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Glaucoma  |   October 2012
A Novel Aptamer Targeting TGF-β Receptor II Inhibits Transdifferentiation of Human Tenon's Fibroblasts into Myofibroblast
Author Affiliations & Notes
  • Xiaoyan Zhu
    From the Department of Ophthalmology and the
  • Lei Li
    Molecular Biology Center Institute of Surgery Research, and the
  • Lingyun Zou
    Bioinformatics Center, College of Basic Medical Sciences, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China.
  • Xudong Zhu
    Molecular Biology Center Institute of Surgery Research, and the
  • Guangjun Xian
    From the Department of Ophthalmology and the
  • Haijun Li
    From the Department of Ophthalmology and the
  • Yan Tan
    Molecular Biology Center Institute of Surgery Research, and the
  • Lin Xie
    From the Department of Ophthalmology and the
  • Corresponding author: Lin Xie, Department of Ophthalmology, Daping Hospital, Third Military Medical University, No. 10, Changjiang Branch Road, Chongqing, People's Republic of China, 400042; xielin_zhou@hotmail.com
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 6897-6903. doi:10.1167/iovs.12-10198
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      Xiaoyan Zhu, Lei Li, Lingyun Zou, Xudong Zhu, Guangjun Xian, Haijun Li, Yan Tan, Lin Xie; A Novel Aptamer Targeting TGF-β Receptor II Inhibits Transdifferentiation of Human Tenon's Fibroblasts into Myofibroblast. Invest. Ophthalmol. Vis. Sci. 2012;53(11):6897-6903. doi: 10.1167/iovs.12-10198.

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

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Abstract

Purpose.: To isolate aptamers that were bound to the extracellular segment of TGF-β receptor II (TβRII) and evaluate their effect on the TGF-β–induced transdifferentiation of fibroblasts.

Methods.: TβRII-binding aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) from a single stranded DNA (ssDNA) library. Human Tenon's fibroblasts (HTFs) were cultured and treated with TGF-β2, TGF-β2 and aptamer S58/68, or aptamer S58/68 alone. Western blot analysis was performed to determine levels of α-smooth muscle actin (α-SMA) and the signaling protein phosphorylated Smad2 (p-Smad2). α-SMA and p-Smad2 subcellular distribution and fibrous actin (F-actin) with rhodamine-phalloidin staining were evaluated by confocal immunofluorescence microscopy. Cell contractility was assessed in collagen gel contraction assays.

Results.: Twenty-one sequences were obtained after eight rounds of selection. Two preferential sequences, aptamer S58 and S68, were isolated and used in the following experiments. Aptamer S58 significantly inhibited α-SMA expression and incorporation into actin stress fibers, as induced by TGF-β2. Aptamer S58 also suppressed TGF-β2–induced cell contraction. Furthermore, aptamer S58 inhibited the TGF-β2–induced phosphorylation and nuclear translocation of Smad2. However, we did not find any effect of aptamer S68 on TGF-β2 activity in vitro.

Conclusions.: Our study revealed that a novel aptamer binding TβRII inhibited TGF-β2–induced myofibroblast transdifferentiation in HTFs.

Introduction
Filtration surgery is principally used for glaucoma when IOP is not well controlled by medication. However, surgery is not always successful due to the occurrence of conjunctival scar at the surgical site. Clinically, mitomycin C (MMC) and 5-fluorouracil (5-FU) have been found to improve surgical outcomes remarkably. 13 However, their applications are limited because of severe side effects, even blindness. Therefore, it is necessary to search for new strategies that are safe and more effective in modulating wound healing and preventing scar formation. 
The cytokine TGF-β has been recognized as a pivotal mediator in wound healing and tissue repair. However, inappropriately high levels of TGF-β activity at wound sites are associated with excessive scarring and fibrosis. 4 Researchers have recognized the importance of antagonizing and blocking TGF-β to modulate wound healing. 58 These findings suggest that inhibiting the effects of TGF-β may reduce scar formation. There are three TGF-β isoforms in humans: TGF-β1, TGF-β2, and TGF-β3, of which TGF-β2 has been found to be the predominant isoform in ocular scarring diseases, such as conjunctival scarring and proliferative vitreoretinopathy. 911 The binding of a TGF-β ligand to a type II receptor dimer initializes the recruitment of the type I receptor dimer to form a heterotetrameric complex and catalyze the phosphorylation of the GS domain of the type I receptor. This leads to a downstream signaling cascade. 1214 Therefore, it may be an effective strategy to impair TGF-β activity through targeting the interaction split between TGF-β ligand and type II receptor. 
Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. 15,16 Aptamers are promising in biotechnological and therapeutic applications because of their high affinity and specificity to target molecules. 17 Recent developments in aptamer-based therapeutics have resulted in the first aptamer treatment approved by the US Food and Drug Administration. Pegaptanib sodium, an RNA aptamer directed against VEGF-165, is offered by OSI Pharmaceuticals (Lansdale, PA) for the treatment of age-related macular degeneration (AMD). 18,19  
The purpose of this study was to screen aptamers targeted against the extracellular domain of TGF-β receptor II (TβRII) and to search for novel molecules that were able to interfere with the binding of TGF-β to its receptor. A dominant sequence called aptamer S58 was isolated in this study. The sequence showed obvious antagonism against TGF-β2–induced myofibroblast (MF) transdifferentiation in human Tenon's fibroblasts (HTFs). Thus, aptamer S58 may provide a novel strategy to relieve scarring after glaucoma filtration surgery. 
Materials and Methods
Reagents
Recombinant TGF-β sRII/Fc was obtained from R&D Systems (Wiesbaden-Nordenstadt, Germany). First Strand cDNA Synthesis Kit, MAXIscript T7 Kit, and SUPERase.IN were purchased from Ambion Company (Austin, TX). Taq DNA polymerase, T4 polynucleotide kinase, pGEM-T Easy Vector System Kit, and DNA extraction kit were purchased from Promega Company (Madison, WI). [γ-32P] adenosine 5′-triphosphate (ATP) was purchased from Beijing Yahui Co., Ltd. (Beijing, China). Cell culture supplies were purchased from Hyclone (Logan, UT) and TGF-β2 was from PeproTech Company (Rocky Hill, NJ). Antibodies raised against the following proteins were used: α-SMA (Abcam, Hong Kong, China), phosphorylated Smad2 (Cell Signaling Technology, Inc., Beverly, MA), total Smad2 (Abcam), horseradish peroxidase-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology Co., Ltd., Peking, China), and conjugated goat anti-mouse (TRITC; Sigma, St. Louis, MO), phalloidin (Alexa-488; Invitrogen, San Diego, CA). All experiments were performed in triplication. 
Construction of Single Strand DNA (ssDNA) Random Library
The ssDNA oligonucleotides contained central region of 40 to 60 random nucleotides flanked by two conserved regions. 20 P108 library: 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-N60-CAGACGACTCGCCCGA-3′ (ESPEC OLIGO SERVICE CORP., Tsukuba, Japan), with a constant sequence at either end and a random sequence at the middle N60. PCR primers: sense 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′; antisense 5′-TCGGGCGAGTCGTCTG-3′. The random ssDNAs and primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). 
Screening of TGF-β sRII/Fc Protein Binding Aptamers
Aptamers were screened using methods previously described. 15,20 Briefly, the constructed ssDNAs (600 pmol) were incubated at 85°C for 15 minutes and placed on ice for 5 minutes. Then, samples were mixed with 20 μg TGF-β sRII/Fc protein in 200 μL binding buffer, including the following: 20 mmol/L Tris-HCl, pH 7.35; 137 mmol/L NaCl; 5 mmol/L KCl; 2 mmol/L CaCl2; and 1 mmol/L MgCl2. The samples were then incubated for 1.5 hours at 37°C. The mixture was filtered and washed with binding buffer three times through a 0.45-μm nitrocellulose membrane on a vacuum filter. Afterwards, the membrane was cut into pieces and processed to recover the ssDNA using 300 μL 7 M urea, 900 μL Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution. A dsDNA library was constructed by the gradient PCR amplification of ssDNA. The PCR system consisted of 0.2 μg ssDNA, 10 μL 10×PCR buffer, 8 μL deoxynucleotide triphosphates (dNTPs) (each 2.0 mmol/L), 8 μL MgCl2, 5 U Taq DNA polymerase, 100 pmol sense primer and 100 pmol antisense primer, which was brought to a total volume of 100 μL with deionized water. The conditions of PCR amplification consisted of 2 minutes of initial denaturation at 94°C, followed by 16 cycles of 30 seconds denaturation at 94°C, 30 seconds annealing at 55°C, 30 seconds extension at 72°C, and a final extension at 72°C for 10 minutes. The PCR amplification products were subjected to 8% urea denaturing PAGE containing 0.5 μg/mL ethidium bromide and viewed on a 260 nm fluoroscopic viewing plate. Once the band was obtained, DNA purification was performed using a DNA purification and recovery kit (Takara Biotech, Dalian, China). Some samples were subjected to asymmetric PCR amplification of double stranded DNA (dsDNA) to construct a ssDNA library, and the remaining DNA samples were stored for later use. The amplified ssDNAs were subjected to screening. The selection was repeated using the enriched pool derived from the first round of selection. 
For the first and second round of screening, the reaction volume was 200 μL, containing 600 pmol of random ssDNAs and 20 μg of TGF-β sRII/Fc. For the fourth to eighth rounds of screening, the reaction volume was 200 μL, containing 200 pmol of random ssDNAs and 2 μg of TGF-β sRII/Fc. 
Cloning and Sequencing
ssDNAs obtained after eight rounds of screening were amplified to dsDNAs by PCR under the same conditions described for screening. dsDNAs were then recovered and purified. The pGEM-T vector was ligated using the DNA ligation kit (Promega Company) according to the manufacturer's instructions, and the resulting products were used to transform Escherichia coli DH5a following the manufacturer's instructions. According to standardized procedure of Blue-white screening, 30 clones were selected randomly and applied to sequencing using ABI Prism 7000 Automated Sequence Detection System (Applied Biosystems, Foster City, CA). We synthesized the most representative sequences, called S58 and S68, according to the DNA sequences analysis. The aptamers were stored at −20°C until use. 
Analysis of Primary and Secondary Structures
The primary structure of oligonucleotide aptamers was analyzed using Clustal W software (in the public domain, http://www.clustal.org/clustal2). These aptamers were preliminarily classified according to sequence alignment results. Secondary structure analysis of some sequences was conducted using the DNA mfold program (in the public domain, http://www.bioinfo.rpi.edu/applications/mfold/). 
Determination of Specific Binding Affinity of Aptamers to TGF-β sRII/Fc
Aptamer sequences were synthesized, and the binding affinity of aptamers to TGF-β sRII/Fc was determined by radiolabeling. The nucleic acid aptamers were labeled with [γ-32P] ATP at the 5′ end. In the blank control group, nucleic acid aptamers (10 pmol) alone were used. In the total control group, aptamers (10 pmol) were added on the nitrocellulose membrane directly without vacuum filtration. Nucleic acid aptamers (10 pmol) and 100 ng/mL TGF-β sRII/Fc protein or bovine serum albumin (BSA) was mixed with binding buffer (reaction volume 100 μL) and incubated for 1 hour at 37°C. The reaction system was subjected to vacuum filtration through the nitrocellulose membrane. The nitrocellulose membrane was then washed with binding buffer, and residual radioactivity was determined using a liquid scintillation counter as counts per minute (CPM) (Beckman LS 5000; Beckman Coulter, Inc., Mississauga, Ontario, Canada). The affinity of ssDNA to the target molecule was calculated as a percentage (P): P = [(BG or TG)-BC]/TC × 100%, where BG is the radioactivity of BSA group remained on the membrane, TG is the remained radioactivity of TGF-β sRII/Fc protein group, BC is the radioactivity of Blank control group, and TC is the radioactivity of Total control group. Each sample was analyzed in triplicate. 
Cell Culture
Small Tenon's biopsy samples were obtained during strabismus surgeries. In total, five patients without a history of ocular surgery and other diseases except for strabismus were selected. The Tenon's biopsies were collected, minced, and placed in 12-well plates (15-mm culture dish) using Dulbecco's modified Eagle's medium (DMEM; Hyclone), which was supplemented with 10% fetal calf serum (Hyclone) and 100 U/mL of penicillin, and 100 μg/mL of streptomycin. HTFs were obtained as an expansion culture of the Tenon's biopsy samples, cells between passages 5 to 10 were used in all experiments. The study has been approved by the Institutional Review Board of Daping hospital, Third Military Medical University, Chongqing, China. All patients provided informed consent. The tenets of the Declaration of Helsinki were followed. 
Aptamers and TGF-β Treatments
Aptamers were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and stored at −20°C until use. Cells were allowed to reach 70% to 80% confluence and starved in serum-free DMEM for 24 hours. The fibroblasts were then treated with 2 ng/mL or 5 ng/mL of TGF-β2 (PEPROTECH) for the indicated time periods. The aptamer solution was diluted in DMEM and added to the cell culture system for 24 hours. 
Western Blot Analysis
After treatment, the cells were rinsed with ice-cold PBS, and total cell protein were extracted using RIPA lysis buffer (20 mM Tris, 150 mM NaCl, 1 Mm EDTA, 1% Triton X-100) containing phosphatase inhibitors and protease inhibitors. Protein concentrations were determined by a bicinchoninicacid (BCA) assay (KMF, Lohmar, Germany). Twenty micrograms of protein extracts were subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham, Braunschweig, Germany) using a Bio-Rad gel-blotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were blocked in 5% milk in Tris-Buffered Saline Tween (TBST; 10 mM Tris HCl, 150 mM NaCl, 0.1% Tween 20) for 1 hour, then incubated with primary antibody overnight at 4°C and with a peroxidase-conjugated secondary antibody for 1 hour at room temperature. After the incubations, membranes were washed in TBST for 30 minutes. Peroxidase was visualized by enhanced chemiluminescence (ECL; Millipore Company, Bedford, MA) and exposure to ECL film. 
Immunofluorescence Confocal Microscopy
HTF cells were plated in serum-free DMEM and incubated on glass coverslips in 6-well chamber slides for 24 hours to allow efficient attachment. Cells were treated with vehicle, TGF-β2, aptamers and TGF-β2, or aptamers alone for 24 hours. Cells were rinsed with ice-cold PBS, fixed in 4% paraformaldehyde for 20 minutes, permeabilized in 0.2% Triton X-100 for 10 minutes, and blocked in 5% normal goat serum for 1 hour. Then, HTFs were incubated with the primary antibody against human α-SMA (diluted 1:50) overnight at 4°C and rinsed with PBS for 30 minutes. A conjugated secondary antibody (TRITC; Zhongshan Goldenbridge Biotechnology Co., Ltd.) against mouse immunoglobulin G (IgG) was used at a 1:100 dilution in blocking buffer. Rhodamine-phalloidin (Alexa Fluor 488; Invitrogen) was used to stain fibrous actin (F-actin) cytoskeleton. Slides mounted with aqueous mounting medium containing 4′,6-diamidimo-2-phenylindole (DAPI) were observed using a laser scanning confocal microscope (TCS SP-2; Leica Microsystems, Bensheim, Germany). 
Cell Contraction Analysis
Fibroblast-populated collagen gels were made following previously described methods. 21 HTFs resuspended in serum-free DMEM (106/mL) were placed on ice. Type I collagen from a rat tail tendon (600 μL) was added to 36 μL of 0.1 mol/L NaOH in a centrifuge tube and then mixed with 69 μL 10×PBS and 2.3 mL of cells suspended DMEM to yield a final concentration of 150,000 cells/mL and 1 mg/mL collagen. The collagen cell suspension was added to 24-well plates, 600 μL in each well, and incubated at 37°C for 1 hour for polymerization. First, HTFs in the collagen solution were treated with vehicle or TGF-β2 at different concentrations (1, 2, 5, 10 ng/mL). Next, HTFs in the collagen solution were treated with vehicle, TGF-β2 (2 ng/mL), or TGF-β2 (2 ng/mL) and aptamers 58/68 (20 nM) for 48 hours. Gels were detached and digitally photo documented. Gels contraction quantification was performed using NIH image software (in the public domain, http://rsb.info.nih.gov/nih-image/index.html). 
Results
Relative Binding Rate of ssDNA Aptamers to TβRII
The affinity of aptamers to TβRII was determined by radioimmunoassay, and the relative binding affinity was proportional to radioactivity (CPM). In addition, the stringency of the selection was controlled by adding BSA. There was a significant difference in the affinity between the TβRII group and the BSA group from the fourth screening round (P < 0.05) (Fig. 1). The affinity of the final enriched library to the TβRII protein markedly increased to 26% compared with the affinity of the initial library, 1.76% (the Table, Fig. 1). After eight rounds of the screening procedure, the CPM value reached its peak, suggesting that TβRII-binding oligonucleotides were significantly enriched (Fig. 1). Thus, the SELEX procedure was terminated after eight screening rounds. 
Figure 1. 
 
Relative binding rate of ssDNA to TβRII. With increasing screening rounds, the residual radioactive nucleic acids on nitrocellulose membrane were enriched; BSA was used as control. There were no significant differences after eight rounds of screening, an indication of approaching saturation. BSA groups had no significant increase in the CPM value from the fourth screening rounds (P < 0.05), indicating no ssDNA enrichment. *P < 0.05 versus BSA group.
Figure 1. 
 
Relative binding rate of ssDNA to TβRII. With increasing screening rounds, the residual radioactive nucleic acids on nitrocellulose membrane were enriched; BSA was used as control. There were no significant differences after eight rounds of screening, an indication of approaching saturation. BSA groups had no significant increase in the CPM value from the fourth screening rounds (P < 0.05), indicating no ssDNA enrichment. *P < 0.05 versus BSA group.
Table. 
 
The Residual Radioactivity on the Nitrocellulose Membrane
Table. 
 
The Residual Radioactivity on the Nitrocellulose Membrane
Group ssDNA-0 ssDNA-2 ssDNA-4 ssDNA-6 ssDNA-8
Blank control 726 ± 126 1,123 ± 422 1,505 ± 233 4,764 ± 1,105 5,441 ± 1,003
BSA 1,949 ± 213 6,705 ± 660 16,168 ± 865 35,097 ± 1,257 32,923 ± 905
TβRII 2,306 ± 645 16,823 ± 1,789 41,900 ± 623 91,280 ± 898 92,410 ± 2,130
Total control 73,450 ± 526 184,630 ± 1,522 212,978 ± 1,748 374,590 ± 2,093 322,477 ± 2,365
Cloning, Sequencing, and Structural Analysis of Aptamers
After eight rounds of SELEX, the remaining variants were cloned and sequenced. After blue-white screening, 30 clones were randomly selected and sequenced, which resulted in 21 sequences that fell into two related families. An analysis of the primary structures demonstrated two sequences that were identical to aptamer S58, and two sequences that were identical to S68 (Fig. 2A). A secondary structure analysis of these sequences showed that the structures differed significantly between the two families. S58 had a special domain that was comprised of a slender stem-loop and a bubble structure formed by 26 bases. Aptamer S68 mainly consisted of two bubble structures linked by a stem-loop (Fig. 2B). 
Figure 2. 
 
Structure analysis of aptamers. The primary structure analysis demonstrated there were two classes superior sequence. There were two sequences identical to aptamer S58, and two sequences identical to S68 (A). The secondary structure analysis of aptamer S58/68 was conducted using the DNA mfold program (B).
Figure 2. 
 
Structure analysis of aptamers. The primary structure analysis demonstrated there were two classes superior sequence. There were two sequences identical to aptamer S58, and two sequences identical to S68 (A). The secondary structure analysis of aptamer S58/68 was conducted using the DNA mfold program (B).
Aptamers Targeting TβRII Impair TGF-β2–Induced SMA Expression in HTFs
To assess the effect of aptamers on TGF-β and its receptor, we measured TGF-β–induced α-SMA protein levels in the presence or absence of aptamers. TGF-β2 (2 ng/mL) significantly induced HTFs to express α-SMA after 24 hours of stimulation (Figs. 3A, 3B). At concentrations of 20 nM and 100 nM, aptamer S58 strongly diminished the TGF-β2–induced increase of α-SMA protein levels at 24 hours (Fig. 3A). However, we found that aptamer S58 alone induced HTFs minimally to express α-SMA at both concentrations of 20 nM and 100 nM (Fig. 3A). In contrast, α-SMA expression stimulated by TGF-β2 was unaffected by aptamer S68 (Fig. 3B). In addition, the scrambled aptamer sequence of S58 or S68, which was used as control, did not show any effect on TGF-β2–induced α-SMA expression (Figs. 3A, 3B). 
Figure 3. 
 
Effect of aptamer S58/68 on TGF-β2–induced α-SMA expression. HTFs were serum-starved overnight and incubated with vehicle, aptamer S58/68 (20 nM/100 nM) or aptamer S58/68 (20 nM/100 nM) with 2 ng/mL TGF-β2 for 24 hours. Scrambled aptamer sequence (Scr*) used as control. The expressions of α-SMA protein were analyzed by Western blot. Aptamer S58 blocked TGF-β2–induced α-SMA expression (A), whereas aptamer S68 did not show any effect (B).
Figure 3. 
 
Effect of aptamer S58/68 on TGF-β2–induced α-SMA expression. HTFs were serum-starved overnight and incubated with vehicle, aptamer S58/68 (20 nM/100 nM) or aptamer S58/68 (20 nM/100 nM) with 2 ng/mL TGF-β2 for 24 hours. Scrambled aptamer sequence (Scr*) used as control. The expressions of α-SMA protein were analyzed by Western blot. Aptamer S58 blocked TGF-β2–induced α-SMA expression (A), whereas aptamer S68 did not show any effect (B).
The Aptamers Prevent the Recruitment of α-SMA to Actin Atress Fibers
Incorporation of α-SMA into actin stress fibers as part of the contractile apparatus is a characteristic feature of MF differentiation. 22 In this study, we visualized the effect of aptamer S58/68 on TGF-β–induced α-SMA recruitment by confocal immunofluorescence microscopy. Strong actin stress fiber formation and recruitment of α-SMA to stress fibers were observed in HTFs after treatment with TGF-β2 (2 ng/mL) at 24 hours (Fig. 4B). Whereas a weak diffused cytoplasmic α-SMA signal and F-actin staining were detected with few intracellular stress fibers in the control group (Fig. 4A). In the presence of aptamer S58, the TGF-β2–induced incorporation of α-SMA into stress fibers and F-actin staining were significantly inhibited (Figs. 4C, 4D). Unlike S58, aptamer S68 only had a weak effect on TGF-β2 (Figs. 4E, 4F). 
Figure 4. 
 
Aptamers prevent the recruitment of α-SMA to actin stress fibers. HTFs were incubated with vehicle (A), 2 ng/mL of TGF-β2 (B), 20 nM of aptamer S58/S68 with TGF-β2 (D, F) or aptamer S58/S68 alone (C, E) for 24 hours. HTFs were stained for nuclei (DAPI, blue), α-SMA (red) or F-actin (Rhodamine-phalloidin, green). Compared with untreated cells (A), TGF-β2–induced α-SMA expression and recruitment to actin stress fibers in most cells that were of hypertrophy and spindle-shaped (B). In the presence of aptamer S58, the TGF-β2–induced incorporation of α-SMA into stress fibers has been significantly prevented and most cells were of thin fusiform (C, D). In contrast, aptamer S68 showed a weaker effect (E, F). Scale bar is 40 μm.
Figure 4. 
 
Aptamers prevent the recruitment of α-SMA to actin stress fibers. HTFs were incubated with vehicle (A), 2 ng/mL of TGF-β2 (B), 20 nM of aptamer S58/S68 with TGF-β2 (D, F) or aptamer S58/S68 alone (C, E) for 24 hours. HTFs were stained for nuclei (DAPI, blue), α-SMA (red) or F-actin (Rhodamine-phalloidin, green). Compared with untreated cells (A), TGF-β2–induced α-SMA expression and recruitment to actin stress fibers in most cells that were of hypertrophy and spindle-shaped (B). In the presence of aptamer S58, the TGF-β2–induced incorporation of α-SMA into stress fibers has been significantly prevented and most cells were of thin fusiform (C, D). In contrast, aptamer S68 showed a weaker effect (E, F). Scale bar is 40 μm.
The Aptamers Suppress TGF-β2–Induced Cell Contraction in Collagen Gels
To further explore the functional effects of the aptamers on TGF-β2, we assessed cell contractility in fibroblast-populated collagen gels. The results showed that TGF-β2 significantly induced the constriction of fibroblast-populated collagen gels in a concentration-dependent manner; however, no marked differences existed among 2, 5, 10 ng/mL concentrations (Figs. 5A, 5B). The untreated gel contracted 37% from its initial size, and TGF-β2 (2 ng/mL) apparently increased cells contractility (22% from the initial size). In contrast, aptamer S58 blocked the TGF-β2–induced increase in contractility (40% from the initial gel size). There were no significant differences between the aptamer S68 group and the TGF-β2 group (P > 0.05) (Figs. 5C, 5D). 
Figure 5. 
 
Aptamers block TGF-β2–induced contraction of HTFs populated collagen gels. HTFs were seeded in neutralized collagen (1 mg/mL) solution and incubated with vehicle or TGF-β2 at different concentrations (A), or with aptamer S58/68 (20 nM) with 2 ng/mL of TGF-β2 (C). Gels were digitally photo documented after 3 days (A, C) and measured as a reduction in gel surface area (B, D).
Figure 5. 
 
Aptamers block TGF-β2–induced contraction of HTFs populated collagen gels. HTFs were seeded in neutralized collagen (1 mg/mL) solution and incubated with vehicle or TGF-β2 at different concentrations (A), or with aptamer S58/68 (20 nM) with 2 ng/mL of TGF-β2 (C). Gels were digitally photo documented after 3 days (A, C) and measured as a reduction in gel surface area (B, D).
Effects of Aptamer S58 on Related Signaling Pathway in HTFs
It has been reported that TGF-β related factors induce a linear signaling pathway from the type II to the type I receptor kinase to Smad activation, which results in ligand-induced transcription. 23 In this study, we explored the influence of aptamer S58/68 on TGF-β–mediated signal transduction through measurement of Smad2 activation in vitro. Following TGF-β2 treatment for 60 minutes, phosphorylated Smad2 proteins (p-Smad2) of HTFs increased significantly. This effect was significantly attenuated by aptamer S58 (Figs. 6A, 6B). Aptamer S68 and the scrambled aptamer sequence of S58 or S68 did not show any effect (P > 0.05 versus TGF group) (Figs. 6A, 6B). Furthermore, we observed nuclear translocation of p-Smad2 on TGF-β2 stimulation influenced by aptamer S58 through confocal immunofluorescence microscopy. These results show that aptamer S58 inhibited the nuclear translocation of p-Smad2 induced by TGF-β2 (Figs. 6C–F). However, p-Smad2 proteins expression was not apparently influenced by aptamer S58 alone (Figs. 6A, 6D). 
Figure 6. 
 
Aptamer S58 reduced TGF-β2–induced p-Smad2 expression in HTFs. HTFs were treated with vehicle, TGF-β2 (2 ng/mL), or aptamer S58/S68 (20 nM) in presence or absence of TGF-β2 for 60 minutes. The scramble sequence of S58 or S68 was used as a control. Protein extracts were prepared and analyzed by Western blot for pSmad-2 expression (A, B; “*” indicates the scrambled sequence). HTFs stained for nuclei (DAPI, blue) and pSmad-2 (red) were visualized by confocal immunofluorescence microscopy (CF). TGF-β2–induced pSmad-2 translocation to the nucleus (E). Aptamer S58 (20 nM) attenuated TGF-β2–induced Smad2 phosphorylation (F). Scale bar is 20 μm.
Figure 6. 
 
Aptamer S58 reduced TGF-β2–induced p-Smad2 expression in HTFs. HTFs were treated with vehicle, TGF-β2 (2 ng/mL), or aptamer S58/S68 (20 nM) in presence or absence of TGF-β2 for 60 minutes. The scramble sequence of S58 or S68 was used as a control. Protein extracts were prepared and analyzed by Western blot for pSmad-2 expression (A, B; “*” indicates the scrambled sequence). HTFs stained for nuclei (DAPI, blue) and pSmad-2 (red) were visualized by confocal immunofluorescence microscopy (CF). TGF-β2–induced pSmad-2 translocation to the nucleus (E). Aptamer S58 (20 nM) attenuated TGF-β2–induced Smad2 phosphorylation (F). Scale bar is 20 μm.
Discussion
In this study, we screened the TβRII binding aptamers through modified SELEX technology from a nucleic acid library for the first time. Our study revealed two families of aptamer sequences. Meanwhile, we found that aptamer S58 significantly inhibited TGF-β2–induced MFs transdifferentiation in HTFs. 
SELEX has been widely used to screen a variety of target molecules. Our previous study reported that LPS activity inhibiting aptamers identified by modified SELEX significantly protected mice with endotoxemia. 20 In this study, aptamers were raised against soluble extracellular fragments of TβRII in vitro, and we evaluated the effects of selected aptamers on TGF-β–induced changes in HTFs. The key process in scar formation is the transdifferentiation of fibroblasts to MFs at wound sites with high contractility. 7,24,25 MFs exert increased contractile activity that is associated with the de novo expression of α-SMA, which is typically expressed in smooth muscle cells. 26,27 So we assessed the expression of α-SMA protein in HTFs, an important marker for MFs. Our results showed that aptamer S58 inhibited both the TGF-β–induced expression of α-SMA and the incorporation of α-SMA into actin stress fibers in HTFs. In addition, to explore the influence of aptamers in a functional assay, we measured the contraction of fibroblast-populated collagen gels. Increased gel contraction exerted by TGF-β–treated fibroblasts was abrogated by aptamer S58. Although the selected aptamers had high affinity for TβRII protein, their effects on TGF-β2 activity in HTFs were extremely different. We found that aptamer S68 did not show any effect. The sequences and structures of aptamers are closely associated with their bioactivity; hence, we postulate that the aptamers' function is related to their secondary structure. However, the relationship between aptamers, TGF-β2, and its receptor is complicated and still unclear. Additionally, aptamer S58 alone could induce α-SMA protein expression in HTFs minimally. This result is most likely due to nonspecific effects or other interactions between the aptamers and the receptor protein. Therefore, further studies on the relationship between structure and function of aptamers are needed to understand the relevant mechanism. 
Furthermore, we studied the signaling pathway that may be involved in the effect of aptamer S58 on TGF-β2 and its receptor. We focus on the TGF-β–induced Smad signaling pathway principally according to the previous researches. 12,13,28 Yamanaka et al. 29 reported that Smad7 gene introduction blocked Smad2/3 nuclear translocation with suppression of α-SMA and modulated injury-induced conjunctival wound healing. Xiao et al. 30 found that the ALK5 inhibitor SB-431542 showed an inhibitory effect against the stimulation of both TGF-β1 and -β2, and SB-431542 inhibited only the increase of p-Smad2. These findings indicate that the Smad signaling pathway could be a major pathway responsible for TGF-β2–induced α-SMA expression. Our study showed that aptamer S58 inhibited the expression of p-Smad2 resulting from TGF-β2. This indicates that aptamer S58 could inhibit TGF-β2 binding to its receptor (TβRII) to reduce its activity. It is worthwhile to point out that the HTFs investigated in this study were from younger donors. It is not clear whether the selected aptamers have similar effects on the HTFs from elderly persons. More studies are needed to address this issue. 
In conclusion, we have identified an aptamer targeted against TβRII that is able to block the activity of TGF-β2 on HTFs. Therefore, aptamer S58 might be useful as an inhibitory agent to investigate TβRII function, and it may have the potential to serve as a therapeutic agent for conjunctival scarring. 
Acknowledgments
The authors thank coworkers at the Molecular Biology Center, Research Institute of Surgery for sharing equipments and reagents. 
References
Krug JH Jr Melamed S. Adjunctive use of delayed and adjustable low-dose 5-fluorouracil in refractory glaucoma. Am J Ophthalmol . 1990;109:412–418. [CrossRef] [PubMed]
Abraham LM Selva D Casson R Leibovitch I. Mitomycin: clinical applications in ophthalmic practice. Drugs . 2006;66:321–340. [CrossRef] [PubMed]
Reinthal EK Denk PO Grub M Besch D Bartz-Schmidt KU. Dose, timing and frequency of subconjunctival 5-fluorouracil injections after glaucoma filtering surgery. Graefes Arch Clin Exp Ophthalmol . 2007;245:369–375. [CrossRef] [PubMed]
Branton MH Kopp JB. TGF-[beta] and fibrosis. Microbes Infect . 1999;1:1349–1365. [CrossRef] [PubMed]
Grisanti S Szurman P Warga M Decorin modulates wound healing in experimental glaucoma filtration surgery: a pilot study. Invest Ophthalmol Vis Sci . 2005;46:191–196. [CrossRef] [PubMed]
Mead AL Wong TTL Cordeiro MF Anderson IK Khaw PT. Evaluation of anti–TGF-beta 2 antibody as a new postoperative anti-scarring agent in glaucoma surgery. Invest Ophthalmol Vis Sci . 2003;44:3394–3401. [CrossRef] [PubMed]
Meyer-Ter-Vehn T Gebhardt S Sebald W p38 inhibitors prevent TGF-beta–induced myofibroblast transdifferentiation in human tenon fibroblasts. Invest Ophthalmol Vis Sci . 2006;47:1500–1509. [CrossRef] [PubMed]
Gabriel VA. Transforming growth factor-beta and angiotensin in fibrosis and burn injuries. J Burn Care Res . 2009;30:471–481. [CrossRef] [PubMed]
Pohlers D Brenmoehl J Loffler I TGF-beta and fibrosis in different organs-molecular pathway imprints. Biochim Biophys Acta . 2009;1792:746–756. [CrossRef] [PubMed]
Cordeiro MF. Role of transforming growth factor beta in conjunctival scarring. Clin Sci . 2003;104:181–187. [CrossRef] [PubMed]
Cordeiro MF Bhattacharya SS Schultz GS Khaw PT. TGF-beta1, -beta2, and -beta3 in vitro: biphasic effects on Tenon's fibroblast contraction, proliferation, and migration. Invest Ophthalmol Vis Sci . 2000;41:756–763. [PubMed]
Derynck R Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature . 2003;425:577–584. [CrossRef] [PubMed]
Song B Estrada KD Lyons KM. Smad signaling in skeletal development and regeneration. Cytokine Growth Factor Rev . 2009;20:379–388. [CrossRef] [PubMed]
Wrana JL Attisano L Wieser R Ventura F Massague J. Mechanism of activation of the TGF-beta receptor. Nature . 1994;370:341–347. [CrossRef] [PubMed]
Tuerk C Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science . 1990;249:505–510. [CrossRef] [PubMed]
Stoltenburg R Reinemann C Strehlitz B. SELEX–A (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng . 2007;24:381–403. [CrossRef] [PubMed]
Mairal T Ozalp VC Lozano Sánchez P Mir M Katakis I O'Sullivan CK. Aptamers: molecular tools for analytical applications. Anal Bioanal Chem . 2008;390:989–1007. [CrossRef] [PubMed]
Ng EWM Shima DT Calias P Cunningham ET Jr Guyer DR Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov . 2006;5:123–132. [CrossRef] [PubMed]
Doggrell SA. Pegaptanib: the first antiangiogenic agent approved for neovascular macular degeneration. Expert Opin Pharmacother . 2005;6:1421–1423. [CrossRef] [PubMed]
Wen AQ Yang QW Li JC Lv FL Zhong Q Chen CY. A novel lipopolysaccharide-antagonizing aptamer protects mice against endotoxemia. Biochem Biophys Res Commun . 2009;382:140–144. [CrossRef] [PubMed]
Meyer-Ter-Vehn T Katzenberger B Han H Grehn F Schlunck G. Lovastatin inhibits TGF-beta–induced myofibroblast transdifferentiation in human tenon fibroblasts. Invest Ophthalmol Vis Sci . 2008;49:3955–3960. [CrossRef] [PubMed]
Junker JPE Kratz C Tollbäck A Kratz G. Mechanical tension stimulates the transdifferentiation of fibroblasts into myofibroblasts in human burn scars. Burns . 2008;34:942–946. [CrossRef] [PubMed]
Derynck R Feng XH. TGF-[beta] receptor signaling. Biochim Biophys Acta . 1997;1333 :F105–F150. [PubMed]
Tomasek JJ McRae J Owens GK Haaksma CJ. Regulation of [alpha]-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-[beta]1 control element. Am J Pathol . 2005;166:1343–1351. [CrossRef] [PubMed]
Meyer-Ter-Vehn T Sieprath S Katzenberger B Gebhardt S Grehn F Schlunk G. Contractility as a prerequisite for TGF-beta-induced myofibroblast transdifferentiation in human tenon fibroblasts. Invest Ophthalmol Vis Sci . 2006;47:4895–4904. [CrossRef] [PubMed]
Hinz B Gabbiani G. Mechanisms of force generation and transmission by myofibroblasts. Curr Opin Biotechnol . 2003;14:538–546. [CrossRef] [PubMed]
Katoh K Kano Y Amano M Onishi H Kaibuchi K Fujiwara K. Rho-kinase–mediated contraction of isolated fibers. J Cell Biol . 2001;153:569–584. [CrossRef] [PubMed]
Yamanaka O Saika S Ohnishi Y Kim-Matsuyama S Kamaraju AK Ikeda K. Inhibition of p38MAP kinase suppresses fibrogenic reaction in conjunctiva in mice. Mol Vis . 2007;13:1730–1739. [PubMed]
Yamanaka O Ikeda K Saika S Miyazaki K Ooshima A Ohnishi Y. Gene transfer of Smad7 modulates injury-induced conjunctival wound healing in mice. Mol Vis . 2006;12:841–851. [PubMed]
Xiao YQ Liu K Shen JF Xu GT Ye W. SB-431542 inhibition of scar formation after filtration surgery and its potential mechanism. Invest Ophthalmol Vis Sci . 2009;50:1698–1706. [CrossRef] [PubMed]
Footnotes
 Supported by grants from the Founding of National Natural Science Foundation of China (81170852), Natural Science Foundation of Military (CWS11J137), and Natural Science Foundation of Chongqing (CSTC, 2010BB5199).
Footnotes
 Disclosure: X. Zhu, None; L. Li, None; L. Zou, None; X. Zhu, None; G. Xian, None; H. Li, None; Y. Tan, None; L. Xie, None
Figure 1. 
 
Relative binding rate of ssDNA to TβRII. With increasing screening rounds, the residual radioactive nucleic acids on nitrocellulose membrane were enriched; BSA was used as control. There were no significant differences after eight rounds of screening, an indication of approaching saturation. BSA groups had no significant increase in the CPM value from the fourth screening rounds (P < 0.05), indicating no ssDNA enrichment. *P < 0.05 versus BSA group.
Figure 1. 
 
Relative binding rate of ssDNA to TβRII. With increasing screening rounds, the residual radioactive nucleic acids on nitrocellulose membrane were enriched; BSA was used as control. There were no significant differences after eight rounds of screening, an indication of approaching saturation. BSA groups had no significant increase in the CPM value from the fourth screening rounds (P < 0.05), indicating no ssDNA enrichment. *P < 0.05 versus BSA group.
Figure 2. 
 
Structure analysis of aptamers. The primary structure analysis demonstrated there were two classes superior sequence. There were two sequences identical to aptamer S58, and two sequences identical to S68 (A). The secondary structure analysis of aptamer S58/68 was conducted using the DNA mfold program (B).
Figure 2. 
 
Structure analysis of aptamers. The primary structure analysis demonstrated there were two classes superior sequence. There were two sequences identical to aptamer S58, and two sequences identical to S68 (A). The secondary structure analysis of aptamer S58/68 was conducted using the DNA mfold program (B).
Figure 3. 
 
Effect of aptamer S58/68 on TGF-β2–induced α-SMA expression. HTFs were serum-starved overnight and incubated with vehicle, aptamer S58/68 (20 nM/100 nM) or aptamer S58/68 (20 nM/100 nM) with 2 ng/mL TGF-β2 for 24 hours. Scrambled aptamer sequence (Scr*) used as control. The expressions of α-SMA protein were analyzed by Western blot. Aptamer S58 blocked TGF-β2–induced α-SMA expression (A), whereas aptamer S68 did not show any effect (B).
Figure 3. 
 
Effect of aptamer S58/68 on TGF-β2–induced α-SMA expression. HTFs were serum-starved overnight and incubated with vehicle, aptamer S58/68 (20 nM/100 nM) or aptamer S58/68 (20 nM/100 nM) with 2 ng/mL TGF-β2 for 24 hours. Scrambled aptamer sequence (Scr*) used as control. The expressions of α-SMA protein were analyzed by Western blot. Aptamer S58 blocked TGF-β2–induced α-SMA expression (A), whereas aptamer S68 did not show any effect (B).
Figure 4. 
 
Aptamers prevent the recruitment of α-SMA to actin stress fibers. HTFs were incubated with vehicle (A), 2 ng/mL of TGF-β2 (B), 20 nM of aptamer S58/S68 with TGF-β2 (D, F) or aptamer S58/S68 alone (C, E) for 24 hours. HTFs were stained for nuclei (DAPI, blue), α-SMA (red) or F-actin (Rhodamine-phalloidin, green). Compared with untreated cells (A), TGF-β2–induced α-SMA expression and recruitment to actin stress fibers in most cells that were of hypertrophy and spindle-shaped (B). In the presence of aptamer S58, the TGF-β2–induced incorporation of α-SMA into stress fibers has been significantly prevented and most cells were of thin fusiform (C, D). In contrast, aptamer S68 showed a weaker effect (E, F). Scale bar is 40 μm.
Figure 4. 
 
Aptamers prevent the recruitment of α-SMA to actin stress fibers. HTFs were incubated with vehicle (A), 2 ng/mL of TGF-β2 (B), 20 nM of aptamer S58/S68 with TGF-β2 (D, F) or aptamer S58/S68 alone (C, E) for 24 hours. HTFs were stained for nuclei (DAPI, blue), α-SMA (red) or F-actin (Rhodamine-phalloidin, green). Compared with untreated cells (A), TGF-β2–induced α-SMA expression and recruitment to actin stress fibers in most cells that were of hypertrophy and spindle-shaped (B). In the presence of aptamer S58, the TGF-β2–induced incorporation of α-SMA into stress fibers has been significantly prevented and most cells were of thin fusiform (C, D). In contrast, aptamer S68 showed a weaker effect (E, F). Scale bar is 40 μm.
Figure 5. 
 
Aptamers block TGF-β2–induced contraction of HTFs populated collagen gels. HTFs were seeded in neutralized collagen (1 mg/mL) solution and incubated with vehicle or TGF-β2 at different concentrations (A), or with aptamer S58/68 (20 nM) with 2 ng/mL of TGF-β2 (C). Gels were digitally photo documented after 3 days (A, C) and measured as a reduction in gel surface area (B, D).
Figure 5. 
 
Aptamers block TGF-β2–induced contraction of HTFs populated collagen gels. HTFs were seeded in neutralized collagen (1 mg/mL) solution and incubated with vehicle or TGF-β2 at different concentrations (A), or with aptamer S58/68 (20 nM) with 2 ng/mL of TGF-β2 (C). Gels were digitally photo documented after 3 days (A, C) and measured as a reduction in gel surface area (B, D).
Figure 6. 
 
Aptamer S58 reduced TGF-β2–induced p-Smad2 expression in HTFs. HTFs were treated with vehicle, TGF-β2 (2 ng/mL), or aptamer S58/S68 (20 nM) in presence or absence of TGF-β2 for 60 minutes. The scramble sequence of S58 or S68 was used as a control. Protein extracts were prepared and analyzed by Western blot for pSmad-2 expression (A, B; “*” indicates the scrambled sequence). HTFs stained for nuclei (DAPI, blue) and pSmad-2 (red) were visualized by confocal immunofluorescence microscopy (CF). TGF-β2–induced pSmad-2 translocation to the nucleus (E). Aptamer S58 (20 nM) attenuated TGF-β2–induced Smad2 phosphorylation (F). Scale bar is 20 μm.
Figure 6. 
 
Aptamer S58 reduced TGF-β2–induced p-Smad2 expression in HTFs. HTFs were treated with vehicle, TGF-β2 (2 ng/mL), or aptamer S58/S68 (20 nM) in presence or absence of TGF-β2 for 60 minutes. The scramble sequence of S58 or S68 was used as a control. Protein extracts were prepared and analyzed by Western blot for pSmad-2 expression (A, B; “*” indicates the scrambled sequence). HTFs stained for nuclei (DAPI, blue) and pSmad-2 (red) were visualized by confocal immunofluorescence microscopy (CF). TGF-β2–induced pSmad-2 translocation to the nucleus (E). Aptamer S58 (20 nM) attenuated TGF-β2–induced Smad2 phosphorylation (F). Scale bar is 20 μm.
Table. 
 
The Residual Radioactivity on the Nitrocellulose Membrane
Table. 
 
The Residual Radioactivity on the Nitrocellulose Membrane
Group ssDNA-0 ssDNA-2 ssDNA-4 ssDNA-6 ssDNA-8
Blank control 726 ± 126 1,123 ± 422 1,505 ± 233 4,764 ± 1,105 5,441 ± 1,003
BSA 1,949 ± 213 6,705 ± 660 16,168 ± 865 35,097 ± 1,257 32,923 ± 905
TβRII 2,306 ± 645 16,823 ± 1,789 41,900 ± 623 91,280 ± 898 92,410 ± 2,130
Total control 73,450 ± 526 184,630 ± 1,522 212,978 ± 1,748 374,590 ± 2,093 322,477 ± 2,365
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