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Purpose: To explore the effect and mechanism of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes on corneal fibrosis., Methods: The wild-type, NLRP3 knockout (KO), and myeloid cell-specific NLRP3 KO (NLRP3 Lyz-KO) C57 mice were used to establish a corneal scarring model. NLRP3 inhibitor, IL-1β neutralizing antibody, and an IL-1R antagonist were used to investigate the role of NLRP3 and IL-1β in corneal fibrosis. The expression of the NLRP3 signaling pathway related proteins, alpha-smooth muscle actin, TGF-β was determined by quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence staining. Flow cytometry was used to detect the infiltration of macrophages during corneal fibrosis., Results: The components of the NLRP3 inflammasomes were elevated and activated during corneal scarring. Additionally, genetic or chemical-mediated blocking of NLRP3 as well as IL-1β significantly alleviated corneal fibrosis. Moreover, neutrophil (CD45+Ly6G+) and macrophage (CD45+ F4/80+) accumulation increased in the cornea during the progression of corneal fibrosis. Intriguingly, the increased concentrations of NLRP3 and IL-1β were prominently colocalized with the infiltrating F4/80+ macrophages. Expectedly, NLRP3 Lyz-KO mice exhibited a marked decrease in their corneal fibrosis symptoms. Mechanistically, the activation of IL-1β or macrophage NLRP3 stimulated the expression of TGF-β1 in the corneal epithelial cells, whereas an NLRP3 deficiency decreased its expression in the corneal epithelium., Conclusions: These observations revealed that the NLRP3 inflammasome activation in infiltrating macrophages contributes to corneal fibrosis by regulating corneal epithelial TGF-β1 expression. Targeting the NLRP3 inflammasome might be a promising strategy for the treatment of corneal scarring.

Purpose: To highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury., Methods: Review of the literature., Results: The regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFβ) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet's membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components-resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet's membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFβ entry and activity, and triggers TGFβ-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency., Conclusions: Injuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet's membrane are key regulators of stromal cellularity through their modulation of TGFβ. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.

Purposes: Trabeculectomy is a surgical procedure for lowering intraocular pressure in glaucoma patients, in which excessive scarring leading to failure of the filtering bleb adversely affects the surgical outcome. Heightened Tenon's capsule fibroblast (TCF) proliferation and extracellular matrix (ECM) deposition are implicated in this process but endogenous factors that regulate TCF functions remain largely elusive. This study sought to elucidate the role of connective tissue growth factor (CTGF) in the regulation of TCF phenotypes and signaling., Methods: Expression of CTGF in scarring and nonscarring Tenon's capsules was measured by real-time PCR and immunofluorescence. Knockdown of CTGC was achieved by lentivirus delivery of small-hairpin RNA. Cell proliferation was measured by CCK8, cell cycle progression, and apoptosis by flow cytometry, adhesion, migration, and invasion of TCF by functional assays in vitro. Proteins and cytokines related to fibrosis were measured by Western blot and ELISA, respectively., Results: Expression of CTGF was significantly upregulated in scarring Tenon's capsules and their isolated fibroblasts when compared with the nonfibrotic counterparts. Functionally, targeting CTGF with lentivirus-delivered small-hairpin RNA inhibited the proliferation, adhesion, migration, and invasion of TCFs, accompanied by downregulation of p38 and nuclear factor-κB as well as matrix metalloproteinase-2, cyclin D1, and collagen I. In addition, lentiviral targeting of CTGF reduced the release of fibrosis-related cytokines from TCFs and inhibited TCF-conditioned, medium-induced macrophage chemotaxis., Conclusions: Our study supports a crucial role of CTGF in the regulation of TCF proliferation and ECM deposition. Targeting CTGF using lentiviral vector may be a promising approach for preventing excessive scarring after trabeculectomy.

Purpose: The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients., Methods: In the trabeculectomy mouse model, intracameral injection(s) of the PlGF inhibitor (5D11D4) were compared to MMC or aflibercept and to the combination of both compounds. Treatment outcome was studied by bleb investigation and by Sirius Red staining. The effect of subconjunctival PlGF administration and topical 5D11D4 on IOP was investigated in normotensive mice and was compared to topical administration of latanoprost, the gold standard for IOP-lowering., Results: Combination of MMC and 5D11D4 was able to significantly improve surgical outcome compared to monotherapy of MMC or 5D11D4 (n = 20; P < 0.001). Compared to combined treatment of MMC with aflibercept, the simultaneous administration of MMC and 5D11D4 was equally efficacious in improving surgical outcome (n = 15; P = 0.88). In normotensive mice, 5D11D4 was able to significantly reduce the IOP-elevation induced by PlGF (n = 10; P < 0.05), whereas no effect of 5D11D4 was seen in naive mice, which was in contrast to latanoprost., Conclusions: The current data suggest that application of MMC together with PlGF inhibition may have complementary effects in the improvement of surgical outcome and is equally efficacious as the combined treatment of MMC and aflibercept. Inhibition of PlGF also might open alternative perspectives as IOP-lowering strategy for glaucoma patients with increased aqueous PlGF levels.

Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring., Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries., Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas., Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Excessive wound contraction can lead to scar formation in the conjunctiva. The effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon fibroblasts (HTFs) cultured in three-dimensional (3D) collagen gels were investigated., Methods: Human Tenon fibroblasts were cultured in 3D gels of type I collagen and in the absence or presence of TGF-β, ATRA, or various inhibitors. Collagen gel contraction was evaluated by measurement of gel diameter. Phosphorylation of various signaling molecules was examined by immunoblot analysis. The formation of actin stress fibers and focal adhesions was detected by laser confocal microscopy., Results: All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. The TGF-β-induced phosphorylation of focal adhesion kinase (FAK) and formation of stress fibers and focal adhesions in HTFs were attenuated by ATRA. All-trans-retinoic acid also inhibited the TGF-β-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as that of c-Jun and Smad2/3. Furthermore, TGF-β-induced collagen gel contraction was blocked by inhibitors of ERK, p38, or JNK signaling., Conclusions: All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs, most likely by attenuating the formation of actin stress fibers and focal adhesions as well as signaling by MAPKs, c-Jun, and Smads. All-trans-retinoic acid may therefore prove effective for inhibition of conjunctival scarring through attenuation of the contractility of Tenon fibroblasts., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo., Methods: Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-β (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 μm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting., Results: The TGF-β activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7)., Conclusions: Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.

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.

Purpose: To investigate type XII collagen expression in corneal scars in vivo., Methods: Type XII collagen protein expression was evaluated by immunohistochemistry in human corneal scars and in a mouse model of corneal scarring at several time points (from day 7 to day 210) after full-thickness excision. Alternative splice variants of the NC3 and NC1 domains of type XII collagen were investigated in the mouse wound-healing model using RT-PCR., Results: Type XII collagen was overexpressed in human corneal scars in areas that were also positive for alpha-smooth muscle actin staining. In a mouse model of corneal wound injury we found that at 14 and 21 days postexcision, type XII collagen was largely concentrated in the subepithelial region of the cornea, especially in and near the wound bed. By 28 days postexcision, expression of type XII collagen decreased but remained higher than that in controls. NC3 short form is the main form expressed in the cornea during the wound-healing process. After injury, the NC1 long splice variant mRNA was the most highly overexpressed variant in the cornea, especially in the epithelium (×2.7, 3.72, and 5.57 at days 7, 14, and 21, respectively, P < 0.01 to 0.001 compared with uninjured samples). Corneal scars from a 7-month-old mouse revealed an overexpression of type XII collagen in the wound area similar to what we observed in human corneal scars., Conclusions: Type XII collagen is overexpressed in permanent human and mouse corneal scars and could represent a new target to treat corneal scarring.

Purpose: To investigate whether topical administration of pirfenidone eye drops could be used to prevent postoperative scarring in a rabbit model of experimental glaucoma filtration surgery., Methods: In a randomized, controlled, masked-observer study, 40 rabbits underwent trabeculectomy in the right eyes and randomly received postoperative administration of 0.1% or 0.5% pirfenidone, perioperative mitomycin C (0.25 mg/mL), or no treatment. Bleb characteristics and functions were evaluated over a period of 4 weeks. The animals were killed on days 7, 14, and 28. Histopathology and immunohistochemistry were performed to determine the amount of scarring and fibrosis. Ocular toxicity was assessed by the Draize test, histopathology, and electron microscope., Results: The four treatment groups were similar with respect to intraocular pressure and anterior chamber depth. Pirfenidone 0.5% significantly prolonged bleb survival, and the blebs were larger and higher than those in the control group (P < 0.05); the 0.1% pirfenidone concentration was less effective. Furthermore, the histology and immunohistology results showed that the 0.5% pirfenidone and mitomycin C groups had less scarring at days 7 to 28 than did the controls. Toxicity assessments showed that pirfenidone did not damage the rabbit eyes., Conclusions: Postoperative use of 0.5% pirfenidone eye drops was associated with improved trabeculectomy bleb survival in a rabbit model. Pirfenidone eye drops may be a safe and effective antiscarring agent in glaucoma filtration surgery.

Purpose: This study evaluated the use of combined bevacizumab with 5-fluorouracil (5-FU) on postoperative scarring and bleb survival after experimental glaucoma filtration surgery in comparison to the agents alone., Methods: Filtration surgery was performed on 26 female New Zealand White rabbits. The rabbits were allocated to one of four treatments: 5-FU combined with bevacizumab, 5-FU alone, bevacizumab alone, or phosphate buffered saline (PBS). The subconjunctival injections were administered immediate postoperatively and weekly for 3 weeks. Clinical assessment and bleb photography were performed. Histologic staining determined the presence of subconjunctvial fibrosis and mRNA expression of collagen I and fibronectin in the tissue was quantified., Results: Bevacizumab in combination with 5-FU resulted in a greater antifibrotic effect compared with monotherapy with 5-FU or bevacizumab alone, as evidenced by the attenuation in fibronectin and mature collagen I expression and deposition (P < 0.05). In addition, this was associated with a 100% bleb survival at day 28 in the combined treatment group compared with monotherapy (50% bevacizumab [P < 0.05] and 25% 5-FU [P < 0.001]). Conjunctival vascularity significantly reduced with bevacizumab treatment both alone and in combination with 5-FU., Conclusions: The results provide compelling evidence that combined bevacizumab and 5-FU offers superior antifibrotic effect over monotherapy in a model of glaucoma filtration surgery, while prolonging bleb survival at the same time. A synergistic effect is suggested to be present.

Purpose: Filtration failure due to excessive postoperative scarring remains a major problem after glaucoma surgery. The authors have investigated whether glaucoma and filtration surgery are associated with increased levels of vascular endothelial growth factor (VEGF), and whether a humanized monoclonal antibody against VEGF, bevacizumab, can reduce postoperative scar formation and improve surgical outcome., Methods: The levels of VEGF in samples of aqueous humor were measured by ELISA. The expression of the VEGF receptors Flt-1 and KDR was analyzed in cultured Tenon fibroblasts by real-time RT-PCR and Western blotting. The effect of VEGF and bevacizumab on Tenon fibroblasts in vitro was determined using a proliferation assay. The in vivo effect of the antibody was investigated in a rabbit model of trabeculectomy by measuring the intraocular pressure (IOP) and bleb area, and by immunohistological analysis of angiogenesis, inflammation, and fibrosis., Results: VEGF levels were increased significantly in the aqueous humor of glaucoma patients and rabbits that had undergone surgery. Both VEGF receptors were expressed on Tenon fibroblasts. Fibroblast proliferation in vitro was stimulated by delivery of VEGF, and was inhibited by administration of bevacizumab. The antibody also reduced angiogenesis and collagen deposition significantly, and improved the outcome of glaucoma surgery in rabbits., Conclusions: VEGF was upregulated in the aqueous humor of glaucoma patients and in the rabbit model, and it stimulated fibroblast proliferation in vitro. This suggests that it is involved in the scarring process after filtration surgery. Bevacizumab reduced the proliferation of fibroblasts in vitro and improved surgical outcome.

Purpose: To explore the inhibitive effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify the potential pharmacologic target(s)., Methods: Twenty-four New Zealand rabbits underwent filtration surgery on the right eye and were divided into a control group and three experimental groups (n=6). Human Tenon's fibroblast monolayer was scraped to generate a single gap, and then the control medium with SB-431542 only or containing 10 microg/L TGF-beta1 and SB-431542 (1-20 microM) was added. The cells were pretreated with SB-431542 or in control medium for 30 minutes before induction with 10 microg/L TGF-beta1 or 1 microg/L TGF-beta2. The expression of alpha-SM-actin, CTGF, and Col I, as well as changes in the Smad, ERK, P38, and AKT signaling pathways were detected., Results: In comparison with the control rabbits, the IOPs in the experimental groups remained at lower levels until day 25 (P<0.05) after the surgery. Histologic profiles showed that there was only a mild deposition of collagen in the subconjunctival space in the experimental groups. The cell growth and migration were inhibited effectively by SB-431542, regardless of whether TGF-beta was present in the culture system. SB-431542 abrogated TGF-beta-induced upregulation of alpha-SM-actin, CTGF, and Col I. It effectively inhibited the phosphorylation of Smad2 stimulated by TGF-beta but not that of the components of the MAPK pathways., Conclusions: SB-431542 inhibits scar formation after glaucoma filtration surgery. The mechanism may be that SB-431542 interferes in the phosphorylation of Smad2, thus abrogating TGF-beta-induced fibroblast transdifferentiation and then decreasing Col I synthesis.

Purpose: To analyze the amounts and distributions of nonsulfated and sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in the interface wound of human postmortem LASIK corneas in comparison with normal control corneas., Methods: Corneal stromal tissue samples from central and paracentral hypocellular primitive stromal interface scars of human LASIK corneas and from similar regions of normal control corneas were collected by laser capture microdissection (LCM) and subsequently were digested with specific glycosidase enzymes. Digests were directly analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS)., Results: Concentrations of both monosulfated GlcNAc(6S)-beta-1,3-Gal (MSD2) and disulfated Gal (6S)-beta-1,4-GlcNAc(6S) (DSD) KS disaccharides from the LASIK interface scars were significantly lower than in normal control corneal stromas. No significant difference was found for the concentration of nonsulfated (NSD) KS disaccharides in LASIK interface scars compared with normal controls. The concentration of DeltaUA-beta-1,3-GalNAc(6S) (Deltadi-6S) CS/DS disaccharides from the LASIK interface scar was significantly higher than normal corneal stroma, whereas concentrations of DeltaUA-beta-1,3-GalNAc(4S) (Deltadi-4S) and nonsulfated Deltadi-0S CS/DS disaccharides demonstrated no significant differences from normal corneas., Conclusions: The profiles of KS and CS/DS disaccharides in LASIK interface scars are significantly different from those in normal cornea stromal tissue, as revealed by LCM and ESI-MS/MS.

Purpose: To compare the effects of the three human isoforms of transforming growth factor (TGF)-beta in vivo using a mouse model of conjunctival scarring, both in normal eyes and after treatment with MMC, with a view to delineating the role of this growth factor in glaucoma filtration surgery., Methods: Application of recombinant human TGF-beta was assessed in a prospective, randomized study of mouse conjunctival scarring, in which subconjunctival TGF-beta1, -beta2, and -beta3 (all 10(-9) M) were compared with control (phosphate-buffered saline [PBS] carrier) and mitomycin C (MMC; 0.4 mg/ml) treatment at 6 hours, and 1, 3, and 7 days after surgery (six eyes/treatment/time point). Effects of TGF-beta2 on eyes previously treated with MMC were also assessed. Histologic studies of enucleated eyes were performed to analyze development of the scarring response, extracellular matrix deposition, and the inflammatory cell profile., Results: All three isoforms of TGF-beta behaved in a similar manner in vivo, being associated with a rapid-onset and exaggerated scarring response compared with control and MMC treatment. TGF-beta-treated eyes showed evidence of an earlier peak in inflammatory cell activity (P < 0.05) and increased collagen type III deposition (P < 0.05). TGF-beta2 treatment significantly stimulated scarring after MMC application (P < 0.05)., Conclusions: TGF-beta1, -beta2, and -beta3 appear to have similar actions in vivo and stimulate the conjunctival scarring response. Application of TGF-beta2 modified the effects of MMC. All TGF-beta isoforms may be potent modulators of the conjunctival scarring response. These studies indicate that TGF-beta2 may naturally modify the antiscarring effects of antimetabolites such as MMC in glaucoma filtration surgery.

Macromolecules from normal rabbit cornea and cornea containing a 2-mm diameter button of scar tissue were biosynthetically labeled with 35S-sulfate and 3H-glucosamine in vivo and in organ culture. Labeled macromolecules, including proteoglycans (PGs) extracted from the normal cornea, scar tissue, and corneal tissue adjacent to the scar with guanidine hydrochloride were chromatographed on DEAE-Sepharose CL-6B columns and eluted with increasing concentrations of NaCl. The elution pattern of corneal macromolecules synthesized in vitro was remarkably similar to that in vivo. In another experiment, corneas having 2-, 4-, and 8-week-old scars were labeled in organ culture and also extracted. Scars synthesized PGs with lower sulfation than those of adjacent corneal tissue. Although PG synthesis in scar decreased with wound age, the synthesis in adjacent cornea remained the same. In a third experiment, PGs extracted from pools of unlabeled 2- and 4-week-old scars, adjacent corneal tissue, and normal corneas were chromatographed on ion-exchange columns and analyzed chemically. The quantity of PGs in scar and adjacent cornea increased with healing time. The ratios of keratan sulfate PG to dermatan sulfate PG in normal cornea, scar, and adjacent cornea was 2.3, 0.6, and 1.5, respectively. The PGs from adjacent corneal tissue had a higher charge density than those from scar. The predominant adjacent-cornea dermatan sulfate PG had a higher charge density than that in normal cornea. The authors conclude that cornea adjacent to the healing wound synthesized PGs measurably different fro those in scar and normal cornea.

Ultrastructural localization of proteoglycans (PGs) in 1-week- to 2-year-old scar was determined by staining with cuprolinic blue dye (CBD) after specific enzymatic digestion of keratan sulfate (KS) glycosaminoglycans (GAGs) or chondroitin sulfate glycosaminoglycans (CSs). High critical electrolyte conditions were maintained for CBD-staining, specific for high-sulfated GAGs. Although KS was detected in the 1-week-old wound, no CBD-stained KS was seen in the anterior stroma adjacent to the wound. The CS was present throughout the 1-week-old wound and adjacent stroma, and PGs were biosynthetically 35SO4-labeled in normal stroma. Subsequently, radioactivity from labeled PGs in normal stroma adjacent to the wound moved into scar tissue during healing. Marked sensitivity of PGs to Chondroitinase ABC indicated an abundance of CS in 2-week-old scars. Punctate CBD-staining and immunohistochemical evidence suggested chemically altered KS is present in the 2-week-old anterior scar. The pattern of CBD-staining in 1- and 2-week scars, after chondroitinase treatment, suggested KS in the younger scar is similar to adult high-sulfated GAG, whereas KS in the 2-week scar contains primarily newly synthesized low-sulfated KS. The latter is consistent with previous immunochemical and biochemical analyses. Cytochemical and immunohistochemical evidence indicated that KS is not present in the 2-week-old posterior scar. By the week 8 of healing, CBD-stained KS was present throughout most of the scar, except along the posterior margin, consistent with earlier stages of healing. The CBD-stained structures in the first 8 weeks of healing were reminiscent of stained GAGs in normal developing cornea. This fetal-like CBD-staining pattern seen in scar, however, changed to that of the normal adult by the 2nd year of healing. The significance of these observations relate to our contention that healing adult cornea recapitulates some ontogenetic events of the normal cornea, and that the nonuniform distribution and chemical properties of GAGs in scar tissue are a function of the movement of existing proteoglycans and de novo synthesis of altered macromolecules.

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.

Normal adult rabbit corneas were digested with 5% pepsin and their collagens extracted with acetic acid. Collagen extracts were fractionated by differential salt precipitation. The 2.5 M NaCl fraction was then redissolved with tris buffer and precipitated with sodium acetate. The precipitate contained a high-molecular-weight disulfide-bonded aggregate which, upon reduction with mercaptoethanol, was converted into three distinct polypeptides having molecular weights between 45 and 66 Kd. These physical characteristics, together with the susceptibility of these polypeptides to collagenase and their amino acid composition, identified the high molecular weight aggregate as type VI collagen. Corneas from neonate rabbits and adult corneas containing 2-week-old scars were organ cultured in the presence of [14C] glycine to incorporate radiolabel into collagen. Tissues were digested with 0.02% pepsin and their collagens extracted with formic acid. The total radioactivity of the extracts and tissue residues was determined before the collagens were separated by SDS-polyacrylamide slab gel electrophoresis. Radioactive collagen polypeptides bands were then stained with Coomassie blue, processed for fluorography, and analyzed by densitometry. The results show that: (1) type VI collagen is synthesized by neonate corneas and healing adult corneas; (2) it is not readily solubilized from either corneal tissue by 0.02% pepsin digestion and formic acid extraction; and (3) the proportion of type VI collagen deposited in scar tissue is markedly lower than that found in neonate corneas.

Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of [14C] glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Corneal keratan sulfate proteoglycan (KSPG) from scar tissue of experimental penetrating corneal wounds in rabbits was analyzed 2-8 weeks after injury using three previously characterized antibodies. Keratan sulfate (KS) was identified in 2 week scars and normal corneal tissue by indirect immunofluorescence using a monoclonal antibody against sulfated KS epitopes. KSPG was measured in unfractionated extracts of scar and of normal corneal tissue using a "sandwich" enzyme-linked immunosorbent assay (ELISA). In extracts of 2 week scars, KSPG molecules reacting with two different anti-KS monoclonal antibodies were 55% and 82% as abundant as in normal tissue extracts. Ion exchange high performance liquid chromatography (HPLC) of tissue extracts found qualitatively similar elution profiles of KSPG antigens from both scar and normal tissues. Direct ELISA of the HPLC-purified KSPG showed identical quantitative binding of antibodies against core protein and KS from normal and scar tissue. KS in the HPLC-purified extracts was sensitive to digestion with endo-beta-galactosidase, whereas core protein antigens were not affected by this enzyme, as expected. Alteration of the antigenic characteristics of the KSPG of scars was detected with a competitive immunoassay using immobilized monoclonal antibodies against KS. KS in extracts from 2, 6, and 8 week scars competed only 5-11% as effectively as KS from normal cornea, although core protein antigens in the scar extracts competed 61-80% as well as those of normal cornea.(ABSTRACT TRUNCATED AT 250 WORDS)