Current Approaches in Treatment of Diabetic Retinopathy and Future Perspectives

DR physiopathology

DR is the most frequent vascular complication of long-term uncontrolled diabetes. High blood glucose level, hypertension, dyslipidemia, duration of diabetic status, ethnic origin, pregnancy, and puberty are among the risk factors. DR is a disease that concerns the vascular level. Since pericyte and endothelial cell loss occur at the center of the physiopathologic process, it can also be named as endotheliopathy of internal retinal microvascular bed. ⁸ At the same time, there is also a dysfunction of vascular smooth muscle. ⁹ The primary reason for these changes is oxidative stress caused by hyperglycemia, inflammation, protein kinase C (PKC), and activation of the renin-angiotensin system. As a result of this process, the VEGF level increases in the retinal environment. ¹⁰ Deterioration of the vascular structure causes leakage of exudative material out of the retinal vein. When this exudative material accumulates on the macula region of the retina, visual acuity decreases.

Retinal neural cell degeneration and dysfunction also contribute to this visual acuity decrease.^4,5 This disruption in the microvascular structure arising from metabolic dysregulation results in aberrant proliferation in vascular endothelial cells in later stages. In this process, the theory that is most widely accepted is the loss of pericytes. Pericytes are placed on the same basal membrane with the vascular endothelial cells, and they are necessary for the endothelial cell vitality. They contribute to the nutrition of endothelial cells and have anti-inflammatory and antiangiogenic activity. Besides, their connection with the endothelial cells contributes to the blood-retina barrier.^9,11 Under diabetic conditions, retinal capillary cells are constantly damaged and in response to this exposure, they are renewed in a continuous turnover status. However, this replicative capacity has a limit called Hayflick limit and compared to normal individuals, this capacity is more limited in diabetic population. ¹²

At the earliest stage, change in the vascular structure manifests itself as a microaneurysm on clinical examination. ¹³ If diabetic control is not achieved, the vascular defect progresses and the existing microaneurysms are accompanied by bleeding, hard exudate (lipid deposits), cottonwool spots (axoplasmic debris accumulation of ganglion cells), venous dilatation, pilling, and intraretinal microvascular abnormalities (dilated capillaries).^13–15 This stage is called nonproliferative DR stage. When the retinal feeding cannot be established at this stage, retinal ischemia occurs and retinal neovascularization will take place. As a result, nonproliferative diabetic retinopathies change to proliferative retinopathy stage. On the other hand, widespread neovascularization foci, which can easily bleed in the retina and at the head of the optic disc, are seen in this phase. These centers may bleed inside the retina and vitreous. In the following stages, fibrovascular proliferation and tractional retinal detachments may develop from these neovascular centers. ¹⁶ While this progression occurs in the stages of DR, edema may occur in the macula due to changes in the vascular structure regardless of the stage of retinopathy.^16,17 And this clinic situation is the most frequently observed source of sight loss in DR.

Treatment of DR

We may classify the treatment of DR under 2 main headings as systemic treatments and local ocular treatments.

As the systemic treatment, tight blood sugar and blood pressure control constitute the 2 main methods of treatment. Two important studies named Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) have shown that tight blood sugar control (Hemoglobin A1c, HbA1c, 7%) has reduced the risk. It is emphasized that, for every percentage decrease in the HbA1c level, the risk of development of retinopathies is reduced by 30%–40%. In another study named Action in Diabetes and Vascular Disease (ADVANCE) Collaborative Group, it is shown that, aggressive glycemic control (HbA1c <6.5%) does not have linear effects on the progress of DR. Similarly, in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study, it is shown that aggressive glycemic control might be related to the mortality.^7,18,19

Epidemiological and clinical studies show the fact that hypertension, which is another systemic risk factor, is a modifiable risk factor for DR development. It is stated that, for every 10 mmHg increase in the systemic systolic blood pressure, the development of DR is increased by 10% and extensively proliferative DR development is increased by 15% at early stage.^7,20,21 On the other hand, in UKPDS, it is seen that tight blood pressure control resulted in 3 times reduction in retinopathy progression, 2 times reduction in loss of sight, and 3 times reduction in the need for laser treatment in type 2 DR. In DCCT study, it was shown that DR progression is directly proportional to triglyceride level and inversely proportional to high-density lipoprotein level. ²²

Among current local treatment of DR, there are laser photocoagulation, IVTA injection, intravitreal anti-anti-VEGF agents, and intravitreal steroid implants. Besides this, new treatment methods, nanomedicine, stem cell, and other systematic treatment methods have been taking part.

Laser photocoagulation

In the treatment of DR, laser treatment is still the mainstay method in preserving the existing level of sight. However, it is accepted that the most important side effect of this treatment is irreversible tissue damage. In DR treatment, 2 types of laser treatment methods are used. While pan-retinal laser photocoagulation is preferred for proliferative type DR treatment, focal laser photocoagulation is preferred for the treatment of macular edema and focal ischemic retina centers. The main purpose of the laser treatment is blocking the release of mediators like VEGF into the retina and vitreous by fully burning the retinal area, which has feeding disorder. Thus, neovascularization formation is prevented. ²³

In the Diabetic Retinopathy Study (DRS) 92 and the Early Treatment Diabetic Retinopathy Study (ETDRS) 91 studies, laser photocoagulation is identified as the main method of treatment for DR.^24,25 As a result of the DRS, it was concluded that pan-retinal photocoagulation reduced the risk of vision loss in proliferative DR by 50% within 5 years and reduced progression risk of less severe DR to severe form compared to the ETDRS. Meanwhile, according to the results of the ETDRS, reasonable focal laser photocoagulation has been shown to halve the risk of moderate vision loss due to clinically significant macular edema. Besides, the Diabetic Retinopathy Clinical Research Network (DRCR.net) reached increased vision levels of patients undergoing macular laser by ∼30% after 2 years (≥10 letters).^7,8,26

Apart from its effect on the level of sight, laser photocoagulation has some ocular side effects and these are as follows: deterioration in dark adaptation (25%), minimal decrease in visual acuity (10%), loss of peripheral visual field (5%), decreased vision at night, and increased visual acuity with impaired color vision, increase in macular edema.^7,8 Due to side effects like these, search for alternative treatment methods to laser photocoagulation continues, while it is effectively used.

Anti-VEGF agents

Vascular changes due to hypoxia during DR cause VEGF release by retinal endothelial cells, pericytes, and retinal pigment epithelium. Increased level of VEGF stimulates angiogenesis and this results in structurally weak new vessel formation (neovascularization). This new capillary web, which is in weak character, is highly permeable and results in leakage of liquid (exudate) into the retina tissue. ²⁷ To block this mechanism, anti-VEGF agents are developed, and in this study, the target is to eliminate both retinal ischemia and retinal edema. With this purpose, 3 agents are frequently being used. These agents are aflibercept (Tarrytown, NY), bevacizumab (Avastin; Genentech, South San Francisco, CA), and ranibizumab (Lucentis; Genentech). ²⁸ They provide their effectiveness by blocking the VEGF-A subtype of VEGF.

Bevacizumab molecule is a recombinant, humanized monoclonal antibody (mAb). It has been originally produced for the treatment of metastatic colorectal cancer by humanization technique. With this technique, the complementary determining regions of a mouse monoclonal antibody (mAb A.4.6.1) transferred to a human antibody and the amino acid sequence of the humanized antibody is 93%–95% human. This new antibody produced from mAb A.4.6.1 monoclonal antibody is Fab 12.^29,30 It blocks all VEGF-A isoforms and has been approved not only for the treatment of metastatic colorectal cancer but also for ovarian and brain cancers. However, it is still used as off-label in retinal disease treatment. ³¹ It has also been shown that bevacizumab is effective in DR and diabetic macular edema (DME).^32–34 Also, many investigations have shown that the injection of intravitreal 1.25 mg bevacizumab is effective in DME treatment.^35–37

Ranibizumab molecule is a recombinant Fab fragment with a weight of 48 kDa and it is obtained from monoclonal bevacizumab antibody (Fab12). It is also originally derived from the murine monoclonal antibody (mAb A4.6.1), but it is not produced in vivo. It necessitates some in vitro recombinant process. After complementarity determining region mutation and affinity selection, the new Fab molecule is Fab-Y0317, and this molecule is ranibizumab. ³⁰ Thanks to its small structure, it has a higher ability to penetrate the tissues. It has been shown that 0.5 or 0.3 mg intravitreal injection is effective in the treatment of DME. ³⁸

On the other hand, aflibercept molecule is a molecule of size 115 kDa and it is a recombinant protein that contains a domain in its structure, which will connect VEGF receptor 1 and 2 linked to IgG Fc fragment. Apart from the other ant-VEGF molecules, it cleans VEGF-A isoforms from the environment before it attaches to their receptors. At the same time, it blocks VEGF-B and placental growth factor-1 (PGF-1) and 2 (PGF-2). Although PGF structurally resembles VEGF-A, it has 4 isoforms. The 2 most important isoforms are PGF-1 and PGF-2. PGF stimulates the migration of endothelial cells by showing their effects on VEGFR-1 and plays an active role in angiogenesis. Blocking this molecule is important for preventing its contribution to the process of neovascularization.^39,40 In many investigations, it has been concluded that it has achieved an increase in the vision level of patients having DME.^41,42 However, the common disadvantage of all 3 agents is that they require repeated intravitreal administration and their effects are transient. Therefore, the necessity of new treatment methods still exists.

Corticosteroid agents

Inflammation, which is the most frequent reason for sight loss of DR, plays an important role as the main mechanism. Although intravitreal corticosteroid injection is used to block this mechanism, the target is to block prostaglandin, proinflammatory cytokines, and VEGF release by blocking proinflammatory cells during inflammation.^43–45 At the same time, another important goal is to increase the vascular barrier function by strengthening the tight junctions between the endothelial cells in the vascular wall.^46,47 Although triamcinolone acetonide is frequently preferred, slow-release dexamethasone and fluocinolone acetonide implants are also used in a current manner.^48–51

Systemic treatment methods

There are some systemic medicines for DR treatment, in addition to systemic glycemic control, and serum lipid and blood pressure regulation. Even though most of them are not in clinical use, their effects have been shown in literature. First of them is PKC inhibitors and it shows an important modulator activity in the expression of VEGF. They act on retinal vascular endothelial cells and show effects by blocking PKC-β (β isoform of PKC). ⁵² Studies have been conducted on the systemic use of Ruboxistaurin. In some clinical studies, it has been showed that Ruboxistaurin has reduced the progression of DME and the need of other treatment method usage.^53–57 However, it is still not used for clinical use and further studies are needed. Another treatment method is growth hormone inhibitors. Growth hormone directly stimulates angiogenesis through endothelial cells. Somatostatin, a growth hormone-inhibiting hormone, binds to its receptors on endothelial cells and blocks these effects at both the receptorial and postreceptorial levels. ⁵⁸ Octreotide, which is a synthetic somatostatin analogue, has been thought for this purpose in the treatment of DR.^59–62

Aldose reductase enzyme is an enzyme in the retina and it has concluded in some animal studies that 2 aldose reductase inhibitors in DR treatment named fiderestat and ranirestat have been reducing the retinal thickness.^63,64 Besides, it has been put forward that anti-inflammatory agents can show effect by decreasing inflammatory mediators and preserving retinal vascular stability. With this aim, it has been studied on intravenous infliximab application. As a result of a randomized study, it was seen that blood-retinal barrier stabilization was provided, but further studies are needed. ⁶⁵

DR is also a disease that progresses with vascular obliteration and retinal hypoxia as a result of a defect in the microvascular structure. Existing hypoxia causes oxidative stress in the cellular level and causes to appear of several oxidant molecules.^66,67 The main ones of them are superoxide (^•O₂⁻), hydroxyl (^•OH), peroxyl (^•RO₂), hydroxiperoxyl (^•HR O₂⁻), hydrogen peroxide (H₂O₂), and peroxynitrite (ONOO⁻). ⁶⁸ In the studies performed, it has been shown that DR can be taken under control by blocking these oxidant molecules with enzymatic and some antioxidant molecules. The studied antioxidant enzymes and molecules are MnSOD (superoxide dismutase), catalase (CAT), glutathione peroxidase (GPx), vitamin E, vitamin C, resveratrol, α-lipoic acid, curcumin, and epigallocatechin gallate (EGCG). They function by decreasing the oxidative stress in retina and retinal pigment epithelium. ⁶⁹ Other treatment modalities include carbonic anhydrase enzyme inhibitors, antiplatelet agents, adenosine receptor agonists, or adenosine reuptake inhibitors. ⁷⁰ The effectiveness of these treatment options and results for their clinical usage are needed to be supported by new studies in the next years.

DR and stem cell treatment

Today, DR is defined as microangiopathy with endothelial damage in its center. The metabolic and physiological changes induce the production of proinflammatory cytokines. The resulting cascade also causes neural cell damage through endothelial cell dysfunction. Current studies suggest that this endothelial damage arises from the loss of pericytes located in the perivascular area. Although pericytes are mainly known to be involved in the formation of the blood-retina barrier, it is also thought to have anti-inflammatory effects on endothelial cell metabolism and perivascular area. ¹³

The presence of endothelial and pericyte cell damage has required stem cell-based researches, which may induce endothelial cell formation. In an animal model study conducted by Asahara et al., it has been shown that bone marrow-sourced CD34⁺ progenitor cells [endothelial progenitor cell (EPC)] have moved toward the ischemic field and contributed to the active angiogenesis process. ⁷¹ In another study, it is suggested that these leading cells, which participate in the active repair process under physiological conditions, both functionally and in the quantity, decrease under diabetic conditions. ⁷² Also, in a study conducted on diabetic mice, it has been shown that mobilization of EPC from the bone marrow and migration toward the ischemic area have reduced. ⁷³ Furthermore, Balaiya et al. showed in their in vitro study that diabetic vitreous and aqueous fluid inhibit migration of CD34⁺ cells, which are derived from healthy humans. This indicates that a number of other growth factors may play a role in diabetic neovascularization. ⁷⁴

There are different EPC types like “circulating angiogenic cells—CAC,” “early endothelial progenitor cells—eEPCs,” and “outgrowth endothelial cells—OECs.” OEC is separated from the other 2 cell types by its more specific maturation to endothelial series and by its CD146 (%99) and Tie2 (%89) surface markers. Medina et al. have shown that OECs can enter into strong intercell connections with endothelial cells by zonula adherence, and become integrated into the retinal vascular web. In this mouse model experimental study, they have identified that OECs play an active role in re-establishment of the circulation and reduction in the number of avascular fields. ⁷⁵ In another in vitro study, it was suggested that common regeneration of endothelium—pericyte complex—could be established by using a mixture of different EPC types (eEPC-OEC), and it could be possible to establish a stronger vascular bed. ⁷⁶

Ritter et al. have examined the behavior of the stem cell in ischemia-based pathologies like DR and premature retinopathies. In their rat model retinopathy studies, they have shown that, under proper conditions (in existence of myeloid-specific hypoxia-inducible factor 1), myeloid progenitor cells showed migration toward avascular retina fields, differentiated to microglial cells, and speeded up the vascularization process. ⁷⁷ Kong et al. have used human umbilical cord mesenchymal stem cells as the source of stem cells and examined the effects of these cells by applying in different doses on the diabetic rat retina at 2nd, 4th, 6th, and 8th weeks. In their studies, after intravitreal application of mesenchymal stem cells, nerve growth factor (NGF) levels were observed. In the diabetic control group, NGF is identified; however, it has reduced within 8 weeks. In stem cell-injected rats, even in the 8th week, NGF continued to rise, and this effect was stronger in the group in which higher concentration of stem cells was applied. ⁷⁸

Another mesenchymal stem cell source is the adipose stromal cell (ASC). It has begun to become a new treatment option and takes part in clinical studies, thanks to recent results. ASCs are progenitor cells originating from multipotent mesenchymal cells, and it has been demonstrated that they are structurally and functionally similar to pericyte found in adipose tissue microvascular beds. In the experiments on rats that are made diabetic by streptozotocin, it is identified that intravitreal applied ASCs showed activity in the perivascular area within 3–6 weeks. They enabled vascular stabilization and showed pericyte activity in vivo.^10,79,80 Besides, it was shown in nude mice that, ASCs play an active role in prevention of the neurodegeneration by excreting different angiogenic and antiapoptotic factors. ⁸¹

In another study, Yang et al. applied ASC intravenously to diabetic rats and examined its impact. In the 1st week after the injection, improvement in blood glucose levels, blood-retina barrier functions, differentiation of photoreceptor (rhodopsin expression), and astrocyte (glial fibrillary acidic protein—GFAP expression) are identified. Systematic application of ASC increases the number and functions of pancreas beta cells and reduces the blood glucose levels. ⁸² For this reason, it is not clear whether the functional healing in the blood-retina barrier can be explained by the differentiation of ASCs or by the reduction in blood glucose levels. However, microvascular changes and increase in the neurotrophic factors after intravitreal application point out the possibility that ASCs have a significant impact on the vascular structure stabilization and protection of retina cells from diabetic damage.

Conventional treatment like laser, intravitreal anti-VEGF, or steroid application targets the VEGF activity. For this purpose, it is aimed to overcome the vascular damage and leakages. It seems like, stem cell applications may become an important alternative in the treatment and prevention of DR complications. In this way, the microvascular structure that lies at the basis of pathology remains functional, especially when it is applied at early stages.

DR treatment through nanomedicine

Nanomedicine is the most popular treatment method in recent years. Its main purpose is to make currently used drugs more effective and to have controllable influence in the target tissue. Nanoparticles that are used for this purpose are in the dimension of 1–1,000 nm. They prevent the inactivation of peptide and protein-based drugs in the biological environment. It provides sustained and controlled drug release, tissue targeting, and increased biousability, and decreases side effect of the drugs.^83,84 Drugs that will be used for treatment purpose are locked into the nanoparticle or attached to its surface. Their releasing features are determined by the biochemical feature of used nanoparticle and their degradation time. Through these features, drug aggregation, and enzymatic and chemical degradation are decreased and drug half-life is increased. At the same time, they increase the access of water-soluble and large biomolecular drugs to the tissues.^83,84

There are mainly 7 types of nanoparticles. These can be ranged as polymeric nanoparticles, liposomes, polyethylene glycol (PEG)-coated liposomes, dendrimers, cationic nanoemulsions, nanostructured lipid carriers (NLC), and solid lipid nanoparticle (SLN). ⁶⁹ Polymers used for polymeric nanoparticle synthesis are divided into 2 groups: synthetic and natural. Among these polymers, chitosan, starch, alginate, and cellulose are in natural structure, and poly β-hydroxybutyrate (PHB), polylactic acid (PLA), polyurethane poly lactic-co-glycolic acid (PLGA), and poly(methyl methacrylate) (PMMA) are synthetic polymers. ⁸⁵ Polymeric nanoparticles that are frequently used for treatment purpose are PLGA, PLA, chitosan, polyvinyl alcohol (PVA), and PMMA. These polymers are certified by Food and Drug Administration (FDA). ⁸⁶ Apart from drug delivery, these polymers are also used as tissue-engineered scaffolds, in gene delivery, vaccine, and cancer diagnosis, and as dental and bone material.^85,87–95

As lipid nanoparticles, SLN, NLC, and liposomes are more preferred and studies on ocular applications are available in the literature.^96–101 Dendrimers frequently consist of polyamidoamine (PAMAM) polymer, whereas cationic nanoemulsions can go into an interaction with human ocular mucosa through electrostatic powers in their structure.^102–104 Besides all these applications, the most important limitations of nanoparticles are that their production requires serious time and cost. Also, they have challenges in industrial applications. At the same time, repetitive applications and slow release may cause pulmonary inflammation, carcinogenicity, and toxicity on the cardiovascular system. The treatment cannot be terminated at any time and tissue targeting may not be fully achieved. ¹⁰⁵

In view of targeting and drug delivery system features of nanoparticles, it came to the fore their usage in ocular neovascularization and DR. Attia et al. designed betamethasone sodium phosphate-loaded nanoparticles for ophthalmic delivery. ¹⁰⁶ Deepa et al. studied plant-based anionic polymers and their nanoparticle system for ocular delivery. ¹⁰⁷ Hirano et al. and Yan et al. worked on betamethasone-loaded chitosan–sodium alginate nanoparticle. They tried to develop nanoparticle-based eye drop and they reached successful results in targeting to the posterior segment of the eye.^108,109

Ohira et al. showed that topical ocular dexamethasone γ-cyclodextrin nanoparticle eye drop treatment is effective in the regression of macular edema in DME. They showed a similar effect to subtenon triamcinolone application. ¹¹⁰ Tanito et al. showed that dexamethasone-cyclodextrin microparticle eye drops have promising effect in DME and result of this study has been supported by the study of Ohira et al. ¹¹¹ Lu et al. showed in diabetic rats that, bevacizumab-chitosan nanoparticles suppress angiogenesis by reducing VEGF expression in DR after intravitreal injection and bevacizumab-chitosan nanoparticles have a longer duration of action. ¹¹² Intravitreal application studies of ranibizumab nanoparticles with chitosan base also have been done. ¹¹³ Deguchi et al. have shown that topical application of nilvadipine nanoparticles in rats could prevent retinal dysfunction. ¹¹⁴

Basic characteristic of SLN is that they do not show biotoxicity. It has been prepared and used by Li et al. as ocular drug delivery of tetrandrine SLN. ¹¹⁵ Lallemand et al. have shown that cationic nanoparticles are extending the drug retention time in ocular mucosa and increasing the tissue targeting the posterior segment of the eye. ¹¹⁶ Araujo et al. have developed lipid carrier-loaded triamcinolone acetonide having the purpose of ocular antiangiogenic activity. ¹⁰¹ Abrishami et al. have developed bevacizumab-covered liposome toward the posterior segment diseases of the eye. At the end of the study, it has been reached to the conclusion that liposomal bevacizumab is reaching a higher concentration compared to non-liposomal form. ¹¹⁷ Kaiser et al. concluded that subconjunctival liposomal minocycline injection in diabetic rats is effective in reaching the posterior segment of the eye. ¹¹⁸ In the light of these information, nanoparticle-related DR and other eye disease treatment studies are promising and new studies are still needed.

DR treatment through extracellular vesicles

In recent years, another title that has a potential role in the treatment of related diseases is extracellular vesicles (EVs) and it is expressed as a biomarker in the diagnosis of diseases. EVs are 40–200 nm size vesicles released from many cells into the extracellular area. They are dispersed from the extracellular area to body fluids such as blood, saliva, urine, seminal fluid, nasal secretion, tear, cerebrospinal fluid, and vitreous.^119–125

This release may be increased in oxidative stress and apoptotic processes as it may occur in all conditions. ¹²⁶ These vesicles dissociate from the cell membrane by exocytosis and contain the antigens of the main cell.^125,127,128 Thanks to these antigens, they can go to target tissues and initiate various interactions. ¹²⁹ In this way, they provide communication between the main cells and target cells.^130,131 Therefore, these vesicles are also called cargos that provide communication between cells.

EVs contain cytokine, growth factors, genetic material such as messenger RNA (mRNA), microRNA (miRNA), proteins, lipids, and immunoglobulins, which vary depending on the cells they are secreted. ¹³² The result of the interaction varies depending on EV content. For example, EVs released from astroglial cells show antiangiogenic properties for ocular tissues, whereas EVs released from retinal pigment cells do not. ¹³³ Therefore, in recent studies, it is emphasized that in many inflammatory and chronic vascular diseases, EVs may have an initiative role. In some studies conducted in the last years, it is also stated that EVs may be shown as an inflammation biomarker for chronic diseases.^134–137 For this reason, EVs constitute a significant potential especially for the diagnosis and treatment of diseases.

DM is another area that EVs are thought to be effective in the diagnosis and treatment of it.^138–141 Previous studies have shown that the EV concentration in plasma of diabetic patients and animals is high and this is related to diabetes-related microvascular complications.^142–144 For the first time, Mazzeo et al. declared that miRNAs carrying EVs are associated with the diagnosis of DR and they are also a biomarker for showing the progression of DR in diabetic patients. ¹⁴³ Huang et al. also concluded that they activate the complement pathway and aggravate the findings of DR. ¹⁴⁴ In another study, it was shown that mesenchymal derived EVs may play a role in the early stages of DR by causing angiogenesis. ¹⁴⁵ In the study of Rondina et al., it was emphasized that EVs originating from different cells in diabetic patients may contain proinflammatory factors that may cause DR in the eye. ¹⁴⁶ In the study of Stepien et al., it was concluded that they may be associated with increased vascular permeability in DR. ¹⁴⁷ It was also stated that EVs could be a biomarker in the diagnosis and treatment of DR. ¹⁴⁸

Considering the researches published in the past, EVs is promising as an important biomarker in the diagnosis of DR. Therefore, it is becoming an important DR treatment target in the future of DM. Further researches are needed for progression monitoring and development of new treatment methods.