Clinical background
Scarring constitutes the major threat to long-term success after most forms of glaucoma filtration surgery (GFS). Successful modulation of scarring increases the percentage of patients achieving final intraocular pressures (IOPs) that are associated with virtually no glaucoma progression. Antifibrotic agents for inhibition of scarring of trabeculectomy blebs are widely used worldwide, and their use is now well established, although they are linked to severe complications such as leakage, infection, hypotony, and endophthalmitis. These complications may lead to irreversible blindness. In addition, as surgery still fails in some individuals, despite maximal doses of current antifibrotics, more effective and selective therapeutic agents are sought.
Pathology
Hemostasis, inflammation, cell proliferation, and remodeling are the main phenomena observed in wound healing. After injury, formation of fibrin clots takes place with activation of the clotting cascade. This mechanism reduces blood loss. At the same time, neutrophils, macrophages, and lymphocytes are attracted to the region (inflammatory phase). Following these phenomena, the proliferative phase occurs: this comprises re-epithelialization and formation of granulation tissue and involves migration of fibroblasts, keratinocytes, and vascular endothelial cells to the wound region from neighboring tissues. Finally, in the remodeling phase, remodeling of the tissue takes place and involves the formation of scar tissue ( Figure 28.1 ). Many different cell types participate in the healing process, including fibroblasts, keratinocytes, endothelial cells, neutrophils, macrophages, lymphocytes, and mast cells.
Pathogenesis
Inflammation
The grading system used in our long-term Medical Research Council trial showed a good correlation between inflammation and long-term outcome ( www.blebs.net ) ( Figure 28.2 ). This finding agrees with many studies that have reported that inflammatory cells and mediators released during and after surgery stimulate the scarring cascade. Topical steroids applied as part of the routine postoperative management are effective in reducing inflammation. Additionally, IOP reduction has been achieved with intrableb triamcinolone acetonate injection at the conclusion of GFS, and this constitutes a relatively safe method for steroid administration. Topical nonsteroidal anti-inflammatory drugs may be effective, but their use is still controversial.
The use of other agents, including ciclosporin and cyclooxygenase-2 inhibitors, against inflammation has been suggested ( Box 28.1 ). For the inhibition of inflammatory cytokines, a novel approach has been attempted with the development of dendrimers: hyperbranched nanomolecules that can be chemically synthesized to have precise structural characteristics. In our in vivo model of GFS, water-soluble conjugates of d (+)-glucosamine and d (+)-glucosamine 6-sulfate with immunomodulatory and antiangiogenic properties applied together enhanced the long-term success of GFS from 30% to 80%. This experimental result is far more effective than that seen with conventional steroids ( Figure 28.3 ).
| •Steroids (intrableb triamcinolone acetonate injection) | Present mainly intracellular activity by alteration of gene expression. Clinically beneficial for intraocular pressure reduction |
| •Synthetic derivatives of glucocorticoids |
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| •Nonsteroidal anti-inflammatory drugs |
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| • d (+)-glucosamine and d (+)-glucosamine 6-sulfate dendrimers | d (+)-glucosamine and d (+)-glucosamine 6-sulfate dendrimers have immunomodulatory and antiangiogenic properties, respectively |
| •Ciclosporin A | Inhibition of lymphocyte-mediated immune responses |
| •Amniotic membrane | Potent anti-inflammatory properties, maintenance of oxygenation and moisture, and mechanical protection of covered tissues |
Fibrin and hemostasis
Fibrin constitutes an important part of wound healing. Fibrinolytic agents are effective in lysing blood clots after surgery, and, in the short term, these agents may lower IOP. The main side-effects that may deter the use of these agents are an increased risk of prolonged bleeding as well as the fact that fibrin breakdown molecules may have a longer-term stimulatory effect on the induction of scarring ( Box 28.2 ).
| •Heparin | Anticoagulant. Inhibits soluble thrombin, not fibrin-bound thrombin |
| •Recombinant Hirudin | Naturally occuring anticoagulant from the leech Hirudo medicinalis. Direct irreversible thrombin inhibitor |
| •Tissue-type plasminogen activator | Lyses blood clots after surgery and may lower intraocular pressure |
| •Urokinase or single-chain urokinase-type plasminogen activator | Thrombolytic (fibrinolytic) agent |
Side-effects of anticoagulants and thrombolytic agents
Risk of further bleeding
Fibrin breakdown molecules may induce scarring
Cytokines, chemokines, and growth factors
Large numbers of growth factors or cytokines are contained in the tissues in a wound and this is the case in GFS and in the aqueous flowing through the bleb ( Box 28.3 ). Transforming growth factor-β (TGF-ß) in wound healing has been shown to be more stimulatory than other growth factors and cytokines found in the aqueous. TGF-ß may even reverse the effect of mitomycin C (MMC) in vivo. Recent finding of enhanced expression of TGF-ß-RII receptors in failed blebs indicates the importance of TGF-ß in scarring after GFS. Modulation of the activity of growth factors may be a useful therapeutic strategy for the inhibition of fibrosis.
| •Recombinant human monoclonal antibody against active transforming growth factor (TGF-ß 2 ) | Inhibition of TGF-ß 2 activity |
| •Small interfering RNA (SiRNA) against TGF-β mRNA | Inhibition of transcription of the mRNA with subsequent inhibition of synthesis of the protein TGF-ß |
| •SiRNA against TGF-β II receptor mRNA | Reduction of inflammation and extracellular matrix deposition in vivo. Increased presence of TGF-β II receptors in the failed blebs |
| •Tranilast (( N -(3′,4′-dimethoxycinnamoyl) anthranilic acid) | Inhibition of TGF-ß activity |
| •Genistein: isoflavone from soy products | Inhibition of TGF-ß activity, tyrosine kinases, matrix metalloproteinases, and angiogenesis |
| •Suramin: polycyclic trypan dye derivative | Inhibition of TGF-ß activity and reduction of fibrosis after experimental glaucoma filtration surgery |
| •Conversion of angiotensin I to II | Angiotensin II regulates TGF-ß 1 expression via angiotensin receptor ligand binding, which contributes to myofibroblast conversion |
| •Chymase inhibitors: chymase is an enzyme released by mast cells | Chymase activates angiotensin I to angiotensin |
| •Smad-7 gene transfer | Suppression of TGF-β action and protection against the development of lung, liver, and renal fibrosis |
| •Rho-associated kinase (ROCK) inhibitors (mainly Y-27632) | Control of GTPase Rho activation, which is triggered by TGF-β. Inhibition of human Tenon’s fibroblasts collagen I contraction in vitro and increase of bleb survival in vivo |
| •Decorin | Small proteoglycan, natural inhibitor of TGF-β. Delay of intraocular pressure increase and decrease of fibrosis after glaucoma filtration surgery |
| •Ribozymes: RNA molecules which can cleave specific bonds in other RNA molecules | Cleavage of TGF-ß-mRNA with subsequent inhibition of synthesis of the protein TGF-ß |
| •Aptamers ARC126 and ARC127 | Platelet-derived growth factor (PDGF) activates ocular fibroblasts. ARC126 and ARC127 bind and block PDGF-B and may improve the success of glaucoma filtration surgery |
| •Adenoviral transfer of a dominant negative p38MAPK gene | Inhibition of Smad3 activation. Reduction of the differentiation of fibroblasts to myofibroblasts in vitro and reduction of conjunctival scarring in vivo |
| •Simvastatin, Inhibitor of the enzyme, HMG-CoA reductase | Inhibition of the connective tissue growth factor gene and protein expression, a downstream mediator of TGF-ß |
| •Lovastatin | Inhibition of TGF-β-induced connective tissue growth factor transcription, α-smooth-muscle actin (α-SMA) transcription and human Tenon’s fibroblasts differentiation to myofibroblasts |
| •Follistatin | Inhibitor of activin A. Reduction of liver and lung fibrosis Potential application in GFS |
Because TGF-ß in the eye seems to be involved in many pathways that are vital for the scarring process, we performed several studies using a variety of biological mechanisms to block TGF-ß activity, including antisense oligonucleotides and a human monoclonal antibody against the active form of human TGF-ß 2 , the predominant isoform in the aqueous (Lerdelimumab, TrabioR, Cambridge Antibody Technology, Cambridge, UK). The theoretical advantage of antibodies includes a self-regulating concept, only working when levels of the target protein are high ( Figure 28.4 ). In an in vivo model of conjunctival scarring, the administration of this antibody significantly improved GFS outcome and appeared much less destructive to local tissue than MMC. A pilot clinical study of this antibody in GFS demonstrated the absence of significant side-effects, inflammatory reaction, and cystic bleb formation. However, two larger randomized controlled trials have not shown a significant effect on the outcome of GFS. Based on the data obtained from an earlier study, we believe that the dose used was not sufficient. Further studies from our lab have shown a significantly enhanced effect with a prolonged dosing regimen, and the data also suggested an enhanced effect in the GFS outcome when the antibody is combined with intraoperative 5-fluorouracil (5-FU).
Small interfering RNA against TGF-ß receptor II mRNA reduced the production of TGF-ß receptor II, the expression and deposition of fibronectin, the migration of human corneal fibroblasts, inflammation and deposition of extracellular matrix (ECM) in an in vivo model of subconjunctival scarring after GFS.
Transglutaminases are calcium-dependent enzymes which cross-link proteins using ϵ-(γ-glutamyl)-lysine bonds. Transglutaminase (tTgase) and its end product ϵ-(γ-glutamyl)-lysine were detected in scarred tissue of failed trabeculectomy blebs. Since vertebrates lack enzymes capable of hydrolyzing these bonds, the protein cross-linking created by transglutaminases seems to be unbreakable. tTgase cross-links fibronectin and collagen-3; these proteins are produced by human Tenon’s fibroblasts (HTFs) in vitro and have been detected in the scar tissue deposited in the bleb area after GFS. In the same study, TGF-ß 2 was shown to stimulate the expression of tTgase and, subsequently, the cross-linking of fibronectin in vitro, leading to the conclusion that inhibition of TGF-ß 2 activity could extend the success of the surgery as this pathway might lead to enhanced cross-linking of the newly formed scar tissue in vivo.
The main intracellular TGF-ß signaling pathway runs through proteins that activate transcription of the genes that encode the Smad proteins. Of particular relevance is Smad-3, which is essential for TGF-ß-induced production of ECM proteins. Targeting intracellular signaling downstream of the TGF-ß receptor could be another effective strategy. Inhibiting Smad3 in immediate postoperative applications might prove beneficial. Smad-7, acting differently from Smad-3, is another potential therapeutic target. As TGF-ß can suppress its action through the induction of Smad-7 (negative-feedback loop), gene transfer of the Smad-7 gene has been shown in animal models to have a protective effect against the development of lung, liver, and renal fibrosis.
P38 MAP kinase (MAPK) is believed to trigger the transcription of Smad-2/3, facilitate the phosphorylation and activation of Smad-3 and, subsequently, the formation of Smad-3/4 complex, which is important for the development of the fibrotic reaction. The Smad-3 signaling pathway is important in retinal fibrosis. Inhibition of Smad-3 is associated with a reduction of a cellular fibrotic reaction. Adenoviral transfer by intravitreal application of a dominant negative p38MAPK gene demonstrated reduced fibrotic reaction of retinal pigment epithelium cells after retinal detachment. In later studies, adenoviral gene transfer of the same gene was applied to in vitro cultured HTFs and in an in vivo conjunctival scarring model in mice. Reduction of the differentiation of fibroblasts to myofibroblasts and decline of the connective tissue growth factor (CTGF) and of the monocyte chemoattractant protein (MCP-1) expression were the main in vitro findings. Inhibition of conjunctival scarring was observed in vivo. As MCP-1 is a chemoattractant for macrophages, one of the main sources of TGF-ß, the reduction in levels of MCP-1 through inhibition of p38MAPK, seems to reduce the levels of TGF-ß; hence, it plays a favorable role in conjunctival scarring. Furthermore, the recent finding that the antiglaucoma drug latanoprost, a prostaglandin F 2α analog, induces formation of stress fibers in HTFs in vitro as well as contraction of collagen I gels mediated by HTFs in vitro is indicative of the important role of MAPKs in subconjunctival scarring. The contraction of collagen I gels by latanoprost was blocked by inhibitors of MAPKs, Rho-activated kinase, myosin light chain (MLC). Moreover, the findings of this in vitro study point out potential induction of scarring by latanoprost in vivo. This conclusion agrees with previous findings that long-term antiglaucoma treatment represents a risk factor in scarring after GFS and that eyes treated with latanoprost after surgery were observed to have smaller decrease of IOP compared to the ones that did not receive latanoprost.
CTGF influences ECM production and subsequent fibrosis. TGF-ß 1 triggers the expression of CTGF, which is also necessary for TGF-ß stimulation of myofibroblast differentiation and collagen contraction. Inhibition of this factor could be a possible future therapeutic target. Lovastatin, a member of the drug class of statins, was shown to inhibit the TGF-ß-induced CTGF transcription, α-smooth-muscle actin (SMA) expression and, subsequently, the HTF differentiation to myofibroblasts as well as collagen contraction in vitro.
Rho is a small GTPase that has been implicated in the formation of stress fibers and focal adhesions, actin cytoskeleton remodeling, and cell contractility. TGF-ß increases cell tension in HTF cultures and contraction in HTF collagen I gels by triggering the activation of Rho. Rho activates the serine-threonine Rho-associated kinase (ROCK), which enhances cytoskeletal tension and results in actomyosin-mediated contraction. ROCK inhibitors reduce cell tension and inhibit the TGF-ß-mediated p-38 activation, α-SMA expression, and HTF development of enhanced contractile abilities characteristic of the so-called myofibroblast phenotype.” ROCK inhibitor Y-27632 inhibited contraction of HTF-seeded collagen I gels and α-SMA expression by HTFs in vitro. In vivo, application of Y-27632 after GFS significantly increased the survival of the blebs compared to controls. Furthermore, reduction of collagen I deposition and scarring in the treated blebs compared to controls was observed in histological analysis. ROCK inhibitors may block TGF-ß-induced scarring by downregulating pathways that are generating mechanical tension and thereby improve the success of GFS.
Myofibroblasts express a platelet-activating factor (PAF) nuclear receptor and tumor necrosis factor-ß (TNF-ß) receptors; PAF and TNF-ß cause time-dependent myofibroblast apoptosis. Future therapeutic approaches may take advantage of the expression of these receptors.
Platelet-derived growth factor (PDGF) has been found to activate certain ocular fibroblasts. Some ocular fibroblasts express high levels of PDGF receptor beta. When stimulated, these fibroblasts proliferate, migrate, produce ECM molecules and cause contraction, fibrosis, and the development of fibrotic membranes such as those observed in proliferative vitreoretinopathy (PVR). Aptamers, nucleic acid-based macromolecules with similar functions as monoclonal antibodies (high affinity and specificity for target proteins), are capable of recognizing, binding, and blocking PDGF-B. Two aptamers, ARC126 and ARC127, have been tested in animal models of PVR, and may also be useful in GFS.
Bone morphogenic proteins (BMPs) are growth factors that are vital for cell proliferation, differentiation, apoptosis, angiogenesis, and other biological functions. Activins are dimeric proteins that participate in cell differentiation and proliferation, apoptosis, inflammation, and neurogenesis. Investigation of the expression of many members of BMPs and activins in normal and scarred conjunctival tissue revealed enhanced mRNA and protein levels of BMP-6 and activin A in scarred compared to normal conjunctiva. The use of follistatin, an inhibitor of activin bioactivity, was shown to be effective in decreasing liver and lung fibrosis. Based on the above findings, the inhibition of BMP-6 and activin A could be a future therapeutic option for the control of scarring after GFS.
Fibroblast proliferation and vascularization
The antimetabolites 5-FU and MMC are the main agents currently used to inhibit scarring and subsequent IOP increase and blindness after trabeculectomy. Lately, mechanisms of prolonged release of 5-FU and MMC are sought ( Box 28.4 ). We have achieved the formulation of tablets that release 5-FU for more than 8 hours in our lab model, which mimics the bleb. Prolonged release of 5-FU may enhance the success of trabeculectomies. Slow-release MMC-loaded hydrogels were shown to inhibit cell proliferation in an in vitro model. In the future, these gels, placed in the bleb, could find an application in GFS in humans. It was recently reported that repeated exposure of HTFs in vitro to MMC increases the expression of P-glycoprotein, a protein that creates multidrug resistance by lowering intracellular concentration of various drugs. This finding may explain failure of repeated trabeculectomies despite the application of MMC. 5-FU seems to be a better option for repeated trabeculectomies as it does not increase the expression of P-glycoprotein.
