1 Background

Cell signaling is being researched as a unique approach for better comprehending molecular processes, establishing intercellular signal network profiles, and finding biomarkers and therapeutic targets associated with cellular functions [1]. It is yet unknown exactly how these transcription factors contribute on a molecular level to the epithelial-mesenchymal transition of the retinal pigment epithelium (EMT of RPE) [2]. Growth factors and other biological agents cause RPE cells to lose their cell polarity and cell-to-cell contact, which triggers EMT through a variety of signaling pathways that encourage cell division, migration, and the synthesis of extracellular matrix (ECM). The ECM is a complex network made up of a number of multidomain macromolecules that are structured in a network and cell/tissue-specific configurations [3]. RPE cells begin to exhibit mesenchymal cell characteristics such as the ability to migrate and proliferate as they become less differentiated. This review focuses on current developments in the field of molecular understanding at the level of cell signaling and presents current thoughts on various approaches by focusing on retinal pigment epithelium-related retinopathies such as proliferative vitreoretinopathy (PVR) and age-related macular degeneration (AMD). The macula, the small region of the central retina that is important for high-acuity vision, breaks down in AMD [4]. One of the problems that poses a major threat to the health of diabetic patients is DR, a microvascular impairment that can result in loss of visual acuity[5, 6]. Proliferative vitreoretinopathy (PVR) is defined by subretinal bands, fibrovascular membranes, and fibrotic alterations in the detached retina [7].

2 Main text

2.1 The role of EMT in retinal pigment epithelium

The Bruch's membrane and photoreceptors are both supported by the retinal pigment epithelium, which plays an essential role in maintaining visual acuity. RPE is terminally differentiated, highly polarised, and located between photoreceptors and choroids [8]. The RPE controls the flow of molecules between the fenestrated choroid capillaries and the photoreceptor layer of the retina to create the outer blood-retinal barrier (BRB). BRB works in many different ways, such as membrane pumps, transporters, channels, passive but selective diffusion, transcytosis, metabolic alteration of solutes in transit, and metabolic modification of solutes after they have left the cell. A monolayered cuboidal epithelium is held together by adherens junctions and tight junctions, which regulate epithelial diffusion via spaces between neighboring cells [9,10,11,12].

EMT is the process through which healthy epithelial cells modify into mesenchymal cells [13]. At four to six weeks of gestation, the human RPE cell undergoes terminal differentiation and thereafter remains mitotically inactive [2]. Additionally, it is a well-studied fact that EMT is highly involved in embryonic organization and re-organization. Furthermore, the EMT can be divided into three types [14] (Fig. 1). There are three basic types of EMT, depending on where the process starts and how it ends. EMTs are divided into three types: type I, which is linked to embryogenesis, type II, which is linked to pathology, and type III, which is linked to carcinogenesis [14, 15]. The study employed many markers to distinguish between epithelial and mesenchymal cells despite the fact that EMT is a dynamic process [16].

Fig. 1
figure 1

Different categories of process of EMT. Based on the place of origin and end of the process, EMT may be classified into three fundamental categories. Type I: EMT (embryogenesis), Type II: pathological EMT, and Type III: cancer-related EMT. Diagrams developed with Microsoft PowerPoint

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The zona-occludens-1 (ZO-1), epithelial cadherin (E-cadherin), and cytokeratin are epithelial markers. N-cadherin, fibronectin, and vimentin are mesenchymal markers [17]. EMT in RPE is considered a fundamental underlying mechanism for severe diseases as it is observed in retinopathies such as AMD and PVR [17, 18]. Additionally, the in vitro, in vivo, and clinical data suggest that RPE cells undergo EMT [19,20,21, 17]. EMT of the RPE is caused by a combination of mechanisms including oxidative stress, pathological inflammation, aging, infections, tight junction loss, ECM breakdown, altered growth factor synthesis, misfolded protein accumulation, and infections [22].

2.2 Cellular signaling of EMT specifically in retinal pigment epithelium

The transformation of epithelial cells into mesenchymal cells, which results in the emergence of new biochemical instructions, requires cellular reprogramming and highly complicated cellular rearrangement [23]. Extracellular signals alter the gene expression of proteins associated with epithelial and mesenchymal tissues during the EMT process in the retinal pigment epithelium. They also regulate a variety of related cellular behaviors, including cell proliferation, migration, and death. This results in the development of PVR and AMD through a network of interconnected signaling pathways [2]. Tight junctions also play a part in the regulation of signaling pathways that control cellular functions including migration, proliferation, and differentiation [2]. Current understanding suggests that the phosphoinositide-3-kinase/Protein kinase B (PI3K/Akt) pathway, TGFβ, Wnt, and Notch are only a few of the regulatory signaling pathways that are associated with the EMT of the RPE, as well as significant interactions between them [24] (Fig. 2).

Fig. 2
figure 2

Signaling cascades involved in EMT process. Many signaling pathways have been implicated in the retinal pigment EMT. either of canonical or non-canonical TGFβ signaling pathways are involved in the EMT of retinal pigment epithelium. Many additional signaling pathways, such as BMP signaling, RTK signaling, Wnt signaling, and jagged-Notch signaling, are implicated in the EMT process in retinal pigment epithelium. Furthermore, the interplay between these pathways is well documented. Diagrams developed with Microsoft PowerPoint

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2.3 Rho signaling pathway

Ras homolog family member A (RhoA/Rho)-kinase is associated with ocular fibrosis. The control of cellular actomyosin cytoskeletal architecture and motility is greatly influenced by two of RhoA's main downstream effectors, the Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rho-associated, coiled-coil-containing kinases, Rho-associated kinase (ROCK) [25]. The Rho pathway has been found to control the assembly and structure of the actin cytoskeleton, as well as associated gene expression. It may be critical for RPE cell fibrotic response [26]. LIM domain kinase (LIMK), an actin-binding protein, phosphorylates cofilin, hence stabilizing actin filaments [3]. Activated RhoA or its downstream effector ROCK increases LIM-kinase activity, which then phosphorylates cofilin in TGFβ1-treated ARPE-19 cells. This phosphorylation reduces cofilin function, which promotes actin polymerization and cytoskeleton remodeling, ultimately leading to fibrosis [26].

TGFβ-induced RhoA activation stimulates cell motility and increases alpha-smooth muscle actin (α-SMA) expression in primary RPE cells [27]. The RhoA/Rho-kinase pathway has been demonstrated to mediate the synthesis of type I collagen by TGFβ2 in human RPE cells [19, 25]. In an in vivo PVR animal model, matrix stiffness enhanced ARPE-19 cell activation via the RhoA/(YAP) pathway, as well as retinal fibrogenesis [28]. Yes-associated protein 1 (YAP1) is a transcription coregulator that increases the expression of genes that regulate cell differentiation and proliferation [3]. Furthermore, thrombin stimulates ROCK and Rho, leading to phosphorylation of the myosin light chain and the production of actin stress fibers in retinal pigment epithelial cells undergoing EMT [29]. Furthermore, recent research suggests that inhibiting RhoA upstream with C3 exoenzyme or inhibiting YAP downstream with verteporfin significantly reduced MMP production and collagen gel contraction in ARPE-19 cells. Blocking RhoA/YAP signaling inhibited the TGFβ/Smad pathway in vivo and reduced PVR-induced retinal fibrogenesis. This paper provides novel PVR treatment approaches that target the RhoA/YAP pathway [28]. Nicotinamide suppresses EMT in the RPE and increases RPE cell differentiation by downregulating ROCK and casein kinase 1 (CK1) [30]. The ROCK inhibitor Y27632 and the RhoA inhibitor simvastatin both decrease TGFβ2-induced type I collagen synthesis in ARPE-19 cells, demonstrating the existence of a connection between the Rho and SMAD pathways [11].

2.4 Signaling cascade Mitogen-activated protein kinase (MAPK)

MAPK is divided into three subfamilies: extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinases (p38s). ERKs are activated by growth factors, whereas JNKs and p38 are activated by cellular stresses or inflammatory cytokines. These signaling pathways regulate several biological processes in the fibrotic process of the eye [30]. The MAPKs cascade, which is regulated by a number of activators, is thought to be involved in the development of the epithelial-to-mesenchymal transition process. The Ras-MAPK pathway activates SNAIL1 and SNAIL2. The early growth response factor-1 (Egr-1) accelerates the transition from epithelial to mesenchymal [31]. TGFβ-induced EMT and fibrosis were mediated by ERK activation in ARPE-19 cells [32]. A recent study of induced pluripotent stem cell (hiPSC)-derived RPE cells found that inhibiting TGFβ and fibroblast growth factor (FGF/MAPK) pathways improved differentiation of RPE[33]. Furthermore, previous in vitro and in vivo studies indicate that the SNAIL is expressed at both the transcription and post-transcription levels in many complex signaling pathways such as integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (P1P3-K), MAPKs, glycogen synthase kinase 3-b (GSK-3b), and nuclear factor kappa B (NF-κB) [34].

Shukal and coworkers demonstrated using an in vitro study that, the anti-epithelial to mesenchymal transition in cells of RPE by a pyruvate analog, the dichloroacetate (DCA) via (MAPK/Erk) and PI3K/Akt pathway [35]. Inhibiting keratin 8 (KRT8) phosphorylation suppresses oxidative stress-mediated epithelial to mesenchymal transition in RPE cells while avoiding potential cell death, indicating that autophagy-mediated KRT8 overexpression combined with MAPK1/3 pathway inhibition could be a potential AMD intervention strategy [18]. Saika and colleagues established the therapeutic efficacy of p38MAPK inhibitor (SB202190) in ARPE-19 cells, with reduced TGFβ2-mediated migration and extracellular matrix production via MAPK signaling [36].

2.5 Notch signaling cascade

It is believed that Notch signaling pathway may be significant in the onset and genesis of many illnesses because the Notch signaling cascade modulates the ratio of cell death to proliferation [37]. The transcriptional regulator retinol-binding proteins (RBP) is necessary for the conventional Notch cascade in the RPE. RBP-J performs a transcriptional factor role in Notch signaling [38, 39]. Throughout the development of the eye, the Notch signaling system plays a role in the regulation of cell fate, differentiation, and patterning [39]. Notch signaling has also been connected to the control of cell proliferation, specification, and differentiation during the retinogenesis and formation of the mammalian ocular lens [39, 40].

In the study of the TGFβ2-induced EMT process in retinal pigment epithelium, it is discovered that the TGFβ-dependent Smad signaling cascade initiates the Jagged/Notch pathway. Further, the blocked Notch pathway inhibited the EMT process effectively [41]. Zhang and coworkers reported that the notch inhibitor (LY411575) blocked the Notch signaling in the PVR model of ARPE-19 cells [42]. One study by Niessen and coworkers targets SNAIL2 as part of Notch signaling and it has been observed that during hypoxia conditions SNAIL1 is directly induced by Notch signaling [43]. The overexpression of Jagged-1 and Notch-3 was observed in the TGFβ2-induced EMT of RPE [44]. The Jagged-1 knockout suppressed TGFβ2-mediated EMT process in RPEvia downregulation of SNAIL, SLUG, and zinc finger E-box binding homeobox 1 (ZEB1) [44]. The collagen-1 (COL1A1 and COL1A2) expression was found regulated by TGFβ2 treatment in cells (ARPE-19), as they are related to Notch/Jagged and MAPK signaling pathway [45]. Another research demonstrates the connection between the Notch/Jagged and MAPK signaling pathways in the EMT process in the RPE caused by the concomitant administration of TGFβ1 and tumor necrosis factor-alpha (TNF-alpha) [45].

2.6 Wnt-β-catenin and Hippo-YAP signaling pathway

An essential component of the Wnt-beta-catenin pathway, beta-catenin is activated by dissociating from its complex and translocating into the nucleus, where it further activates the genes SNAIL and other EMT-related genes [16]. The Wnt signaling mechanism is crucial for cancer, aging, and post-natal stem cell regeneration in addition to regulating tissue differentiation during embryogenesis [46]. Beta-catenin binds to the cytoplasmic domain of cadherins, illustrating a point at which the Wnt pathway and the cadherin adhesion mechanism converge [46, 47]. Important for the EMT, the phosphorylation statuses of beta-catenin, a key Wnt mediator, and GSK-3, which is positioned upstream of beta-catenin, are altered. By changing the expression or active status of its transcriptional regulators Snail and Smads, the Wnt/beta-catenin pathway, as well as the pathways started by FGF and TGFβ, govern the EMT process in RPE. The junction of many pathways is where GSK-3 is found [46, 48].

The Wnt-beta-catenin pathway is activated by laser-mediated coagulation, and this furthers the EMT process in RPE outcomes, according to an earlier investigation by Han and colleagues [49]. The overexpression of beta-catenin was prevented in one in vitro experiment using ARPE-19 cells by the XAV939 (Wnt-beta downregulator). The increased expression of EMT markers is caused by the overexpression of beta-catenin [50]. The stimulation of EMT by the Wnt-beta-catenin signaling pathway was also demonstrated by further excessive light exposure on the retina [51]. Pentraxin 3 (HC-HA/PTX3) is a potent, non-toxic inhibitor that, in a dose-dependent manner, inhibits Wnt signaling to suppress the EMT process in RPE [52]. So, as already established, laser photocoagulation triggers a Wnt/beta-catenin signal transduction pathway, which in turn encourages the proliferation and conversion of the epithelial cells of the retinal pigment epithelium into mesenchymal cells. It may be favorable for the regeneration of the RPE to therapeutically regulate Wnt/beta-catenin signaling.

Contact inhibition and EMT, which govern organ size, are regulated by the Hippo signaling system [50]. The Hippo signaling cascade controls the transcription factors YAP and TAZ, which link the EMT process to cell proliferation [53, 54]. Hippo-YAP was shown to be associated with cadherin and the phosphorylation of Src family members [56]. The in vitro investigation demonstrated the role of EMT in RPE suppression by reducing tight junction disintegration [17]. The Hippo-YAP pathway, whose activity is mostly dependent on tight and adheren junctions, maintains RPE differentiation [16]. The Yap, on the other hand, was not found in the primary cells (RPE) of mice [17]. More research is needed to completely understand the facts surrounding the Hippo-YAP pathway, which is linked to the EMT process in the RPE.

2.7 Transforming growth factor-beta (TGFβ) signaling pathway

TGFβ and its intracellular cascade are particularly important in the EMT process in RPE [2]. TGFβ is a crucial cytokine as an anti-inflammatory compound. Its production is associated with wound injury and inflammation [16]. TGFβ showed a role in both normal physiological as well as abnormal pathological conditions [17]. The presence of TGFβ1, TGFβ2, and TGFβ3 are reported in the human eye [55]. In general, the concentration of TGFβ cytokine is increased as an inflammatory response but if this condition remains for a prolonged period then it leads to EMT [56]. The drastic increased TGFβ2 favors a significant loss of cell–cell attachment in RPE [57]. The expression of TGFβ, epidermal growth factor (EGF), insulin-like growth factor (IGF-II), or FGF-2 initiates the process of digesting the basement membrane by binding with epithelial receptors and starts kinase activities [58]. Among these factors, the most interesting cytokine is the EGF and its receptor, epidermal growth factor (EGFR). Many research efforts have established its significance in the induction, maintenance, and control of cell proliferation, differentiation, and migration [59]. Further, it should be noted that TGFβ is a known inducer of EMT, and that EGFR has been observed to rise in EMT-affected cellular microenvironment where EGF-assisted cell plasticity occurs [60].

In previous studies, it has been found that the galactoside-binding lectin family protein (Galactin-1) is crosstalk with TGFβ signaling, in a knockout mouse it shows decreased choroidal neovascularization (CNV) severity and suppression of EMT process in RPE [61] (Table 1). Some other studies also represent the crosstalk between Smad-dependent signaling and ERK1/2 molecular interaction in RPE [41]. Troglitazone and pioglitazone, peroxisome proliferator-activated receptor-gamma (PPAR-γ) suppresses phosphorylation of Smad and thus inhibit TGFβ2-mediated EMT process in RPE [62, 63]. Inhibition of sub-retinal fibrosis is shown by the retinoic acid receptor gamma (RAR-γ) agonist through the TGFβ pathway [64].

Table 1 List of regulators of EMT process in RPE targeting via different signaling cascades
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Additionally, there are many agents such as bradykinin (BK) [65], fucoidan [13], bone morphogenic protein (BMP7) [66], BMP4 [67], LY-364947 (TGFβRI- inhibitor) [68], Baicalein [69], LYTAK1 (TAK1 inhibitors) [70], salinomycin [71], protein kinase-A inhibitor (H89) [72], and resveratrol [73] showed suppression of EMT process in RPE either in vivo or in vitro investigations. Recent studies on induced pluripotent stem cell (hiPSC)-derived retinal cells discovered that suppressing the protein kinase C or BMP signaling pathways, as well as reducing FGF/MAPK signaling, improved RPE differentiation [33]. Additionally, in the Smad3-mutated mouse PVR model, TGFβ signaling triggered the downregulation of the EMT process [74].

Some other therapeutic agents are involved in the inhibition of the EMT process in RPE but by interacting with multiple signaling such as curcumin suppressing the Akt, MAPK, and TGFβ pathways in RPE cells (Table 1). The mammalian target of rapamycin (mTOR) suppressor (Trichostatin A) hindered the EMT process in RPE by down-regulating the Jagged/Notch pathway, non-canonical TGF/Akt, MAPK, and ERK1/2, as well as the standard Smad signaling pathways [75]. It has been demonstrated that several intravitreal anti-vascular endothelial growth factor (anti-VEGF) medications can diminish retinal fibrosis in patients with active neovascular AMD (n-AMD) [19, 76, 77]. Choroidal neovascularization, which results in exudation, leakage, and finally fibrosis with photoreceptor loss, can be used to characterize n-AMD [3]. In recent safety phase II research, subretinal fibrosis in n-AMD patients receiving the combination of platelet-derived growth factor [Fovista®(E10030)] and an anti-VEGF drug was investigated. Additionally, a controlled phase II trial in n-AMD is now being conducted to examine the impact of either the FGF2 antagonist RBM-007 alone or in conjunction with the anti-VEGF drug on subretinal fibrosis [19].

3 Conclusions

Age-related macular degeneration and proliferative vitreoretinopathy activate several signaling cascades in RPE cells, including the SMAD, the Rho, the MAPK, the Jagged/Notch, and the Wnt/-catenin pathways, according to studies done in vitro and in vivo. The most active signaling cascade in the process of the retinal pigment epithelium's epithelial to mesenchymal conversion is discovered to be TGFβ, namely its intracellular cascade and both SMAD and non-SMAD pathways. There are no medications on the market right now that aim to treat the EMT of the retinal pigment epithelium. Retinopathies, in particular those caused by the epithelial to the mesenchymal conversion of retinal pigment epithelium cells, may only be treated with antimetabolite pharmaceuticals, however, these drugs have severe, occasionally blinding side effects.

A link between the molecular targets and the regulators of the EMT of RPE must be established in order to choose the best candidate for retinopathy therapy. To treat retinopathies, especially those brought on by the epithelial-mesenchymal transition of retinal pigment epithelium cells, it is critical to investigate to explore potential pharmaceutical treatments.