deposition and scarring in the treated blebs compared to controls (Honjo et al., 2007). Application of ROCK inhibitors may block TGF-b-induced scarring by downregulating pathways generating mechanical tension and thereby improve the success of GFS.

Another growth factor that activates certain ocular fibroblasts is platelet-derived growth factor (PDGF). Some ocular fibroblasts express high levels of PDGF-b. When stimulated, these fibroblasts proliferate, migrate, and produce ECM molecules, causing contraction, fibrosis, and development of fibrotic membranes such as those observed in proliferative vitreoretinopathy (PVR; Nagineni et al., 2005). 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 (Akiyama et al., 2006), and may also be useful in glaucoma surgery.

The potential involvement of p38 mitogen-acti- vated protein kinase (p38MAPK) in the induction of fibrogenic reaction has also been investigated. p38MAPK is believed to trigger the transcription of Smad2/3, facilitate the phosphorylation and activation of Smad3, and subsequently the formation of Smad3/4 complex, which is important for the development of the fibrotic reaction. The Smad3 signaling pathway is important in retinal fibrosis. Inhibition of Smad3 is associated with a reduction of a cellular fibrotic reaction (Saika et al., 2004). Adenoviral transfer by intravitreal application of a dominant negative p38MAPK gene demonstrated reduced fibrotic reaction of RPE cells after retinal detachment (Saika et al., 2005). In later studies, adenoviral gene transfer of the same dominant negative p38MAPK gene was applied to in vitro cultured HTFs (Meyer-ter-Vehn et al., 2006) and to an in vivo conjunctival scarring model in mice (Yamanaka et al., 2007). Reduction of the differentiation of fibroblasts to myofibroblasts and decline of the 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

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sources of TGF-b, the reduction in levels of MCP-1 through inhibition of p38MAPK seems to reduce the levels of TGF-b; hence its favorable role in conjunctival scarring (Cordeiro et al., 1999b; Dewald et al., 2005; Saika, 2006; Yamanaka et al., 2007). Therefore, the inhibition of p38MAPK has been proposed as a possible future therapeutic option.

Bone morphogenic proteins (BMPs) are growth factors with an important role not only in embryonic development and morphogenesis but also in postnatal life. They are vital for cell proliferation, differentiation, apoptosis, angiogenesis, and other biological functions (Reddi, 1997a, b; Chen et al., 2004). Activins are dimeric proteins that participate in the mechanisms of cell differentiation and proliferation, apoptosis, inflammation and neurogenesis (Tsuchida, 2004). 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 (Andreev et al., 2006). The use of follistatin, an inhibitor of activin bioactivity, was shown to be effective in decreasing liver and lung fibrosis (Aoki et al., 2005; Patella et al., 2006). 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 (Andreev et al., 2006).

Cellular proliferation and vascularization

The main agents used to inhibit scarring and subsequent IOP increase after trabeculectomy are the anticancer agents 5-FU and MMC. Recently, an interesting approach of a slow release mechanism of MMC was published. 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 (Blake et al., 2006). It has been reported recently 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.

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5-FU seems to be a better option for repeated trabeculectomies as it does not increase the expression of P-glycoprotein (Hueber et al., 2007). Beta-irradiation, which we have shown to have similar effects on controlling the proliferation of fibroblasts (Khaw et al., 1991), significantly improved the success of GFS in a large African trial using a dose based on our laboratory studies (Kirwan et al., 2003, 2006) and may also be useful in pediatric GFS (Akimoto et al., 1998).

Additional future interesting experimental strategies to modulate proliferation include overexpressing genes that inhibit fibroblast proliferation, such as p21 WAF-1/CIP-1 introduced via an adenovirus system (Perkins et al., 2002), antagonizing integrins and their receptors (Paikal et al., 2000), or altering intracellular gene transcription (Akimoto et al., 1998). Paclitaxel, an antineoplasmatic agent that prevents mitosis by blocking the depolymerization of intracellular microtubules, has been reported to inhibit HTF proliferation and prolong the success of GFS. But as paclitaxel is hydrophobic, its administration in the subconjunctival area is not easy. Polyanhydride disks (Jampel et al., 1993) and Carbopol 980 hydrogel (Koz et al., 2007) containing paclitaxel have been tested with encouraging results comparable to the antiscarring effects of MMC, although the development of severe inflammation, especially in the case of polyanhydride disks, has been reported (Jampel et al., 1993).

Photodynamic therapy with a diffuse blue light coupled with a photosensitizing agent to kill fibroblasts may also be another way to control healing in the area of treatment (Grisanti et al., 2000). Over the past decade, many studies have demonstrated that photodynamic therapy reduces neovascularization and subsequent maturation of scar tissue after glaucoma filtration. Intraoperative photodynamic therapy using preoperative subconjunctival application of ethyl etiopurin (Hill et al., 1997) and 2u,7u-bis-(2-carboxyethyl)-5-(and-6)-car- boxyfluorescein acetoxymethyl ester (BCECF-AM) as photosensitizers (Grisanti et al., 1999) has proven to be safe and effective against scarring after GFS in vivo. Pilot clinical trials of intraoperative photodynamic therapy with locally administered BCECF-AM have shown promising results

(Diestelhorst and Grisanti, 2002; Jordan et al., 2003). Furthermore, postoperative photodynamic therapy has been reported to be effective against neovascularization and scarring as well. Intravenous administration of the photosensitizer verteporfin in the early postoperative period to occlude the newly formed capillaries in the bleb area had favorable results in the modulation of wound healing in rabbits after GFS (Stasi et al., 2006). More recently the VEGF antagonist Bevacizumab has been used clinically in GFS to reduce vascularity, which we have found greatly increases the risk of GFS failure using a masked scoring system.

Cell motility, matrix contraction and synthesis

Bleb analysis using carefully graded masked digital photographs reveals that tissue contraction is a critical component of filtration surgery failure. We have developed a more detailed understanding of the processes that occur during tissue contraction and have recently been able to image cells and matrix simultaneously during the process of contraction. The matrix metalloproteinases (MMPs) are enzymes that degrade ECM. Based on laboratory observations, we discovered that cell-mediated collagen contraction could be inhibited using MMP inhibitors (Daniels et al., 2003). In an experimental model of glaucoma surgery, the use of an MMP inhibitor led to a dramatic reduction of scarring, with retention of normal tissue morphology. This action is equivalent to MMC, but without the deleterious side effects (Wong et al., 2003, 2005). A number of agents can also affect the cytoskeleton of the cell and hence inhibit migration. Taxol and etoposide (microtubulestabilizing agents) have been used in models of filtration surgery, and they prolong bleb survival (Jampel and Moon, 1998). b-Aminopropionitrile and D-penicillamine interfere with molecular crosslinking of collagen, and there is experimental and clinical evidence that they may work in filtration surgery (Jampel et al., 1998). MMP inhibition also surprisingly results in a reduction of collagen synthesis in vitro (Daniels et al., 2003), which may help to explain the dramatic reduction in scar tissue formation in vivo (Wong et al., 2003).