January 2003
Volume 44, Issue 1
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Retina  |   January 2003
Tissue Transglutaminase as a Modifying Enzyme of the Extracellular Matrix in PVR Membranes
Author Affiliations
  • Siegfried G. Priglinger
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
  • Christian A. May
    Department of Anatomy, Friedrich Alexander University, Erlangen, Germany.
  • Aljoscha S. Neubauer
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
  • Claudia S. Alge
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
  • Carl-L. Schönfeld
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
  • Anselm Kampik
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
  • Ulrich Welge-Lussen
    From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; and the
Investigative Ophthalmology & Visual Science January 2003, Vol.44, 355-364. doi:10.1167/iovs.02-0224
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      Siegfried G. Priglinger, Christian A. May, Aljoscha S. Neubauer, Claudia S. Alge, Carl-L. Schönfeld, Anselm Kampik, Ulrich Welge-Lussen; Tissue Transglutaminase as a Modifying Enzyme of the Extracellular Matrix in PVR Membranes. Invest. Ophthalmol. Vis. Sci. 2003;44(1):355-364. doi: 10.1167/iovs.02-0224.

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

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Abstract

purpose. Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes.

methods. PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product ε-(γ-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor, interleukin-6, and interleukin-1β. Their effect was studied using immunohistochemistry and Northern and Western blot analyses.

results. Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with ε-(γ-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-β2 treatment.

conclusions. The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-β2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy.

Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and is the major complication in rhegmatogenous retinal detachment. 1 2 3 It is characterized by the formation of scarlike fibrocellular membranes on the retinal surface, in the vitreous and the subretinal space. Similar to evolving scars, these membranes can contract and membrane contraction subsequently may result in recurrent detachment of the retina. The precise pathogenic mechanism involved in the formation of epi- and subretinal membranes is not completely understood. Dissemination of retinal pigmented epithelial (RPE) cells from their normal site on Bruch’s membrane to multiple loci on the detached neuroretina is thought to be a key pathologic event in the genesis of the epi- and subretinal membranes associated with the development of PVR. 3 4 5 6 7 8 Once in this new environment, RPE cells dedifferentiate into fibroblast-like cells. With the breakdown of the blood–retinal barrier, cytokines such as transforming growth factor (TGF)-β2, basic fibroblast growth factor (bFGF), interleukin (IL)-6, and IL-1β are released into the vitreous cavity 9 and stimulate the proliferating cells to produce the numerous extracellular matrix (ECM) components that form the fibrocellular PVR membranes. 10 11 12 13 14  
ECM accumulation is paralleled by the release of matrix-degrading enzymes such as proteases and matrix metalloproteinases (MMPs). 15 16 One major function of these enzymes is to degrade ECM proteins to prevent excess ECM deposition. 17 MMPs have been identified in PVR membranes, 16 but despite the presence of these degrading enzymes, ECM continues to accumulate. This accumulation may result from either increased protein synthesis or a slower rate of protein degradation. 
Recent studies regarding scar formation in skin demonstrated transglutaminase-induced irreversible cross-links of ECM, which cannot be digested by any known enzyme. 18 19 In keeping with this, excessive accumulation of ECM in PVR membranes could result from the formation of irreversible ECM cross-links in fibrocellular membranes. 
Transglutaminases are calcium-dependent enzymes that catalyze the posttranslational modification of proteins through an acyl transfer reaction between the γ-carboxamide group of a peptide-bound glutaminyl residue and various amines. Covalent cross-linking using ε-(γ-glutamyl)-lysine bonds is stable and resistant to enzymatic, chemical, and mechanical disruption. 20 Endopeptidases capable of hydrolyzing the ε-(γ-glutamyl)-lysine cross-links formed by transglutaminases have not been described in vertebrates, and even lysosomes do not contain enzymes capable of splitting the ε-(γ-glutamyl)-lysine bonds. 21 22 23 tTgase (type II) is the most widespread member of this family and is present with diverse functions in many different cell types and tissues. 24 25 26 The enzyme plays a role in programmed cell death, 23 cell adhesion, 27 and interaction between the cell and its ECM through the cross-linking of proteins such as fibronectin, 28 vitronectin, 29 laminin-nidogen complexes, 24 30 and collagen type III. 31 Most of these components have been shown to be present in the ECM of PVR membranes. 13  
The present study was designed to find out whether extracellular tTgase and irreversible ε-(γ-glutamyl)-lysine cross-links formed by tTgase are present in human PVR membranes. 
RPE cell culture is a well-accepted in vitro model of PVR. 7 8 In vitro RPE cells have been shown to be capable of synthesizing both ECM components 32 and degrading enzymes such as MMPs. 33 34 In the present study we investigated the capacity of native as well as cultured RPE cells to synthesize tTgase. 
Because PVR-associated breakdown of the blood–retinal barrier leads to a release of several cytokines, including TGF-β2, bFGF, IL-6, and IL-1β, into the vitreous cavity 9 we investigated the influence of these cytokines on synthesis of tTgase by cultured human RPE cells. The activity of extracellular tTgase was demonstrated by its ability to cross-link fibronectin, an ECM component that has been shown in PVR membranes. 13  
Materials and Methods
Tissue Samples
Thirty-three samples of epi- or subretinal PVR membranes were obtained from patients who were undergoing vitreoretinal surgery for PVR in the Department of Ophthalmology of Ludwig-Maximilians-University (Munich, Germany). Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki. All cases of PVR involved previous rhegmatogenous retinal detachment. 
Operations were performed by different surgeons who used the same technique. Conventional vitreous surgery was performed with a three-port system. Epi- and subretinal membranes were separated from the retina by peeling whole tissues. 
Membranes were put into phosphate-buffered saline (PBS, pH 7.4) during the operation and either snap frozen in liquid nitrogen for mRNA extraction or mounted in optimal cutting temperature (OCT) medium (Merck, Darmstadt, Germany) and then stored in liquid nitrogen for cryostat sections. 
In Situ tTgase Assay for Enzyme Activity of tTgase in PVR Specimens
Unfixed 5-μm cryostat sections from eight PVR membranes were preincubated with 1% bovine serum albumin (BSA; Roche, Mannheim, Germany) in 0.1 M Tris/HCl (pH 8.2) for 30 minutes at room temperature (RT). Transglutaminase activity was detected by subsequent incubation in Tris buffer containing 12 μM monodansylcadaverine (MDC; Sigma, Deisenhofen, Germany) and 5 mM CaCl2. Control sections were incubated with MDC and 2 mM putrescine or MDC and 10 mM EDTA instead of CaCl2. Parallel sections were incubated in the same buffer containing 15 μg/mL guinea pig liver transglutaminase (Sigma). Both, endogenous and exogenous enzyme reactions were allowed to proceed for 1 hour at RT and then were stopped by washing the slides for 5 minutes in PBS and 10 mM EDTA, with two further washes in plain PBS. Incorporated dansyl label was finally detected by incubation with polyclonal antidansyl serum (Mobi-Tec, Göttingen, Germany) for 1 hour at RT. After three washes in PBS, bound antibody was detected with Cy3-coupled swine anti-rabbit IgG (Dianova, Hamburg, Germany) diluted 1:100 in PBS for 30 minutes. Preparations were mounted in Kaiser gelatin (Merck). Localization of Tgase activity was observed by fluorescence microscope (Leica, Wetzlar, Germany). 
Immunohistochemical Staining of Tissue Sections
Immunohistochemical double staining for extracellular tTgase and fibronectin as well as combined staining of tTgase and ε-(γ-glutamyl)-lysine isopeptide was performed in sections obtained from 13 PVR membranes. Unfixed PVR membranes were cut at a thickness of 8 μm. After a wash in Tris-buffered saline (TBS, pH 7.2–7.4) and preincubation with dry-milk solution (Blotto; Merck) to minimize nonspecific staining, the sections were incubated for 2 hours at RT with mouse anti-tTgase (Cub7402; Quartett, Berlin, Germany), goat anti-tTgase (TG100; Biomol, Hamburg, Germany), mouse anti ε-(γ-glutamyl)-lysine isopeptide (CovalAb, Oullins, France), and rabbit anti-fibronectin (Sigma). All antibodies used were diluted 1:100 in TBS containing 3% BSA. After a wash in TBS, the sections were incubated with goat anti-mouse IgG Cy-2, goat anti-mouse IgG Cy-3, swine anti-rabbit IgG Cy-2 and anti-goat IgG Cy-3 (Dianova) diluted 1:100 in blocking buffer for 2 hours at RT. 
Control sections were either incubated with BSA-TBS replacing the primary antibody or with a combination of 1:200 diluted primary antibody plus a fivefold weight excess of guinea pig tTgase (Sigma). A confocal microscope (Bio-Rad, London, UK) was used to study the stained sections. 
To demonstrate the distribution of the immunofluorescence staining phase, contrast microscopy images of the tissue sections were performed. 
Isolation of Human RPE Cells
Sixteen human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 4 to 16 hours after death. The donors ranged in age between 15 and 73 years. None of the donors had a known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki. Human RPE (hRPE) cells were harvested by using a procedure that has been described previously. 32 In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% polyvinyl pyrrolidone iodine, and rinsed again in the NaCl solution. The anterior segment from each donor eye was removed and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera with fine forceps. The eyecup was rinsed with Ca2+- and Mg2+-free Hank’s balanced salt solution, and treated with 0.25% trypsin (Gibco, Karlsruhe, Germany) for 1 hour at 37°C. The trypsin was aspirated and the eyecup filled with Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS; Biochrom). The medium was gently agitated with a pipette, to release the hRPE into the medium and avoid damage to Bruch’s membrane. 
For RT-PCR analysis, the hRPE cells from six donors were released from Bruch’s membrane by gently pipetting 0.5× PBS solution into the eye. Suspended hRPE cells were transferred to a 1.5-mL microcentrifuge tube and centrifuged for 5 minutes at 800 rpm. After centrifugation, the supernatant was removed and replaced by RNA extraction solution. 
Human RPE Cell Culture
The hRPE cell suspension was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL DMEM supplemented with 20% FCS, and maintained at 37°C and 5% carbon dioxide. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin 35 with a pan-cytokeratin antibody (Sigma; data not shown). The cells were tested and found free of contaminating macrophages (anti-CD11; Sigma) and endothelial cells (anti-von Willebrand factor; Sigma; data not shown). 
For growth factor experiments, second- to fifth-passage hRPE cells were grown to confluence. hRPR cells were then washed, incubated overnight in serum-free medium, and subsequently incubated in serum-free DMEM supplemented with either 2.0 ng/mL TGF-β2 (R&D Systems, Wiesbaden, Germany), 200 pg/mL bFGF (Peprotech, Rocky Hill, NJ), 320 pg/mL IL-6 (Peprotech) and 5 pg/mL IL-1β (Peprotech) for 24 hours. Control cultures were incubated under identical conditions without growth factors in the medium. Highly differentiated RPE cells were established by the method of Campochiaro and Hackett 33 using 75-cm2 flasks coated with laminin. RPE cell cultures were maintained in DMEM supplemented with 10% FBS and 10 ng/mL bFGF. The medium was changed every 2 days. These cells exhibited a cobblestone epithelial morphology with areas of dense pigmentation and expressed RPE65 mRNA, a marker for differentiation of RPE cells. 37 38 The expression of RPE65 in highly differentiated RPE cells was demonstrated by Northern blot analysis (data not shown). 
Immunohistochemistry of Cell Cultures
For detection of extracellular tTgase, confluent hRPE cells grown in four-well plastic chamber slides were incubated for 2.5 hours with serum-free DMEM containing 0.75 μg/mL monoclonal antibody to tTgase. Cells were then washed in PBS and fixed in 4% paraformaldehyde in PBS. After blocking with BSA, cells were incubated with anti-mouse IgG-Cy2 for 2 hours at RT and then washed in PBS before mounting. For double staining of tTgase and fibronectin, cells were first stained for extracellular tTgase, as described earlier. After blocking in 5% BSA, the cells were incubated for 15 hours at 4°C with rabbit anti-fibronectin antibody (Sigma) diluted 1:50 in blocking buffer. Samples were then washed with PBS and incubated with goat anti-mouse IgG Cy-2 and swine anti-rabbit IgG Cy-3 diluted 1:100 in blocking buffer for 2 hours at RT. 
RT-PCR Analyses of PVR Membranes and Native and Cultured Human RPE Cells
Total mRNA of 12 PVR membranes and native and cultured hRPE cells of epithelial and fibroblast-like differentiation status was extracted with a micro-RNA kit (peqGOLD RNAPure; peqLab, Erlangen, Germany). After confirming the structural integrity of the total RNA samples of each PVR membrane and hRPE cells by electrophoresis on 1% agarose gels and subsequent staining with 0.5 μmol/mL ethidium bromide, RNA samples were treated with 3 U of RQ RNase-free DNase (Promega, Madison, WI) for 35 minutes at 37°C to remove traces of contaminating genomic DNA. The content of RNA was measured by photometric measurement and the RNA-concentration was adjusted. Using Moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo(dT)-17 primer (Gibco) first-strand complementary DNA (cDNA) was prepared from total RNA. The quality of RNA and cDNA synthesis was shown by amplification of the housekeeping gene glyceraldehyde-3-phosphatedehydrogenase (GAPDH). 
PCR was performed on the same quantity of total cDNA in a total volume of 50 μL with 1 U native Taq polymerase (Eppendorf, Hamburg, Germany). PCR was started with a hot start: 10 minutes for 94°C to denature DNA, followed by 36 cycles of 1 minute of melting at 94°C, 1 minute of annealing at 59.7°C, and 2 minutes of extension at 72°C in a thermocycler (Mastercycler Gradient; Eppendorf). After the last cycle, the polymerization step was extended for a further 10 minutes to complete all strands. Each PCR reaction was repeated at least twice. 
The primers for tTgase (forward, 5′-CAGAACAGCAACCTTCTCATCGAG-3′; reverse, 5′-TTGGACTCCGTAAGGCAGTCAC-3′; positions 1054–1811; product size, 758 bp; annealing temperature, 59.7°C) and GAPDH (forward. 5′-CCTGCTTCACCACCTTCTTG-3′; reverse, 5′-CATCATCTCTGCCCCCTCTG-3′; positions, 416–852; product size, 437 bp; annealing temperature, 59.7°C) were purchased from Metabion (Munich, Germany). The specificity of the PCR product was analyzed by sequencing (Sequiserve, Vaterstetten, Germany). PCR performed on each sample of RNA that had not been reverse transcribed to cDNA was used as negative control and showed no amplified product. 
For semiquantitative PCR, the number of cycles was optimized by checking amplification after each cycle from cycles 23 to 36 for tTgase and from 20 to 33 for GAPDH. This showed that the 29th cycle was in the geometric phase for tTgase and GAPDH. The band intensity was measured with an imaging workstation (LAS-1000 Imager; RayTest, Pforzheim, Germany). Quantification was performed with the accompanying software (AIDA; RayTest). The final amount of PCR product was expressed as the ratio of the tTgase gene amplified to that of the GAPDH gene. 
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from confluent hRPE cultures in 10-cm Petri dishes by guanidinium thiocyanate-phenol-chloroform extraction method (RNA isolation kit; Stratagene, Heidelberg, Germany). Total RNA (3 μg per lane) was denatured and size fractionated by gel electrophoresis in 1% agarose gels containing 2.2 M formaldehyde. The RNA was then vacuum blotted onto a nylon membrane (Roche) and cross-linked (1600 μJ, Stratalinker; Stratagene). The amount and quality of the RNA was assessed by staining the membrane with methylene blue, and images were taken with the LAS-1000 Imager (RayTest). Prehybridizations, hybridizations, and chemiluminescence detection of the digoxigenin riboprobe were performed as described previously. 39 In brief, after hybridization the membrane was washed twice with 2× SSC and 0.1% sodium dodecyl sulfate (SDS) at RT, followed by two washes in 0.1× SSC, 0.1% SDS for 15 minutes at 68°C. After hybridization and posthybridization washes, the membrane was washed for 5 minutes in washing buffer (100 mM maleic acid, 150 mM NaCl [pH 7.5], and 0.3% Tween 20) and incubated for 60 minutes in blocking solution. Blocking solution contained 100 mM maleic acid (pH 7.5), 150 mM NaCl, and 1% blocking reagent (Roche). Anti-digoxigenin alkaline phosphatase (Roche) was diluted 1:10,000 in blocking solution and used to incubate the membrane for 30 minutes. The membrane was then washed four times for 15 minutes each in washing buffer. The membrane was equilibrated in detection buffer (100 mM Tris-HCl and 100 mM NaCl [pH 9.5]) for 10 minutes. For chemiluminescence detection, the luminescent agent (CDP-star; Roche) was diluted 1:100 in detection buffer and used to incubate the filter for 5 minutes at RT. After air drying, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected with the imaging workstation (LAS-1000 Imager; RayTest) with exposure times ranging from 10 minutes to 1 hour. Chemiluminescent signal quantification was performed with the accompanying software (AIDA; RayTest). 
Western Blot Analysis of tTgase
Cells grown in 35 mm tissue culture dishes were washed twice with PBS, collected, and lysed in NP-40 (150 mM NaCl, 50 mM Tris [pH 8.0], and 1% NP-40) cell lysis buffer. The samples for gel analysis were boiled for 5 minutes and protein content was measured by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Proteins were loaded (2 μg/lane) and separated by electrophoresis on a 5% polyacrylamide stacking gel and an 8% separating gel. 40 After gel electrophoresis, the proteins were transferred with semidry blotting onto a PVDF-membrane (Roche). The membrane was incubated with PBS containing 0.1% Tween 20 (PBST, pH 7.2) and 5% PBS for 1 hour. The primary antibody (tTgase 1:2000, Cub7402; Quartett) was then added and allowed to react overnight at RT. After the membrane was washed three times in PBST, it was incubated with an alkaline phosphatase–conjugated swine anti-mouse antibody (Dianova; diluted 1:20,000) for 30 minutes. Visualization of the alkaline phosphatase was achieved by chemiluminescence. The luminescent agent (CDP-star; Roche) was diluted 1:100 in detection buffer, and the filter was incubated for 5 minutes at RT. After air drying, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected as described for Northern blot analysis. 
tTgase Activity of Cultured Human RPE Cells
tTgase activity was measured by the incorporation of biotinylated cadaverine into fibronectin. 41 For this assay, 96-well plates were precoated with plasma fibronectin (5 μg/mL; Sigma) and incubated overnight at 4°C. Twenty-four hours before seeding, hRPE cells were treated with 2.0 ng/mL TGF-β2 (R&D Systems), 200 pg/mL bFGF, 320 pg/mL IL-6, or 5 pg/mL IL-1β. Untreated and cytokine-treated hRPE cells were then plated at a density of 2 × 105 cells/mL in 100 μL serum-free DMEM in the presence of 0.1 mM biotinylated cadaverine (Mobi-Tec). Cells were allowed to incubate on the fibronectin-coated plates for different periods (0, 5, 10, 20, 40, 60, 90, or 120 minutes) at 37°C, then washed twice with PBS, containing 3 mM EDTA. As a negative control, fibronectin-coated 96-well plates were incubated with 100 μL serum-free DMEM containing 0.1 mM biotinylated cadaverine alone. The detergent solution (100 μL) consisting of 0.1% deoxycholate in PBS containing 3 mM EDTA was then added to each well and was incubated with gentle shaking for 20 minutes. The supernatant was discarded, and the remaining fibronectin layer was washed three times with Tris-HCl. Reaction was then blocked with 3% BSA in Tris-HCl buffer for 30 minutes at 37°C and washed three times with Tris-HCl buffer. The incorporated biotinylated cadaverine was revealed with a 1:5000 dilution of peroxidase conjugate (Extravidin; Sigma) which was incubated for 1 hour at 37°C. After washing three times with Tris-HCl the fibronectin layer was incubated for 20 minutes at RT in 200 μL of substrate solution (a mixture of H2O2 and tetramethylbenzidine). Color development was stopped by adding 50 μL stop solution to each well. The optical density was determined with an ELISA reader (Molecular Devices, Garching, Germany) set at 450 nm. 
Results
PVR Membranes
Transglutaminase Activity.
By incorporation of fluorescein-dansyl-cadaverine into unfixed frozen sections of PVR membranes, marked ECM transglutaminase activity was shown (Fig. 1A) . Cadaverine acts as a competitive primary amine-bound ε-amino lysine group and therefore becomes incorporated into endogenous γ-glutamyl residues if transglutaminase is active. In control experiments we used additional putrescine, which acts as a competitive amine to cadaverine (Fig. 1B) or EDTA to bind calcium and therefore block the calcium-dependent enzymes (Fig. 1C) . Substitution of guinea pig tTgase showed that almost the whole PVR membrane consisted of substrates for tTgase (Fig. 1D) . All investigated epi- and subretinal membranes showed similar staining patterns. 
tTgase mRNA and Protein.
Using the RT-PCR technique, we demonstrated the presence of tTgase mRNA in all investigated epi- and subretinal PVR membranes (Fig. 2) . The actual size of the PCR-product of tTgase was found to be close to the theoretically expected value (on the basis of the primer position 758 bp) and showed the expected sequence (data not shown). 
Immunohistochemical staining with antibodies against tTgase revealed specific staining in all PVR membrane sections studied (Figs. 3A 4A 4E) . Epi- and subretinal PVR membranes showed no differences in staining pattern. In both, staining was present throughout the ECM of the entire PVR membrane, but was most intense at the rim of the membrane, where staining for the enzyme lined the entire membrane. Within the membrane, staining occurred in a patchy pattern. In areas where pigmented cells were present (visualized by autofluorescence), staining for the enzyme was nearly absent (Fig. 3A)
Colocalization of tTgase and Fibronectin.
Staining for fibronectin was present throughout the entire ECM of the PVR membrane (Fig. 4B) . In some regions the rim of the membrane showed more intense staining than the central area of the PVR membrane. At intensely stained rim areas, a colocalization of tTgase and fibronectin was visible (Fig. 4C) . No differences between epi- and subretinal membranes were found. 
Reaction Product of tTgase and Colocalization to the Enzyme.
The staining pattern of the tTgase-catalyzed reaction product ε-(γ-glutamyl)-lysine bond (Figs. 3C 4F) was similar to the staining pattern of the enzyme (Figs. 3A 4E) . The monoclonal antibody against the end product of tTgase showed strong staining at the rim of the PVR membrane, whereas in the central area, the membrane showed only weak staining (Fig. 3C 4F) . No staining was visible around the pigmented cells (Fig. 3C) . Double staining for tTgase and ε-(γ-glutamyl)-lysine bonds showed a colocalization of the enzyme and its end product (Fig. 4G) . No differences in staining pattern between epi- and subretinal membranes were found. 
All control sections incubated without the primary antibody or with a combination of primary antibody and fivefold excess of tTgase were unstained (data not shown). 
Cell Culture of Human RPE-Cells
tTgase mRNA and Protein.
RT-PCR from native and cultured human RPE cells revealed a single band for tTgase cDNA (Fig. 5) . Semiquantitative analysis showed that the expression of tTgase cDNA in cultured dedifferentiated hRPE cells was five times higher than in freshly prepared native hRPE cells. Long-term cultured, differentiated hRPE cells showed a significant decrease in tTgase expression similar to native RPE cells (Fig. 5) . All investigated RPE preparations and cell cultures showed similar results. PCR performed on negative control samples, when the reverse transcriptase step was omitted, produced negative results (data not shown). 
tTgase Induction by TGF-β2.
Treatment of cultured hRPE cells with TGF-β2 markedly increased the level of tTgase mRNA (Figs. 6A 6B) . Northern blot analysis of untreated hRPE cells showed a single faint band after hybridization with an antisense tTgase RNA probe, which was 3.5 kb in length. The levels of tTgase mRNA in TGF-β2–treated RPE cells were approximately four to five times higher than those detected in the untreated control cells. In contrast, bFGF, IL-6, and IL-1β had no effect on tTgase expression in hRPE cells. The same results were obtained at the protein level. A single band was detected in Western blot analysis at the molecular mass of approximately 80 kDa (Fig. 6C) . Treatment with TGF-β2 increased the amount of tTgase in cultured RPE cells approximately fourfold. Treatment of the cells with bFGF, IL-6, and IL-1β had no effect. 
Extracellular tTgase Activity.
In the test for extracellular tTgase activity, a basal incorporation of biotinylated cadaverine into fibronectin was found after 10 to 120 minutes (Fig. 7) . This incorporation increased markedly, when hRPE cells were pretreated for 24 hours with 2.0 ng/mL TGF-β2 before seeding (Fig. 7) . Treatment of hRPE cells with bFGF, IL-6, and IL-1β for 24 hours before seeding had no effect on the extracellular tTgase activity compared with untreated control cells (Fig. 7)
Colocalization of tTgase and Fibronectin.
To demonstrate extracellular tTgase immunohistochemically, anti-tTgase antibody was added to the culture medium of unfixed hRPE cells. 42 Using this method, only a weak staining of tTgase was present in untreated hRPE cells (Fig. 8A) . Treatment with TGF-β2 increased the amount of extracellular tTgase (Fig. 8B) . Fibronectin also increased after TGF-β2 treatment (Fig. 8D) . Confocal imaging of double-stained sections revealed a partial colocalization of extracellular tTgase and fibronectin (Figs. 8C 8D)
Discussion
This study demonstrates transglutaminase activity and the presence of tTgase in PVR membranes. The almost identical staining pattern of tTgase and its end product ε-(γ-glutamyl)-lysine in PVR membranes together with the in situ incorporation of dansyl cadaverine indicates that the tTgase present in PVR membranes was functionally active. Active tTgase cross-links fibronectin, but also other ECM components such as vitronectin, laminin, nidogen, and collagen type III. 24 28 29 30 31 Because tTgase-catalyzed bonds are resistant to any known mammalian enzyme, they are resistant to the action of collagenases and other matrix-degrading enzymes such as MMPs. 21 22 23 In PVR membranes, the presence of degrading enzymes such as serine proteases and MMPs has been demonstrated. 43 44 45 The functional role of MMPs in PVR membrane formation includes degradation of matrix components and, subsequently, support of the invasion of cells into the membrane. 8 We found that tTgase staining was most prominent at the rim of the membranes, but was nearly absent around pigmented cells. It is therefore possible that invasion of RPE cells into the center of the PVR membrane is not inhibited by tTgase activity. Conversely, the rim and parts of the stroma showed colocalization of tTgase, ε-(γ-glutamyl)-lysine, and fibronectin, indicating that cross-linking of ECM occurred in these areas. This could explain the discrepancy between elevated degrading enzymes and an accumulation of ECM components in PVR membranes. 
Immunohistochemical staining pattern for tTgase and its end product, ε-(γ-glutamyl)-lysine, did not reveal any differences between epi- and subretinal PVR membranes. The most abundant cells in epiretinal membranes are pigment epithelial cells, 46 macrophage-like cells, glial cells, and fibroblast-like cells. 46 47 In contrast, subretinal membranes have been presumed to originate exclusively from RPE cells. 8 However, immunohistochemical analysis suggested the presence of glial cells beside RPE cells in subretinal PVR membranes. 48  
The finding that epi- and subretinal PVR membranes exhibit similar staining pattern for tTgase suggests that tTgase expression may be independent of the cellular composition of the PVR membranes. One explanation for this finding may be that all known cellular components of PVR membranes, such as macrophages, fibroblasts, astrocytes, and RPE cells, have been shown to be capable of transglutaminase synthesis. Macrophages and fibroblasts are known to produce tTgase. 49 50 Previous studies have shown that tTGase is expressed in brain astrocytes in vitro and is further induced by inflammation-associated cytokines. 51  
The finding that both, tTgase protein expression and activity, was observed in all PVR membranes suggests that the tTgase gene is continuously activated. Regulatory elements of the tTgase gene appear to be responsive to several cytokines. 52 53 54 In eyes affected with PVR, TGF-β2, bFGF, IL-6, and IL-1β are increased. 9 Cultured hRPE cells, a well-established in vitro model for PVR, 7 8 exposed to these factors showed increased tTgase expression and enhanced cross-linking of the ECM component fibronectin only after treatment with TGF-β2. The other cytokines had no effect on the expression or activity of tTgase. Previously, it has been shown that TGF-β also inhibits the expression of matrix degrading enzymes (plasminogen activators and MMP). 55 The combination of TGF-β−mediated increase in tTgase activity in context with TGF-β mediated decrease of MMP enzymes may underline the importance of this factor in the formation of PVR membranes. Finally, tTgase can activate latent TGF-β. 56 All these findings together indicate that a vicious circle may occur during the formation of PVR membranes. A constant level of active TGF-β2 seems most likely to be responsible for the increased tTgase activity, which in turn supports the activation of TGF-β. 
RT-PCR revealed the presence of a basal level of tTgase mRNA in native, differentiated RPE cells of normal human donors. Cultured, dedifferentiated hRPE cells showed a significant increase in tTgase mRNA, whereas differentiation of hRPE cells under culture conditions lead to a decrease in expression of tTgase mRNA to levels comparable to native hRPE cells. These results suggest that the expression of tTgase changes with the grade of hRPE cell differentiation and that low levels of tTgase in native hRPE cells may not be due to culturing trauma. These results further lead to the assumption that dedifferentiation of hRPE cells, as found in PVR membranes, may contribute to an increase in tTgase activity. 
In addition to the cross-linking of ECM, tTgase has some other functions including a support for cell adhesion 57 and a regulation of cell growth. 58 If transformation of epithelial RPE cells to fibroblast-like cells in vivo is also correlated with a marked increase in tTgase, the enzyme may also play an important role in the onset of proliferative vitreoretinopathy. 
Previously, it has been reported that topical treatment of incisional skin wounds with putrescine, a competitive inhibitor of transglutaminase cross-linking, caused decreased wound-breaking strength. 19 Furthermore, systemic administration of MDC or spermidine, also competitive inhibitors of transglutaminase activity, decreased healing of gastric and duodenal stress ulcers. 59 Some pharmaceutical compounds seem to inhibit transglutaminase, not only competitively, but irreversibly. 60 The use of such components may prevent or decrease the stable scarlike formation of ECM in PVR membranes and therefore offer new specific therapeutic strategies for protection against this major complication in rhegmatogenous retinal detachment. 
 
Figure 1.
 
Visualization of transglutaminase activity in an epiretinal PVR membrane. Transglutaminase activity was detected in unfixed PVR cryostat sections by incubation with the transglutaminase amine donor MDC in the presence of Ca2+. Incorporated MDC was visualized using an antidansyl antiserum and Cy3-conjugated secondary antibodies. (A) MDC was incorporated mainly at the border of the membrane. (B) Competition of MDC with putrescine or (C) inhibition of endogenous transglutaminase by EDTA prevents MDC incorporation to a large extent. (D) In the presence of exogenous tTgase (MDC+tTgase) incorporation of MDC is found in the whole PVR membrane, indicating the presence of substrates for this enzyme. Magnification: (A, B) ×150; (C, D) ×120.
Figure 1.
 
Visualization of transglutaminase activity in an epiretinal PVR membrane. Transglutaminase activity was detected in unfixed PVR cryostat sections by incubation with the transglutaminase amine donor MDC in the presence of Ca2+. Incorporated MDC was visualized using an antidansyl antiserum and Cy3-conjugated secondary antibodies. (A) MDC was incorporated mainly at the border of the membrane. (B) Competition of MDC with putrescine or (C) inhibition of endogenous transglutaminase by EDTA prevents MDC incorporation to a large extent. (D) In the presence of exogenous tTgase (MDC+tTgase) incorporation of MDC is found in the whole PVR membrane, indicating the presence of substrates for this enzyme. Magnification: (A, B) ×150; (C, D) ×120.
Figure 2.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from epiretinal (lane A) and subretinal (lane B) PVR membranes with a primer specific for tTgase. Total mRNA (1 μg) was used as template for first-strand synthesis, and cDNA was then amplified by PCR. Typical results are shown for experiments repeated at least two times. All investigated membranes showed similar results. PCR performed on negative controls, when the reverse transcriptase step was omitted, were negative (data not shown). MW: DNA standard.
Figure 2.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from epiretinal (lane A) and subretinal (lane B) PVR membranes with a primer specific for tTgase. Total mRNA (1 μg) was used as template for first-strand synthesis, and cDNA was then amplified by PCR. Typical results are shown for experiments repeated at least two times. All investigated membranes showed similar results. PCR performed on negative controls, when the reverse transcriptase step was omitted, were negative (data not shown). MW: DNA standard.
Figure 3.
 
Confocal micrographs showing immunohistochemical staining for tTgase (A) and the tTgase reaction product ε-(γ-glutamyl)-lysine isopeptide (C) in PVR membranes. The most intense staining was found at the rim of the membrane. Autofluorescence of granules within the RPE indicating pigmented cells stained green. In these areas staining for the enzyme and the isopeptide bond was nearly absent. Corresponding phase-contrast images are shown (B, D). Magnification, ×135.
Figure 3.
 
Confocal micrographs showing immunohistochemical staining for tTgase (A) and the tTgase reaction product ε-(γ-glutamyl)-lysine isopeptide (C) in PVR membranes. The most intense staining was found at the rim of the membrane. Autofluorescence of granules within the RPE indicating pigmented cells stained green. In these areas staining for the enzyme and the isopeptide bond was nearly absent. Corresponding phase-contrast images are shown (B, D). Magnification, ×135.
Figure 4.
 
Confocal micrographs showing immunohistochemical staining for (A, E) tTgase, (B) fibronectin (FN), and the (F) tTgase product ε-(γ-glutamyl)-lysine isopeptide in PVR membranes. The enzyme tTgase is colocalized to its substrate FN (C) and the enzyme reaction product ε-(γ-glutamyl)-lysine (G; yellow). Corresponding phase-contrast images are shown (D, H). Magnification, ×120.
Figure 4.
 
Confocal micrographs showing immunohistochemical staining for (A, E) tTgase, (B) fibronectin (FN), and the (F) tTgase product ε-(γ-glutamyl)-lysine isopeptide in PVR membranes. The enzyme tTgase is colocalized to its substrate FN (C) and the enzyme reaction product ε-(γ-glutamyl)-lysine (G; yellow). Corresponding phase-contrast images are shown (D, H). Magnification, ×120.
Figure 5.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from native, dedifferentiated fibroblast-like and long-term cultured, highly differentiated human RPE cells of passage 3 with a primer specific for tTgase. The mRNA expression of tTgase in hRPE was measured by semiquantitative RT-PCR. The optical density of the tTgase PCR products was expressed as the ratio to the GAPDH amplicon of the same cDNA sample. The number below each band shows the ratio of the optical density of the tTgase PCR product normalized to the GAPDH amplicon of the same cDNA. MW: DNA standard.
Figure 5.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from native, dedifferentiated fibroblast-like and long-term cultured, highly differentiated human RPE cells of passage 3 with a primer specific for tTgase. The mRNA expression of tTgase in hRPE was measured by semiquantitative RT-PCR. The optical density of the tTgase PCR products was expressed as the ratio to the GAPDH amplicon of the same cDNA sample. The number below each band shows the ratio of the optical density of the tTgase PCR product normalized to the GAPDH amplicon of the same cDNA. MW: DNA standard.
Figure 6.
 
(A) Northern blot analysis of tTgase mRNA in confluent human RPE cells 24 hours after treatment with either 2.0 ng/mL TGF-β2, 200 pg/mL bFGF, 320 pg/mL IL-6, or 5 pg/mL IL-1β. (B) Methylene blue staining of the 28 and 18S rRNA bands demonstrated relative integrity and loading of RNA. (C) Western blot analysis of tTgase in RPE monolayers treated as described for Northern blot. Lysates from approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band shows the chemiluminescent measurement. Co, control; RDI, relative densitometric intensity (normalized to 28S rRNA).
Figure 6.
 
(A) Northern blot analysis of tTgase mRNA in confluent human RPE cells 24 hours after treatment with either 2.0 ng/mL TGF-β2, 200 pg/mL bFGF, 320 pg/mL IL-6, or 5 pg/mL IL-1β. (B) Methylene blue staining of the 28 and 18S rRNA bands demonstrated relative integrity and loading of RNA. (C) Western blot analysis of tTgase in RPE monolayers treated as described for Northern blot. Lysates from approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band shows the chemiluminescent measurement. Co, control; RDI, relative densitometric intensity (normalized to 28S rRNA).
Figure 7.
 
Cell-mediated incorporation of biotinylated cadaverine into fibronectin by tTgase, using either untreated (Co.) or treated human RPE cells. Treated cells were incubated for 24 hours under serum-free conditions in the presence of TGF-β2, bFGF, IL-6, or IL-1β. Human RPE cells were plated (2 × 104 cells/well) in complete DMEM without serum in the presence of 0.1 mM biotinylated cadaverine. Cells were allowed to incubate in the fibronectin-coated plates for different periods at 37°C, and reaction were stopped by washing cells with PBS containing 3 mM EDTA. Color development was determined by using an ELISA plate reader set to 450 nm. Data are expressed as the mean results ± SEM of nine experiments with three different RPE cell cultures.
Figure 7.
 
Cell-mediated incorporation of biotinylated cadaverine into fibronectin by tTgase, using either untreated (Co.) or treated human RPE cells. Treated cells were incubated for 24 hours under serum-free conditions in the presence of TGF-β2, bFGF, IL-6, or IL-1β. Human RPE cells were plated (2 × 104 cells/well) in complete DMEM without serum in the presence of 0.1 mM biotinylated cadaverine. Cells were allowed to incubate in the fibronectin-coated plates for different periods at 37°C, and reaction were stopped by washing cells with PBS containing 3 mM EDTA. Color development was determined by using an ELISA plate reader set to 450 nm. Data are expressed as the mean results ± SEM of nine experiments with three different RPE cell cultures.
Figure 8.
 
Confluent human RPE cultures double stained for tTgase (red) and fibronectin (green). Staining was performed in cells after treatment with 2.0 ng/mL TGF-β2 for 24 hours (B, D) and in untreated control cultures (A, C). Extracellular staining for tTgase was much less pronounced in the untreated (A) than in the TGF-β2–treated (B) cells. After treatment with TGF-β2 the amount of extracellular fibronectin (green) increased markedly (D) compared with that in untreated control cells (C). In TGF-β2–treated cells numerous yellow-stained strands were observed between the cells, indicating an increase in colocalization of tTgase and fibronectin (D). Magnification, ×120.
Figure 8.
 
Confluent human RPE cultures double stained for tTgase (red) and fibronectin (green). Staining was performed in cells after treatment with 2.0 ng/mL TGF-β2 for 24 hours (B, D) and in untreated control cultures (A, C). Extracellular staining for tTgase was much less pronounced in the untreated (A) than in the TGF-β2–treated (B) cells. After treatment with TGF-β2 the amount of extracellular fibronectin (green) increased markedly (D) compared with that in untreated control cells (C). In TGF-β2–treated cells numerous yellow-stained strands were observed between the cells, indicating an increase in colocalization of tTgase and fibronectin (D). Magnification, ×120.
The authors thank Elke Lütjen-Drecoll for fruitful discussion and comments on the manuscript and Katja Obholzer and Harald Kroehn for expert technical assistance. 
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Figure 1.
 
Visualization of transglutaminase activity in an epiretinal PVR membrane. Transglutaminase activity was detected in unfixed PVR cryostat sections by incubation with the transglutaminase amine donor MDC in the presence of Ca2+. Incorporated MDC was visualized using an antidansyl antiserum and Cy3-conjugated secondary antibodies. (A) MDC was incorporated mainly at the border of the membrane. (B) Competition of MDC with putrescine or (C) inhibition of endogenous transglutaminase by EDTA prevents MDC incorporation to a large extent. (D) In the presence of exogenous tTgase (MDC+tTgase) incorporation of MDC is found in the whole PVR membrane, indicating the presence of substrates for this enzyme. Magnification: (A, B) ×150; (C, D) ×120.
Figure 1.
 
Visualization of transglutaminase activity in an epiretinal PVR membrane. Transglutaminase activity was detected in unfixed PVR cryostat sections by incubation with the transglutaminase amine donor MDC in the presence of Ca2+. Incorporated MDC was visualized using an antidansyl antiserum and Cy3-conjugated secondary antibodies. (A) MDC was incorporated mainly at the border of the membrane. (B) Competition of MDC with putrescine or (C) inhibition of endogenous transglutaminase by EDTA prevents MDC incorporation to a large extent. (D) In the presence of exogenous tTgase (MDC+tTgase) incorporation of MDC is found in the whole PVR membrane, indicating the presence of substrates for this enzyme. Magnification: (A, B) ×150; (C, D) ×120.
Figure 2.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from epiretinal (lane A) and subretinal (lane B) PVR membranes with a primer specific for tTgase. Total mRNA (1 μg) was used as template for first-strand synthesis, and cDNA was then amplified by PCR. Typical results are shown for experiments repeated at least two times. All investigated membranes showed similar results. PCR performed on negative controls, when the reverse transcriptase step was omitted, were negative (data not shown). MW: DNA standard.
Figure 2.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from epiretinal (lane A) and subretinal (lane B) PVR membranes with a primer specific for tTgase. Total mRNA (1 μg) was used as template for first-strand synthesis, and cDNA was then amplified by PCR. Typical results are shown for experiments repeated at least two times. All investigated membranes showed similar results. PCR performed on negative controls, when the reverse transcriptase step was omitted, were negative (data not shown). MW: DNA standard.
Figure 3.
 
Confocal micrographs showing immunohistochemical staining for tTgase (A) and the tTgase reaction product ε-(γ-glutamyl)-lysine isopeptide (C) in PVR membranes. The most intense staining was found at the rim of the membrane. Autofluorescence of granules within the RPE indicating pigmented cells stained green. In these areas staining for the enzyme and the isopeptide bond was nearly absent. Corresponding phase-contrast images are shown (B, D). Magnification, ×135.
Figure 3.
 
Confocal micrographs showing immunohistochemical staining for tTgase (A) and the tTgase reaction product ε-(γ-glutamyl)-lysine isopeptide (C) in PVR membranes. The most intense staining was found at the rim of the membrane. Autofluorescence of granules within the RPE indicating pigmented cells stained green. In these areas staining for the enzyme and the isopeptide bond was nearly absent. Corresponding phase-contrast images are shown (B, D). Magnification, ×135.
Figure 4.
 
Confocal micrographs showing immunohistochemical staining for (A, E) tTgase, (B) fibronectin (FN), and the (F) tTgase product ε-(γ-glutamyl)-lysine isopeptide in PVR membranes. The enzyme tTgase is colocalized to its substrate FN (C) and the enzyme reaction product ε-(γ-glutamyl)-lysine (G; yellow). Corresponding phase-contrast images are shown (D, H). Magnification, ×120.
Figure 4.
 
Confocal micrographs showing immunohistochemical staining for (A, E) tTgase, (B) fibronectin (FN), and the (F) tTgase product ε-(γ-glutamyl)-lysine isopeptide in PVR membranes. The enzyme tTgase is colocalized to its substrate FN (C) and the enzyme reaction product ε-(γ-glutamyl)-lysine (G; yellow). Corresponding phase-contrast images are shown (D, H). Magnification, ×120.
Figure 5.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from native, dedifferentiated fibroblast-like and long-term cultured, highly differentiated human RPE cells of passage 3 with a primer specific for tTgase. The mRNA expression of tTgase in hRPE was measured by semiquantitative RT-PCR. The optical density of the tTgase PCR products was expressed as the ratio to the GAPDH amplicon of the same cDNA sample. The number below each band shows the ratio of the optical density of the tTgase PCR product normalized to the GAPDH amplicon of the same cDNA. MW: DNA standard.
Figure 5.
 
Agarose gel electrophoresis and ethidium bromide staining of PCR products amplified from native, dedifferentiated fibroblast-like and long-term cultured, highly differentiated human RPE cells of passage 3 with a primer specific for tTgase. The mRNA expression of tTgase in hRPE was measured by semiquantitative RT-PCR. The optical density of the tTgase PCR products was expressed as the ratio to the GAPDH amplicon of the same cDNA sample. The number below each band shows the ratio of the optical density of the tTgase PCR product normalized to the GAPDH amplicon of the same cDNA. MW: DNA standard.
Figure 6.
 
(A) Northern blot analysis of tTgase mRNA in confluent human RPE cells 24 hours after treatment with either 2.0 ng/mL TGF-β2, 200 pg/mL bFGF, 320 pg/mL IL-6, or 5 pg/mL IL-1β. (B) Methylene blue staining of the 28 and 18S rRNA bands demonstrated relative integrity and loading of RNA. (C) Western blot analysis of tTgase in RPE monolayers treated as described for Northern blot. Lysates from approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band shows the chemiluminescent measurement. Co, control; RDI, relative densitometric intensity (normalized to 28S rRNA).
Figure 6.
 
(A) Northern blot analysis of tTgase mRNA in confluent human RPE cells 24 hours after treatment with either 2.0 ng/mL TGF-β2, 200 pg/mL bFGF, 320 pg/mL IL-6, or 5 pg/mL IL-1β. (B) Methylene blue staining of the 28 and 18S rRNA bands demonstrated relative integrity and loading of RNA. (C) Western blot analysis of tTgase in RPE monolayers treated as described for Northern blot. Lysates from approximately equal amounts of protein (2 μg) were separated by SDS-PAGE and blotted for immunochemical detection of tTgase content. The number below each band shows the chemiluminescent measurement. Co, control; RDI, relative densitometric intensity (normalized to 28S rRNA).
Figure 7.
 
Cell-mediated incorporation of biotinylated cadaverine into fibronectin by tTgase, using either untreated (Co.) or treated human RPE cells. Treated cells were incubated for 24 hours under serum-free conditions in the presence of TGF-β2, bFGF, IL-6, or IL-1β. Human RPE cells were plated (2 × 104 cells/well) in complete DMEM without serum in the presence of 0.1 mM biotinylated cadaverine. Cells were allowed to incubate in the fibronectin-coated plates for different periods at 37°C, and reaction were stopped by washing cells with PBS containing 3 mM EDTA. Color development was determined by using an ELISA plate reader set to 450 nm. Data are expressed as the mean results ± SEM of nine experiments with three different RPE cell cultures.
Figure 7.
 
Cell-mediated incorporation of biotinylated cadaverine into fibronectin by tTgase, using either untreated (Co.) or treated human RPE cells. Treated cells were incubated for 24 hours under serum-free conditions in the presence of TGF-β2, bFGF, IL-6, or IL-1β. Human RPE cells were plated (2 × 104 cells/well) in complete DMEM without serum in the presence of 0.1 mM biotinylated cadaverine. Cells were allowed to incubate in the fibronectin-coated plates for different periods at 37°C, and reaction were stopped by washing cells with PBS containing 3 mM EDTA. Color development was determined by using an ELISA plate reader set to 450 nm. Data are expressed as the mean results ± SEM of nine experiments with three different RPE cell cultures.
Figure 8.
 
Confluent human RPE cultures double stained for tTgase (red) and fibronectin (green). Staining was performed in cells after treatment with 2.0 ng/mL TGF-β2 for 24 hours (B, D) and in untreated control cultures (A, C). Extracellular staining for tTgase was much less pronounced in the untreated (A) than in the TGF-β2–treated (B) cells. After treatment with TGF-β2 the amount of extracellular fibronectin (green) increased markedly (D) compared with that in untreated control cells (C). In TGF-β2–treated cells numerous yellow-stained strands were observed between the cells, indicating an increase in colocalization of tTgase and fibronectin (D). Magnification, ×120.
Figure 8.
 
Confluent human RPE cultures double stained for tTgase (red) and fibronectin (green). Staining was performed in cells after treatment with 2.0 ng/mL TGF-β2 for 24 hours (B, D) and in untreated control cultures (A, C). Extracellular staining for tTgase was much less pronounced in the untreated (A) than in the TGF-β2–treated (B) cells. After treatment with TGF-β2 the amount of extracellular fibronectin (green) increased markedly (D) compared with that in untreated control cells (C). In TGF-β2–treated cells numerous yellow-stained strands were observed between the cells, indicating an increase in colocalization of tTgase and fibronectin (D). Magnification, ×120.
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