Abstract
Tissue contraction and fibrosis are major causes of morbidity in the human body. In the eye in particular, fibrosis and scarring are responsible for the pathogenesis or failure of treatment of all major blinding diseases, with postoperative wound healing responses posing a major problem for most ocular surgery on a worldwide scale. This is one of the largest areas of unmet need in ophthalmology, with currently no antifibrotic treatments available clinically. This review focuses on the ubiquitous myocardin-related transcription factor/serum response factor (MRTF-A/SRF) transcription pathway as a potential novel therapeutic target in fibrotic eye diseases. It describes how the MRTF-A/SRF pathway is intricately linked to all the key regulators and pathways in ocular fibrosis, and how it could potentially lead to a new avenue of antifibrotic therapies in the future.
Fibrosis and scarring are responsible for the pathogenesis or failure of treatment of all major blinding diseases, including corneal scarring, failure of glaucoma filtration surgery, capsule fibrosis after cataract surgery, age-related macular degeneration, diabetic retinopathy, and failure of retinal detachment surgery due to proliferative vitreoretinopathy.
Ocular fibrosis leads to significant visual impairment and blindness in millions of people worldwide, and is one of the largest areas of unmet need in clinical ophthalmology and medicine. Yet the only treatments currently available are anticancer drugs that have significant potentially blinding side effects, such as tissue damage and infection. There is thus an urgent need to identify novel targets to prevent scarring and postsurgical fibrosis in the eye.
Serum response factor (SRF) is a ubiquitous transcription factor that controls growth–factor regulated immediate early genes such as c-fos and actin, as well as muscle-specific genes.
1,2 The two principal families of signal-regulated SRF cofactors are the ternary complex factor (TCF) family and more recently, the myocardin-related transcription factor (MRTF) family.
3,4
Myocardin-related transcription factor A (MRTF-A) is a member of the MRTF family of SRF coactivators, and its activity responds to variations in the cellular concentration of G-actin, which it binds through N-terminal RPEL motifs.
1,2 There is increasing evidence that the MRTF-A/SRF pathway plays a key role in fibroblast activation, and MRTF-A knockout models have shown reduced fibrosis and scarring in various diseases, such as heart fibrosis,
5 lung fibrosis,
6 dermal fibrosis,
7 and vascular fibrosis.
8 In this review, we will discuss the emerging role of the MRTF-A/SRF transcription pathway in different types of fibrotic eye diseases, and also potential novel therapeutic approaches to prevent scarring and promote healthy cell regeneration for diseases and traumas that affect the front and back of the eye.
Tissues where fibrosis affects the anterior segment of the eye include the cornea, trabecular meshwork, and conjunctiva. Corneal diseases can be inherited (e.g., corneal dystrophies), or acquired secondary to infection (e.g., herpetic keratitis) or inflammation (e.g., pterygium). The final common pathway in all these corneal diseases is often inflammatory changes leading to corneal edema, neovascularization, and scarring of the corneal stroma.
9
In glaucoma, progressive fibrosis and malfunctioning of the trabecular meshwork, in particular the aberrant production of extracellular matrix, leads to increased resistance to aqueous outflow and glaucomatous damage.
10 Long-term success after glaucoma filtration surgery also depends directly on the wound healing response, and scarring over the drainage site is the main cause of postoperative increase in intraocular pressure and surgical failure.
11
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade components of the extracellular matrix and play a key role in modulating ocular scarring.
12–14 Expression of MMPs is low in normal cells to allow for healthy connective tissue remodeling, and overexpression of MMPs can lead to fibrosis, cancer,
15 arthritis,
16 and arteriosclerosis.
17 Neutralization of MMP activity also leads to a reduction in cell-mediated collagen contraction
14 and scarring in vivo.
13
Several MMPs, including MMP-2, MMP-9, MMP-13, and MMP-14, are expressed in normal and wounded corneas, and play a critical role in the balance of extracellular matrix turnover during corneal wound healing.
18,19 In cases of corneal vascularization following trauma, Kvanta et al.
20 have shown increased expression of MMP-2 in the limbal region and corneal stroma. Azar et al.
21 have also reported increased MMP-2 and MMP-9 levels during corneal wound healing after LASIK and excimer laser photorefractive keratectomy. In glaucoma, MMPs have been identified in human trabecular meshwork cells, and the balance between MMP and inhibitor activity plays a crucial role in modulating trabecular extracellular matrix and aqueous outflow.
22,23 McCluskey et al.
24 have also reported increased MMP-1, MMP-2, and MMP-3 expression in the bleb capsules surrounding glaucoma implants.
There is now increasing evidence of a link between the MRTF-A/SRF pathway and MMP expression.
25–28 In a recent paper, Esnault et al.
25 have shown that MMP-2, MMP-9, and MMP-14 are all SRF targets in NIH3T3 fibroblasts. Kim et al.
27 and Zhe et al.
28 have also reported that SRF overexpression led to an increase in MMP-2 and MMP-9 levels in hepatocellular cancer and lung fibroblasts respectively. Moreover, Howard et al.
26 have shown that MRTF-A knockdown led to the upregulation of MMP-2 in the presence of transforming growth factor β (TGF-β).
The MRTF-A/SRF transcription pathway is an important upstream regulator of MMP expression in ocular fibrosis (
Fig.). Serum response factor can regulate the gene expression of profibrotic targets, such as MMPs, by both direct and indirect routes. Through the indirect route, SRF can act through the activator protein 1 (AP-1) to modulate the expression of multiple MMP genes.
29,30
Growth factors play a pivotal role in ocular fibrosis. Sotozono et al.
31 have shown that topical keratinocyte growth factor accelerated corneal epithelial wound healing in vivo by increasing cell proliferation at the corneal limbus. Chandrasekher et al.
32 have also reported that hepatocyte growth factor (HGF) promoted epithelial wound closure in a corneal organ culture model. In a randomized multicenter study, Pastor et al.
33 have shown a significant increase in corneal wound healing time using topical epidermal growth factor (EGF). Iyer et al.
34 have also reported that connective tissue growth factor (CTGF) modulated extracellular matrix synthesis, actin cytoskeletal dynamics, and contractile force in trabecular meshwork cells.
One of the most important growth factors in ocular fibrosis is TGF-β, which increases fibroblast proliferation,
35 controls extracellular matrix synthesis,
36 and accelerates myofibroblast differentiation.
37 It is expressed in high levels in corneal cells, and is important for corneal wound healing and maintenance of corneal transparency.
38 Transforming growth factor-β is also found in high concentrations in the aqueous humor of glaucoma patients
39 and in the trabecular meshwork, which suggests a role in aqueous outflow regulation.
40
Previous studies have reported that subconjunctival injection of a monoclonal antibody to TGF-β decreased conjunctival scarring,
41 but did not prolong bleb survival after glaucoma filtration surgery in a randomized clinical trial.
42 Mead et al.
43 have suggested that an alternate prolonged dosing regimen might be required. Spitzer et al.
44 have also reported that tranilast, which is an antiallergic drug that inhibits TGF-β expression, improved the surgical outcome of glaucoma filtration surgery when combined with cross-linked hyaluronic acid.
The MRTF-A/SRF pathway is an important downstream regulator of TGF-β (
Fig.). Crider et al.
7 have shown that MRTF-A/MRTF-B are key regulators of TGF-β–induced fibroblast to myofibroblast differentiation, and knockdown of MRTF-A/MRTF-B decreases the expression of α-smooth muscle actin and contractile force. Tsapara et al.
45 have also shown that the guanine nucleotide exchange factor (GEF-H1) is a crucial target and functional effector of TGF-β, by orchestrating Rho signaling to regulate gene expression and cell migration.
Cytokines play a crucial role in the development of fibrosis, and several cytokines, particularly interleukin-1 (IL-1), have well-defined proinflammatory and profibrotic characteristics.
46 IL-1, IL-6, and tumor necrosis factor (TNF) are produced by corneal cells and inflammatory cells, and regulate the growth and migration of corneal epithelial cells during wound healing.
38 Previous studies have also reported high levels of IL-1 in the trabecular meshwork of glaucoma patients,
47 and increased IL-6 levels after TGF-β treatment.
48
Vincenti et al.
49,50 have shown that IL-1 is an important upstream regulator of MMP expression through the mitogen activated protein kinase (MAPK) pathway (
Fig.). The MAPK family consists of the c-Jun N-terminal kinases (JNKs), the extracellular signal-regulated kinases (ERKs), and the p38 kinases.
51 The JNKs and p38 kinases are activated by cytokines and apoptotic signals,
52 while the ERKs respond to cytokines and growth factors.
53 Upon activation, MAPKs translocate to the nucleus to phosphorylate various transcription factors—namely AP-1, which dimerizes with c-fos to drive the transcription of multiple human MMP genes.
54
Angiogenesis plays a key role in wound healing.
55 Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, and stimulates both fibroblasts and endothelial cells in wound healing.
56,57
Several studies have shown promising results of anti-VEGF therapies (bevacizumab, ranibizumab) in preventing corneal vascularization.
58–60 Lee et al.
61 have reported that subconjunctival bevacizumab injections accelerated regeneration of the basement membrane after chemical injury. Cho et al.
62 have also shown that knocking down VEGF improved corneal graft survival by decreasing angiogenesis and lymphangiogenesis.
The role of anti-VEGF therapies in reducing scarring and fibrosis after glaucoma filtration surgery remains controversial. Several anti-VEGF studies have shown potential in decreasing scar formation and prolonging bleb survival in glaucoma surgery.
56,63,64 However, Rodríguez-Agirretxe et al.
65 have recently reported that combined mitomycin C (MMC) and bevacizumab implants decreased intraocular pressure to a lesser extent than MMC alone, and that bevacizumab could interact with MMC. In the first randomized clinical trial comparing MMC and bevacizumab, Nilforushan et al.
66 have also reported that the MMC group had better intraocular pressure control but similar bleb morphology to the bevacizumab group. Larger randomized clinical trials are needed to investigate their efficacy and safety in glaucoma filtration surgery.
The MRTF-A/SRF pathway is a key downstream regulator of VEGF in ocular fibrosis (
Fig.). Vascular endothelial growth factor acts through the Rho-actin pathway to upregulate the MRTF-A/SRF pathway, and stimulates fibroblasts and endothelial cells.
67 The MRTF-A/SRF transcription pathway thus represents a potential novel therapeutic target to modulate angiogenesis and fibrosis in the eye.
Cataract is the leading cause of reversible blindness worldwide. Posterior capsular opacification represents the most common complication of cataract surgery, and affects 20% to 40% of patients within 2 to 5 years after surgery.
68 Posterior capsular opacification has an even higher incidence after congenital cataract surgery, and carries a significant risk of amblyopia and visual impairment in young children.
69
Cataract surgery induces a wound healing response in the lens. Residual lens epithelial cells (LECs) proliferate, migrate to the posterior aspect of the lens capsule, undergo epithelial-to-mesenchymal (EMT) transition, deposit collagen, and regenerate new lens fibers, resulting in capsular opacification of the visual axis.
70,71
Growth factors, MMPs, and cytokines all play a crucial role in the pathogenesis of posterior capsular opacification (
Fig.). Migration of LECs onto the posterior lens capsule is important for the remodeling of the extracellular matrix and capsular contraction, and is associated with MMP activity in the lens.
72 Cytokines, such as IL-1, are also synthesized by LECs in vitro and stimulate mitosis, collagen synthesis, and production of proinflammatory prostaglandins.
73
In addition, TGF-β plays a pivotal role in the cell biology of posterior capsular opacification by inducing EMT transition in LECs, and formation of extracellular matrix.
73–75 Gupta et al.
76 have recently shown that TGF-β-induced EMT transition strongly correlates with the nuclear translocation of MRTF-A in LECs, confirming the key role of MRTF-A as a downstream regulator of TGF-β in posterior capsular opacification (
Fig.).
Currently, the standard treatment for posterior capsular opacification is the Nd:YAG laser capsulotomy that involves clearing the central visual axis in the opacified posterior capsule. This represents a significant economic burden and also carries a small risk of sight-threatening complications, such as macular edema, retinal detachment, intraocular lens damage, raised intraocular pressure, and endophthalmitis.
77
Various research groups have studied the intraocular use of cytotoxic and therapeutic drugs in the anterior chamber to prevent capsular opacification, namely mitomycin,
78 fluorouracil,
79 MMP inhibitor (ilomastat),
72 daunomycin,
80 diclofenac and cyclosporin A,
81 cyclooxygenase 2 inhibitors,
82 and saporin.
83 However, the risks of their toxic effects on the corneal endothelial cells, iris, ciliary body epithelial cells, and retina have restricted their clinical use in humans.
There is a large unmet need for more targeted and safer therapies in capsular opacification and fibrosis, and new drug delivery techniques are also being developed. A sealed capsule irrigation device (PerfectCapsule; Milvella Ltd., North Sydney, NSW, Australia) has shown potential as it is a small flexible device that can be inserted into the anterior chamber after removal of the cataractous lens, to form a sealed-capsule irrigation system over the anterior lens capsule.
84,85
Future areas of research will focus on the design of new drugs to selectively target lens epithelial cells intraoperatively, with minimal risk of toxicity to surrounding ocular structures. There is now emerging evidence that the MRTF-A/SRF pathway could represent a potential novel therapeutic target to modulate lens epithelial cells and prevent capsular opacification and fibrosis after cataract surgery.
76
The major blinding retinal diseases are age-related macular degeneration (AMD),
86 diabetic retinopathy,
87 retinopathy of prematurity,
88 and failure of retinal detachment surgery due to proliferative vitreoretinopathy.
89 The final common pathophysiological mechanism in all these diseases is the wound healing response of the retina, leading to fibrovascular scarring and fibrosis.
Angiogenesis is a critical component of the wound healing response in the retina, and occurs in response to ischemia, inflammation, and trauma.
90 However, these abnormal new blood vessels tend to leak fluid, causing retinal edema, retinal and vitreous hemorrhage, fibrovascular scarring, and tractional retinal detachment in more advanced cases.
Retinal vascular endothelial cells (ECs) line the arborizing retinal microvasculature that supplies the neural retina, and play a crucial role in angiogenesis.
91 Angiogenesis is tightly regulated by the balance of angiogenic stimulators including VEGF, TGF-β, and TNF-α, and antiangiogenic inhibitors such as interferon α, thrombospondin-1, and endostatin on the retinal microvasculature.
90 When this delicate balance is upset, this leads to proliferation, migration, and differentiation of ECs.
Weinl et al.
92 have recently shown that knocking down SRF or its cofactors (MRTF-A/MRTF-B) in ECs caused retinal hypovascularization in the postnatal mouse, but intraretinal neovascularization in the adult mouse. Both of these contrasting retinal phenotypes resembled blinding human retinopathies, namely developmental hypovascularization in early-onset familial exudative vitreoretinopathy and Norrie disease,
93 versus the adult-onset neovascular macular degeneration,
86 retinal angiomatous proliferation,
94 and macular telangiectasia.
95
In addition, Franco et al.
96 have shown that SRF plays a key role in sprouting angiogenesis and small vessel integrity, and is essential for the expression of actin and VE-cadherin in ECs. Other authors have also shown that MRTF-A is crucial for vascular remodeling,
8 and that MRTF-B plays an important role in normal vascular development and smooth muscle gene expression.
97
Vascular endothelial growth factor is the dominant angiogenic stimulus in retinal neovascularization.
98 Chai et al.
67 have shown that SRF knockdown inhibited VEGF-stimulated migration and proliferation of ECs, and impaired VEGF-induced actin polymerization and early gene expression. Chai et al.
67 have also reported that VEGF increased SRF activity in ECs through the Rho-actin and MEK-ERK signaling pathways.
Recent studies have identified SRF as an important downstream mediator of VEGF signaling in endothelial cells, and a critical requirement for VEGF-induced angiogenesis.
67,92 (
Fig.). The MRTF-A/SRF transcription feedback circuit, which links cytoplasmic actin dynamics with nuclear gene regulation, thus represents a potential new therapeutic target in the treatment of fibrovascular retinal diseases.
Ocular fibrosis is a complex multifactorial process, and there is increasing evidence that the MRTF-A/SRF transcription pathway plays a pivotal role in the pathogenesis or failure of treatment of blinding fibrotic eye diseases. Recent studies offer new insights into the molecular and genetic basis of wound healing, inflammation, and angiogenesis, and have identified matrix metalloproteinases, growth factors, cytokines, and angiogenic factors as key upstream or downstream regulators of the MRTF-A/SRF transcription pathway (
Fig.).
Several research groups have studied the use of MMP inhibitors in ocular fibrosis. Wong et al.
13 have shown that ilomastat decreased conjunctival scarring after glaucoma filtration surgery, and reduced capsular fibrosis after cataract surgery.
72 Das et al.
99 have shown that retinal neovascularization can be suppressed by the synthetic MMP inhibitor BB-94. Ozerdem et al.
100 have also reported that progression of proliferative vitreoretinopathy can be significantly delayed by intravitreal prinomastat injections. The MRTF-A/SRF pathway is an important upstream regulator of MMP expression, and thus represents a potential novel target to inhibit MMP expression and prevent ocular scarring.
Our increased understanding of the molecular machinery of Rho signaling has identified new potential pharmacological approaches to fibrosis, based on small molecules targeting the posttranslational modifications of Rho itself, the Rho effector kinase ROCK, the formin family of F-actin nucleators, or the activity of SRF reporters.
101–104 For example, ROCK inhibition has been shown to have anti-glaucoma effects in a rabbit model by relieving intraocular pressure.
105
In this regard, the development of apparently specific inhibitors of MRTF/SRF signaling, such as CCG-1423
104 and CCG-203971,
106 is of particular interest. For example, CCG-1423 can inhibit TGF-β induced myofibroblast differentiation in a pulmonary fibrosis model.
107,108 The mechanism by which these agents act remains to be unequivocally validated. A recent report suggests that CCG-1423 controls nuclear G-actin levels through inhibition of MICAL-2, a member of the MICAL family of actin monooxygenases,
109 but there is also evidence that CCG-1423 acts directly on RPEL-family proteins to prevent their nuclear import.
110 Although CCG-1423 and its derivatives may therefore affect other proteins as well as the MRTFs, it will be of interest to explore their use in the ocular fibrosis setting.
Effective ocular drug delivery techniques represent another major hurdle in the translation of new antifibrotic therapies into clinical practice. The efficiency of ilomastat in glaucoma filtration surgery currently depends on continuous exposure to the drug, which could trigger a reactive overexpression of MMPs and side effects due to cumulative doses. New slow release drug delivery techniques, such as sustained release implants and in situ gels, are thus currently being developed in glaucoma filtration surgery.
111 The sealed-capsule irrigation system (Milvella Ltd.) have shown promising results to prevent capsular fibrosis after cataract surgery, with minimal risk of toxicity to surrounding ocular structures.
84 Intravitreal injections also represent a safe and effective drug delivery technique to treat retinal diseases, but there is a need for more sustained release drugs to decrease the need for multiple injections.
112
An innovative and exciting approach in the future will be the genetic modulation of cells responsible for ocular fibrosis and scarring.
113–115 Small interfering RNAs, microRNAs, and short hairpin RNAs are small RNA transcripts that play a key role in posttranscriptional gene silencing, and potentially represent one of the most important advances in next generation therapeutics.
116,117 Small interfering RNA-027 provides proof-of-concept that siRNAs are well tolerated in the eye.
118 Animal models of glaucoma filtration surgery and proliferative vitreoretinopathy in the mouse and rabbit could also be used to test new antifibrotic therapies in vivo.
13,119–121
Future research will also focus on identifying the groups of patients at risk of scarring after surgery, by improving their genotyping and phenotyping using modern tissue and cellular imaging and biomarkers. Being able to predict a patient's risk of scarring according to their genetic profile holds great potential to the development of a more personalized and stratified therapy in ocular fibrosis. Combination therapies targeting several pathways might in fact be more efficacious than monotherapies in the high-risk groups. A tantalizing goal in the future will be the development of novel and personalized anti-fibrotic gene therapeutics to prevent scarring and promote healthy cell regeneration after disease, trauma, or surgery in the eye.
Supported by a Francis Crick Institute Clinical Research Training Fellowship from the University College London (UCL) National Institute for Health Research (NIHR) Biomedical Research Centre BRC (CY); the NIHR BRC at Moorfields Eye Hospital and UCL Institute of Ophthalmology (PTK, MB); and the Cancer Research UK London Research Institute/Francis Crick Institute (RT).
The authors alone are responsible for the content and writing of the paper.
Disclosure: C. Yu-Wai-Man, None; R. Treisman, None; M. Bailly, None; P.T. Khaw, None