Inflammatory mediators of proliferative vitreoretinopathy: hypothesis and review

The TGF-β superfamily of multifunctional mediators not only regulates cell growth and differentiation, but also promotes fibrosis and proliferation [27, 28]. Three isoforms of TGF-β, namely TGF-β1, TGF-β2 and TGF-β3, are expressed in the anterior segment of the human eye [29]. In addition, TGF-β1 was reported to be stored or activated by photoreceptors [30]. TGF-β2 is observed in the long ciliary arteries and photoreceptor outer segments [30]. In choroid and retina, TGF-β3 is expressed in isolated individual cells [30]. Hoerster et al. [31] reported that in rabbit models of PVR, TGF-β1 and TGF-β2 were dramatically up-regulated in both aqueous humor (AH) and vitreous. In addition, Kon et al. [20] observed significant elevation of TGF-β2 in human vitreous PVR samples. RPE is considered to play an essential role in the formation and contraction of PVR membranes [15]. Of note, Yao et al. [32] pointed out that PVR progression was related to TGF-β-induced epithelial–mesenchymal transition (EMT) in RPE cells. Also, Dvashi et al. [33] showed that TGF-β1 played a pivotal role in EMT of RPE via activation of transforming growth factor beta-activated kinase 1 (TAK1). Rojas et al. [34] highlighted that Smad7 was associated with PVR development and considered as a novel target for the treatment of PVR. In addition, up-regulation of Smad7, an inhibitor of TGF-β, could suppress RPE cells’ fibrogenic response to EMT [35]. Consistent with these results, pirfenidone inhibited TGF-β1-induced fibrogenesis by blocking the nuclear translocation of Smads in a human PRE cell line, ARPE-19 [36]. Later, Yang et al. [37] explored the function of long noncoding RNA (lncRNA) MALAT1 in regulating EMT in RPE cells induced by TGF-β1. Increased expression of lncRNA MALAT1 was apparently observed in RPE cells incubated with TGF-β1 [37]. However, knockdown of MALAT1 could result in the inhibition of TGF-β1-induced EMT and proliferation of RPE cells partially via the activation of Smad2/3 signaling [37]. Consequently, they believed that lncRNA MALAT1 played an important role in TGF-β1-induced EMT in human RPE cells, which might shed light on the targeted therapy of PVR [37]. Researchers explored the distribution of selected cytokine gene polymorphisms in PVR patients and attempted to identify potential genetic markers [38]. Through the detection of single-nucleotide polymorphism (SNP), they found significant difference in genotype distribution of TGF-β1 codon 10 polymorphism between PVR patients and RD patients [38]. In comparison with controls, a statistical difference in TGF-β1 codon 25 in PVR patients was observed [38]. Therefore, they considered that TGF-β1 genetic profile was associated with PVR development [38]. Furthermore, Carrington et al. [15] confirmed that TGF-β2 could stimulate RPE-mediated contraction of the retina. Additionally, neutralizing antibodies against TGF-β2 effectively inhibited RPE cell-mediated contraction [15]. Thus, TGF-β2 may represent a potential target for PVR therapies [15]. Recently, Chen and his colleagues have investigated the function of Jagged/Notch signaling in TGF-β2-mediated EMT in RPE cells [39]. They reported that knockdown of Jagged-1 expression and blockade of the Notch signaling pathway could suppress EMT in RPE cells induced by TGF-β2, which might be associated with the formation of PVR [39]. During the process of TGF-β2-mediated EMT in RPE cells, a total of 304 miRNAs were reported to have changed, of which 119 were up-regulated and 185 were down-regulated, suggesting that miRNAs might be associated with EMT in RPE cells [40]. Among them, the expression of miRNA-29b was down-regulated more than 80% [40]. Thus, Cao et al. [41] have investigated the roles of mechanical stretch and TGF-β2 in EMT in human RPE cells and the association between miRNA-29b and PVR progression. Their observations confirmed that TGF-β2 could not only induce EMT in RPE cells, but also suppress the expression of miRNA-29b in time–dose dependence [41]. Therefore, they speculated that miRNA-29b might have potential as part of a clinical strategy for PVR treatment [41]. Recently, Liu et al. [42] have investigated the effect of mouse double minute 2 (MDM2) on TGF-β2-mediated EMT in RPE cells. Importantly, their experiments demonstrated that dCas9/MDM2-sgRNA could block TGF-β2-mediated expression of MDM2 and EMT biomarkers in RPE cells [42]. In vitro, TGF-β2 contributes to EMT, collagen production and fibrosis, while decorin antagonizes TGF-β and exhibits independent anti-fibrosis properties [43]. Therefore, decorin, as an antifibrotic agent, might be a promising candidate for the inhibition of RPE fibrosis induced by TGF-β2 [43]. Mony et al. [44] explored the association between TGF-β2-mediated EMT in RPE cells and altered Na, K-ATPase expression, which was observed on the apical membrane in RPE cells. Of note, their experiments revealed that the lack of expression of Na, K-ATPase β1 subunit might be related to TGF-β2-mediated EMT and fibrosis in RPE cells [44]. After the activation of TGF-β2 signaling, EMT biomarkers were found to be induced, such as fibronectin, α-SMA pressure and actin fibers, while the decreased expression of β1 subunit was observed [44]. Interestingly, knockdown of β1 subunit in RPE cells could contribute to the mesenchymal cell morphology and induction of EMT biomarkers, indicating that the lack of Na, K-ATPase β1 subunit might be a potential trigger of TGF-β2-mediated EMT in RPE cells [44]. In addition, the reduction of Na, K-ATPase β1 mRNA was negatively correlated with the level of HIF-1α [44]. It has been reported that the binding of HIF-1α with Na, K-ATPase β1 promoter and the inhibition of HIF-1α activity could block the decrease of Na, K-ATPase β1 mediated by TGF-β2, suggesting that HIF-1α might participate in the regulation of Na, K-ATPase β1 during the EMT in RPE cells [44]. Due to the abnormal expression of TGF-β in PVR, and the efficacy of TGF-β inhibitors to prevent fibrogenic response, we agree that TGF-β is likely to be associated with the development of PVR and propose TGF-β as a candidate target for PVR therapies. However, this hypothesis still needs to be validated in a more precise system, such as an animal model of PVR in which TGF-β is knocked out or knocked down.

Like TGF-β, many laboratories have demonstrated that PDGF and PDGF receptor (PDGFR) might participate in PVR development. Cui et al. [45] and Robbins et al. [21] confirmed that levels of both PDGF and PDGFR were significantly higher in PVR membranes than in control membranes. Both PDGF and PDGFR could be secreted by RPE and glial cells [21, 45]. In addition, subsequent studies indicated that the level of PDGF was dramatically elevated in the vitreous of PVR patients [46]. Moreover, animal experiments supported the hypothesis that PDGF and PDGFR might participate in the complex process of PVR development [46, 47]. Most rabbit models of PVR are established by injection of fibroblasts into the vitreous [48]. In these animals, PDGF is undetectable in the vitreous of the control group, while overexpression of PDGF, especially PDGF-C, is observed in the vitreous of model rabbits [46]. Coincidentally, vitreous is reported to contain the protease necessary for PDGF-C activation [47]. Furthermore, inhibition of PDGFR expression could reduce cellular PVR formation [49, 50]. Among three isoforms of PDGFR, namely PDGFRα, PDGFRβ and PDGFRαβ, PDGF-C can activate PDGFRα and PDGFRαβ [46, 51]. Previous findings indicated that PDGFRα could be capable of triggering the events that ultimately lead to experimental PVR [52]. Recently, Zhang et al. [53] have demonstrated that crocetin could act as an effective inhibitor of PDGF-BB-induced proliferation and migration of RPE. Taken together, PDGF-C and its receptor PDGFRα are considered more important in the formation of PVR. Actually, besides PDGF, non-PDGF factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and insulin could be capable of activating PDGFR indirectly [2, 4]. Direct activation of PDGFRα is driven by PDGF, causing subsequent activation of receptor’s intrinsic kinase [4]. Non-PDGFs could activate PDGFRα through reactive oxygen species (ROS) and Src family kinases (SFKs) [2, 4]. Non-PDGFs could engage its receptors and increase the level of ROS, resulting in the activation of SFK and phosphorylation of PDGFRα [2, 4]. Indirect activation of PDGFRα pathway by Non-PDGFs could prolong the activity of signaling enzymes such as PI3K/Akt, inhibit the expression of p53 gene and thereby promote cell survival and proliferation [2, 4]. In recent years, ultrasound-targeted microbubble destruction (UTMD) and RNA interference (RNAi) have shed light on gene therapy of eye diseases and become a new research hotspot [54]. Interestingly, studies have shown that the combination of UTMD and recombinant adeno-associated virus (rAAV)-mediated RNAi targeting TGFβ2 and PDGF-B could represent a novel approach to inhibit PVR proliferation [54]. As mentioned above, both clinical studies and animal experiments support the theory that PDGF and PDGFR are implicated in the occurrence and development of PVR. These studies highlight more therapeutic targets for PVR prevention. However, a targeted therapy has not yet reached clinical trials.

Interleukin can be secreted by a variety of cells and stimulate cellular responses. In addition to activating and regulating immune cells, interleukin is a critical signal for abnormal proliferation, differentiation of T- and B-lymphocytes and enhancement of inflammation [18, 55, 56]. In recent years, interleukin has been associated with the occurrence and development of PVR [18, 19, 22]. As a marker of acute inflammatory reaction, IL-6 exhibits a wide range of biologic activities. Not only can IL-6 attract chemokines, it can also recruit leukocytes to local inflammatory sites [18, 57]. Using a multiplex bead-based immunoassay, Ricker et al. [18] investigated interleukin levels in the subretinal fluid of 21 PVR patients and 54 RRD patients. A significant increase in IL-6 was observed in the PVR cases when compared with controls [18]. Consistent with these data, Symeonidis et al. [19] reported that IL-6 was up-regulated in both vitreous and subretinal fluid in the PVR group. Most importantly, they demonstrated that IL-6 levels were positively correlated with the extent and duration of RRD and PVR grade [19]. Previous findings have shown that RPE cells, forming part of the blood–retinal barrier, were potent producers of various inflammatory cytokines including IL-6 [58]. Also, IL-6 could be secreted by other cell types. Similarly, Kon et al. [20] and EI-Ghrably et al. [59] demonstrated that IL-6 was a critical mediator in the complex process of postoperative PVR development. The function of IL-6 signal transduction depends on the gp130 family [60]. Recently, Lumi et al. [61] have assessed the differences in genotype distributions of single-nucleotide polymorphisms (SNPs) between PVR cases and RRD cases. Interestingly, the significant difference in genotype distribution of rs1800795 (IL-6) could be observed between PVR cases and controls [61]. Furthermore, in IL-6 (rs1800795), RRD patients with the G allele possibly had a higher risk of progressing into PVR than patients with the C gene [61]. Unfortunately, after adjustment by multiple hypothesis testing, genotype distribution of IL-6 (rs1800795) had no statistical difference between two groups [61]. Herein, in order to clarify the genetic contribution to PVR, further investigations are needed. In addition to the recruitment and activation of neutrophils, IL-8 plays a pivotal role in the regulation of inflammatory responses and mediation of angiogenesis. Interestingly, the IL-8 levels were elevated in PVR cases [62, 63]. However, IL-8 levels did not correlated with PVR grade [64]. Later, Rasier et al. [22] measured the levels of IL-8 in 22 RRD patients and 12 cases of epiretinal membranes and macular holes. Similarly, IL-8 levels were significantly elevated in the RRD group [22]. Thus, Rasier et al. [22] speculated that IL-8 might contribute to the development of PVR. Previous report has indicated that the mRNA encoding IL-8 was obviously observed in subretinal fluid and vitreous in PVR patients [65]. Perhaps in the future, neutralizing the expression of cytokines may inhibit development of PVR. Of course, this requires further exploration.

TNF-α, a member of the TNF ligand superfamily, has a wide range of biologic activities and is implicated in the regulation of inflammatory response, apoptosis and cellular proliferation [66‐68]. Limb et al. [23] investigated the distribution of TNF-α within epiretinal membranes in 26 PVR patients. They reported that approximately 85% of the 26 epiretinal membranes contained TNF-α not intracellularly and in the extracellular matrix [23]. Herein, they maintained that cytokine-mediated inflammatory pathways were critical for the development of PVR [23]. In line with this result, the vitreous levels of soluble TNF-receptors (sTNF-Rs, sTNF-RI, sTNF-RII) and TNF-α were found to be higher in 30 PVR cases than in 30 RRD cases [69]. Importantly, levels of sTNF-Rs were correlated with the duration of retinal detachment (RD) and grade of severity. Therefore, they hypothesized that sTNF-Rs-mediated regulation of TNF-α activity might effectively limit severity of retinal proliferation [69]. Previous studies have shown that adhesion and migration of RPE cells to provisional extracellular matrices(ECM) played a critical role in the formation of epiretinal membrane in PVR [70]. Jin and his colleagues investigated the effects of TNF-α on adhesion and migration of RPE cells to ECM and explored the mechanism of this response [70]. They pointed out that TNF-α could regulate RPE cells to ECM through the activation of MAPK pathway [70]. Therefore, they believed that TNF-α and MAPK might be the potential target for PVR therapy [70]. Recently, Wang et al. [71] have reported that it was through the activation of Akt/mTORC1 signaling that TNF-α could promote the migration of RPE cells by inducing the expression of matrix metallopeptidase (MMP)-9. Moreover, the expression of TNF-α mRNA-positive cells was obtained in epiretinal membranes in PVR patients, which might provide new direction for the treatment of PVR [72]. Although the association between various SNPs in TNF, such as rs1800629 (TNF), and the increased risk of PVR was reported, previous results could not be replicated [61]. Based on these results, we believe that TNF-α is implicated in the development of PVR and thus represents a therapeutic target. It will be interesting to investigate whether TNF-a-specific inhibitors, such as infliximab (remicade), could prevent or reduce PVR.

Currently, surgery is the standard treatment for PVR [9]. The aim of PVR surgery is to reattach the retina and achieve satisfactory visual acuity. Although tremendous advances in vitreoretinal surgical techniques, such as 27-G transconjunctival sutureless vitrectomy system and noncontact wide-angle viewing system [57, 81], have been made over recent decades, both anatomic success and functional success rates are still unsatisfactory. In prospective studies, postoperative PVR occurs in 4% to 34% of cases [82]. Herein, novel therapeutic strategies are required to prevent or halt the progression of PVR.

Several pharmacological adjuvant therapies have been applied to improve surgical outcomes, including anti-inflammatory, anti-proliferative, antineoplastic, anti-growth factor agents and statins. (Table 1) The clinical trials (RCTs) for the evaluation of pharmacological adjuvant therapies of PVR are summarized in Table 2.