Modulation of wound healing during and after glaucoma surgery

The process of wound healing

Wound healing is a cascade of overlapping processes that include hemostasis, inflammation, cell proliferation, and remodeling. After injury, blood coagulation takes place and fibrin clots are formed to reduce blood loss. This is followed by an inflammatory phase where neutrophils, macrophages, and lymphocytes are attracted to the

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region. This leads to a proliferative phase where fibroblasts migrate into the site of injury and re-epithelialization, angiogenesis, and formation of granulation tissue occur. Finally, remodeling of the tissue takes place and involves the formation of scar tissue. Many different cell types take part in wound healing, including fibroblasts, keratinocytes, endothelial cells, neutrophils, macrophages, lymphocytes, and mast cells (Lobmann et al., 2004).

Several treatments and surgical approaches have been developed to successfully modulate scarring after glaucoma filtration surgery (GFS). Steroids applied topically and systemically reduce inflammation and fibrosis. Anticancer agents such as

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mitomycin-C (MMC) inhibit fibroblast function and survival when applied locally. However, as these agents have significant side effects, safer and more effective drugs are required. In this chapter, we review current and future agents in development to modulate healing and scarring in glaucoma surgery.

Using surgical and anatomical principles to modify therapy

By revisiting the basic principles of surgery and fluid flow through the bleb, we have shown that simple changes in surgical and antimetabolite application technique can dramatically lower side effects even when the same concentrations of antimetabolites are used (Wells et al., 2003, 2004) (Figs. 1–3). This stresses the importance of combining new therapies with appropriate surgical techniques to maximize the benefit of any therapy. For instance, techniques that minimize tissue damage are clearly important.

The principle of using spacers is well established with the use of tube implants, with plate spacers to keep the subconjunctival space open. Physical spacers using tissue such as amniotic membrane may increase the success of GFS. Human amniotic membrane appears to have antiangiogenic, antiinflammatory, and antifibrotic characteristics. The effects of amniotic membrane have been tested both in animal models of GFS (Barton et al., 2001; Demir et al., 2002) and in humans (Fujishima et al., 1998; Bruno et al., 2006) with encouraging results. An interesting approach to the application of amniotic membrane in GFS was reported in trabeculectomies of high-risk patients, who had undergone at least two or more trabeculectomies with MMC. Two pieces of amniotic membrane soaked in MMC were sutured, one under the scleral flap and one into the subconjunctival space. After 6–18 months, the IOP was found to be significantly reduced (Drolsum et al., 2006). A bioequivalent gel may in future perform the function of amniotic membranes. Furthermore, amniotic membrane transplantation can be used instead of conjunctival advancement to repair lateonset bleb leakage (Rauscher et al., 2007). Gas, such as perfluoropropane or sodium hyaluronate 2.3%, may improve subconjunctival drainage

Fig. 1. Simple changes in surgical technique, which have markedly reduced bleb-related complications.

Fig. 2. Focal cystic bleb after exposure to mitomycin C before the changes in surgical technique.

Fig. 3. Improvement in bleb morphology using the new technique and same dose of mitomycin C.

spaces with resultant creation of more diffuse blebs (Wong et al., 1999; Lopes et al., 2006).

In a different area of surgery in the sub-Tenon’s space, strabismus surgery, polyurethane sheets, with or without a dexamethasone sustained drug delivery system, significantly reduce adhesions (Kim et al., 2004). Polytetrafluoroethylene (PTFE), Seprafilm (a biodegradable membrane made of sodium hyaluronate and carboxymethylcellulose), ADCON-L (a polyglycan ester), Interceed (a cellulose matrix), sodium hyaluronate and polyglactin mesh, and future variations may also be used in GFS in the future. Devices made of relatively inert materials already used in other spaces such as the suprachoroidal space may also facilitate aqueous outflow by keeping the surgical field free of scar tissue. However, in many cases the continuous inflammatory reaction due to the presence of the biomaterial leads to excessive scarring and poor postoperative results.

Blood clotting and fibrin formation

Based on the principle that fibrin is a critical part of healing, agents that break down fibrin, such as tissue plasminogen activator, are effective in lysing blood clots after surgery (WuDunn, 1997). Although, in the short term, fibrinolytic agents may lower intraocular pressure (IOP), one of the side effects that has inhibited further use of such agents is the increased risk of prolonged bleeding. Furthermore, fibrin breakdown molecules may have a longer-term stimulatory effect on the induction of scarring (Gray et al., 1993).

Inflammatory cells and mediators

Inflammatory cells and mediators released during and after surgery stimulate scarring. The grading system we developed in our long-term Medical Research Council (MRC) trial showed a good correlation between inflammation and the longterm outcome (www.blebs.net; Fig. 4). There is good evidence that topical steroids, used as part of routine postoperative management, are effective in reducing inflammation (Kent et al., 1998). Intrableb triamcinolone acetonide injection at the conclusion of GFS has been reported to be

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clinically beneficial with regards to IOP reduction and a relatively safe method for steroid administration (Tham et al., 2006). Topical nonsteroidal anti-inflammatory drugs (NSAIDs) may also be effective (Kent et al., 1998), but their use is still controversial.

Other agents reducing inflammation include cyclosporine and cyclooxygenase-2 inhibitors, although the effect of intraoperative or postoperative application of cyclosporine in an animal model was not conclusive (Lattanzio et al., 2005). A novel approach to the inhibition of inflammatory cytokines is 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 from 30 to 80% (Shaunak et al., 2004). This experimental result is very encouraging and far more effective than that seen with intensive topical steroid drops (Fig. 5).

Growth factors

The tissues in a wound and specifically in GFS as well as the aqueous flowing through the bleb contain a large number of growth factors or cytokines (Chang et al., 2000). Transforming growth factor beta (TGF-b) has been shown to be more stimulatory than other growth factors and cytokines found in the aqueous (Khaw et al., 1994). TGF-b may even reverse the effect of MMC in vivo (Khaw et al., 1994). Therapeutic strategies that modulate the activity of growth factors including TGF-b may be useful inhibitors of fibrosis. Tranilast ((N-(3u,4u)-dimethoxycinnamoyl) anthranilic acid)) is effective against TGF-b activity and has antiscarring potential when used in GFS. TGF-b activity is also inhibited by genistein and suramin. Suramin reduces postoperative scarring in an experimental model of GFS (Mietz et al., 1998), and an early pilot study has been encouraging (Mietz and Krieglstein, 2001). Interferon alpha (IFN-a), a cytokine with antifibrotic action, also reduces scarring activity of

fibroblasts, although a clinical trial did not show it to be significantly better than antimetabolites (Gillies et al., 1999).

As, in the eye, TGF-b seems more important than other growth factors (Khaw et al., 1994), we have used a variety of biological mechanisms to block TGF-b activity, including antisense oligonucleotides (Cordeiro et al., 2003) and a human monoclonal antibody against the active form of human TGF-b2, the predominant isoform in the aqueous (lerdelimumab, Trabios, Cambridge Antibody Technology, Cambridge, UK). Unlike antimetabolites, one of the theoretical advantages of the monoclonal antibody is its target specificity: it only acts if there is TGF-b2 in the wound, minimizing the risk of adverse events such as hypotony. In an in vivo model of conjunctival scarring, it significantly improved the outcome of GFS (Cordeiro et al., 1999a) 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, often observed after use of antimetabolites. However, two larger randomized controlled trials have not shown a significant effect on the outcome of GFS (Khaw et al., 2007). Based on the data obtained from an earlier study (Cordeiro et al., 1999a), we believe that the dose used was not sufficient. Subsequent experiments from our laboratory have shown a significantly enhanced effect with a prolonged dosing regimen (Mead et al., 2003), and the data also suggested an enhanced effect in the GFS outcome when the antibody

is combined with intraoperative 5-fluorouracil (5-FU). Due to the encouraging results, further studies are planned with different dosing regimens.

The importance of blocking TGF-b2 in order to control scarring after GFS was shown in a recent study in which tissue transglutaminase (tTgase) and its end product e-(g-glutamyl)-lysine were

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detected in scarred tissue of failed trabeculectomy blebs. tTgases are calcium-dependent enzymes that cross-link proteins using e-(g-glutamyl)-lysine bonds. Since vertebrates lack enzymes capable of hydrolyzing these bonds, the protein cross-linking created by tTgases seems to be unbreakable (Priglinger et al., 2006). tTgase cross-links fibronectin and collagen 3; these proteins are produced by human Tenon’s layer fibroblasts (HTFs) in vitro and have been detected in the scar tissue deposited in the bleb area after GFS. TGF-b2, known for enhancing conjunctival scarring after GFS, was shown in the same study to trigger the expression of tTgase and subsequently the crosslinking of fibronectin in vitro (Priglinger et al., 2006). In vivo, this pathway might lead to enhanced cross-linking of the newly formed scar tissue and subsequent failure of the GFS, leading again to the conclusion that inhibition of TGF-b2 could extend the success of the surgery.

Alternative inhibitors of TGF-b include decorin, a small proteoglycan, which is a natural TGF-b- inhibitor. In an in vivo experimental model of GFS, the outcome of preoperative and postoperative application of decorin was encouraging as it decreased fibrosis and delayed the increase of IOP (Grisanti et al., 2005). In vitro, silencing RNA (siRNA) against TGF-b receptor II–mRNA reduced the production of TGF-b receptor II as well as the production and deposition of fibronectin and the migration of human corneal fibroblasts. The application of the same siRNA molecule reduced inflammation and deposition of extracellular matrix (ECM) in an in vivo model of subconjunctival scarring after GFS (Nakamura et al., 2004). Furthermore, encapsulated siRNA molecules in poly (D,L-lactide-co-glycolide) microspheres, targeting TGF-b2, applied as a single injection after trabeculectomy led to 100% survival of the bleb for more than a month (Gomes dos Santos et al., 2006).

Targeting intracellular signaling downstream of the TGF-b receptor could be another effective strategy. Within the cell, the main TGF-b signaling pathway runs through proteins that activate transcription of genes that encode the Smad proteins. Of particular relevance is Smad3, which is essential for TGF-b-induced production of

ECM proteins (Chen et al., 1999; Massague, 1999). Inhibiting Smad3 in immediate postoperative applications might prove beneficial (Leask and Abraham, 2004). Smad7, acting differently from Smad3, is another potential therapeutic target. As TGF-b can suppress its action through the induction of Smad7 (negative feedback loop), gene transfer of the Smad7 gene has been shown in animal models to have a protective effect against the development of lung, liver, and renal fibrosis (Schiller et al., 2004).

Connective tissue growth factor (CTGF) is a factor that influences ECM production and subsequent scar formation and fibrosis. TGF-b1 triggers the expression of CTGF, which is also necessary for TGF-b stimulation of myofibroblast differentiation and collagen contraction (Garrett et al., 2004). Inhibition of this factor could be a possible future therapeutic target (Wu et al., 2006). Myofibroblasts express a platelet-activating factor (PAF) nuclear receptor and TNF-b receptors; PAF and TNF-b cause time-dependent myofibroblast apoptosis. Future therapeutic approaches may take advantage of the expression of these receptors (He and Bazan, 2006).

TGF-b was recently shown to increase cell tension in HTF cultures and contraction in HTF collagen I gels by triggering the activation of GTPase Rho, which regulates actin cytoskeleton remodeling and cell contractility. Rho activates the serine–threonine kinase, Rho-associated kinase (ROCK), that enhances cytoskeletal tension and results in actomyosin-mediated contraction. ROCK inhibitors reduced cell tension and inhibited the TGF-b-mediated p38 activation, alpha smooth muscle actin (alpha-SMA) expression, and HTF development of enhanced contractile abilities characteristic of the so-called myofibroblast phenotype, not differentiation (Meyer-ter-Vehn et al., 2006). The effectiveness of ROCK inhibitor Y-27632 has been tested in vitro and in vivo (Honjo et al., 2007). In vitro, Y-27632 inhibited contraction of HTF-seeded collagen I gels and alpha-SMA expression by HTFs. In vivo, application of Y-27632 after GFS in rabbits resulted in significant increase in the survival of the blebs compared to controls. Additionally, histological analysis revealed reduction of collagen I