated in human endothelial cell. An in vivo model of retinal microvasculature from diabetic mice overexpressing sod2 was used to study effect of deamination on increased base mismatch.

Inhibition of DNA methylation, regulation of cytosine deamination, significantly inhibited an increase in base mismatch and prevented mitochondrial dysfunction. Overexpression of sod2 in mice also prevented diabetes induced D-loop hypermethylation and increase in base mismatch. Inhibition of DNA methylation limits the availability of methylated cytosine for deamination suggesting cross talk between DNA methylation and base mismatch. The crosstalk continued even after termination of hyperglycemia, suggesting its role in metabolic memory. Ying Zhu, et al. concluded DNMT 1 mediated PPAR-alpha methylation promotes apoptosis and increase ROS level of human retinal cell pericytes obtained from experimental animal model and causes damage of retinal tissue in DR mice [84].

Similar studies done by Yaki Li, et al. for THBS1, He Yui, et al. for MEG3, etc. suggested a close relation of DNA methylation and Diabetic retinopathy [85].

Conclusion

In summary, oxidative stress, inflammation, neovascularization, and metabolic memory are only a few of the intricate pathophysiological processes whereby DNA methylation plays a crucial role in the onset and progression of DR. It may function as a biomarker for DR early detection as well as a possible target for treatment and prevention. However, treatments for DR are still somewhat new and mostly target the neovascularization of PDR patients, while research on DNA methylation is still in its early stages. In the future, DNMTT inhibitors may be used as therapeutics to treat DR. It will be crucial to determine which kind of Dnmt inhibitor controls the antioxidant genes and the precise hypermethylation CpG site of the genes, though, as the majority of these inhibitors that are presently being researched are nonselective. Are there any negative effects, such as myelosuppression and neurological damage, that are linked to cancer treatment when these inhibitors are used to treat DR? Do these inhibitors profoundly alter the retina's morphology and functionality? Do people with diabetes grow tolerant to sudden suppression of retinal Dnmts? Is it possible for DNMTS inhibitors to compensate for the lack of anti-VEGF medications and cortisol implants?Future investigations must take into account each of these queries. Therefore, it is anticipated that additional study on DNA methylation will lead to new discoveries in the therapy of DR and provide people who are blind from DR with hope again.

Authors Contributions and Authors Information

All authors searched for the literature and wrote and edited the manuscript. All authors have read and agreed to the published version of the manuscript..

Conflict of Interest Funding

None.

Funding

Not applicable.

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