Free
Glaucoma  |   August 2013
The Role of LOX and LOXL2 in Scar Formation After Glaucoma Surgery
Author Affiliations & Notes
  • Tine Van Bergen
    The Laboratory of Ophthalmology, Katholieke Universiteit Leuven, Leuven, Belgium
  • Derek Marshall
    Gilead Sciences, Foster City, California
  • Sara Van de Veire
    The Laboratory of Ophthalmology, Katholieke Universiteit Leuven, Leuven, Belgium
  • Evelien Vandewalle
    The Laboratory of Ophthalmology, Katholieke Universiteit Leuven, Leuven, Belgium
  • Lieve Moons
    Unit of Animal Physiology and Neurobiology, Biology Department, Katholieke Universiteit Leuven, Leuven, Belgium
  • Jean Herman
    Interface Valorization Platform (IVAP), Katholieke Universiteit Leuven, Leuven, Belgium
  • Victoria Smith
    Gilead Sciences, Foster City, California
  • Ingeborg Stalmans
    The Laboratory of Ophthalmology, Katholieke Universiteit Leuven, Leuven, Belgium
  • Correspondence: Ingeborg Stalmans, University Hospitals Leuven, Department of Ophthalmology, Kapucijnenvoer 33, B-3000, Leuven, Belgium; ingeborg.stalmans@uzleuven.be
Investigative Ophthalmology & Visual Science August 2013, Vol.54, 5788-5796. doi:10.1167/iovs.13-11696
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Tine Van Bergen, Derek Marshall, Sara Van de Veire, Evelien Vandewalle, Lieve Moons, Jean Herman, Victoria Smith, Ingeborg Stalmans; The Role of LOX and LOXL2 in Scar Formation After Glaucoma Surgery. Invest. Ophthalmol. Vis. Sci. 2013;54(8):5788-5796. doi: 10.1167/iovs.13-11696.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: The aim of this study was to elucidate the role of lysyl oxidase (LOX) and lysyl oxidase like (LOXL) 2 in pathologic wound healing after glaucoma surgery. We therefore investigated the expression of LOX and LOXL2 and evaluated the therapeutic potential of anti-LOX (GS-639556, formerly M64) and anti-LOXL2 (GS-607601, formerly AB0023) antibodies in a rabbit model of glaucoma trabeculectomy.

Methods.: Ocular expression of LOX and LOXL2 was investigated by immunohistologic staining at different time points after trabeculectomy. Treatment with GS-639556 or GS-607601 was initiated in rabbits immediately after trabeculectomy by giving both intracameral and subconjunctival injections. Thereafter, the antibodies were given twice a week subconjunctivally until day 30 after surgery (day of euthanization). Treatment outcome was studied by clinical investigation of the bleb and by immunohistochemical analysis of angiogenesis, inflammation, and collagen deposition.

Results.: LOX and LOXL2 were both upregulated in Tenon's capsule and the conjunctiva after glaucoma surgery. Repeated administration of LOX- or LOXL2-targeting monoclonal antibodies increased bleb area and bleb survival. Analyses of immunohistologic stainings showed that both antibodies significantly decreased fibrosis, whereas the anti-LOXL2 antibody also significantly reduced blood vessel density and inflammation.

Conclusions.: Targeting LOXL2 with an inhibitory monoclonal antibody (GS-607601) reduced pathologic angiogenesis, inflammation, and fibrosis. These results suggest that LOXL2 could be an appealing target for treatment of scar formation after glaucoma surgery, and point to the potential therapeutic benefits of simtuzumab, a humanized monoclonal antibody derived from GS-607601.

Introduction
The lysyl oxidase family is an important class of extracellular matrix (ECM) crosslinking enzymes. Lysyl oxidase (LOX) is the best characterized member of this family and is known as an amine oxidase that catalyzes crosslinking of collagen and elastin in the ECM via generation of aldehydes on lysine residues. 1,2 Several additional lysyl oxidase family members have been identified (LOXL1, ‐2, ‐3 and ‐4) and are fully functional. 3 LOX and LOXL proteins are expressed during development and maintenance in the skin, aorta, heart, lung, liver, cartilage, kidney, stomach, small intestine, colon, ovaries, testis, and brain of the mouse. 4 Given their roles in disease-associated modification of the ECM, 1,2 LOX family enzymes have also been described as critical contributors to the development of a variety of fibrotic-related diseases, including liver and lung fibrosis, 5 tumor progression, 5,6 and neurodegenerative and cardiovascular diseases. 7,8 Targeting LOXL2 with an inhibitory monoclonal antibody 9 has been shown to be efficacious in rodent models of cancer, as well as in liver and lung fibrosis models. 5 Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts and endothelial cells, decreased production of growth factors and cytokines, and decreased TGF-β pathway signaling. 5  
In healthy ocular tissues, lysyl oxidase activity is present in the vitreous, iris, ciliary body, lens, choroid, retinal pigment epithelium, and retina. 2 Although a majority of blinding ocular diseases are associated with a disruption of the tissue architecture in the eye caused by vascular leakage and fibrosis, 10 little information is available about LOXL involvement in ocular diseases. The vitreous levels of lysyl oxidase activity were significantly decreased in proliferative diabetic retinopathy and rhegmatogenous retinal detachment. These reduced lysyl oxidase levels might be associated with an inadequate cross-linking that causes ECM changes, known to be present during these diseases. 2 An indirect effect of LOX in corneal wound healing is suggested by Schultz et al., who showed that administration of TGF-β on human ocular fibroblasts could increase LOX expression as well as synthesis of ECM components, resulting in ECM remodeling. 11 Recent studies showed that polymorphisms of LOXL1 are associated with a significantly increased risk of exfoliation glaucoma, 12 suggesting a potential role for the LOX family in the pathogenesis of this disease. Indeed, a very recent study showed that LOX expression was upregulated in trabecular meshwork (TM) cells derived from glaucoma patients. This suggests that increased LOX activity might be responsible for an increased aqueous humor outflow resistance by inducing deposition of ECM material within the TM. 13 Progressive fibrosis is not only associated with the pathogenesis of glaucoma, but can also be a consequence of surgical treatment to lower the IOP. Surgical failure is indeed characterized by an excessive postoperative wound healing response with subsequent scarring. 14,15 The role of LOXL in the process of wound healing after glaucoma surgery, however, is still unknown. 
The first aim of this study was to characterize the expression of LOX and LOXL2 in pathologic wound healing after glaucoma filtration surgery. Next, we investigated the therapeutic antifibrotic potential of anti-LOX (GS-639556) and anti-LOXL2 (GS-607601) antibodies in a rabbit model of glaucoma filtration surgery. In addition, the possible anti-angiogenic and anti-inflammatory effects of both antibodies were analyzed. 
Materials and Methods
All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The Institutional Animal Care and Research Advisory Committee of the Katholieke Universiteit (KU) Leuven approved all experimental animal procedures. 
Rabbit Model of Filtration Surgery
Female New Zealand rabbits (animal facility of KU Leuven), 12 to 14 weeks of age and weighing 2 to 3 kg, were used. Before surgery, the IOP was measured in both eyes with a tonometer (Tono-Pen; Medtronic Solan, Jacksonville, FL) under 4 mg/mL oxybuprocaine hydrochloride topical anesthesia (Unicaine; Thea Pharma, Schaffhaussen, Switzerland). Three recordings per eye were averaged. General anesthesia was induced by intramuscular injection of 1.2 mL ketamine (Ketalar, 50 mg/mL; Pfizer, Ann Arbor, MI) and 0.8 mL xylazine (Rompun, 2%; Bayer Health Care, Pittsburgh, PA). Surgery was performed on both eyes, using a technique previously described. 16 In a first experiment, four rabbits (8 eyes) were used to determine LOX and LOXL2 localization on days 3, 8, 14, and 30 after surgery by immunohistochemistry. In a second experiment of 12 rabbits, six rabbits were treated with the anti-LOX antibody (GS-639556, formerly M64; Gilead Sciences, Foster City, CA), whereas the other six rabbits were treated with the anti-LOXL2 antibody (GS-607601, formerly AB0023; Gilead Sciences), immediately after surgery. In all rabbits, one eye was injected with the antibody and the contralateral eye was used as a control and received an injection of PBS. For each eye, 200 μL (0.6 mg) were injected in the anterior chamber (AC) and 100 μL (0.3 mg) were injected subconjunctivally (SC) into the filtration bleb immediately after surgery. Thereafter, repeated injections (0.3 mg/injection) were given twice a week subconjunctivally until day 30 after trabeculectomy (day of euthanization). Rabbits were clinically examined on day 1 after surgery and then every 2 days until they were killed. IOP, bleb area (width × length) and height were analyzed under topical anesthesia using calipers. Bleb survival was taken as the endpoint of the study; bleb failure was defined as the appearance of a scarred and flat bleb. The examiner of the rabbits was masked to the treatment allocation. 
Immunohistochemistry
On the day of euthanization, rabbits were killed using a lethal intravenous injection of sedative. Both eyes were enucleated: a superior piece of the bleb (fibrotic conjunctiva) and an inferior part of the conjunctiva and Tenon's capsule (nonfibrotic) were collected. The tissues were fixed overnight in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Serial sections (7 μm) were cut and subjected to different (immuno) histochemical stainings. Hematoxylin and Eosin (H&E; Merck, Darmstadt, Germany) staining was performed on the first serial section to locate the bleb. To investigate LOX and LOXL2 expression at the protein level, immunohistologic stainings were performed. Rabbit sections were incubated overnight at room temperature with a monoclonal mouse anti-human LOX (1:1000) or LOXL2 antibody (1:200). Inflammation was analyzed by anti-CD45 staining and anti-CD31 staining was performed to identify the blood vessels. The samples were incubated overnight with mouse anti-rabbit CD45 antibody (1:100, MCA808; AbDSerotec, Oxford, UK) or mouse anti-human CD31 antibody (1:200, M0823; Dakocytomation, Copenhagen, Denmark), respectively. The following day, all sections were incubated with peroxidase-labeled rabbit anti-mouse secondary antibodies and tyramide signal amplification (Renaissance TSA Indirect, NEL700; PerkinElmer, Waltham, MA) with diaminobenzidine (DAB, 32750; Sigma-Aldrich, St. Louis, MO) as a chromogen. Deposition of collagen was analyzed in both groups by Sirius Red staining. 
Imaging and Analysis
Images were obtained using a microscope (Leica Microsystems, Wetzlar, Germany) with a digital camera (Axiocam MrC5; Carl Zeiss, Oberkochen, Germany) at a magnification of 20× and a resolution of 2584 × 1936 pixels. Morphometric analyses were performed using commercial software (Axiovision; Carl Zeiss). The density of blood vessels was determined by calculating the CD31-positive area (area of neovascularization) as a proportion of the total wound area of the bleb. The density of leukocytes was quantified by counting the CD45-positive cells, expressed in number per millimeter squared of the total area. LOX and LOXL2 expression was quantified by calculating the LOXL2 positive area as a percentage of the total area. Fibrosis was evaluated using Sirius Red staining and was determined by measuring the percentage of the area of mature collagen fibers to the total wound area. Polarized light was used to distinguish mature from immature collagen fibers. Mature type I collagen fibers appear bright yellow or orange, whereas immature collagen fibers appear green. 
Expression of Different Wound Healing–Related Factors
Since it is known that different factors in the aqueous humor can influence the process of ocular wound healing, 17 the expression of a variety of such molecules was measured by multi-analyte bead-based immunoassay. Rabbit aqueous humor samples (200 μL) were obtained from rabbits treated with either antibody, at 30 days after surgery. All samples were immediately transferred to and stored at −80°C until analysis. Samples were analyzed by Myriad RBM (Austin, TX) with their Human Inflammation MAP 1.0 panel, whereby levels of various markers including α1-antitrypsin, β-2 microglobulin, intercellular adhesion molecule (ICAM)-1, IL-1β, IL-1 receptor antagonist (IL-1ra), matrix metalloproteinase (MMP)-3, and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) were determined. 
Statistical Analysis
All histologic data were analyzed using the Student's t-test for independent samples. Data at individual time points were analyzed using mixed model analysis for repeated measures and overall P values were calculated (GraphPad Prism 5; GraphPad Software, San Diego, CA). Kaplan-Meier survival analysis was performed for bleb failure using the logrank test (GraphPad Prism 5). Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing–related factors and the bleb area on day 30 (GraphPad Prism 5). P values less than or equal to 0.05 were considered to be statistically significant. Data are represented as mean ± SEM. 
Results
Ocular Expression of LOX and LOXL2
To investigate the localization and the expression of LOX and LOXL2 proteins in the rabbit eye, immunohistochemical staining was performed at different time points after trabeculectomy. At every time point assessed from day 1 to 30 after surgery, LOX and LOXL2 were found to be expressed in the conjunctiva and Tenon's capsule, both in the inferior (nonfibrotic, nonbleb conjunctiva) and in the superior (fibrotic bleb) samples (Figs. 1A, 1B). LOX protein levels were upregulated in the fibrotic tissues as compared with the nonfibrotic tissues at multiple time points after surgery: on day 3 (2.11-fold; P = 0.03), day 14 (6.95-fold; P = 0.02), and day 30 (3.20-fold; P = 0.03) (N = 8; overall P: P = 0.007; Fig. 1C). An upregulation was also measured for LOXL2 protein in the fibrotic tissues. LOXL2 expression was increased compared with nonfibrotic samples at day 3 (5.35-fold; P = 0.02), 14 (3.06-fold; P = 0.01), and at day 30 (2.76-fold; P = 0.001) (N = 8; overall P: P = 0.008; Fig. 1D). In both groups, no significant upregulation was seen at postoperative day 8. 
Figure 1
 
Quantification of the level of LOX and LOXL2 in Tenon's capsule and conjunctiva. Images show representative pictures of the immunostainings for LOX (A) and LOXL2 (B) on day 30 after surgery in the superior conjunctiva (conj) and Tenon. Both LOX (C) and LOXL2 (D) proteins were upregulated in the fibrotic samples as compared with the nonfibrotic tissues (control) on different time points after glaucoma filtration surgery in rabbits (*P < 0.05).
Figure 1
 
Quantification of the level of LOX and LOXL2 in Tenon's capsule and conjunctiva. Images show representative pictures of the immunostainings for LOX (A) and LOXL2 (B) on day 30 after surgery in the superior conjunctiva (conj) and Tenon. Both LOX (C) and LOXL2 (D) proteins were upregulated in the fibrotic samples as compared with the nonfibrotic tissues (control) on different time points after glaucoma filtration surgery in rabbits (*P < 0.05).
Thus, LOX and LOXL2 were both upregulated in Tenon's capsule and conjunctiva after trabeculectomy. Importantly, since LOX and LOXL2 levels were upregulated as early as day 3 after glaucoma surgery, both proteins could play an important role in the early stages of postoperative wound healing. 
Effect of GS-639556 and GS-607601 in a Rabbit Trabeculectomy Model
To determine whether inhibition of LOX or LOXL2 impacted glaucoma surgery outcome in vivo, antibodies targeting each protein were assessed for efficacy in preservation of bleb area and height in a surgical rabbit trabeculectomy model. One group of rabbits received repeated injections of the anti-LOX antibody (GS-639556; 0.6 mg AC and 0.3 mg SC immediately after surgery, 0.3 mg SC twice a week thereafter). Another group of rabbits was treated with repeated injections of the anti-LOXL2 antibody (GS-607601; 0.6 mg AC and 0.3 mg SC immediately after surgery, 0.3 mg twice a week thereafter). Bleb area, height, and survival were analyzed as a measure of filtration surgery outcome. The animals were killed at day 30 after trabeculectomy. 
Figure 2 shows the typical appearance of the bleb after treatment on postoperative day 30. GS-639556 and GS-607601 administration were associated with a large bleb (Figs. 2B, 2D, respectively) compared with a flat and scarred bleb in their respective control groups (Figs. 2A, 2C). Treatment with GS-639556 resulted in a postsurgical increase in bleb area (N = 6; P < 0.0001; Fig. 3A) and bleb height (N = 6; P < 0.0001; Fig. 3B) from day 17 onwards. Repeated injections of GS-639556 significantly prolonged bleb survival after filtration surgery, compared with control, as shown in the Kaplan-Meier survival curve (N = 6; P = 0.0007; Fig. 3C). All blebs had failed in the control group by day 23, whereas all blebs survived until day 30 (day of euthanization) in the GS-639556 group. There was no evidence that inhibition of LOXL2 affected the IOP, as the pressure in both groups remained similar over a period of 30 days (P = NS; data not shown). The immunohistochemical stainings on day 30 showed a significant reduction of 21 ± 5% in collagen deposition in the bleb of the treated eyes compared with control (N = 6; P = 0.0009; Figs. 4C, 4G). There were no significant differences in blood vessel density and inflammation within the treated and control eyes (N = 6; Figs. 4A, 4B, 4G). In the group treated with GS-607601, antibody administration significantly increased bleb area (N = 6; P < 0.0001; Fig. 3D) and bleb height (N = 6; P < 0.0001; Fig. 3E) from day 19 onwards. Anti-LOXL2 antibody prolonged bleb survival in treated eyes compared with control eyes, as shown in the Kaplan-Meier survival curve (N = 6; P = 0.0005; Fig. 3F). All blebs had failed in the control group by day 23, whereas 83% of the blebs survived until day 30 in the GS-607601 treated group (one of the 6 blebs failed at day 27). Thirty days after surgery, immunohistochemical stainings showed a significant reduction of 44 ± 6% in blood vessel density (N = 6; P = 0.01; Figs. 4D, 4G), 32 ± 5% decrease in inflammation (N = 6; P = 0.01; Figs. 4E, 4G), and 16 ± 4% reduction of collagen deposition (N = 6; P = 0.01; Figs. 4F, 4G) in the blebs of the treated eyes compared with controls. Of note, in both groups the inferior conjunctiva showed no difference in blood vessel density, inflammation, or collagen deposition in the treated versus control eyes (Figs. 4A–F; nonbleb bars). 
Figure 2
 
Macroscopic postoperative photographs of rabbit eyes after surgery. Macroscopic photographs of rabbit eyes at day 30, after repeated anti-LOX (B) or anti-LOXL2 (D) antibody injections show surviving blebs that remain diffusely elevated compared with their respective controls (A, C).
Figure 2
 
Macroscopic postoperative photographs of rabbit eyes after surgery. Macroscopic photographs of rabbit eyes at day 30, after repeated anti-LOX (B) or anti-LOXL2 (D) antibody injections show surviving blebs that remain diffusely elevated compared with their respective controls (A, C).
Figure 3
 
Clinical investigation of rabbit eyes after surgery and administration of anti-LOX(L2) antibodies. Treatment with anti-LOX (GS-639556) and anti-LOXL2 (GS-607601) significantly improved the outcome of surgery by increasing the bleb area ([A, D] P < 0.0001) and bleb height ([B, E] P < 0.0001) from day 17 to 19 onwards, respectively, compared with control. As shown in the Kaplan-Meier survival curve (C, F), both antibodies significantly prolonged bleb survival after filtration surgery, compared with control.
Figure 3
 
Clinical investigation of rabbit eyes after surgery and administration of anti-LOX(L2) antibodies. Treatment with anti-LOX (GS-639556) and anti-LOXL2 (GS-607601) significantly improved the outcome of surgery by increasing the bleb area ([A, D] P < 0.0001) and bleb height ([B, E] P < 0.0001) from day 17 to 19 onwards, respectively, compared with control. As shown in the Kaplan-Meier survival curve (C, F), both antibodies significantly prolonged bleb survival after filtration surgery, compared with control.
Figure 4
 
Neovascularization, inflammation, and collagen deposition in rabbit eyes. The density of blood vessels and leukocytes was determined by calculating the CD31-positive area and by counting the CD45-positive cells, respectively, as a proportion of the total wound area of the bleb. Fibrosis was stained by Sirius Red and was determined by measuring the percentage of the area of mature collagen fibers in the total wound area of the bleb using polarized light. (AC) Treatment with anti-LOX antibody (GS-639556) showed no significant differences in neovascularization and inflammation compared with PBS-treated eyes. The process of collagen deposition was significantly decreased (by 21%) after inhibition of LOX at the filtration site on day 30 as compared with the control eyes. (DF) Treatment of anti-LOXL2 (GS-607601) showed significant reductions of 44% in neovascularization, 32% in inflammation, and 16% in collagen deposition in the bleb of the treated eyes compared with controls. In both groups the inferior conjunctiva showed no difference in blood vessel density, inflammation, or collagen deposition in the treated and control eyes (*P < 0.05). (G) The images show representative pictures of immunostainings of eyes treated with the anti-LOX or anti-LOXL2 antibody or control eyes, at 30 days after surgery (magnification of ×20).
Figure 4
 
Neovascularization, inflammation, and collagen deposition in rabbit eyes. The density of blood vessels and leukocytes was determined by calculating the CD31-positive area and by counting the CD45-positive cells, respectively, as a proportion of the total wound area of the bleb. Fibrosis was stained by Sirius Red and was determined by measuring the percentage of the area of mature collagen fibers in the total wound area of the bleb using polarized light. (AC) Treatment with anti-LOX antibody (GS-639556) showed no significant differences in neovascularization and inflammation compared with PBS-treated eyes. The process of collagen deposition was significantly decreased (by 21%) after inhibition of LOX at the filtration site on day 30 as compared with the control eyes. (DF) Treatment of anti-LOXL2 (GS-607601) showed significant reductions of 44% in neovascularization, 32% in inflammation, and 16% in collagen deposition in the bleb of the treated eyes compared with controls. In both groups the inferior conjunctiva showed no difference in blood vessel density, inflammation, or collagen deposition in the treated and control eyes (*P < 0.05). (G) The images show representative pictures of immunostainings of eyes treated with the anti-LOX or anti-LOXL2 antibody or control eyes, at 30 days after surgery (magnification of ×20).
Thus, repeated administration of a LOX inhibitor (GS-639556) and a LOXL2 inhibitor (GS-607601) improved surgical outcome by increasing bleb area, bleb height, and bleb survival. Anti-LOX antibody reduced collagen deposition, whereas anti-LOXL2 antibody significantly reduced blood vessel density, inflammation, and collagen deposition in the blebs at postoperative day 30. 
Upregulation of Different Wound Healing–Related Factors in Aqueous Humor
On day 30, aqueous humor was collected from both rabbit eyes after treatment. The expression level of different factors related to wound healing in aqueous humor was analyzed by multi-analyte bead-based immunoassay. Anti-LOX treatment increased the expression of β-2 microglobulin (1.26-fold; P = 0.002), ICAM-1 (2.18-fold; P = 0.04), and RANTES (2.10-fold; P = 0.04) in the aqueous humor compared with control (Figs. 5A–C). Alpha1-antitrypsin (5.17-fold; P = 0.02), β-2 microglobulin (1.18-fold; P = 0.04), ICAM-1 (3.60-fold; P = 0.02), IL-1β (7.15-fold; P = 0.02), IL-1ra (3.49-fold; P = 0.04), and MMP-3 (4.62-fold; P = 0.03) were upregulated in the aqueous humor after anti-LOXL2 treatment compared with control eyes (Figs. 5D–I). 
Figure 5
 
Upregulation of different wound healing related factors in rabbit aqueous humor. The expression level of different factors related to wound healing in aqueous humor was analyzed by Myriad RBM with their Human Inflammation MAP 1.0 panel. (AC) Anti-LOX treatment (GS-639556) increased the expression levels of β-2 microglobulin (P = 0.002), ICAM-1 (P = 0.04), and RANTES (P = 0.04) in the rabbit aqueous humor compared with control. (DI) Beta-2 microglobulin (P = 0.04), ICAM-1 (P = 0.02), α1-antitrypsin (P = 0.02), IL-1β (P = 0.02), IL-1 ra (P = 0.04), and MMP-3 (P = 0.03) were upregulated after anti-LOXL2 treatment (GS-607601) compared with control eyes.
Figure 5
 
Upregulation of different wound healing related factors in rabbit aqueous humor. The expression level of different factors related to wound healing in aqueous humor was analyzed by Myriad RBM with their Human Inflammation MAP 1.0 panel. (AC) Anti-LOX treatment (GS-639556) increased the expression levels of β-2 microglobulin (P = 0.002), ICAM-1 (P = 0.04), and RANTES (P = 0.04) in the rabbit aqueous humor compared with control. (DI) Beta-2 microglobulin (P = 0.04), ICAM-1 (P = 0.02), α1-antitrypsin (P = 0.02), IL-1β (P = 0.02), IL-1 ra (P = 0.04), and MMP-3 (P = 0.03) were upregulated after anti-LOXL2 treatment (GS-607601) compared with control eyes.
Although an upregulation of different factors was seen in the antibody-treated groups, several treatment groups had a large distribution of the results, whereas the PBS-treated eyes had more tightly outcomes. Moreover, this variation was also seen in the values of the bleb area, especially in the anti-LOXL2 treated eyes (Fig. 3D). Therefore, Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing related factors and the bleb area on day 30 after anti-LOXL2 treatment. Significant correlations between different factors and bleb features were found. Wound healing promoting factors were negatively correlated with the bleb area. Thus, eyes with a high/low expression of wound healing promoting factors were correlated to small/large blebs, respectively. The wound healing inhibitory factors were positively correlated with the bleb area after anti-LOXL2 treatment. Thus, eyes with a high/low expression of wound healing inhibitory factors were correlated to large/small blebs, respectively. The significant correlations between bleb area on day 30 and expression of different wound healing factors (β-2 microglobulin, ICAM-1, α1-antitrypsin, IL-1β, IL-1ra, and MMP3) are illustrated in Figure 6
Figure 6
 
Correlations between different factors and bleb features in the anti-LOXL2–treated eyes. Scatterplots showing significant correlations between different factors and bleb area on day 30 after anti-LOXL2 treatment. Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing related factors and the bleb area. (AB) Wound healing promoting factors were negatively correlated with the bleb area: β-2 microglobulin (r = −0.92; P = 0.02) and ICAM-1 (r = −0.87; P = 0.03). (CF) The wound healing inhibitory factors were positively correlated with the bleb area after anti-LOXL2 treatment: α1-antitrypsin (r = 0.94; P = 0.02), IL-1β (r = 0.92; P = 0.02), IL-1ra (r = 0.94; P = 0.02), and MMP-3 (r = 0.94; P = 0.02).
Figure 6
 
Correlations between different factors and bleb features in the anti-LOXL2–treated eyes. Scatterplots showing significant correlations between different factors and bleb area on day 30 after anti-LOXL2 treatment. Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing related factors and the bleb area. (AB) Wound healing promoting factors were negatively correlated with the bleb area: β-2 microglobulin (r = −0.92; P = 0.02) and ICAM-1 (r = −0.87; P = 0.03). (CF) The wound healing inhibitory factors were positively correlated with the bleb area after anti-LOXL2 treatment: α1-antitrypsin (r = 0.94; P = 0.02), IL-1β (r = 0.92; P = 0.02), IL-1ra (r = 0.94; P = 0.02), and MMP-3 (r = 0.94; P = 0.02).
Thus, treatment with LOX- or LOXL2-targeting antibodies led to an aqueous upregulation of different factors after glaucoma surgery, which can influence the process of wound healing. Moreover, significant correlations between different factors and bleb features in the anti-LOXL2–treated eyes were found. 
Discussion
Filtering surgery (trabeculectomy) is the most effective treatment for glaucoma, and is therefore a crucial procedure in the management of this blinding disease. 14 Unfortunately, excessive postoperative wound healing with subsequent scarring frequently leads to surgical failure. 15 Peroperative administration of antimitotic agents, such as mitomycin-C (MMC) and 5-Fluorouracil (5-FU) can improve surgical outcome, 18 but these antimitotics carry a risk of vision-threatening complications. Furthermore, blocking TGF-β, which seemed promising in animal models, 19 was not effective in a human clinical trial. 20 Since the LOXL family plays an important role in the fibrotic process by cross-linking collagen and elastin, we hypothesized that these enzymes might be good target candidates for antifibrotic adjunctive strategies in glaucoma surgery. To investigate the role of LOX and LOXL2 in the wound healing process and to explore the therapeutic potential of blocking antibodies, the standard rabbit model for glaucoma surgery was used. 
We showed for the first time that LOX and LOXL2 were upregulated in Tenon's capsule and conjunctiva after surgery. Therefore, LOX and LOXL2 could play an important role in aberrant ocular wound healing after surgery. Indeed, treatment with either anti-LOX or -LOXL2 targeting antibody significantly increased bleb area and bleb survival compared with control. Moreover, inhibition of LOXL2 reduced blood vessel density and inflammation, whereas both antibodies decreased collagen deposition after surgery. These results are comparable with the study of Barry-Hamilton et al., who observed LOXL2 induction in disease-associated fibrosis and neovascularization, and efficacy in models of fibrosis and cancer after GS-607601 treatment. 5 Importantly, a recent study showed that LOXL2 was expressed in angiogenic endothelial cells as a hypoxia-target and accumulated in the endothelial ECM. 21 Moreover, it is known that induction of angiogenesis regulators, such as VEGF, occurs with similar kinetics as LOXL2 in mouse post ischemic revascularization, suggesting that LOXL2 plays an important role in the endothelial cell angiogenic response. 22  
Karalekas et al. demonstrated that the presence of several molecules in the aqueous humor can influence the process of wound healing, which may increase the risk of scarring after filtration surgery. 17 Therefore, aqueous humor was collected at day 30 and the levels of these proteins after treatment were analyzed by multi-analyte bead-based immunoassay. From literature, we know that aqueous levels of β-2 microglobulin, α1-antitrypsin, IL-1, and MMP are upregulated after glaucoma surgery (Rodriguez-Agirretxe I, et al. IOVS 2012;53:ARVO E-Abstract 2514). 23 Therefore, we believe that those levels were likely induced in the PBS group over naïve rabbits due to surgery. However, anti-LOX antibody treatment further increased the expression of β-2 microglobulin, ICAM-1, and RANTES, factors that are known to stimulate the process of wound healing by increasing inflammation and/or blood vessel formation. 2428 β-2 microglobulin and ICAM-1 were also upregulated after anti-LOXL2 treatment. However, other inhibitory factors, such as α1-antitrypsin, IL-1β, IL-1ra, and MMP-3 also increased, and could have had opposing effects on the wound healing process by inhibiting blood vessel, elastin, and collagen formation. 2935 Moreover, a significant correlation was observed between the expression of these factors and bleb features after anti-LOXL2 treatment. Eyes with high/low expression of wound healing promoting factors were correlated to small/large blebs, respectively, whereas eyes with a high/low expression of wound healing inhibitory factors were correlated to large/small blebs, respectively. Therefore, these data provide one possible explanation for the difference in efficacy between the two monoclonal antibodies. Targeting LOX did not affect blood vessel density and inflammation, since treatment with GS-639556 primarily increased the expression of wound healing promoting factors. On the other hand, GS-607601(anti-LOXL2 antibody) primarily induced factors that inhibit wound healing and therefore could be improving surgical outcome by inhibiting the angiogenic, inflammatory, and profibrotic component of the wound healing process. 
Conclusions
We showed that LOX and LOXL2 play an important role in wound healing after glaucoma filtration surgery. Targeting LOXL2 with an inhibitory monoclonal antibody (GS-607601) had a broader efficacy than targeting LOX, reducing angiogenesis and inflammation, as well as fibrosis. These results render LOXL2 an appealing target for scar formation after glaucoma surgery, and point to the potential therapeutic benefits of the clinical candidate monoclonal antibody simtuzumab. 
Acknowledgments
The authors thank Sofie Beckers, Magda Bressinck, and Ann Verbeek for their technical support. 
Supported by grants from Fonds voor Wetenschappelijk Onderzoek Vlaanderen and by Fund for Research in Ophthalmology. 
Disclosure: T. Van Bergen, P; D. Marshall, Gilead Sciences (E), P; S. Van de Veire, None; E. Vandewalle, None; L. Moons, None; J. Herman, None; V. Smith, Gilead Sciences (E), P; I. Stalmans, Gilead Sciences (F), P 
References
Rodriguez C Rodriguez-Sinovas A Martinez-Gonzalez J. Lysyl oxidase as a potential therapeutic target. Drug News Perspect . 2008; 21: 218–224. [CrossRef] [PubMed]
Coral K Angayarkanni N Madhavan J Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment. Invest Ophthalmol Vis Sci . 2008; 49: 4746–4752. [CrossRef] [PubMed]
Molnar J Fong KS He QP Structural and functional diversity of lysyl oxidase and the LOX-like proteins. Biochim Biophys Acta . 2003; 1647: 220–224. [CrossRef] [PubMed]
Hayashi K Fong KS Mercier F Boyd CD Csiszar K Hayashi M. Comparative immunocytochemical localization of lysyl oxidase (LOX) and the lysyl oxidase-like (LOXL) proteins: changes in the expression of LOXL during development and growth of mouse tissues. J Mol Histol . 2004; 35: 845–855. [CrossRef] [PubMed]
Barry-Hamilton V Spangler R Marshall D Allosteric inhibition of lysyl oxidase-like-2 impedes the development of a pathologic microenvironment. Nat Med . 2010; 16: 1009–1017. [CrossRef] [PubMed]
Akiri G Sabo E Dafni H Lysyl oxidase-related protein-1 promotes tumor fibrosis and tumor progression in vivo. Cancer Res . 2003; 63: 1657–1666. [PubMed]
Lopez B Gonzalez A Hermida N Valencia F de Teresa E Diez J. Role of lysyl oxidase in myocardial fibrosis: from basic science to clinical aspects. Am J Physiol Heart Circ Physiol . 2010; 299: H1–H9. [CrossRef] [PubMed]
Vadasz Z Kessler O Akiri G Abnormal deposition of collagen around hepatocytes in Wilson's disease is associated with hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2. J Hepatol . 2005; 43: 499–507. [CrossRef] [PubMed]
Rodriguez HM Vaysberg M Mikels A Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor. J Biol Chem . 2010; 285: 20964–20974. [CrossRef] [PubMed]
Friedlander M. Fibrosis and diseases of the eye. J Clin Invest . 2007; 117: 576–586. [CrossRef] [PubMed]
Schultz G Chegini N Grant M Khaw P MacKay S. Effects of growth factors on corneal wound healing. Acta Ophthalmol Suppl . 1992; 60–66.
Schlotzer-Schrehardt U. Molecular pathology of pseudoexfoliation syndrome/glaucoma–new insights from LOXL1 gene associations. Exp Eye Res . 2009; 88: 776–785. [CrossRef] [PubMed]
Sethi A Mao W Wordinger RJ Clark AF. Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells. Invest Ophthalmol Vis Sci . 2011; 52: 5240–5250. [CrossRef] [PubMed]
Lama PJ Fechtner RD. Antifibrotics and wound healing in glaucoma surgery. Surv Ophthalmol . 2003; 48: 314–346. [CrossRef] [PubMed]
Addicks EM Quigley HA Green WR Robin AL. Histologic characteristics of filtering blebs in glaucomatous eyes. Arch Ophthalmol . 1983; 101: 795–798. [CrossRef] [PubMed]
Khaw PT Doyle JW Sherwood MB Grierson I Schultz G McGorray S. Prolonged localized tissue effects from 5-minute exposures to fluorouracil and mitomycin C. Arch Ophthalmol . 1993; 111: 263–267. [CrossRef] [PubMed]
Karalekas DHA Rosenberg L Ruderman J Krupin T. Effects of human glaucomatous and non-glaucomatous aqueous humor on fibroblast proliferation in vitro. Invest Ophthalmol Vis Sci . 1994; 35: 1898–1898.
Katz GJ Higginbotham EJ Lichter PR Mitomycin C versus 5-fluorouracil in high-risk glaucoma filtering surgery. Extended follow-up. Ophthalmology . 1995; 102: 1263–1269. [CrossRef] [PubMed]
Cordeiro MF Mead A Ali RR Novel antisense oligonucleotides targeting TGF-beta inhibit in vivo scarring and improve surgical outcome. Gene Ther . 2003; 10: 59–71. [CrossRef] [PubMed]
Khaw P Grehn F Hollo G A phase III study of subconjunctival human anti-transforming growth factor beta(2) monoclonal antibody (CAT-152) to prevent scarring after first-time trabeculectomy. Ophthalmology . 2007; 114: 1822–1830. [CrossRef] [PubMed]
Bignon M Pichol-Thievend C Hardouin J Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood . 2011; 118: 3979–3989. [CrossRef] [PubMed]
Couffinhal T Silver M Zheng LP Kearney M Witzenbichler B Isner JM. Mouse model of angiogenesis. Am J Pathol . 1998; 152: 1667–1679. [PubMed]
Anshu A Price MO Richardson MR Alterations in the aqueous humor proteome in patients with a glaucoma shunt device. Mol Vis . 2011; 17: 1891–1900. [PubMed]
Nomura T Huang WC Zhau HE Beta2-microglobulin promotes the growth of human renal cell carcinoma through the activation of the protein kinase A, cyclic AMP-responsive element-binding protein, and vascular endothelial growth factor axis. Clin Cancer Res . 2006; 12: 7294–7305. [CrossRef] [PubMed]
Byeseda SE Burns AR Dieffenbaugher S Rumbaut RE Smith CW Li Z. ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing. Am J Pathol . 2009; 175: 571–579. [CrossRef] [PubMed]
Bhatwadekar AD Glenn JV Curtis TM Grant MB Stitt AW Gardiner TA. Retinal endothelial cell apoptosis stimulates recruitment of endothelial progenitor cells. Invest Ophthalmol Vis Sci . 2009; 50: 4967–4973. [CrossRef] [PubMed]
Kagawa S Matsuo A Yagi Y Ikematsu K Tsuda R Nakasono I. The time-course analysis of gene expression during wound healing in mouse skin. Leg Med (Tokyo) . 2009; 11: 70–75. [CrossRef] [PubMed]
Frank S Kampfer H Wetzler C Stallmeyer B Pfeilschifter J. Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression. Biochem J . 2000; 347; 265–273. [CrossRef] [PubMed]
Hoya T. Effects of porcine pancreatic elastase-1 on the elastin in human trabecular meshwork–immunohistochemical studies: report 1 [in Japanese]. Nihon Ganka Gakkai Zasshi . 1993; 97: 1011–1027. [PubMed]
Johansson A Olerud J Johansson M Carlsson PO. Angiostatic factors normally restrict islet endothelial cell proliferation and migration: implications for islet transplantation. Transpl Int . 2009; 22: 1182–1188. [CrossRef] [PubMed]
Chong HC Tan MJ Philippe V Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing. J Cell Biol . 2009; 184: 817–831. [CrossRef] [PubMed]
Thomay AA Daley JM Sabo E Disruption of interleukin-1 signaling improves the quality of wound healing. Am J Pathol . 2009; 174: 2129–2136. [CrossRef] [PubMed]
Ishida Y Kondo T Kimura A Matsushima K Mukaida N. Absence of IL-1 receptor antagonist impaired wound healing along with aberrant NF-kappaB activation and a reciprocal suppression of TGF-beta signal pathway. J Immunol . 2006; 176: 5598–5606. [CrossRef] [PubMed]
Mamiya K Ohguro H Ohguro I Effects of matrix metalloproteinase-3 gene transfer by electroporation in glaucoma filter surgery. Exp Eye Res . 2004; 79: 405–410. [CrossRef] [PubMed]
Steinbrech DS Longaker MT Mehrara BJ Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation. J Surg Res . 1999; 84: 127–133. [CrossRef] [PubMed]
Figure 1
 
Quantification of the level of LOX and LOXL2 in Tenon's capsule and conjunctiva. Images show representative pictures of the immunostainings for LOX (A) and LOXL2 (B) on day 30 after surgery in the superior conjunctiva (conj) and Tenon. Both LOX (C) and LOXL2 (D) proteins were upregulated in the fibrotic samples as compared with the nonfibrotic tissues (control) on different time points after glaucoma filtration surgery in rabbits (*P < 0.05).
Figure 1
 
Quantification of the level of LOX and LOXL2 in Tenon's capsule and conjunctiva. Images show representative pictures of the immunostainings for LOX (A) and LOXL2 (B) on day 30 after surgery in the superior conjunctiva (conj) and Tenon. Both LOX (C) and LOXL2 (D) proteins were upregulated in the fibrotic samples as compared with the nonfibrotic tissues (control) on different time points after glaucoma filtration surgery in rabbits (*P < 0.05).
Figure 2
 
Macroscopic postoperative photographs of rabbit eyes after surgery. Macroscopic photographs of rabbit eyes at day 30, after repeated anti-LOX (B) or anti-LOXL2 (D) antibody injections show surviving blebs that remain diffusely elevated compared with their respective controls (A, C).
Figure 2
 
Macroscopic postoperative photographs of rabbit eyes after surgery. Macroscopic photographs of rabbit eyes at day 30, after repeated anti-LOX (B) or anti-LOXL2 (D) antibody injections show surviving blebs that remain diffusely elevated compared with their respective controls (A, C).
Figure 3
 
Clinical investigation of rabbit eyes after surgery and administration of anti-LOX(L2) antibodies. Treatment with anti-LOX (GS-639556) and anti-LOXL2 (GS-607601) significantly improved the outcome of surgery by increasing the bleb area ([A, D] P < 0.0001) and bleb height ([B, E] P < 0.0001) from day 17 to 19 onwards, respectively, compared with control. As shown in the Kaplan-Meier survival curve (C, F), both antibodies significantly prolonged bleb survival after filtration surgery, compared with control.
Figure 3
 
Clinical investigation of rabbit eyes after surgery and administration of anti-LOX(L2) antibodies. Treatment with anti-LOX (GS-639556) and anti-LOXL2 (GS-607601) significantly improved the outcome of surgery by increasing the bleb area ([A, D] P < 0.0001) and bleb height ([B, E] P < 0.0001) from day 17 to 19 onwards, respectively, compared with control. As shown in the Kaplan-Meier survival curve (C, F), both antibodies significantly prolonged bleb survival after filtration surgery, compared with control.
Figure 4
 
Neovascularization, inflammation, and collagen deposition in rabbit eyes. The density of blood vessels and leukocytes was determined by calculating the CD31-positive area and by counting the CD45-positive cells, respectively, as a proportion of the total wound area of the bleb. Fibrosis was stained by Sirius Red and was determined by measuring the percentage of the area of mature collagen fibers in the total wound area of the bleb using polarized light. (AC) Treatment with anti-LOX antibody (GS-639556) showed no significant differences in neovascularization and inflammation compared with PBS-treated eyes. The process of collagen deposition was significantly decreased (by 21%) after inhibition of LOX at the filtration site on day 30 as compared with the control eyes. (DF) Treatment of anti-LOXL2 (GS-607601) showed significant reductions of 44% in neovascularization, 32% in inflammation, and 16% in collagen deposition in the bleb of the treated eyes compared with controls. In both groups the inferior conjunctiva showed no difference in blood vessel density, inflammation, or collagen deposition in the treated and control eyes (*P < 0.05). (G) The images show representative pictures of immunostainings of eyes treated with the anti-LOX or anti-LOXL2 antibody or control eyes, at 30 days after surgery (magnification of ×20).
Figure 4
 
Neovascularization, inflammation, and collagen deposition in rabbit eyes. The density of blood vessels and leukocytes was determined by calculating the CD31-positive area and by counting the CD45-positive cells, respectively, as a proportion of the total wound area of the bleb. Fibrosis was stained by Sirius Red and was determined by measuring the percentage of the area of mature collagen fibers in the total wound area of the bleb using polarized light. (AC) Treatment with anti-LOX antibody (GS-639556) showed no significant differences in neovascularization and inflammation compared with PBS-treated eyes. The process of collagen deposition was significantly decreased (by 21%) after inhibition of LOX at the filtration site on day 30 as compared with the control eyes. (DF) Treatment of anti-LOXL2 (GS-607601) showed significant reductions of 44% in neovascularization, 32% in inflammation, and 16% in collagen deposition in the bleb of the treated eyes compared with controls. In both groups the inferior conjunctiva showed no difference in blood vessel density, inflammation, or collagen deposition in the treated and control eyes (*P < 0.05). (G) The images show representative pictures of immunostainings of eyes treated with the anti-LOX or anti-LOXL2 antibody or control eyes, at 30 days after surgery (magnification of ×20).
Figure 5
 
Upregulation of different wound healing related factors in rabbit aqueous humor. The expression level of different factors related to wound healing in aqueous humor was analyzed by Myriad RBM with their Human Inflammation MAP 1.0 panel. (AC) Anti-LOX treatment (GS-639556) increased the expression levels of β-2 microglobulin (P = 0.002), ICAM-1 (P = 0.04), and RANTES (P = 0.04) in the rabbit aqueous humor compared with control. (DI) Beta-2 microglobulin (P = 0.04), ICAM-1 (P = 0.02), α1-antitrypsin (P = 0.02), IL-1β (P = 0.02), IL-1 ra (P = 0.04), and MMP-3 (P = 0.03) were upregulated after anti-LOXL2 treatment (GS-607601) compared with control eyes.
Figure 5
 
Upregulation of different wound healing related factors in rabbit aqueous humor. The expression level of different factors related to wound healing in aqueous humor was analyzed by Myriad RBM with their Human Inflammation MAP 1.0 panel. (AC) Anti-LOX treatment (GS-639556) increased the expression levels of β-2 microglobulin (P = 0.002), ICAM-1 (P = 0.04), and RANTES (P = 0.04) in the rabbit aqueous humor compared with control. (DI) Beta-2 microglobulin (P = 0.04), ICAM-1 (P = 0.02), α1-antitrypsin (P = 0.02), IL-1β (P = 0.02), IL-1 ra (P = 0.04), and MMP-3 (P = 0.03) were upregulated after anti-LOXL2 treatment (GS-607601) compared with control eyes.
Figure 6
 
Correlations between different factors and bleb features in the anti-LOXL2–treated eyes. Scatterplots showing significant correlations between different factors and bleb area on day 30 after anti-LOXL2 treatment. Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing related factors and the bleb area. (AB) Wound healing promoting factors were negatively correlated with the bleb area: β-2 microglobulin (r = −0.92; P = 0.02) and ICAM-1 (r = −0.87; P = 0.03). (CF) The wound healing inhibitory factors were positively correlated with the bleb area after anti-LOXL2 treatment: α1-antitrypsin (r = 0.94; P = 0.02), IL-1β (r = 0.92; P = 0.02), IL-1ra (r = 0.94; P = 0.02), and MMP-3 (r = 0.94; P = 0.02).
Figure 6
 
Correlations between different factors and bleb features in the anti-LOXL2–treated eyes. Scatterplots showing significant correlations between different factors and bleb area on day 30 after anti-LOXL2 treatment. Spearman correlation coefficient (r) was calculated to evaluate the strength of association between expression of wound healing related factors and the bleb area. (AB) Wound healing promoting factors were negatively correlated with the bleb area: β-2 microglobulin (r = −0.92; P = 0.02) and ICAM-1 (r = −0.87; P = 0.03). (CF) The wound healing inhibitory factors were positively correlated with the bleb area after anti-LOXL2 treatment: α1-antitrypsin (r = 0.94; P = 0.02), IL-1β (r = 0.92; P = 0.02), IL-1ra (r = 0.94; P = 0.02), and MMP-3 (r = 0.94; P = 0.02).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×